Berberine inhibits NLRP3 inflammasome activation and proinflammatory macrophage M1 polarization to accelerate peripheral nerve regeneration

Abstract

Berberine (BBR) has demonstrated potent anti-inflammatory effects by modulating macrophage polarization. Nevertheless, the precise mechanisms through which berberine regulates post-injury inflammation within the peripheral nerve system remain elusive. This study seeks to elucidate the role of BBR and its underlying mechanisms in inflammation following peripheral nerve injury (PNI). Adult male C57BL/6J mice subjected to PNI were administered daily doses of berberine (0, 60, 120, 180, 240 ​mg/kg) via gavage from day 1 through day 28. Evaluation of the sciatic function index (SFI) and paw withdrawal threshold revealed that BBR dose-dependently enhanced both motor and sensory functions. Immunofluorescent staining for anti-myelin basic protein (anti-MBP) and anti-neurofilament-200 (anti–NF–200), along with histological staining comprising hematoxylin-eosin (HE), luxol fast blue (LFB), and Masson staining, demonstrated that BBR dose-dependently promoted structural regeneration. Molecular analyses including qRT-PCR, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence confirmed that inactivation of the NLRP3 inflammasome by MCC950 shifted macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, while also impeding macrophage infiltration. Furthermore, BBR significantly downregulated the expression of the NLRP3 inflammasome and its associated molecules in macrophages, thereby mitigating NLRP3 inflammasome activation-induced macrophage M1 polarization and inflammation. In summary, BBR's neuroprotective effects were concomitant with the suppression of inflammation after PNI, achieved through the inhibition of NLRP3 inflammasome activation-induced macrophage M1 polarization.

Graphical abstract

Introduction

Despite the peripheral nervous system (PNS) having higher regenerative potentials after injury compared to the central nervous system (CNS), post-injury excessive inflammation stress severely hinders the regeneration of PNS and is accompanied by the attendant high disability and bad prognosis [1].

Inflammation in PNI are primarily mediated by macrophages [2,3]. Two types of macrophages existed in the injury site of PNS: resident macrophages derived from the yolk sac and embryonic liver tissue, proliferation and self-renew, acting as the first line of intrinsic immune defense after PNI, along with neutrophils and Schwann cells (SCs); and infiltrating macrophages derived from bone marrow under injury stimulation [3,4]. At 3 ​h after injury, non-resident macrophages infiltrated the damaged sciatic nerve and peaked on day 3, mainly with M1 phenotype. At 3–14 days, the infiltrating macrophages gradually convert from the M1 phenotype (pro-inflammation) to the M2 phenotype (anti-inflammation) [[5], [6], [7], [8]]. Dynamic conversion of macrophage M1 to M2 sub-phenotypes plays a pivotal role in regulating nerve regeneration after PNI [3,5]. Despite M1 macrophage mainly responding for debris clearance through inducing excessive inflammation after PNI, neutrophils, and Schwann cells can compensate for this process well after macrophage depletion [[9], [10], [11]]. Meanwhile, M2 performs dominant pro-regenerative potentialities after PNI(13). Thus, shortening M1 macrophage infiltration to attenuate inflammatory injury via promoting macrophage sub-phenotype skewing from M1 to M2 could be a promising therapeutic strategy for PNI.

The inflammasome, a cytoplasmic polymer protein complex comprising a nucleotide-binding oligomerization domain (NOD)-like receptor, leucine-rich repeat/LRR- and pyrin domain-containing proteins (NLRPs), or a PYHIN receptor, along with an adaptor protein ASC (apoptosis-associated speck-like protein with a CARD), and an effector caspase (pro-caspase-1), can be activated by damage-associated molecular patterns (DAMPs) and pattern recognition receptors (PRRs), playing a crucial role in orchestrating inflammatory responses [13,14]. NLRP3 inflammasome is the most studied and involved in various inflammatory responses of CNS, such as cerebral ischemia/reperfusion injury, memory, cognitive impairment, depression, etc. [[15], [16], [17], [18]]. NLRP3 also was recently reported to be involved in peripheral nervous inflammation of diabetes-related lesions, neuropathic pain, and neurodegenerative diseases [[19], [20], [21]], but the effects and underlying mechanisms of NLRP3 activation on the inflammatory response, particularly mediated by macrophage after mechanic PNI remain elucidative.

