Porcine decellularized nerve matrix hydrogel attenuates neuroinflammation after peripheral nerve injury by inhibiting the TLR4/MyD88/NF-κB axis

Abstract

Peripheral nerve injury causes severe neuroinflammation and has become a global medical challenge. Previous research has demonstrated that porcine decellularized nerve matrix hydrogel exhibits excellent biological properties and tissue specificity, highlighting its potential as a biomedical material for the repair of severe peripheral nerve injury; however, its role in modulating neuroinflammation post–peripheral nerve injury remains unknown. Here, we aimed to characterize the anti-inflammatory properties of porcine decellularized nerve matrix hydrogel and their underlying molecular mechanisms. Using peripheral nerve injury model rats treated with porcine decellularized nerve matrix hydrogel, we evaluated structural and functional recovery, macrophage phenotype alteration, specific cytokine expression, and changes in related signaling molecules in vivo. Similar parameters were evaluated in vitro using monocyte/macrophage cell lines stimulated with lipopolysaccharide and cultured on porcine decellularized nerve matrix hydrogel–coated plates in complete medium. These comprehensive analyses revealed that porcine decellularized nerve matrix hydrogel attenuated the activation of excessive inflammation at the early stage of peripheral nerve injury and increased the proportion of the M2 subtype in monocytes/macrophages. Additionally, porcine decellularized nerve matrix hydrogel negatively regulated the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB axis both in vivo and in vitro. Our findings suggest that the efficacious anti-inflammatory properties of porcine decellularized nerve matrix hydrogel induce M2 macrophage polarization via suppression of the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB pathway, providing new insights into the therapeutic mechanism of porcine decellularized nerve matrix hydrogel in peripheral nerve injury.

Introduction

Peripheral nerve injury (PNI) is a prevalent condition that results in high morbidity and severe disability worldwide. It has been estimated that approximately one million patients with PNI per year globally suffer from persistent motor and sensory deficits and chronic pain, which greatly affects their quality of life (Noble et al., 1998; Taylor et al., 2008). Although the peripheral nervous system harbors a remarkable potential for regeneration, complete functional recovery after PNI is difficult to achieve (Nocera and Jacob, 2020; Ding et al., 2024; Cong et al., 2025). Presently, autologous nerve graft is the most frequent clinical approach for the reconstruction of segmental peripheral nerve defects (Sarker et al., 2018; Houshyar et al., 2019). However, this repair strategy has many critical drawbacks, including an inadequate supply of autologous nerve donor tissue, limited functional recovery, and potential for neuroma formation (Evans, 2000; Sarker et al., 2018; Beris et al., 2019). With the rise in neural tissue engineering technology, various biomaterials have been widely applied in biomedicine, such as hydrogels, and may serve as a substitute for nerve autografts (Li et al., 2021c; Bernard et al., 2023).

Native tissue–derived hydrogels are highly hydrophilic with cross-linked polymeric networks (Xia and Chen, 2022), acting as a hybrid/modified material for the delivery of stem cells and/or bioactive molecules to fill irregularly shaped defects and maintain tissue homeostasis (Xing et al., 2021; Saldin et al., 2017). Furthermore, natural hydrogels retain large amounts of decellularized extracellular matrix (ECM) components, including collagen, glycosaminoglycans, and proteoglycans, which provide adequate nutritional support for inducing cellular growth, migration, and differentiation (Giobbe et al., 2019; Brown et al., 2022). Natural hydrogels are becoming increasingly versatile for biomedical applications, with prepared scaffold products (e.g., AlloPatch® and Surgisis®) that achieve encouraging results in clinical practice (Crapo et al., 2011; Spang and Christman, 2018; Yao et al., 2019). Previously, we prepared an ECM hydrogel harvested from a xenogeneic porcine peripheral nerve source (porcine decellularized nerve matrix hydrogel [pDNM-gel]) that exhibited superior biocompatibility, bioactivity, and biodegradability (Lin et al., 2018). In vitro, pDNM-gel induced axonal elongation, Schwann cell proliferation, and stem cell differentiation (Li et al., 2021b; Wang et al., 2022). In the animal sciatic nerve injury model, it facilitated locomotor function recovery, nerve conduction velocity, and neurite myelination (Li et al., 2021b; Rao et al., 2021). Furthermore, these beneficial effects were enhanced by reconstituting pDNM-gel into a nerve guidance conduit (Zheng et al., 2021). Despite the therapeutic outcomes of pDNM-gel in the treatment of PNI, the molecular mechanisms underlying its neuroprotective effect are largely unexplored.

PNI triggers the Wallerian degeneration process, which involves a series of structural and morphological changes, including axonal disruption and myelin disintegration (Zhao et al., 2022). Following such degeneration, macrophages are recruited to phagocytose large amounts of cellular debris that accumulate in the distal injured nerve segment (Chen et al., 2015b). Meanwhile, the functional phenotypes of macrophages change to regulate inflammatory reactions. Generally, the pro-inflammatory response is induced by classically activated M1-subtype macrophages secreting various pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, aggravating neurotoxicity and tissue damage (Chimal-Ramírez et al., 2016; Shapouri-Moghaddam et al., 2018). Conversely, the anti-inflammatory response is mediated by alternatively activated M2-subtype macrophages that produce large amounts of anti-inflammatory molecules, such as IL-4, IL-10, and transforming growth factor (TGF)-β, reducing local neuroinflammation and facilitating nerve regeneration (Ganta et al., 2017; Shapouri-Moghaddam et al., 2018). Thus, macrophages play a distinct role in neuroimmunomodulation (Xiong et al., 2016). The conversion of macrophage polarization from an M1 to an M2 phenotype to exert anti-inflammatory and pro-healing functions is optimal for promoting axonal regeneration and neuronal remyelination in neurotraumatic diseases (Liu et al., 2021b; Tang et al., 2023).

Inflammation is affected by a variety of signaling pathways, including the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) pathway (Sun, 2017). Upon exogenous stimulation with factors such as lipopolysaccharide (LPS), TLR4 molecules dimerize and interact with MyD88 to activate IκB, leading to NF-κB separation, activation, and nuclear translocation, and ultimately enhancing pro-inflammatory gene transcription (Kuzmich et al., 2017). Many studies have established the involvement of the TLR4/MyD88/NF-κB axis in various conditions, including tumorigenesis, aging, tissue damage, and infection (Ropert et al., 2008; Lu et al., 2022; Liu et al., 2023). In the injured adult mammalian peripheral nerve system, the TLR4/MyD88/NF-κB axis participates in macrophage phenotype transformation and immunoregulation (Wang et al., 2023c). Specifically, in an active state, it induces M1 macrophage polarization and increases pro-inflammatory cytokine secretion, forming an inflammatory microenvironment to prevent nerve regeneration (Zhao et al., 2021a). Pharmacological blockade of this signaling pathway can inhibit macrophage polarization towards M1 and protect peripheral nerves from excessive inflammation-induced neuronal damage and functional deficits (Boivin et al., 2007). As such, selective intervention in the TLR4/MyD88/NF-κB axis may be a promising strategy to ameliorate neuroinflammation and nerve degeneration.

Reports on the regulatory effects of pDNM-gel on post-PNI neuroinflammation are rare. In this study, we examined the effects of pDNM-gel on monocyte/macrophage polarization and cytokine secretion. Additionally, we explored the participation of the TLR4/MyD88/NF-κB axis in these activities to better understand the molecular mechanisms of pDNM-gel in PNI repair.

Methods

Ethics statement

All experimental protocols involving animals followed the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, were approved by the Institutional Animal Care and Use Committee of Peking University on August 11, 2022 (Approval No. ER-0023-035), and are reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments; Percie du Sert et al., 2020). The rats were reared and surgeries were conducted in strict accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals.

Preparation of porcine decellularized nerve matrix hydrogel

The raw sciatic nerves used in this study were obtained from a total of 10 male adult miniature pigs (age, 12 months; body weight, 30–40 kg) purchased from the Guangdong Medical Laboratory Animal Center in Foshan, China (License No. SCXK (Yue) 2022-0002). The pDNM-gel was prepared as previously described (Kim et al., 2004; Kasper et al., 2020). Briefly, fresh sciatic nerves were removed from residual fat and connective tissue, washed with deionized water three times, and sliced into ~5-cm pieces. Any remaining cellular components were removed via infusion with 3.0% Triton X-100 and 4.0% sodium deoxycholate in a solvent mixture containing dichloromethane and ethanol (2:1, v/v). After freeze drying, the porcine decellularized nerve matrices were smashed into powder using a Thomas Wiley Mini-Mill (Thomas Scientific, Swedesboro, NJ, USA), followed by digestion in pepsin solution (1 mg/mL) at 25°C for 24 hours. The solution was centrifuged at 95,589 × g for 30 minutes at 4°C and the upper layer of solution was collected and stored –80°C for up to 1 year. To obtain pDNM-gel, HCl (0.1 M), NaOH (0.1 M), and 10 × phosphate-buffered saline were added to the digested solution and gelled at 37°C for 10 minutes.