Berberine (BBR), a natural isoquinoline alkaloid extracted from the rhizome and root of Chinese medicinal plants, possesses multiple therapeutic effects on metabolic disorders, dyslipidemia, diabetes, and inflammation et al. [[22], [23], [24], [25], [26]]. Berberine can penetrate the blood-brain barrier (BBB) and keep it stable in the brain after oral administration [27]. Thus, based on these properties, the neuroprotective effects of berberine on central nervous system lesions with inflammatory disorders have been explored, for example, attenuating neuroinflammation in ischemic and hemorrhagic stroke, spinal cord injury, and traumatic brain injury [[28], [29], [30], [31], [32], [33]]. Additionally, in PNS, berberine has also been reported to promote post-injury neurite extension, axon regeneration, and remyelination [34,35]. However, the potential effects and mechanisms by which berberine regulates inflammatory response after PNI remain unclear.

Here, we found that NLRP3 inflammasome activation induced macrophagic pro-inflammation M1 polarization after PNI. BBR attenuated the inflammation, which is tightly related to the M1-polarized macrophage inhibition and M2-polarized macrophage promotion after PNI, and the anti-inflammation role of BBR is intimately linked to NLRP3 inflammasome inactivation.

Materials and Methods

Animals

All work was conducted with the formal approval of the Institutional Review Board and Ethics Committee of South China Agricultural University (No.2022F119). All the experiments on animals in this study were carried out according to the guidelines from the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health).

In this work, specific-pathogen-free male and female C57BL/6J mice were used to avoid the neuroprotective effect of estrogen. Six-week-old male and female C57BL/6J mice, weighing 20 ​g–30 ​g, were purchased from the Guangdong Sijia Jingda Biotechnology Co. LTD (China, license No. SCXK (Yue) 2020-0052). All mice were kept in a room with a constant humidity of 55 ​± ​5%, temperature of 24 ​± ​2 ​°C, and a 12 ​h light/dark cycle. The sciatic nerve crush injury mouse model was established as previously described [5,36,37]. Briefly, mice were deeply anesthetized with intraperitoneal injection of 1% pentobarbital sodium at a dosage of 40 ​mg/kg. After shaving and skin preparation, mices were fixed on the operating table and disinfected 3 times. A 2 ​cm longitudinal skin incision was made on the dorsal side of the thigh root, and the sciatic nerve was exposed and bluntly freed from surrounding subcutaneous fascia, muscles, and blood vessels. The lift side was set as the control side. A medium-sized straight hemostatic forceps was used to press the sciatic nerve to the last grid of the same hemostatic forceps with about 54 ​N compressive force (5 ​mm distal from the sciatic notch) for three times in three different directions for 10 ​s per crush. They were randomly grouped into seven groups per different treatments as follows: Sham, saline, 60 ​mg/kg berberine, 120 ​mg/kg berberine, 180 ​mg/kg berberine, 240 ​mg/kg berberine.

Cell and culture

Macrophage strain RAW264.7 were purchased from the Cell Bank of the Chinese Academy of Sciences, and cultured in DMEM culture supplemented with 1% penicillin-streptomycin-mixed and 10% fetal calf serum (FBS) at 37 ​°C in a 5% CO2 incubator.

Regents

BBR was purchased from Solarbio Co. (CAS#2086-83-1 Beijing, China). BBR was dissolved in dimethyl sulfoxide (DMSO, Sigma, Louis, MO, USA) and stored at 4 ​°C. BBR was dissolved in 0.9% saline with 0.5% sodium carboxymethylcellulose at different concentrations (60 ​mg/kg, 120 ​mg/kg, 180 ​mg/kg, 240 ​mg/kg) and administered via gastric gavage manner.