Sciatic nerve crush injury model, administration, and grouping

A total of 74 specific pathogen-free level adult male Sprague-Dawley (SD) rats weighing 200–220 g were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. in Jiaxing, China (Animal License No. SYXK (Yue) 2020-0029). To avoid the potential for data variability owing to the estrus cycle of female rats, only male rats were used in this study (Shansky, 2019). Before the operation, rats were raised three per cage under a 12-hour light/dark cycle and given regular chow and water ad libitum for 1 week. The sciatic nerve crush injury model was established in accordance with our previous description (Li et al., 2017). Briefly, after anesthetization via intraperitoneal injection with 4% pentobarbital sodium (50 mg/kg; Sigma, St. Louis, MO, USA), the skin and muscles were opened with a tissue scissor (12.5 cm) to expose the right sciatic nerve. Next, two vascular clips (30 g force; Oscar, Shanghai, China) were used to clamp the sciatic nerve for 2 minutes. After removing the clips, the dissected musculature and the skin were sutured layer by layer using 4–0 silk sutures. To avoid infection, the surgical rats received an intraperitoneal injection of 8000 units of penicillin for 3 consecutive days.

The rats used in these experiments were divided into the following three groups using the random number table method: sham operation (Control group), PNI group, and PNI plus pDNM-gel treatment (pDNM-gel group). Each group contained ≥ 8 rats. Following the surgery, the pDNM-gel group rats were immediately administered a single-dose orthotopic injection of 200 μL pDNM-gel solution (1%) (Li et al., 2021b). Both the Control and PNI group rats received the same equal volume of normal saline. In the Control group, the sciatic nerve was exposed, but not clamped. At 3, 7, and 21 days post-surgery, the sciatic nerves were isolated and harvested for histological and pathological analyses. Additionally, to examine the role of the TLR4/MyD88/NF-κB axis in regulating pDNM-gel-treated nerve regeneration, we administered 0.25 mg/kg CRX-527 (Yang et al., 2020b) or 0.5 μg/kg CL075 (Jenei et al., 2023) to the damaged site in pDNM-gel-treated rats via orthotopic injection at 0, 7, and 14 days post-injury (dpi). At 21 dpi, the regenerated nerve fibers were harvested and examined via immunofluorescence and transmission electron microscopy (TEM). A study flow chart and experimental design, including the animal groups, treatment interventions, and applied methods, are provided in Additional Figures 1 ^{(2.8MB, tif)} and 2 ^{(1.7MB, tif)} .

Walking track analysis

To assess lower limb functional recovery, the walking behavior of each animal was measured pre-operatively (0 days), and at 3, 7, 14, and 21 dpi. Footprints were used to calculate the sciatic function index (SFI), which was previously described (de Medinaceli et al., 1982). SFI scores varied from 0 to –100, with an SFI of 0 ±11 representing normal sciatic nerve function and an SFI of –100 indicating total paralysis (Wang et al., 2023a). The two observers recording the results of this procedure were unaware of the group assignments of the rats.

Electrophysiological test

Electrophysiological analysis was conducted as described previously (Li et al., 2021b). Briefly, at 21 dpi, rats were anesthetized and the right sciatic nerve was carefully exposed. Two bipolar stimulating electrodes were placed on the proximal and distal ends of the regenerating nerve trunk. A recording electrode was inserted into the gastrocnemius muscle. The stimulating mode was set at supramaximal stimulation intensity, and the motor nerve conduction velocity and the compound muscle action potentials were measured using a BL-420N Data Acquisition & Analysis System (TECHMAN SOFT, Chengdu, China).

Transcriptome analysis

At 7 dpi, sciatic nerve tissues were collected, immediately immersed in TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA, Cat# 15596026), stored in dry ice, and sent to Shanghai APEXBIO Corporation (Shanghai, China) for RNA sequencing. The cDNA libraries were constructed using the TruSeq Stranded Total RNA LT Kit (Illumina Inc., San Diego, CA, USA). The gene expression levels of each sample in the Control and PNI groups (n = 3/group) were stabilized with log2(transcripts per million [TPM] + 1), and the differentially expressed genes (DEGs) were filtered using |log2(FoldChange)| > 1.5 and an adjusted P-value of < 0.05. Heatmap and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Seurat R package (version 4.1.1; https://www.r-project.org/) and the KEGG platform (https://www.genome.jp/kegg/), respectively.

Cell culture and treatment

The RAW264.7 macrophage cell line, which has been extensively used to study macrophage/monocyte differentiation, immunomodulation, and their potential mechanisms (Hartley et al., 2008), was purchased from the Cell Bank of Chinese Academy of Sciences Shanghai, China (Cat# 91062702, RRID: CVCL_0493) and cultured under previously described conditions (An et al., 2023).

At passage 3, the cells were divided into four treatment groups: Control, LPS, LPS + pDNM-gel, and pDNM-gel. The Control group was grown in the fresh RPMI 1640 medium (Gibco, Waltham, MA, USA, Cat# 11875093) containing 10% fetal bovine serum (Cat# 10270-106, Gibco) and 1% penicillin/streptomycin (P/S, 10,000 μg/mL, Sigma, Cat# P0781), which was termed normal culture medium, the LPS group in normal culture medium containing 100 ng/mL LPS (Sigma, Cat# L5293), the LPS + pDNM-gel group in normal culture medium containing LPS and pDNM-gel, and the pDNM-gel group in normal culture medium containing pDNM-gel. The RAW264.7 cells in these four groups were seeded in 6-well or 24-well plates. Before seeding cells, each well in the LPS + pDNM-gel and pDNM-gel groups was coated with 0.5% pDNM-gel solution and placed in an incubator (Thermo Fisher Scientific) at 37°C for 10 minutes to form gels. For 6-well plates, each well was pre-coated with 20 μL pDNM-gel solution. For 24-well plates, we first placed a glass coverslip in each well, which was then coated with 5 μL pDNM-gel. After growing the cells to 60%–70% density, they were incubated with LPS for 24 hours (Additional Figure 3 ^{(1.6MB, tif)} ).

To verify the participation of the TLR4/MyD88/NF-κB axis in macrophage phenotypic alteration and changes in cytokine secretion in pDNM-gel-treated cells, the RAW264.7 macrophage cells were seeded on the 24-well plate which had precoated-with pDNM-gel and cultured overnight. Then the medium was replaced with the fresh normal culture medium that contained 100 ng/mL LPS and the cells were cultured in this condition for another 24 hours. Then, the medium was added with one of the following agonists/inhibitors and treated the cells for a further 24 hours: 500 µM CRX-527 (InvivoGen, Toulouse, France, Cat# tlrl-crx527), 100 nM TAK242 (MedChemExpress, Monmouth Junction, NJ, USA, Cat# HY-11109), 2 µM CL075 (MedChemExpress, Cat# HY-117066), or 100 µM T6167923 (MedChemExpress, Cat# HY-19744; Additional Figure 3 ^{(1.6MB, tif)} ). In the LPS + pDNM-gel group, they were treated with LPS for 48 hours after culturing on pDNM-gel overnight.

Flow cytometry

The phenotype switch of RAW264.7 macrophages was detected by flow cytometry, using procedures that were previously described in detail (Liu et al., 2020). The cells were incubated with the following fluorescent antibodies for 1 hour at 37°C in a dark room: FITC anti-mouse CD68 (1:200, BioLegend, Beijing, China, Cat# 137005, RRID: AB_10575475), PE anti-mouse CD86 (1:500, BioLegend, Cat# 105007, RRID: AB_313150), and APC anti-mouse CD206 (1:500, BioLegend, Cat# 141707, RRID: AB_10896057). Data were obtained using a CytoFLEX flow cytometer (Attune NxT, Thermo Fisher Scientific). Double staining for CD86/CD68 and CD206/CD68 was quantified by manipulating BD FlowJo^TM 10 software (Treestar, Ashland, OR, USA).

Quantitative reverse transcription-polymerase chain reaction

The transcriptional changes in anti-inflammatory and pro-inflammatory genes in the RAW264.7 cells under different treatments were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA extraction, reverse transcription, amplification, and relative quantitation of the target gene were performed as described in our previous study (Li et al., 2021a). Briefly, total RNA was isolated from RAW264.7 cells using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Reverse transcription to synthesize cDNA was carried out on a CFX-Connect System (Bio-Rad, Hercules, CA, USA) using the RNA PCR kit (Life Technologies). The qRT-PCR conditions were as follows: denaturation at 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 45 seconds. The expression of target genes was calculated using the comparative ΔΔCt method and normalized to the β-actin gene. Gene-specific primer sequences are shown in Table 1. The reactions were performed in triplicate.

Table 1.

Primer sequences for quantitative reverse transcription-polymerase chain reaction

Gene	Primer sequence (5'–3')	Product size (bp)
TNF-α	F: CAG GCG GTG CCT ATG TCT C	89
	R: CGA TCA CCC CGA AGT TCA GTA G
IL-1β	F: GAA ATG CCA CCT TTT GAC AGT G	116
	R: TGG ATG CTC TCA TCA GGA CAG
IL-6	F: CTG CAA GAG ACT TCC ATC CAG	131
	R: AGT GGT ATA GAC AGG TCT GTT GG
IL-4	F: GGT CTC AAC CCC CAG CTA GT	102
	R: GCC GAT GAT CTC TCT CAA GTG AT
IL-10	F: CTT ACT GAC TGG CAT GAG GAT CA	101
	R: GCA GCT CTA GGA GCA TGT GG
TGF-β	F: TCT GCA TTG CAC TTA TGC TGA	100
	R: AAA GGG CGA TCT AGT GAT GGA
β-actin	F: GGC TGT ATT CCC CTC CAT CG	154
	R: CCA GTT GGT AAC AAT GCC ATG T

F: Forward; R: reverse.

Enzyme-linked immunosorbent assay

The levels of anti-inflammatory and pro-inflammatory cytokines in the culture supernatant or sciatic homogenate were quantified by enzyme-linked immunosorbent assay (ELISA) kits from Multi Sciences (Hangzhou, China), following the manufacturer’s standard protocol. A microplate reader (Thermo Fisher Scientific) was used to detect the optical density at 450 nm. The following ELISA kits were used to assay the indicated cytokines in cell culture medium: mouse TNF-α (Cat# EK282/4-AW1), mouse IL-1β (Cat# EK201B/3-AW1), mouse IL-10 (Cat# EK210/4-AW1), and mouse TGF-β (Cat# EK981-BW1). For assays of the rat sciatic nerve tissue, we used the following: rat TNF-α (Cat# EK382/3-96), rat IL-1β (Cat# EK301B/4-96), rat IL-10 (Cat# EK310/2-96), and rat TGF-β (Cat# EK981-96).

Immunofluorescence detection

The procedures for immunofluorescence staining of RAW264.7 cells and sciatic nerve longitudinal sections (10 μm), namely immobilization, permeabilization, blocking, sequential addition of primary and secondary antibodies (used only once in this study), nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), and image capture, were described in our previous study (Li et al., 2020). The primary and secondary antibodies used here were as follows: mouse anti-neurofilament-H (1:5000, Cell Signaling Technology, Danvers, MA, USA, Cat# 69485, RRID: AB_828308); rabbit anti-myelin basic protein (MBP; 1:1000, Abcam, Cambridge, UK, Cat# ab7349, RRID: AB_305869); mouse anti-CD68 (1:1000, Abcam, Cat# ab201340, RRID: AB_2920880); rabbit anti-inducible nitric oxide synthase (iNOS; 1:1000, Abcam, Cat# ab178945, RRID: AB_2861417); rabbit anti-arginase 1 (Arg1; 1:1000, Abcam, Cat# ab96183, AB_10678968); mouse anti-TLR4 (1:1000, Abcam, Cat# ab22048, RRID: AB_446735); rabbit anti-phosphorylated nuclear factor-κB (p-NF-κΒ; 1:5000, Abcam, Cat# 3033, RRID: AB_331284); rabbit anti-MyD88 (1:1000, Thermo Fisher Scientific, Cat# PA5-19919, RRID: AB_11154287); goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor^TM 488 (1:1000, Thermo Fisher Scientific, Cat# A-11008, RRID: AB_143165); and donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor^TM 568 (1:1000, Thermo Fisher Scientific, Cat# A10037, RRID: AB_11180865). Tissue sections and cells were incubated with primary antibodies overnight at 4°C, washed three times, then incubated with the corresponding secondary antibodies for 1 hour at 25°C. Under high‐power magnification (200× for tissues and 400× for cells), images were screened and captured under a Nikon confocal laser microscope (A1 PLUS, Nikon, Tokyo, Japan).

Transmission electron microscopy

Approximately 1 mm of sciatic nerve tissue within the crush site was immobilized in a 2.5% glutaraldehyde solution for 24 hours, then stained with 1% osmium tetroxide solution and 1% uranyl acetate for 1 hour each. After gradient dehydration with descending concentrations of acetone, the crushed portion of each sample was embedded in epoxy resin and cut into ultra-thin (50 nm) sections to receive staining with 1% uranium acetate citric acid. The staining sections were observed under a transmission electron microscope (H-600, HITACHI, Tokyo, Japan). The number and thickness of myelin sheaths in the different groups were calculated using ImageJ software (version 1.8.0, National Institutes of Health, Bethesda, MD, USA).

Image quantification

The fluorescence intensity and positive area were measured and quantified using ImageJ. To quantify the areas positive for neurofilament-H and MBP in each group, we measured the immunoreactivity to each within an area of 100 μm² using ImageJ. The percentages of iNOS⁺ and Arg1⁺ macrophages in injured sciatic nerve tissues were calculated by dividing the number of iNOS⁺ or Arg1⁺ cells by CD68⁺ cells. All cells labeled CD68⁺, iNOS⁺, and Arg1⁺ were counted using ImageJ. To obtain data on the relative fluorescence intensity of specific proteins, we used ImageJ to analyze the integrated density of iNOS, Arg1, TLR4, MyD88, and p-NF-κΒ positivity. The values were then normalized to the average value of the Control or LPS + pDNM-gel groups. At the tissue level, at least three randomly selected fields per section (n = 6 sections per animal) were used for statistical analysis. At the cellular level, six random photomicrographs per glass coverslip were captured, and each experiment was performed in triplicate.

Western blotting

The sciatic nerves in the lesion region and its distal zone were harvested at 3, 7, and 21 dpi. These tissues were homogenized using a PhD-LM25 homogenizer (Saint Paul, MN, USA), and the cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China, Cat# P0013B). Next, cellular and tissue protein concentrations were measured using a BCA assay kit (Beyotime Biotechnology, Cat# P0010). The protein suspensions of each sample were subjected to western blotting (WB) in accordance with our previously published protocols (Li et al., 2020). The following antibodies were used: rabbit anti-peripheral myelin protein 22 (Pmp22; 1:1000, Abcam, Cat# ab270400, RRID: AB_2376574); rabbit anti-growth-associated protein-43 (GAP43; 1:500, Affinity Biosciences, Jiangsu, China, Cat# DF7766, RRID: AB_2841232); rabbit anti-microtubule-associated protein-2 (MAP-2; 1:500, Affinity Biosciences, Cat# AF4081, RRID: AB_2835352); mouse anti-MBP (1:500, Affinity Biosciences, Cat# BF8010, RRID: AB_2356243); rabbit anti-iNOS (1:500, Affinity Biosciences, Cat# AF0199, RRID: AB_2833391); rabbit anti-CD86 (1:500, Affinity Biosciences, Cat# P42081, RRID: AB_2539526); rabbit anti-Arg1 (1:500, Affinity Biosciences, Cat# DF6657, RRID: AB_2838619); rabbit anti-CD206 (also called MRC1, 1:500, Affinity Biosciences, Cat# DF4149, RRID: AB_2836514); rabbit anti-TLR4 (1:500, Affinity Biosciences, Cat# AF7017, RRID: AB_2835322); rabbit anti-MyD88 (1:500, Affinity Biosciences, Cat# AF5195, RRID: AB_2837681); rabbit anti-p-NF-κB (1:500, Affinity Biosciences, Cat# AF2006, RRID: AB_2834435); mouse anti-NF-κB (1:500, Affinity Biosciences, Cat# BF8005, RRID: AB_2846809); rabbit-anti-p-IκBα (1:500, Affinity Biosciences, Cat# AF2002, RRID: AB_2834433); rabbit-anti-IκBα (1:500, Affinity Biosciences, Cat# AF5002, RRID: AB_2834792); mouse anti-GAPDH (1:1000, Proteintech, Philadelphia, PA, USA, Cat# 60004-1-Ig, RRID: AB_2107436); mouse anti-β-actin (1:1000, Proteintech, Cat# 81115-1-RR, RRID: AB_2923704); goat anti-rabbit IgG(H+L) HRP-linked secondary antibody (1:5000, Multi Sciences, Cat# GAR007, RRID: AB_2827833); and goat anti-mouse IgG(H+L) HRP-linked secondary antibody (1:5000, Multi Sciences, Cat# GAM007, RRID: AB_2927718). Blotting bands were detected using electrochemiluminescence (ECL) reagents (Thermo Fisher Scientific, Cat# 35050). Densitometric quantification was performed by ImageJ software and relative protein expression was normalized to GAPDH or β-actin.

Statistical analysis

No statistical methods were used to predetermine sample sizes; however, we employed sample sizes that were similar to those reported in previous publications (Wang et al., 2023b; Li et al., 2024). Data are expressed as mean ± standard error of the mean (SEM). Statistical analysis of data was carried out using GraphPad Prism 5 Software (GraphPad Software, San Diego, CA, USA). Procedures used for detailed analyses were described in our previous study (Li et al., 2020). Briefly, one-way analysis of variance followed by Bonferroni’s post hoc test was performed to compare multiple groups (n ≥ 3), and an unpaired t-test was used to analyze two groups. A P-value < 0.05 was considered to indicate a statistically significant difference.

Results

Porcine decellularized nerve matrix hydrogel alleviates peripheral nerve injury–induced axonal degeneration and functional deficits

First, we investigated the therapeutic effect of pDNM-gel on peripheral nerve recovery in terms of locomotor function, nerve conduction and structure, and related molecular changes. A walking track analysis was performed to obtain SFI values at 0, 3, 7, 14, and 21 days post-surgery. As shown in Figure 1A, all rats displayed normal pre-operative sciatic nerve function, but exhibited several hindlimb paralyses after PNI. Over the next few weeks, their locomotor function gradually improved. At 21 dpi in particular, model rats in the pDNM-gel group had significantly higher SFI values than those in the PNI group (P < 0.05; Figure 1A). Electrophysiological analysis revealed that, compared with the PNI group, the pDNM-gel group exhibited faster nerve conduction velocity (P < 0.05 for motor nerve conduction velocity; Figure 1B and C) and better recovery of the gastrocnemius muscle (P < 0.01 for CAMP; Figure 1B and D). Next, we co-stained the sciatic nerve tissue sections with the axon marker neurofilament-H and the myelin marker MBP. Immunological staining of the epicenter regions of the regenerated nerves showed that immunoreactivity to neurofilament-H and MBP in the pDNM-gel group was significantly stronger compared with that in the PNI group, but was weaker than that in the Control group (Figure 1E). Statistical analysis further revealed that the percentage of the area positive for neurofilament-H and MBP staining was significantly larger in the pDNM-gel group than in the PNI group (P < 0.001; Figure 1F and G). Similarly, there were more areas of positivity for neurofilament-H and MBP in both the proximal and distal sites of the injured nerves in the pDNM-gel group than in the PNI group, suggesting more effective repair of injured nerves under pDNM-gel treatment (P < 0.001; Additional Figures 4 ^{(3.2MB, tif)} and 5 ^{(4.9MB, tif)} ).