Histological staining

The isolated regenerating nerves were prefixed with 4% paraformaldehyde (Sigma, USA) for 24 ​h. Next, samples were postfixed with 1% osmium tetroxide for 1 ​h, and washed with PBS 3 times. All of the nerves were sequentially dehydrated in 50%, 75%, 85%, 95%, and 100% ethanol, and immersed in 100% propylene oxide for 30 ​min to avoid shrinkage. Dehydrated samples were immersed in a mixed solution with 1:1 of Epon and propylene oxide for 2 ​h, followed by being embedded in pure Epon and baked overnight at 65 ​°C. Then, samples were sliced into 2 ​μm semi-thin sections, attached to glass slides, and stained with hematoxylin-eosin (HE) and Masson to observe the morphology and histological changes of the sciatic nerve, and LFB to the myelination of axon under an optical microscope. Three sections per sample were randomly chosen and six images per section were randomly taken and analyzed at 20 ​× ​magnification.

Immunofluorescent staining (IF)

The slicing processing and IF staining for the sciatic nerve were performed as previously reported with slight modification [5,38]. In brief, 1 ​cm of injured sciatic nerve segment was collected and fully fixed with 4% paraformaldehyde for 24 ​h at 4 ​°C, next embedded in the paraffin block, and then sliced into 5 ​μm thickness slices. After deparaffinized, hydrated, and antigen retrieval, these sections were blocked in 10% bovine serum albumin (BSA) for 1 ​h at room temperature (RT). Next, primary antibodies were used to incubate sections overnight at 4 ​°C as follows: anti-IBA (1:500, Abcam, Rabbit, UK) for overall macrophages, anti-iNOS (1:500, Abcam, Rabbit, UK) for M1 macrophage and anti-CD206 (1:500, Abcam, Rabbit, UK) for M2 macrophage, anti-neurofilament-200 (NF-200, 1:200, Cell Signaling Technology, Danvers, MA, USA) for regenerative nerve fibers, and anti-myelin basic protein (MBP, 1:50, Cell Signaling Technology, Danvers, MA, USA) for regenerative myelin. Then, after being washed with blocking buffer 3 times, the secondary antibody was incubated for 1 ​h at RT and light-proof wetting box. After being washed again, DAPI was used to stain the nuclei for 5 ​min at RT. Images were acquired using a confocal microscope (ZEISS, Germany).

Sciatic functional index (SFI) measurements

The SFI was used to assess the motor function recovery of the injured sciatic nerve and was carried out before the crush injury and on postoperative weeks 2, 4, 6, and 8 weeks as in our previous study [5]. Three parameters, including toe spread (TS, first to the fifth toe), intermediate toe spread (ITS, second to the fourth toe), and total print length (PL, tip of the third toe to heel) were calculated to determine SFI as follows (E: experimental lateral; N: normal lateral):

$$\text{SFI}=109\times \frac{\mathrm{T}{\mathrm{S}}_{\mathrm{E}}-\mathrm{T}{\mathrm{S}}_{\mathrm{N}}}{\mathrm{T}{\mathrm{S}}_{\mathrm{N}}}+13.3\times \frac{\text{IT}{\mathrm{S}}_{\mathrm{E}}-\text{IT}{\mathrm{S}}_{\mathrm{N}}}{\text{IT}{\mathrm{S}}_{\mathrm{N}}}-38.3\times \frac{\mathrm{P}{\mathrm{L}}_{\mathrm{E}}-\mathrm{P}{\mathrm{L}}_{\mathrm{N}}}{\mathrm{P}{\mathrm{L}}_{\mathrm{N}}}－8.8$$

Paw withdraw threshold assay

Painful mechanical sensibility for sensory function recovery evaluation was assessed by an electronic Von Frey algesimeter (Bioseb, Chaville, France) as previously reported [39]. Briefly, mouse were placed on a plastic-enclosed wire mesh platform. Firstly, the planter surface of each hind paw was set with 0.88 ​mm diameter spring wire, and the attendant was connected to a force sensor. As the pressure gradually increased, the value of pressure was recorded until the rat withdrew its paw. This process was repeated in triplicate with a 15-min interval time at each testing day (weeks 1–4), and the mean of the three values was applied to calculate the percentage of injuries to the contralateral healthy hind paw.

Acetone test

The acetone test was used to detect cold hypersensitivity as previously reported [40]. Briefly, we applied 100 ​μL of acetone to the paw surface of mice and counted the sum of the number of times the mice lifted and licked their paws within 1 ​min, three times in total, with a 5-min intervals, and calculated the average value of the three times.