Figure 1.

pDNM-gel improves nerve regeneration and functional restoration in vivo.

(A) Quantitative analysis of SFI scores at 0, 3, 7, 14, 21 dpi in sciatic nerves from the Control, PNI, and pDNM-gel groups (n = 8/group). (B) Representative electrophysiological measurements in the three groups at 21 dpi (n = 8/group). (C, D) Quantitative analyses of MNCV and CMAP from B. (E) Representative immunofluorescence photomicrographs of the epicenter region of regenerative sciatic nerves in each group using anti-neurofilament-H (green) and anti-MBP (red) antibodies. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (F, G) Quantitation of neurofilament-H-positive and MBP-positive areas per 100 μm² in each group from E (n = 5/group). (H) WB detection of Pmp22, GAP43, MAP-2, and MBP. (I–L) Quantification of the expression of different proteins from H. WB experiments were performed in triplicate. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. CMAP: Compound muscle action potential; DAPI: 4′,6-diamidino-2-phenylindole; dpi: days post-injury; GAP-43: growth-associated protein-43; MAP-2: microtubule-associated protein-2; MBP: myelin basic protein; MNCV: the motor nerve conduction velocity; pDNM-gel: porcine decellularized nerve matrix hydrogel; Pmp22: peripheral myelin protein 22; PNI: peripheral nerve injury; SFI: sciatic function index; WB: western blotting.

The levels of growth regulation and remyelination-related proteins, namely Pmp22, GAP43, MAP-2, and MBP, were dramatically higher in regenerated nerves from the pDNM-gel group than in those from the PNI group (P < 0.01 for Pmp22, GAP43, and MAP-2; P < 0.05 for MBP; Figure 1H–L).

These findings indicated that pDNM-gel has a neuroprotective ability, as demonstrated by the efficient facilitation of morphological and functional recovery following PNI.

Porcine decellularized nerve matrix hydrogel induces M2 macrophage polarization and reduces the inflammatory response in vivo

Next, we evaluated the regulatory effects of pDNM-gel on macrophage polarization within the epicenter region of injured sciatic nerves at 3 and 7 dpi. Immunofluorescence analysis at each time point following PNI showed that, as the number of iNOS⁺CD68⁺ M1-type macrophages gradually decreased, the number of Arg1⁺CD68⁺ M2-type macrophages remarkably increased (Figure 2A). Under the administration of pDNM-gel, this phenotypic transformation trend was more pronounced (Figure 2A). Consistently, the percentage of iNOS⁺ macrophages was lower in pDNM-gel group rats than in PNI group rats (P < 0.05 at 3 dpi; P < 0.001 at 7 dpi; Figure 2B), while the percentage of Arg1⁺ macrophages in these two groups showed the opposite trend (P < 0.01 at 3 dpi; P < 0.001 at 7 dpi; Figure 2C). Furthermore, the trends in macrophage polarization at the proximal and distal sites of the injured nerves were consistent with those at the epicenter region. Specifically, samples from PNI rats with or without pDNM-gel treatment had a higher percentage of M2-subtype macrophages at 7 dpi than at 3 dpi, and there were more M2 macrophages in pDNM-gel rats than in PNI rats at the same time points (Additional Figures 6 ^{(3.8MB, tif)} and 7 ^{(3.8MB, tif)} ).

Figure 2.

pDNM-gel attenuates M1 macrophage activation and forms an anti-inflammatory microenvironment to promote peripheral nerve regeneration.

(A) Macrophage subtype analysis of immunofluorescence photomicrographs of longitudinal sciatic nerve sections within the epicenter region from PNI rats treated with/without pDNM-gel for 3 and 7 days. M1-subtype macrophages were iNOS⁺ (green) and CD68⁺ (red), M2-subtype macrophages were Arg1⁺ (green) and CD68⁺ (red), and M0 macrophages were CD68⁺ (red). Nuclei were stained with DAPI (blue). Scale bars: 10 µm. (B, C) Quantification showing the percentages of iNOS⁺ and Arg1⁺ macrophages in the different groups from A (n = 5/group). (D) Representative immunoblotting bands of iNOS, CD86, Arg1, and CD206 from PNI rats treated with/without pDNM-gel for 3 and 7 days. (E–H) Quantification of iNOS, CD86, Arg1, and CD206 data from D. (I, J) Quantification of ELISAs of the levels of various inflammatory cytokines from PNI rats treated with/without pDNM-gel for 3 and 7 days (n = 5/group). WB experiments were performed in triplicate. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Arg1: Arginase 1; CD206: cluster of differentiation protein 206; CD68: cluster of differentiation protein 68; CD86: cluster of differentiation protein 86; ELISA: Enzyme-linked immunosorbent assay; iNOS: inducible nitric oxide synthase; pDNM-gel: porcine decellularized nerve matrix hydrogel; PNI: peripheral nerve injury; WB: western blotting.

In WB analysis, the patterns of changes in the expression of M1 phenotypic markers (iNOS and CD86) and M2 phenotypic markers (Arg1 and CD206) between the PNI and pDNM-gel groups were similar to those in immunofluorescence analysis (Figure 2D). Specifically, at 3 dpi, PNI rats treated with pDNM-gel showed decreased protein levels of M1 macrophage markers compared with the PNI group (P < 0.05 for iNOS; P < 0.01 for CD86; Figure 2E and F) and increased protein levels of M2 macrophage markers compared with the PNI group (P < 0.001 for Arg1; P < 0.001 for CD206; Figure 2G and H). At 7 dpi, these differences were even more apparent (pDNM-gel group versus PNI group: P < 0.001 for iNOS, CD86, Arg1, and CD206; Figure 2E–H).

We also detected the levels of inflammatory cytokines at 3 and 7 dpi using ELISAs. As depicted in Figure 2I and J, the indicated inflammatory cytokines were upregulated at the early stage in all PNI rats, but were altered to varying degrees by pDNM-gel treatment. Specifically, the concentrations of pro-inflammatory cytokines TNF-α and IL-1β were significantly lower in PNI + pDNM-gel rats than in PNI rats (TNF-α: P < 0.001 at 3 dpi and P < 0.001 at 7 dpi; IL-1β: P < 0.01 at 3 dpi and P < 0.01 at 7 dpi; Figure 2I). In contrast, the concentrations of anti-inflammatory cytokines TGF-β and IL-10 were significantly higher in PNI + pDNM-gel rats than in PNI rats (TGF-β: P < 0.001 at 3 dpi and P < 0.001 at 7 dpi; IL-1β: P < 0.001 at 3 dpi and P < 0.001 at 7 dpi; Figure 2J).

Taken together, our data indicated that treatment with pDNM-gel boosts M2 macrophage polarization, thus minimizing the inflammatory response in rats suffering from sciatic nerve injury.

Porcine decellularized nerve matrix hydrogel inhibits activation of the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB axis in vivo

RNA sequencing and WB analyses were performed to identify potential molecular pathways by which pDNM-gel moderates macrophage polarization and the inflammatory response. KEGG enrichment analysis of DEGs between the Control and the PNI groups revealed the involvement of several signaling pathways, with the NF-κB signaling pathway in the top 10 most highly enriched pathways (Figure 3A). Furthermore, among the genes known to be involved in this pathway, all but Tnf were more highly expressed in the PNI group than in the Control group (Figure 3B). These genes included those with direct action on NF-κB (Malt1, Nfkb2, Nfkbia, and Tnfaip3), upstream mediators of NF-κB signaling (Traf1, Gadd45a, Gadd45b, and Ticam2), and downstream regulators of NF-κB signaling (Cxcl2, Il1r1, Eda2r, and Ticam2), with the remaining genes involved in immune and inflammatory responses via triggering by NF-κB related-signaling. Similarly, WB detected higher expression of TLR4/MyD88/NF-κB axis-related proteins, including TLR4, MyD88, p-NF-κB, and p-IκBα, in the PNI group compared with that in the Control group (Figure 3C). Administration of pDNM-gel markedly attenuated the detection of these signaling markers in WB, which was confirmed in quantitative analysis (pDNM-gel group versus PNI group: P < 0.05 for TLR4 and MyD88, and P < 0.01 for p-NF-κB/NF-κB ratio and p-IκBα/IκBα ratio; Figure 3D–G). These data indicated that pDNM-gel administration may assist with attenuating the activation of the TLR4/MyD88/NF-κB signaling pathway in vivo.

Figure 3.

TLR4/MyD88/NF-κB axis is involved in pDNM-gel-mediated macrophage polarization in vivo.

(A) Top 10 enriched pathways from KEGG analysis of DEGs in sciatic nerves from Control and PNI group rats (7 dpi; n = 3/group). (B) Heatmap of DEGs associated with the NF-κB signaling pathway in the Control and PNI groups. (C) Representative immunoblotting bands of TLR4, MyD88, p-NF-κB, NF-κB, p-IκBα, and IκBα in rat sciatic nerves from the Control, PNI, and pDNM-gel groups at 7 dpi. (D–G) Quantification of TLR4, MyD88, and the ratios of p-NF-κB to NF-κB and p-IκBα to IκBα from C. WB experiments were performed in triplicate. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. DEGs: Differentially expressed genes; IκBα: nuclear factor kappa B inhibitory protein alpha; KEGG: kyoto encyclopedia of genes and genomes; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; p-IκBα: phosphorylated nuclear factor kappa B inhibitory protein alpha; p-NF-κB: phosphorylated nuclear factor-κB; PNI: peripheral nerve injury; TLR4: Toll-like receptor 4; WB: western blotting.