Western blotting

Injured sciatic nerve segments and macrophages were harvested and lysed using RIPA buffer (Thermofisher Scientific Inc., Massa Chusetts, American) containing 1% proteinase inhibitor cocktail, 1% PMSF, and 1% deoxycholate. A bicinchoninic acid assay (BCA) kit (Beyotime Biotechnology, Haimen, China) was used to quantify the protein concentration. Next, 1/3 volume of 4x Loading buffer (Thermo Scientific, USA) was added, and mixed protein suspensions were denatured in a 70 ​°C water bath for 10 ​min. Western blotting was performed as standard protocol. In brief, after 7.5%–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the samples were transferred to a polyvinylidene fluoride (PVDF) membrane followed by blocking with 5% skim milk in TBST solution (Tris-HCl buffered saline with 0.05% tween 20) for 1 ​h at the room. After washed 3 times with TBST solution, samples were incubated with primary antibodies as follows at 4 ​°C overnight: anti-NLRP3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat#13158), anti-ASC (1:1000, Biorbyt, St Louis, MO, USA, Cat#orb338943), anti-caspase-1 (1:1000, Proteintech Group, Wuhan, China, Cat# 22915-1-AP), anti-IL-1β (1:1000, Biorbyt, St Louis, MO, USA, Cat#orb101745), anti-IL-18 (1:1000, Proteintech Group, Wuhan, China, Cat# 10663-1-AP), anti-GSDMAD-N (1:1000, Biorbyt, St Louis, MO, USA, Cat#orb422737), and anti-β-actin (1:1000, Sigma, Cat# A5441, RRID: AB_476744). After being washed 3 times with TBST solution, samples were incubated with secondary antibodies at 37 ​°C for 1 ​h as follows: Horseradish peroxidase (HRP)-conjugated affineur goat anti-rabbit/mouse IgG (H ​+ ​L) (1:5000, Proteintech Group, Cat#SA00001-2, Cat#SA00001-1). The immunoreactive signals were visualized using a gel imaging analysis system (Kodak, Tokyo, Japan).

Enzyme-linked immunosorbent Assay（ELISA）

Sciatic nerve segments were harvested and washed with pre-cooled PBS (0.02 ​mol/L, PH 7.1–7.2) 3 times, and added into glass homogenizer with 200 ​μl pre-cooled PBS for further fully grinding. Next, sciatic segments homogenization was collected and centrifuged at 5000×g for 5 ​min and the supernatant was retained for testing according to the manufacturer's protocols of corresponding ELISA kits, including TGF-β1 (Maiman, Jiangsu, China), IL-10 (Maiman, Jiangsu, China), IL-4 (Maiman, Jiangsu, China), Arg-1 (Maiman, Jiangsu, China), IL-6 (Maiman, Jiangsu, China), TNF-α (Maiman, Jiangsu, China), IFN-γ (Maiman, Jiangsu, China), iNOS (Maiman, Jiangsu, China) and IL-1β (Maiman, Jiangsu, China).

Statistical analysis

The measurement data are shown as mean ​± ​standard deviation (mean ​± ​SD). The data results were calculated and analyzed by statistical SPSS 20.0 software. Student's t-test was used to assess the significance of differences between groups, and one-way ANOVA or ANOVA with post hoc Bonferroni correction followed by the Scheffé test was used to assess multiple group comparisons. P ​< ​0.05 was set as a threshold level of statistical significance. The levels of significance were defined as follows: ∗P ​< ​0.05, ∗∗P ​< ​0.01, and ∗∗∗P ​< ​0.001.

Results

BBR dosage-dependently promotes axon and myelin regeneration, and motor and sensory function recovery after PNI

The regenerative effect of BBR after sciatic nerve injury has been previously shown in a rat model focusing on morphological changes [34]. Here, we aimed to expand these results by assessing the functional recovery of the sciatic nerve in a mouse model. In addition, we explored the mechanisms of this effect by testing the possible role of anti-inflammatory effects of BBR, as shown in Fig. 1A. Paw withdraw thresholds (Von Frey) and cold hypersensitivity (acetone test) assay showed that berberine improved the sensory function of the injured sciatic nerve (Fig. 1B and C). Sciatic nerve function (SFI) assay showed that both male and female mice treated with higher berberine concentration had a better motor function recovery (Fig. 1D–F). In addition, the effect of different dosage BBR on the body weight of both male and female mice have no significant difference (Fig.S1).