Porcine decellularized nerve matrix hydrogel induces M2 polarization in RAW264.7 macrophages and enhances their anti-inflammatory response in vitro

To demonstrate that pDNM-gel could also accelerate M2 polarization of macrophages in vitro, we employed the immortalized murine RAW264.7 macrophage cell line, stimulated by LPS, with or without administration of pDNM-gel. Cell polarization and cytokine accumulation were then detected via immunofluorescence staining, flow cytometry, qRT-PCR, and ELISA. Immunofluorescence staining images showed that LPS induced abundant iNOS expression and blocked Arg1 production, but the reverse effect was observed when LPS-stimulating RAW264.7 cells were grown on the pDNM-gel surface (LPS + pDNM-gel group versus LPS group: P < 0.01 for iNOS immunoreactivity and P < 0.001 for Arg1 immunoreactivity; Figure 4A–D). Similarly, flow cytometry analysis demonstrated that culturing on pDNM-gel increased the percentage of M2 phenotype cells and decreased the percentage of M1 phenotype cells under LPS-stimulation (LPS + pDNM-gel group versus LPS group: P < 0.001 for CD86⁺/CD68⁺ cells and P < 0.001 for CD206⁺/CD68⁺ cells; Figure 4E–H).

Figure 4.

pDNM-gel inhibits M1 macrophage polarization and pro-inflammatory cytokine production in vitro.

(A, B) Dual immunofluorescence staining for iNOS (M1-subtype marker, green), Arg1 (M2-subtype marker, green), and CD68 (pan-macrophage marker, red) in RAW264.7 cells treated with/without LPS (100 ng/mL) and with/without culturing on pDNM-gel (0.5%) for 24 hours. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (C, D) Quantitative analysis of the fluorescence intensity of iNOS and Arg1 (relative to that in the Control group) in the groups from A and B. (E, F) Flow cytometry analysis to distinguish classically activated M1-phenotype from alternatively activated M2-phenotype macrophages. (G, H) Quantification of the proportions of CD86⁺/CD68⁺ (M1-phenotype) and CD206⁺/CD68⁺ (M2-phenotype) macrophages in the Control, LPS, LPS+pDNM-gel, and pDNM-gel groups from E and F. (I, J) qRT-PCR analysis of the mRNA expression levels of the TNF-α, IL-1β, IL-6, IL-4, IL-10, and TGF-β genes in the different groups. (K, L) ELISAs of the levels of TGF-β, IL-10, TNF-α, and IL-1β in RAW264.7 cell supernatants. All of these experiments were repeated at least three times. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Arg1: Arginase 1; CD206: cluster of differentiation protein 206; CD68: cluster of differentiation protein 68; CD86: cluster of differentiation protein 86; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; IL-10: interleukin-10; IL-1β: interleukin-1β; IL-4: interleukin-4; IL-6: interleukin-6; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharides; n.s.: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; qRT-PCR: quantitative reverse transcription polymerase chain reaction; TGF-β: transforming growth factor-beta; TNF-α: tumor necrosis factor-alpha.

The levels of pro- and anti-inflammatory genes and cytokines were evaluated to detect the classical features of macrophage polarization and their corresponding impacts. qRT-PCR analysis showed that LPS stimulation of RAW264.7 cells largely activated M1-like phenotype genes (Figure 4I). Specifically, expression levels of the TNF-α and IL-1β genes increased by nearly 8-fold, while those of the IL-6 gene increased by approximately 15-fold (LPS group versus Control group: P < 0.001). By contrast, under pDNM-gel protection, LPS-stimulated RAW264.7 cells showed upregulation of M2-related phenotype genes, including IL-4, IL-10, and TGF-β (LPS + pDNM-gel versus LPS group: P < 0.001 for IL-4, IL-10, and TGF-β; Figure 4J).

In agreement with the qRT-PCR analysis, ELISA data also showed that, compared with LPS-stimulated RAW264.7 cells, those treated with LPS and cultured on pDNM-gel exhibited significantly enhanced production of anti-inflammatory cytokines (P < 0.001 for TGF-β and IL-10; Figure 4K), and notably restricted secretion of pro-inflammatory cytokines (P < 0.01 for TNF-α and IL-1β; Figure 4L).

We also used the THP-1 monocyte cell line to evaluate the immunomodulatory effects of pDNM-gel. See Additional file 1 ^{(103.3KB, pdf)} for the cell culture and treatment details. Immunofluorescence analysis showed that cells in all of the experimental treatment groups were labeled with CD11b, indicating full differentiation of THP-1 monocytes into macrophages under phorbol myristate acetate induction. The fluorescence intensity of CD80⁺ (an M1-specific marker) was significantly enhanced by LPS stimulation compared with that in untreated Control cells, but this LPS effect was weakened in cells pre-cultured on pDNM-gel (Additional Figure 8 ^{(4MB, tif)} A). Interestingly, exposure to either pDNM-gel or IL-4 alone did not notably alter CD80 expression in THP-1 macrophages (Additional Figure 8 ^{(4MB, tif)} A). Positive fluorescence signals for CD206 (an M2-like marker) were stronger in cells exposed to LPS + pDNM-gel or IL-4 than in cells treated with or without LPS or pDNM-gel (Additional Figure 8 ^{(4MB, tif)} B). Additionally, compared with the Control, pDNM-gel, and IL-4 groups, the relative fluorescence intensity of CD80 was around 4.5-fold and 2.4-fold higher in the LPS and LPS + pDNM-gel groups, respectively (Additional Figure 8 ^{(4MB, tif)} C). The relative fluorescence intensity of CD206 was around 1.9-fold and 2.3-fold higher in the LPS + pDNM-gel and IL-4 groups compared with that in the Control group (Additional Figure 8 ^{(4MB, tif)} D). Similarly, we observed high secretion of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in THP-1 cells exposed to LPS (Additional Figure 8 ^{(4MB, tif)} E). By contrast, THP-1 cells cultured under LPS + pDNM-gel conditions induced abundant levels of the anti-inflammatory cytokines IL-10 and TGF-β, which were further increased by IL-4 stimulation (Additional Figure 8 ^{(4MB, tif)} F).

Collectively, these findings implied that culturing on pDNM-gel can drive monocyte/macrophage polarization towards an M2-like phenotype, activating anti-inflammatory effects in vitro.

Porcine decellularized nerve matrix hydrogel attenuates the lipopolysaccharide-mediated activation of the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB axis in vitro

As a canonical inflammatory transcriptional pathway, the TLR4/MyD88/NF-κB axis is essential for inducing M1 macrophage polarization and pro-inflammatory cytokine secretion (Abdollahi et al., 2023; Geng et al., 2023). Thus, we speculated that pDNM-gel-mediated activation, polarization, and functioning of macrophages might be involved in arresting this pathway. To confirm this hypothesis, we employed WB and immunofluorescence staining to detect the influence of pDNM-gel on this pathway in RAW264.7 cells. WB results showed that the protein levels of TLR4 and MyD88, and the phosphorylation of NF-κB and IκBα, were significantly induced by LPS stimulation, while pre-coating culture wells with pDNM-gel reduced these effects (Figure 5A). Quantitative analysis also revealed that the ratios of TLR4 to GAPDH, MyD88 to GAPDH, p-NF-κB to NF-κB, and p-IκBα to IκBα were markedly lower in the LPS + pDNM-gel group than in the LPS group (P < 0.001 for TLR4/GAPDH, MyD88/GAPDH, p-NF-κB/NF-κB, and p-IκBα/IκBα; Figure 5B–E). Similarly, immunofluorescence analysis of LPS-stimulated RAW264.7 cells revealed that culturing on pDNM-gel resulted in markedly lower percentages of positivity for TLR4, MyD88, and p-NF-κB (Figure 5F–I; LPS + pDNM-gel group versus LPS group: P < 0.001 for TLR4, MyD88, and p-NF-κB immunoreactivities). These results confirmed that pDNM-gel could suppress the activity of the TLR4/MyD88/NF-κB axis in LPS-stimulated RAW264.7 cells.

Figure 5.

Changes in expression of TLR4/MyD88/NF-κB signaling pathway components in RAW264.7 cells cultured on pDNM-gel and stimulated by LPS.

(A) Representative immunoblotting bands of TLR4, MyD88, p-NF-κB, NF-κB, p-IκBα, and IκBα in RAW264.7 cells treated with/without LPS (100 ng/mL) and with/without culturing on pDNM-gel (0.5%) for 24 hours. (B–E) Relative quantification of TLR4 and MyD88 protein content and p-NF-κB/NF-κB and p-IκBα/IκBα ratios from A. (F) Immunofluorescence staining of TLR4 (green), MyD88 (green), and p-NF-κB (green) in the Control, LPS, LPS + pDNM-gel, and pDNM-gel groups. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (G–I) Quantification of the fluorescence intensity of TLR4, MyD88, and p-NF-κB from F. Data are expressed as mean ± SEM from three independent assays in vitro; ***P < 0.001. DAPI: 4′,6-Diamidino-2-phenylindole; IκBα: nuclear factor kappa B inhibitory protein alpha; LPS: lipopolysaccharides; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; pDNM-gel: porcine decellularized nerve matrix hydrogel; p-IκBα: phosphorylated nuclear factor kappa B inhibitory protein alpha; p-NF-κB: phosphorylated nuclear factor-κB; TLR4: Toll-like receptor 4.