Fig. 1.

Berberine (BBR) dosage-dependently facilitated motor and sensory function recovery after PNI. A: the timing of the experimental procedures. B-C: Mechanical and cold withdraw thresholds assay showed that berberine improved the sensory function of the injured male and female sciatic nerve in a dose-dependent manner (n = 5/group). D. Sciatic nerve function index (SFI) assay showed that BBR significantly accelerated post-injury motor function repair of the sciatic nerve (All of the high dosage group P < 0.05, n = 5/group). E: The paw of mice at post-injury 1W. F: The paw of mice at post-injury 6W. Notes: Data are presented as means ± SD, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 10.001.

The structural reconstruction was assessed by histological staining and immunofluorescent staining (IF). LFB and HE staining at the 3 ​d after PNI showed that BBR reduced myelin disintegration and inflammatory cell infiltration with a dose-dependent manner (Fig.S2). The result of IF at the 2 week after PNI showed that berberine dose-dependently accelerated anti-neurofilament 200 (NF-200)-marked axon regeneration (Fig. 2A and B) and anti-myeline basic protein (MBP)-marked remyelination (Fig. 2A–C). HE and Masson staining were implemented to observe the morphology and histological changes of the sciatic nerve and its fibers. Furthermore, these results showed that a higher concentration of berberine led to a more orderly and tightly arranged structure, less vacuolization and edema, reduced fibrosis, and enhanced revascularization (Fig. 2D). LFB staining showed that berberine dose-dependently increased the number of myelin (Fig. 2D).

Fig. 2.

Berberine (BBR) dosage-dependently facilitated post-injury structural regeneration. A: Immunofluorescence staining for sciatic nerves showed that BBR intensively increased the fluorescence density of MBP (myelin marker) and NF-200 (axon marker) in a dose-dependent manner (Blue: DAPI; Green: MBP; Red: NF-200; Scale bar ​= ​20 ​μm). Sciatic nerve function index (SFI) assay showed that BBR significantly accelerated post-injury motor function repair of the sciatic nerve (All of the high dosage group P ​< ​0.05, n ​= ​5/group). B, C: The analysis of IF results in A. D: HE, Masson staining showed higher-concentration BBR had more order and tight structure arrangement and more revascularization, as well as less vacuolization, edema, and fibrosis; LFB for myeline staining showed a more remarkable increase of the myelin number. Notes: Data are presented as means ​± ​SD, ∗P ​< ​0.05, ∗∗P ​< ​0.01 and ∗∗∗P ​< ​0.001.

In sum, these data suggest that berberine accelerated the structural reconstruction, and motor and sensory functional recovery of the injured sciatic nerve in a dose-dependent manner.

BBR ameliorated inflammatory intensity by regulating macrophage polarization after PNI

The polarized macrophagic sub-phenotype was intimately linked to the extent of inflammatory infiltration after PNI [3,12,41]. To clarify the M1 polarization of macrophages, IF staining was performed on the M1 macrophage's marker of iNOS and CD86, where iNOS ​+ ​or CD86⁺ macrophages (M1) were lower with the increase of berberine concentration (Fig. 3A and B and Fig.S3A, B). On the other hand, to clarify the M2 polarization of macrophages, IF staining was performed on the M2 macrophagic marker of CD206 and Arg-1, and this showed that CD206+ or Arg-1+ macrophages (M2) were higher with the increase of berberine concentration (Fig. 3A, B and Fig. S3A, B). Thus, BBR dose-dependently increased the M2/M1 ratio which could benefit the regenerative microenvironment of damaged nerve localization (Fig. 3A–C and Fig.S3 C). Interestingly, berberine administrations also reduced macrophage infiltration (Fig. 3A–D). Also as verified by ELISA, the secretion of other pro-inflammation M1 macrophage markers (IL-6, IFN-γ, TNF-α, IL-1β, iNOS) were lower (Fig. 3E) and anti-inflammation M2 macrophage marker (IL-10, IL-4, TGF-β1, Arg-1) were higher at injury sit in high-concentration berberine administration after PNI (Fig. 3F). Further, we tested the inhibition of macrophage inflammation by BBR in vitro, and the results showed that BBR dose-dependently attenuated LPS-induced macrophage proinflammatory cytokines production (Fig. S4).

Fig. 3.

Berberine promotes macrophage M2 polarization and reshapes the anti-inflammation microenvironment. A: Immunofluorescence staining for the CD206+ cells (M2 macrophage) and decreased iNOS ​+ ​cells (M1 macrophage) in sciatic nerves slices (Blue: DAPI; Green: CD206; Red: iNOS; Scale bar ​= ​50 ​μm, n ​= ​5/group). B, C: Statistical analysis for A showed that BBR dose-dependently decreased M1 macrophage counts, as well as increased M2 macrophage counts and M2/M1 ratio. D: The analysis of A suggests BBR dose-dependently reduced macrophage infiltration, where 120 ​mg/kg reached a plateau at the injury site. E: ELISA assay shows BBR dose-dependently increased anti-inflammation cytokines IL-10, IL-4, TGF-β1 release, and the M2 macrophage marker expression of Arg-1 (n ​= ​5/group). F: ELISA assay shows BBR dose-dependently reduced pro-inflammation cytokines IL-6, IFN-γ, IL-1β release, and the expression of iNOS (n ​= ​5/group). Notes: Data are presented as means ​± ​SD, ∗P ​< ​0.05, ∗∗P ​< ​0.01 and ∗∗∗P ​< ​0.001.

Our data determine, after PNI, that BBR significantly attenuated inflammation by reducing macrophagic infiltration and boosting M2 polarization.

BBR inhibits NLRP3 inflammasome activation thus inducing macrophage M2 polarization after PNI

NLRP3 inflammasome is a main activator of trauma-induced inflammation in both CNS and PNS. The western blotting results showed that the expression of NLRP3 and its linked molecules, including pro-IL-1β, IL-1β, pro-caspase-1, caspase-1, and ASC was remarkably upregulated after PNI (Fig. 4A and B). To identify the effects of NLRP3 on macrophage polarization after PNI, the specific antagonist MCC950 was used. The IF result showed that MCC950 decreased iNOS ​+ ​cells (M1 macrophage) and increased CD206+ macrophages (M2) (Fig. 4C and D), and accompanied an increase of M2/M1 ratio (Fig. 4C–E), all of which suggested MCC950 promoted macrophage M2 polarization after PNI. The ELISA results showed that NLRP3 inactivation by MCC950 significantly decreased the expression of other M1 macrophagic markers (IL-6, TNF-α, CD80) (Fig. 4F), and increased the expression of other M2 macrophagic markers (IL-10, IL-4, TGF-β1, Arg-1) (Fig. 4G). These data indicate that the NLRP3 inflammasome plays a crucial role in promoting M1 macrophage polarization after PNI. Additionally, while macrophages are significant cytokine producers, the contributions of other cell types to the inflammatory response should not be overlooked.

Fig. 4.

NLRP3 inflammasome activation promotes macrophage M1 polarization and injury-site inflammation after PNI. A: Western blotting showed that NLRP3 and its linked molecules, including ASC, pro-IL-1β, IL-1β, pro-caspase-1, caspase-1 (n ​= ​5/group). B: The analysis of A. C: Immunofluorescence staining for the CD206+ cells (M2 macrophage) and decreased iNOS ​+ ​cells (M1 macrophage) in sciatic nerves slices (Blue: DAPI; Green: CD206; Red: iNOS; Scale bar ​= ​50 ​μm, n ​= ​5/group). D: Statistical analysis for C showed that MCC950 decreased iNOS ​+ ​M1 macrophage as well as increased CD206+ M2 macrophage counts and (E) M2/M1 ratio. F: ELISA assay shows MCC950 reduced anti-inflammation cytokine IL-6 and TNF-α release, and the M1 macrophage marker of CD80 expression (n ​= ​5/group). G: ELISA assay shows MCC950 increased pro-inflammation cytokines IL-10, IL-4, TGF-β1 release, and iNOS expression (n ​= ​5/group). Notes: Data are presented as means ​± ​SD, ∗P ​< ​0.05, ∗∗P ​< ​0.01 and ∗∗∗P ​< ​0.001.