TLR4/MyD88/NF-κB pathway intervention affects pDNM-gel-mediated anti-inflammatory effects and nerve regeneration

Having determined that pDNM-gel could inhibit the TLR4/MyD88/NF-κB axis in vivo and in vitro, we next sought to identify whether components of this axis could override the pDNM-gel-mediated macrophage phenotypic switch and anti-inflammatory effects. First, we added TLR4/MyD88/NF-κB signaling agonists (CRX-527 and CL075) or inhibitors (TAK242 and T6167923) to the medium of RAW264.7 cell cultures. The toxicity profiles of these compounds were determined via Cell Counting Kit (CCK)-8 assays. As shown in Additional Figures 9 ^{(1.6MB, tif)} , 11 ^{(2.1MB, tif)} , 13 ^{(1.6MB, tif)} , and 15 ^{(1.8MB, tif)} , there was no significant cytotoxicity of CRX-527, TAK242, CL075, or T6167923 on RAW264.7 cells at concentrations below 1000 μM, 400 nM, 16 µM, and 400 nM, respectively. Furthermore, we used WB analysis to determine that the optimal doses of these four compounds for activating or suppressing the TLR4/MyD88/NF-κB signaling pathway were 500 μM for CRX-527, 100 nM for TAK242, 2 μM for CL075, and 100 μM for T6167923 (Additional Figures 10 ^{(2.7MB, tif)} , 12 ^{(2.1MB, tif)} , 14 ^{(2.2MB, tif)} , and 16 ^{(581.3KB, tif)} ). Under these treatment conditions, we found that the TLR4 agonist CRX-527 and the TLR4 inhibitor TAK242 increased and decreased the levels of TLR4 and its downstream molecules, including MyD88, p-NF-κB, and p-IκBα, respectively (Figure 6A–E). Similarly, the MyD88 agonist CL075 and the MyD88 inhibitor T6167923 promoted and inhibited the levels of MyD88, p-NF-κB, and p-IκBα, respectively (Figure 6A–E). Subsequently, we examined macrophage polarization in each group via co-immunostaining and flow cytometry. The addition of the agonists CRX-527 and CL075 increased the fluorescence intensity of iNOS and reduced that of Arg1 (Figure 6F–I), while the addition of inhibitors TAK242 and T6167923 had a tendency for the opposite effect (Figure 6F–I). The flow cytometry assays of the percentages of M1-like macrophages (CD86⁺CD68⁺) and M2-like macrophages (CD206⁺CD68⁺) were consistent with the results of co-immunostaining (Figure 6J–M). Finally, qRT-PCR and ELISA analyses showed that stimulation with CRX-527 and CL075 significantly upregulated the expression of pro-inflammatory-related genes (Figure 6N) and the secretion of corresponding cytokines (Figure 6P), while downregulating the expression of anti-inflammatory-related genes (Figure 6O) and the secretion of anti-inflammatory cytokines (Figure 6Q). These effects were reversed in RAW264.7 cells treated with TAK242 and T6167923 (Figure 6N–Q).

Figure 6.

TLR4/MyD88/NF-κB pathway plays an indispensable role in regulating pDNM-gel-mediated macrophage polarization and anti-inflammatory reactions in vitro.

(A) WB detection of TLR4, MyD88, p-NF-κB, NF-κB, p-IκBα, and IκBα in RAW264.7 cells treated with LPS (100 ng/mL) + pDNM-gel (0.5%) for 24 hours, followed by the addition of CRX-527 (500 µM), TAK242 (100 nM), CL075 (2 µM), or T6167923 (100 µM) for another 24 hours. (B–E) Quantitative analysis of the expression levels of these four proteins from A. (F, G) Co-staining of CD68 (red) with iNOS (green) or Arg1 (green) in the LPS + pDNM-gel, LPS + pDNM-gel + CRX-527, LPS + pDNM-gel + TAK242, LPS + pDNM-gel + CL075, and LPS + pDNM-gel + T6167923 treatment groups. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (H, I) Quantification of the fluorescence intensity of iNOS and Arg1 from F and G. (J, K) Flow cytometry detection of M1-type and M2-type macrophages in each group. (L, M) Quantitative analysis of the percentages of double-positive CD68⁺CD86⁺ and CD68⁺CD206⁺ cells from J and K. (N, O) qRT-PCR analysis of the mRNA expression of pro-inflammatory signature genes TNF-α, IL-1β, and IL-6, and anti-inflammatory signature genes IL-4, IL-10, and TGF-β, in each group. (P, Q) ELISAs of inflammatory cytokine levels in RAW264.7 cell culture supernatants in each group. Data are expressed as mean ± SEM from three independent assays in vitro; *P < 0.05, **P < 0.01, ***P < 0.001. Arg1: Arginase 1; CD206: cluster of differentiation protein 206; CD68: cluster of differentiation protein 68; CD86: cluster of differentiation protein 86; DAPI: 4′,6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; IL-10: interleukin-10; IL-1β: interleukin-1β; IL-4: interleukin-4; IL-6: interleukin-6; iNOS: inducible nitric oxide synthase; IκBα: nuclear factor kappa B inhibitory protein alpha; LPS: lipopolysaccharides; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; n.s,: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; p-IκBα: phosphorylated nuclear factor kappa B inhibitory protein alpha; p-NF-κB: phosphorylated nuclear factor-κB; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; TGF-β: transforming growth factor-beta; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-alpha.

Next, we sought to examine the effects of these agonists and inhibitors of TLR4/MyD88/NF-κB signaling in vivo, using immunofluorescence and TEM. Immunofluorescence analysis showed that the positive effects of pDNM-gel on axonal and myelin regeneration were markedly suppressed by treatment of rats with CRX-527 or CL075 (Figure 7A). Statistical analysis revealed larger areas of positive staining for neurofilament-H and MBP in sciatic nerve sections of rats treated with pDNM-gel compared with those treated with pDNM-gel plus CRX-527 or CL075 at 21 dpi (Figure 7C and D; neurofilament-H: P < 0.001; MBP: P < 0.001). Consistently, TEM of the ultrastructure of sciatic nerve tissues showed that both agonists significantly increased the number and thickness of newborn myelin sheaths in nerve-contused rats receiving pDNM-gel treatment (Figure 7B, E, and F; number of myelin sheaths: P < 0.001; thickness of myelin sheaths: P < 0.01).

Figure 7.

Promotional effect of pDNM-gel on nerve regeneration is abolished using TLR4/MyD88/NF-κB agonists in vivo.

(A) Immunofluorescence staining for neurofilament-H (green) and MBP (red) in sciatic nerves from rats treated with pDNM-gel, pDNM-gel + CRX-527, or pDNM-gel + CL075 at 21 dpi (n = 5/group). Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (B) Representative TEM images of regenerated sciatic nerve cross-sections from the three groups at 21 dpi. Scale bars: 10 μm. (C, D) Calculated area (%) of positive staining of neurofilament-H and MBP per 100 μm² in the immunofluorescence images for the three groups (n = 5/group). (E, F) Statistical analysis of the number and thickness of myelin sheaths in the three groups using ImageJ software (n = 3/group). Data are expressed as mean ± SEM; **P < 0.01, ***P < 0.001. DAPI: 4′,6-Diamidino-2-phenylindole; dpi: days post-injury; MBP: myelin basic protein; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; pDNM-gel: porcine decellularized nerve matrix hydrogel; PNI: peripheral nerve injury; SEM: standard error of the mean; TEM: transmission electron microscopy; TLR4: Toll-like receptor 4.

Taken together, our findings suggested that pDNM-gel-mediated anti-inflammatory reactions and nerve regeneration were negatively regulated by the TLR4/MyD88/NF-κB axis.

Discussion

Although previous preclinical studies have demonstrated the promise of pDNM-gel as a regenerative biomaterial for repairing PNI (Liu et al., 2021a; Zheng et al., 2021), whether this beneficial effect was related to immunoregulation remains unknown. In the present study, we revealed that pDNM-gel possesses anti-inflammatory activity, manifesting as suppressed secretion of pro-inflammatory genes and cytokines, and enhanced expression of anti-inflammatory genes and cytokines. Furthermore, we found that this activity results from augmented M2 macrophage polarization, which likely results from suppressed activation of the TLR4/MyD88/NF-κB axis. These findings offer a new mechanistic insight into pDNM-gel-mediated enhancement of peripheral nerve regeneration.

Neuroinflammation is thought to play a crucial role in the progression of PNI (Gaudet et al., 2011). An excessive or prolonged inflammatory response forms an unbalanced microenvironment that aggravates neuronal apoptosis, axonal degeneration, demyelination, and neuropathic pain (Sommer et al., 2018; Kalinski et al., 2020). Thus, alleviating the local inflammatory microenvironment in the injured nerve segment is an effective therapeutic approach for improving the structural and functional recovery of peripheral nerves (Chen et al., 2015b; Zhang et al., 2020). In the peripheral nervous system, the degree of the inflammatory response is mediated by a variety of immune cells, including macrophages, monocytes, and microglia, with macrophages playing a crucial role (Wu et al., 2017; Zigrnond and Echevarria, 2019). Macrophages exert distinct functions through alteration of their phenotypes, with the M1-phenotype exerting pro-inflammatory functions and the M2 phenotype inhibiting inflammatory reactions (Chen et al., 2015b; Liu et al., 2019). Polarization of macrophages into these two phenotypes can produce different pro- and anti-inflammatory cytokines, which are crucial for regulating the dynamic changes of the PNI microenvironment (Cheng et al., 2021; Fu et al., 2024; Kou et al., 2024). Modulation of macrophage phenotypes to construct an appropriate microenvironment in injured areas has become a promising therapeutic solution for PNI repair (Tomlinson et al., 2018; Dervan et al., 2021). For instance, Li et al. (2022) found that LPS-preconditioned mesenchymal stem cells could effectively shift the pro-inflammatory M1 phenotype into a pro-regenerative M2 phenotype to promote injured nerve regeneration. Additionally, a bioceramic-based nerve guidance conduit showed excellent therapeutic effects on axonal regeneration via increasing M2-like macrophage infiltration in a 10-mm critically sized rat nerve defect model (Xuan et al., 2024). Interestingly, in this study, we found that pDNM-gel could effectively induce M2 macrophage polarization and increase the expression of anti-inflammatory cytokines at the early stage of PNI. In vitro, pDNM-gel showed a similar function, enhancing macrophage/monocyte phenotype transition from M1/M0 to M2 and decreasing the excessive secretion of pro-inflammatory cytokines. These findings demonstrated that pDNM-gel has anti-inflammatory properties that can assist in PNI repair.

Next, we attempted to elucidate how pDNM-gel exerts its anti-inflammatory activity. From the perspective of ingredient analysis, pDNM-gel retains an abundance of ECM proteins and some residual growth factors (Meder et al., 2021). These biomacromolecules are considered to influence its immunomodulatory and inflammatory suppressive functions. As one type of ECM protein, collagen VI has been demonstrated to induce macrophage migration and M2 subtype transformation (Chen et al., 2015a). Previously, we detected fibroblast growth factor 2 in the pDNM-gel using mass spectrometry-based proteomics (Li et al., 2021b). This mitogenic protein belongs to the growth factor family and is highly effective in promoting macrophage polarization from M1 to M2 for wound repair (Wu et al., 2021). Thus, we suspected that these specific protein components and other unknown components retained within pDNM-gel might be highly favorable for promoting macrophage polarization and attenuating the inflammatory cascade. While decellularized ECM-derived biomaterials contain some immunogenic substances (e.g., residual DNA and matrix antigens) that could be recognized by immune cells to provoke an adverse immune response, these undesired components are almost completely absent in pDNM-gel, ensuring its good biocompatibility (Lin et al., 2018; Li et al., 2021b).

Lastly, we recognized that a molecular understanding of the pDNM-gel-mediated modulation of macrophage polarization and inflammatory response could provide novel insights into the development of pharmaceuticals to treat PNI. Although these pathophysiological processes are regulated by several transcription factors, intervention in the activation of the TLR4/MyD88/NF-κB transduction pathway has become particularly crucial (Kfoury et al., 2014; Zhao et al., 2021b). Accumulating evidence illustrates that the TLR4/MyD88/NF-κB signaling pathway positively regulates macrophage polarization towards an M1 phenotype, whereas downregulation of this axis initiates M2 macrophage polarization and anti-inflammatory activity (Luo et al., 2022; You et al., 2022; Sharma et al., 2023). Recently, Yang et al. (2020a) reported that a deficiency of MyD88 alleviates neuropathic pain and promotes nerve regeneration after PNI. Our findings showed that treatment with pDNM-gel significantly attenuated the activated signaling of the TLR4/MyD88/NF-κB pathway and upregulation of related proteins. Additionally, pharmacological inhibition of TLR4 and MyD88 expression enhanced macrophage transformation towards the M2 subtype and the anti-inflammatory response in macrophages stimulated with LPS and cultured on pDNM-gel; reactivation of either factor resulted in the opposite effects. Hence, we concluded that pDNM-gel-mediated suppression of the TLR4/MyD88/NF-κB axis was vital for macrophage phenotype switching and inflammatory factor expression.

Our discovery that pDNM-gel attenuates neuroinflammation highlights an important component of its role in the functional recovery and improved nerve regeneration after PNI. However, the retention of different active components within the pDNM-gel and its unique nanofibrous structure also provide a suitable microenvironment for the effective repair of peripheral nerve damage. These extracellular macromolecules found in pDNM-gel, including laminin, fibronectin, and collagens, provide biological guides for neuronal growth and Schwann cell proliferation (Szynkaruk et al., 2013; Zilic et al., 2016). Furthermore, various bioactive growth factors within the pDNM-gel, including brain-derived neurotrophic factor, nerve growth factor, and vascular endothelial growth factor, may be effective in promoting endogenous neural stem cell differentiation and angiogenesis (Badylak, 2007; Lin et al., 2018). Additionally, its three-dimensional ultrastructure comprising cross-linked polymeric nanofibers provides a supportive organizational microenvironment specific for axonal extension and nerve remodeling (Li et al., 2021b; Rao et al., 2021). These attributes all contribute to the neuroprotective capability of pDNM-gel.

This study has some limitations. First, we did not identify which one or more components of pDNM-gel play a crucial role in attenuating neuroinflammation after PNI. Considering the complex composition of pDNM-gel, proteomic analysis is needed to identify the specific components involved in regulating inflammation, followed by separate biochemical analyses to evaluate their inflammatory regulatory effects. Second, we did not use primary macrophages derived from bone marrow (BMDMs) to evaluate the immunomodulatory effects of pDNM-gel. Although primary BMDMs might have provided more conclusive data compared with RAW264.7 cells, they are subject to source constraints and donor variability. Thus, further studies on primary BMDMs are required.

Taken together, our findings revealed that pDNM-gel can be applied to effectively stimulate macrophage transformation into the M2 subtype, increasing anti-inflammatory cytokine secretion and reducing pro-inflammatory cytokine expression to construct a suitable microenvironment for neurological regeneration and functional restoration following PNI. Furthermore, the molecular mechanism underlying this therapeutic effect of pDNM-gel may involve attenuated activation of TLR4/MyD88/NF-κB-signaling. Reduced neuroinflammation via pDNM-gel offers an alternative neuroprotective method to ameliorate damaged peripheral nerve structure and function.

Additional files:

Additional Figure 1 ^{(2.8MB, tif)} : Study flow chart and numbers of rats in each treatment group and each assessment.

Additional Figure 1

Study flow chart and numbers of rats in each treatment group and each assessment.

ELISA: Enzyme-linked immunosorbent assay; pDNM-gel: porcine decellularized extracellular matrix hydrogel; PNI: peripheral nerve injury; RNA-seq: ribonucleic acid sequencing; TEM: transmission electron microscopy

Additional Figure 2 ^{(1.7MB, tif)} : Timeline of the in vivo study.

Additional Figure 2

Timeline of the in vivo study.

CMAP: Compound muscle action potentials; ELISA: Enzyme-linked immunosorbent assay; IF: immunofluorescence; MNCV: motor nerve conduction velocity; pDNM-gel: porcine decellularized extracellular matrix hydrogel; RNA-seq: ribonucleic acid sequencing; SD: Sprague-Dawley; TEM: transmission electron microscopy; WB: western blotting.

Additional Figure 3 ^{(1.6MB, tif)} : Timeline of the in vitro study.

Additional Figure 3

Timeline of the in vitro study.

ELISA: Enzyme-linked immunosorbent assay; IF: immunofluorescence; LPS: lipopolysaccharides; pDNM-gel: porcine decellularized extracellular matrix hydrogel; qRT-PCR: quantitative reverse transcription polymerase chain reaction; WB: western blotting.

Additional Figure 4 ^{(3.2MB, tif)} : pDNM-gel promotes nerve regeneration at the proximal site.

Additional Figure 4

pDNM-gel promotes nerve regeneration at the proximal site.

(A) Representative images of co-staining of the proximal portion of harvested nerve sections with neurofilament-H (green) and MBP (red) at 21 dpi in the PNI and pDNM-gel groups. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (B, C) Calculated area (%) of positive staining of neurofilament-H and MBP per 100 μm² in the immunofluorescence images from the two groups (n = 5/group). Data are expressed as mean ± SEM; ^***P < 0.001. dpi: Days post-injury; MBP: myelin basic protein; DAPI: 4’,6-Diamidino-2-phenylindole; PNI: peripheral nerve injury; pDNM-gel: porcine decellularized nerve matrix hydrogel, SEM: standard error of the mean.

Additional Figure 5 ^{(4.9MB, tif)} : pDNM-gel promotes nerve regeneration at the distal site.

Additional Figure 5

pDNM-gel promotes nerve regeneration at the distal site.

(A) Representative images of co-staining of the distal portion of harvested nerve sections with neurofilament-H (green) and MBP (red) at 21 dpi in the PNI and pDNM-gel groups. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (B, C) Calculated area (%) of positive staining of neurofilament-H and MBP per 100 μm² in the immunofluorescence images from the two groups (n = 5/group). Data are expressed as means ± SEM; * P < 0.05, ***P < 0.001. dpi: Days post-injury; MBP: myelin basic protein; DAPI: 4’,6-Diamidino-2-phenylindole; PNI: peripheral nerve injury; pDNM-gel: porcine decellularized nerve matrix hydrogel, SEM: standard error of the mean.

Additional Figure 6 ^{(3.8MB, tif)} : pDNM-gel accelerates macrophage polarization towards the M2 subtype in the proximal region of the injured sciatic nerves.

Additional Figure 6

pDNM-gel accelerates macrophage polarization towards the M2 subtype in the proximal region of the injured sciatic nerves.

(A) Immunofluorescence photomicrographs of the proximal portion of the harvested nerves to analyze M1-subtype (labeling iNOS⁺/CD68⁺) and M2-subtype (labeling Arg1⁺/CD68⁺) macrophages from PNI rats treated with/without pDNM-gel for 3 and 7 days, respectively (n = 5/group). iNOS (green), Arg1 (green), and CD68 (red) staining M1-, M2-, and M0 macrophages, respectively. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (B, C) Quantification showing the percentage of NOS⁺ and Arg1⁺ macrophages in the PNI and PNI + pDNM-gel groups at 3 and 7 dpi. Data are expressed as mean ± SEM; **P < 0.01, ***P < 0.001. Arg1: Arginase 1; CD68: cluster of differentiation protein 68; DAPI: 4’,6-diamidino-2-phenylindole; dpi: days post-injury; iNOS: inducible nitric oxide synthase; pDNM-gel: porcine decellularized nerve matrix hydrogel; PNI: peripheral nerve injury; SEM: standard error of the mean.

Additional Figure 7 ^{(3.8MB, tif)} : pDNM-gel accelerates macrophage polarization towards the M2 subtype in the distal region of the injured sciatic nerves.

Additional Figure 7

pDNM-gel accelerates macrophage polarization towards the M2 subtype in the distal region of the injured sciatic nerves.

(A) Immunofluorescence photomicrographs of the distal portion of the harvested nerves to analyze M1-subtype (labeling iNOS⁺/CD68⁺) and M2-subtype (labeling Arg1⁺/CD68⁺) macrophages from PNI rats treated with/without pDNM-gel for 3 and 7 days, respectively (n = 5/group). iNOS (green), Arg1 (green), and CD68 (red) staining M1-, M2-, and M0 macrophages, respectively. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (B, C) Quantification showing the percentage of NOS⁺ and Arg1⁺ macrophages in the PNI and PNI + pDNM-gel groups at 3 and 7 dpi. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001. Arg1: Arginase 1; CD68: cluster of differentiation protein 68; DAPI: 4’,6-diamidino-2-phenylindole; dpi: days post-injury; iNOS: inducible nitric oxide synthase; pDNM-gel: porcine decellularized nerve matrix hydrogel; PNI: peripheral nerve injury; SEM: standard error of the mean.

Additional Figure 8 ^{(4MB, tif)} : pDNM-gel induces differentiation of phorbol myristate acetate-primed THP-1 cells to the M2 macrophage phenotype.

Additional Figure 8

pDNM-gel induces differentiation of phorbol myristate acetate-primed THP-1 cells to the M2 macrophage phenotype.

(A, B) Double staining with CD80 (M1 phenotype, green)/CD11b (THP-1 macrophage phenotype, red) or CD206 (M2 phenotype, green)/CD11b in THP-1 macrophage cells stimulated with LPS (100 ng/mL) and with/without culturing on pDNMgel (0.5%) for 24 hours. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (C, D) Relative fluorescence intensity of CD80 and CD206 quantified by Image J. (E, F) ELISAs of the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, and anti-inflammatory cytokines IL-10 and TGF-β in the Control, LPS, LPS + pDNM-gel, and pDNM-gel groups. Data are expressed as mean ± SEM, with all experiments performed in triplicate; *P < 0.05, **P < 0.01, ***P < 0.001. CD11b: cluster of differentiation protein 11b; CD206: cluster of differentiation protein 206; CD80: cluster of differentiation protein 80; DAPI: 4’,6-diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; IL-10: interleukin-10; IL-1β: interleukin-1β; IL-6: interleukin-6; LPS: lipopolysaccharide; n.s.: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; SEM: standard error of the mean; TGF-β: transforming growth factor-beta; TNF-α: tumor necrosis factoralpha.

Additional Figure 9 ^{(1.6MB, tif)} : Cytotoxicity of CRX-527 in RAW264.7 cells.

Additional Figure 9

Cytotoxicity of CRX-527 in RAW264.7 cells.

CCK-8 assay of cytotoxicity of CRX-527 in RAW264.7 cells treated with the indicated concentrations of CRX-527 (0, 30, 60, 125, 250, 500, 1000, 2000, and 4000 μM) for 24 hours (n = 3).

Additional Figure 10 ^{(2.7MB, tif)} : Determination of the optimal concentration of CRX-527 for inducing TLR4 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

Additional Figure 10

Determination of the optimal concentration of CRX-527 for inducing TLR4 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

(A) Immunoblots of TLR4 in cultured RAW264.7 cells treated with different concentrations of CRX-527 (0, 250, 500, and 1000 μM). β-actin was probed as a loading control. (B) Quantification of the level of TLR4 protein relative to β-actin. Data expressed as means ± SEM (n = 3); ^***P < 0.001. LPS: Lipopolysaccharides; n.s.: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; SEM: standard error of the mean; TLR4: Toll-like receptor 4.

Additional Figure 11 ^{(2.1MB, tif)} : Cytotoxicity of TAK242 in RAW264.7 cells.

Additional Figure 11

Cytotoxicity of TAK242 in RAW264.7 cells.

CCK-8 assay of cytotoxicity of TAK242 in RAW264.7 cells treated with the indicated concentrations of TAK242 (0, 12.5, 25, 50, 100, 200, 400, 800, and 1600 nM) for 24 hours (n = 3).

Additional Figure 12 ^{(2.1MB, tif)} : Determination of the optimal concentration of TAK242 for suppressing TLR4 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

Additional Figure 12

Determination of the optimal concentration of TAK242 for suppressing TLR4 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

(A) Immunoblots of TLR4 in cultured RAW264.7 cells treated with different concentrations of TAK242 (0, 25, 50, 100, 200, and 400 nM). GAPDH was probed as a loading control. (B) Quantification of the level of TLR4 protein relative to GAPDH. Data expressed as mean ± SEM (n = 3); ***P < 0.001. LPS: Lipopolysaccharides; n.s.: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; SEM: standard error of the mean; TLR4: Toll-like receptor 4.

Additional Figure 13 ^{(1.6MB, tif)} : Cytotoxicity of CL075 in RAW264.7 cells.

Additional Figure 13

Cytotoxicity of CL075 in RAW264.7 cells.

CCK-8 assay of cytotoxicity of CL075 in RAW264.7 treated with the indicated concentrations of CL075 (0, 1, 2, 4, 8, 16, 32, 64 μM) for 24 hours (n = 3).

Additional Figure 14 ^{(2.2MB, tif)} : Determination of the optimal concentration of CL075 for inducing MyD88 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

Additional Figure 14

Determination of the optimal concentration of CL075 for inducing MyD88 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

(A) Immunoblots of MyD88 in cultured RAW264.7 cells treated with different concentrations of CL075 (0, 1, 2, 4, 8, and 16 μM). GAPDH was probed as a loading control. (B) Quantification for the level of MyD88 protein relative to GAPDH. Data expressed as mean ± SEM (n = 3); ^***P < 0.001. LPS: Lipopolysaccharides; MyD88: myeloid differentiation factor 88; n.s.: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; SEM: standard error of the mean.

Additional Figure 15 ^{(1.8MB, tif)} : Cytotoxicity of T6167923 in RAW264.7 cells.

Additional Figure 15

Cytotoxicity of T6167923 in RAW264.7 cells.

CCK-8 assay of cytotoxicity of T6167923 in RAW264.7 cells treated with different concentrations of T6167923 (0, 6.25, 25, 100, 400, and 1600 μM) for 24 hours (n = 3).

Additional Figure 16 ^{(581.3KB, tif)} : Determination of the optimal concentration of T6167923 for inhibiting MyD88 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

Additional Figure 16

Determination of the optimal concentration of T6167923 for inhibiting MyD88 expression in RAW264.7 cells cultured on pDNM-gel and stimulated with LPS.

(A) Immunoblots of MyD88 in cultured RAW264.7 cells treated with different concentrations of T6167923 (0, 6.25, 25, 100, and 400 μM). β-actin was probed as a loading control. (B) Quantification of the level of MyD88 protein relative to β-actin. Data expressed as means ± SEM (n = 3); ^***P < 0.001. LPS: Lipopolysaccharides; MyD88: myeloid differentiation factor 88; n.s.: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; SEM: standard error of the mean.

Additional file 1 ^{(103.3KB, pdf)} : Additional methods.

Additional file 1

Additional methods

Funding Statement

Funding: This study was supported by the Shenzhen Hong Kong Joint Funding Project, No. SGDX20230116093645007 (to LY); the Shenzhen Science and Technology Innovation Committee International Cooperation Project, No. GJHZ20200731095608025 (to LY); Shenzhen Development and Reform Commission’s Intelligent Diagnosis, Treatment and Prevention of Adolescent Spinal Health Public Service Platform, No. S2002Q84500835 (to LY); Shenzhen Medical Research Fund, No. B2303005 (to LY); Team-based Medical Science Research Program, No. 2024YZZ02 (to LY); Zhejiang Provincial Natural Science Foundation of China, No. LWQ20H170001 (to RL); Basic Research Project of Shenzhen Science and Technology from Shenzhen Science and Technology Innovation Commission, No. JCYJ20210324103010029 (to BY); Shenzhen Second People’s Hospital Clinical Research Fund of Guangdong Province High-level Hospital Construction Project, Nos. 2023yjlcyj029 (to BY), 2023yjlcyj021 (to LL); Guangdong Basic and Applied Basic Research Foundation, No. 2022A1515110679 (to LL); and China Postdoctoral Science Foundation, No. 2022M722203 (to GL).

Data availability statement:

All relevant data are within the paper and its Additional files.