To further determine the effect of BBR on inflammasome, different concentrations (0, 60, 120, 180, 240 ​mg/kg) of BBR were administrated after PNI. The qRT-PCR results showed that BBR dose-dependently inhibited the mRNA expression of NLRP3 and its linked molecules, including IL-1β, caspase-1, ASC, and GSDMAD (Fig. 5A). Consistently, the immunofluorescent staining (IF) for the colocation of IBA ​+ ​cells and NLRP3+ cells showed that BBR significantly reduced the expression of NLRP3 in the overall damaged nerve segments (Fig. 5B and C) and the macrophages (NLRP3+ and IBA+) (Fig. 5B–D) after PNI. These data suggested that NLRP3 inflammasome inactivation at least partially responds to the BBR-mediated macrophagic anti-inflammation M2 polarization after PNI.

Fig. 5.

BBR dose-dependently inhibits the expression of NLRP3 inflammasome in PNI. A: qRT-PCR results showed that BBR dose-dependently reduced NLRP3, ASC, GSDMAD, IL-1β, and caspase-1 expression in the injury site after PNI. B: Immunofluorescence staining (IF) for the NLRP3 and IBA (macrophage marker) in sciatic nerve slices (Blue: DAPI; Green: NLRP3; Red: IBA; Scale bar = 50 μm, n = 5/group). C: Analysis for B showed that BBR dose-dependently reduced the fluorescent intensity of NLRP3 in the whole injured nerve segments, and (D) the expression of NLRP3 in injury-site macrophages. Notes: Data are presented as means ± SD, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

Discussion

In the present study, we determined that BBR, a natural isoquinoline alkaloid with prominent anti-inflammation characteristics, could alleviate the intense inflammation after PNI via accelerating the shift of macrophage M1 polarized phenotype to M2, promoting post-injured nerve injury and functional recovery. Meanwhile, we found that NLRP3 inflammasome activation induced macrophage M1 polarization, which could be the main causative factor of inflammation in peripheral nerve injury. Further, we unveiled that BBR alleviates inflammation and accelerates nerve regeneration at a considerable fraction of through inactivating NLRP3 inflammasome.

BBR has widely performed anti-inflammation potential in a series of diseases. BBR resolved the inflammatory response of viral myocarditis and that in infracted myocardium [25,42]. BBR could improve colitis and Alzheimer's disease via triggering aryl hydrocarbon receptor (AhR) activation and gut host-microbiota [[43], [44], [45]]. In the nervous system, BBR also performed potent anti-inflammation potentialities. BBR could mitigate the neuroinflammation of CNS including that induced by intracerebral hemorrhage, ischemic/refusion, and trauma [30,31,33,46], and that in a series of neurodegenerative diseases such as Parkinson's, Alzheimer's, Huntington's disease, and diabetes cognitive-impairment, etc. [45,[47], [48], [49]]. BBR also performs the neuroprotective function in different neuropathic pain of PNS via regulating inflammation [[50], [51], [52]]. This work further determined that BBR also possesses anti-inflammation potentiality in mechanic PNI, in which BBR accelerated the conversion of macrophage M1 sub-phenotype to M2. Our work might also reveal why BBR performed potent pro-regenerative function after mechanic PNI [34,53]. Additionally, BBR not only attenuates inflammatory cell activity but also reduces neuronal oxidative stress injury to promote post-injury regeneration of PNS [54,55].

NLRP3 inflammasome was intimately linked to acute and chronic inflammation in CNS and central neuropathic pain caused by PNI [[56], [57], [58], [59]]. In PNS, after injury, the elements of NLRP3 inflammasome in the injury site and the corresponding dorsal root ganglion of the L4-L5 segments of the spinal cord were remarkably upregulated, which intensively impeded nerve regeneration [19,20]. Despite the pro-inflammation effect of NLRP3 inflammasome in CNS being widely studied [16,[56], [57], [58], [59]] and also demonstrating a blocking effect on nerve regeneration through initiating intense neuroinflammatory response [19,20], its effect on macrophagic inflammatory phenotype was not further explored. In the present study, we found that NLRP3 inflammasome was remarkably activated and induced macrophage pro-inflammation M1 polarization after PNI, which might respond most to the delay of nervous regeneration and function recovery.

Recently, some studies determined NLRP3 inflammasome could be inactivated by BBR to attenuate intense inflammatory responses in different diseases, including suppressing epithelial-to-mesenchymal transition to protect diabetes neuropathy [60], ameliorating influenza viral pneumonia-induced pulmonary inflammation [61], remedying Ca2+ signaling disorder-induced endothelial junction dysfunction [62], attenuating autophagic impairment in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease [63], and inhibiting the neuroinflammation and neuroplasticity disruption in corticosterone-induced depression-like mice model [28]. In this work, we also found the BBR dose-dependently attenuated the mechanic injury-induced NLRP3 inflammasome activation-induced inflammation, accelerating nerve regeneration and functional recovery after PNI. NLRP3 inflammasome activation was one of the factors for macrophagic chemotaxis [64,65]. Interestingly, In this study, we found that BBR had no effects on macrophagic infiltration at the injury site of the peripheral nerve, which was consistent with those effects as the intracerebral hemorrhage (ICH) and spinal cord injury from previous studies [30,66]. In vitro, BBR was also determined to inhibit LPS-induced macrophage migration in an AMPK-dependent manner [67]. The above discrepant results in vivo and vitro suggested that BBR regulates macrophage migration in vivo with multiple cross-linking mechanisms.

Macrophage infiltration and activation following PNI are integral yet complex components of both the early and later stages of the inflammatory cascade. Activation of macrophages may commence soon after PNI, at times coinciding with or even preceding initial inflammatory responses. Notably, macrophages are capable of rapidly migrating to the site of injury, as early as 3 ​h post-injury, where they initiate the clearance of cellular debris and release cytokines that regulate the inflammatory milieu [1,3]. This observation underscores the potential for macrophage activation to occur in the acute phase of nerve damage, potentially before the comprehensive onset of inflammation. Moreover, it is acknowledged that macrophage recruitment and activation are modulated by early inflammatory signals, such as the emanation of damage-associated molecular patterns (DAMPs) and pro-inflammatory cytokines from the damaged tissue. These factors not only attract macrophages to the injury locus but also catalyze their activation [3,19,20]. Thus, while the infiltration and activation of macrophages can be initiated shortly after a peripheral nerve injury, these processes are intricately influenced by antecedent inflammatory events. The dynamic interplay among various immune cells and signaling entities plays a pivotal role in orchestrating the inflammatory response and facilitating the tissue repair mechanisms. However, delineating the precise causal relationships among the activations of central and peripheral inflammasomes remains a challenge, representing a limitation of current studies.

In summary, we determined the anti-inflammation and neuroprotective effects of BBR on PNI. Further, we found that NLRP3 inflammasome activation mediated macrophagic pro-inflammation M1 polarization at the injury site after PNI, and BBR remarkably attenuated this pathological process by inactivating NLRP3 inflammasome, providing new clues for the development of strategies for pharmacological therapy by modulating excessive inflammation.

Funding

This study was supported by the “Five-five” project construction project of the Third Hospital of Sun Yat-sen University (No. 2023WW504); the National Natural Science Foundation of China (No. 81571202); the Guangzhou Science and Technology Project, grant number (No. 202201020578); the Guangdong Basic and Applied Basic Research Foundation, grant number (No. 2022A1515012433).

Availability of data and materials

The data and materials supporting the conclusions of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All the animal studies were carried out according to the approved protocols and guidelines of the Institutional Animal Ethical Care Committee of South China Agricultural University Experimental Animal Center.

Consent for publication

Not applicable.

Author Contributions

Jun Sun designed the study. Jun Sun, Qiuhua Zeng, and Zhimin Wu performed the animal experiments. Cong Ling, Lixin Huang and Tao Sun helped implement Western blot and RT-qPCR experiments. Jun Sun and Qiuhua Zeng performed the statistical analysis and figure production. Jun Sun drew the manuscript. Baoyu Zhang, Cong Ling, Chuan Chen and Hui Wang revised the paper. Hui Wang supplied fundings. All authors have read and agree to the published version of the manuscript.

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: