Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

Abstract

The M1/M2 phenotypic shift of microglia after spinal cord injury plays an important role in the regulation of neuroinflammation during the secondary injury phase of spinal cord injury. Regulation of shifting microglia polarization from M1 (neurotoxic and proinflammatory type) to M2 (neuroprotective and anti-inflammatory type) after spinal cord injury appears to be crucial. Tryptanthrin possesses an anti-inflammatory biological function. However, its roles and the underlying molecular mechanisms in spinal cord injury remain unknown. In this study, we found that tryptanthrin inhibited microglia-derived inflammation by promoting polarization to the M2 phenotype in vitro. Tryptanthrin promoted M2 polarization through inactivating the cGAS/STING/NF-κB pathway. Additionally, we found that targeting the cGAS/STING/NF-κB pathway with tryptanthrin shifted microglia from the M1 to M2 phenotype after spinal cord injury, inhibited neuronal loss, and promoted tissue repair and functional recovery in a mouse model of spinal cord injury. Finally, using a conditional co-culture system, we found that microglia treated with tryptanthrin suppressed endoplasmic reticulum stress–related neuronal apoptosis. Taken together, these results suggest that by targeting the cGAS/STING/NF-κB axis, tryptanthrin attenuates microglia–derived neuroinflammation and promotes functional recovery after spinal cord injury through shifting microglia polarization to the M2 phenotype.

Introduction

Neuroinflammation is a hallmark of neurodegenerative and traumatic diseases in the central nervous system (CNS) (Si et al., 2023). Microglia are the primary components of the innate immune system in the CNS (Xu et al., 2016), and they polarize into the M1 (neurotoxic and proinflammatory type) or M2 (neuroprotective and anti-inflammatory type) phenotype in response to microenvironmental disruptions. M1 microglia (characterized by upregulation of cluster of differentiation 86 [CD86]) induce neuroinflammation by releasing proinflammatory factors, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α). Conversely, M2 microglia (characterized by upregulation of CD206 and arginase-1 [Arg-1]) inhibit neuroinflammation by secreting anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, and transforming growth factor-β1. The shift in microglial M1/M2 phenotypes has been associated with the prognosis of traumatic CNS disorders, such as spinal cord injury (SCI), and neurodegenerative diseases, such as Alzheimer’s disease (David and Kroner, 2011; Tang and Le, 2016). Therefore, a cell-based therapy targeting microglial polarization is an ideal therapeutic strategy for neuroinflammation-related diseases.

SCI is a grievous pathological condition characterized by damage to the spinal cord resulting from traumatic or non-traumatic etiologies, and it can lead to temporary or permanent changes in motor, sensory, and other physiological functions below the injured level (Ahuja et al., 2017; Gedde et al., 2019; Anjum et al., 2020; Al-Sammarraie et al., 2023; de Almeida et al., 2023). Traumatic SCI rapidly triggers a dominant proinflammatory response by resident microglia (Devanney et al., 2020, Papa et al., 2016). After traumatic SCI, resting microglia polarize into M1 and M2 subpopulations. M1 microglia are predominant and have detrimental effects on neuroprotection, whereas M2 microglia suppress the secondary inflammatory response and exhibit neuroprotective effects (Fan et al., 2019; Gaojian et al., 2020; Fan et al., 2020; Kobashi et al., 2020). An increasing number of studies have sought to attenuate the microglia-derived neuroinflammation after SCI by using compounds, lentivirus, and other methods to ultimately promote neuroprotection and functional recovery in rats and mice (Chen et al., 2018; Zeng et al., 2019; Liu et al., 2021a). Thus, understanding the underlying mechanisms of the inflammatory response and identifying methods to effectively modulate microglia polarization are necessary for prospective therapeutic strategies for SCI.

Traumatic CNS diseases can trigger cellular homeostasis disruption, leading to cytoplasmic double-stranded DNA (dsDNA) accumulation. Subsequently, cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, is stimulated by dsDNA and activates downstream endoplasmic reticulum (ER) protein stimulator of interferon genes (STING) via catalyzing cyclic GMP-AMP (cGAMP) synthesis (Li et al., 2020). STING dimers translocate from the ER to other perinuclear structures, especially the Golgi body, and are phosphorylated by TANK-binding kinase 1 (TBK1). Interferon regulatory factor 3 (IRF3) binds to phosphorylated STING (p-STING) and is then phosphorylated by TBK1 to form an active transcription factor complex (Guo et al., 2019). Additionally, the cGAS-STING axis was shown to trigger TBK1-dependent or TBK1-independent nuclear factor-κB (NF-κB) pathway activation, which is considered an additional function independent of interferon regulatory factor 3-mediated interferon response and is different from the conventional NF-κB activation route via inhibitor of kappa B kinase recruitment (Abe and Barber, 2014; de Oliveira Mann et al., 2019). NF-κB plays a crucial role as a transcription factor in enhancing proinflammatory cytokine expression levels and facilitating microglial polarization into the M1 phenotype (Wang et al., 2014). Therefore, targeting the cGAS/STING/NF-κB pathway is a potential preclinical treatment strategy for regulating neuroinflammation and eventually promoting neuroprotection after traumatic SCI.

Tryptanthrin (Tryp), an alkaloid belonging to the indoloquinazoline class, can be obtained through sublimation of several plants, such as indigo, and Isatis, polygonim and Wrightia species (G et al., 2021). Recent studies have shown that Tryp has a variety of therapeutic bioactivities, including antibacterial, antiviral, antiparasitic, anti-inflammatory and antineoplastic activity (Mitscher and Baker, 1998; Danz et al., 2002a; Liao and Leung, 2013; Onambele et al., 2015; Lin et al., 2020; Tsai et al., 2020). Research on the role of Tryp in the CNS is scarce. A previous study reported Tryp possessed high blood–brain barrier permeation potential, which provides a basis for its effects in the CNS (Jahne et al., 2016). However, the roles of Tryp on microglia-mediated neuroinflammation and its underlying mechanisms after SCI remain unclear.

In this study, we showed that the cGAS/STING/NF-κB axis played a crucial role in microglial polarization and neuroinflammation both in vitro and in an SCI mouse model. Moreover, we emphasized the importance of targeting the cGAS/STING/NF-κB axis by Tryp in microglia-derived neuroinflammation and for functional recovery after SCI. The findings suggest a potential pharmacological therapeutic strategy for SCI.

Methods

Cell culture and viability assay

Microglia cell line BV2 (to mimic microglial characteristics in vitro, Cat# GDC0311, RRID: CVCL_0182) and Neuro-2a (N2a) cell line (to mimic neuronal characteristics in vitro, Car# CCL-131, RRID: CVCL_0470) were provided by Zhejiang University, Hangzhou, China. Both of the cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) under controlled conditions of 95% air and 5% CO₂ at 37°C. Cell experiment analyses were carried out according to the experimental timeline as shown in Additional Figure 1 ^{(2.7MB, tif)} . Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) cell counting kit (A311-01/02, Vazyme Biotech, Nanjing, China). BV2 cells were seeded into 96-well plates with an estimated density of 3000 cells per well. The cells were starved for 24 hours in serum-free media, and then subjected to incubation with different concentrations of Tryp (0, 2.5, 5.0, 10.0, 20.0, and 50.0 μM) for varying durations (3, 6, 12, and 24 hours). Then, 10 μL of CCK-8 solution was added to every well and incubated at 37°C for 2 hours. The optical density of the cells was measured at 450 nm using a microplate reader (Varioskan Flash, Thermo Fisher Scientific, Waltham, MA, USA).

Lipopolysaccharide-induced inflammatory model in vitro

The lipopolysaccharide (LPS)-induced inflammatory model of BV2 cells was established as described previously (Ban et al., 2023). Briefly, BV2 cells (4 × 10⁵/mL) were pretreated with LPS (1 μg/mL; Solarbio, Beijing, China) for 24 hours to induce a proinflammatory condition. BV2 cells were divided into four groups: Control (Ctrl) group (treated with 0.1% dimethyl sulfoxide [DMSO]), LD-treated group (treated with LPS + 0.1% DMSO), LT-treated group (treated with LPS + Tryp [10 μM]), and LT + SR-717-treated group (treated with LPS + Tryp [10 μM] + SR-717 [20 μΜ]).

Fluorescent phallotoxin staining

The staining procedure for phallotoxin (SF633-conjugated, Solarbio) was described previously (Riveline and Nurse, 2009). Briefly, BV2 cells were washed with phosphate-buffered saline (PBS) three times and then fixed with 3.7% formaldehyde solution in PBS for 10 minutes at room temperature (RT). Subsequently, the cells were washed with PBS three times and permeabilized with 0.1% Triton X-100 (Solarbio) for 5 minutes. Next, the BV2 cells were stained with phallotoxin solution (1:40) for 20 minutes at RT. When BV2 cells were subjected to coimmunostaining of ionized calcium-binding adapter molecule 1 (Iba1) and phallotoxin, the cells were initially stained with Iba1 and its appropriate secondary antibody, and then with phallotoxin immediately after. All images were acquired using identical settings on an Olympus SLIDEVIEW™ VS200 microscope (Olympus, Tokyo, Japan) and analyzed using ImageJ (Version Fiji) software (Media Cybernetics, Silver Springs, MD, USA) and Photoshop (Version 2021, Adobe, San Jose, CA, USA).

Immunostaining

For cultured cell immunostaining, BV2 cells, N2a cells and primary cultured microglia were rinsed with PBS and fixed in 4% paraformaldehyde (PFA) for 20 minutes. Subsequently, the cells were blocked and permeabilized using 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS containing 0.1% Triton X-100 (Solarbio) for 1 hour at RT. The cells were then incubated with primary antibodies (Additional Table 1) overnight at 4°C. Subsequently, the cells were washed with PBS three times and incubated with the respective fluorescence-conjugated secondary antibody (Additional Table 1) for 1 hour at RT.

Additional Table 1.

Antibodies used for immunostaining

Antibody	Dilution	Supplier	Cat#	RRID
Mouse anti-NeuN	1: 500	Cell Signaling Technology (Danvers, MA, USA)	94403	AB_2904530
Mouse anti-Ibal	1: 200	Santa Cruz Biotechnology (Santa Cruz, CA, USA)	sc-32725	AB_667733
Mouse anti-MBP	1: 500	Abcam (Cambridge, MA, USA)	ab62631	AB_956157
Mouse anti-CD68	1: 3000	Proteintech (Wuhan, China)	66231-2-Ig	AB_2881622
Rabbit anti-NF	1: 500	Abcam	ab8135	AB_306298
Rabbit anti-Iba1	1: 500	Abcam	ab178846	AB_2636859
Rabbit anti-CD86	1: 500	Proteintech	6903-1-AP	AB_2880677
Rabbit anti-CD206	1: 200	Proteintech	18704-1-AP	AB_10597232
Rabbit anti-CD206	1: 500	Cell Signaling Technology	24595	AB_2892682
Rabbit anti-cGAS	1: 1500	Proteintech	26416-1-AP	AB_2880507
Rabbit anti-p-STING	1: 1000	Cell Signaling Technology	50907	AB_2827656
Rabbit anti-cleaved caspase-3	1: 500	Cell Signaling Technology	9661	AB_2341188
Rabbit anti-CHOP Goat anti-rabbit IgG (H+L)	1: 1000	Cell Signaling Technology	2895 A-11008	AB_2089254
secondary antibody, Alexa Fluor™ 488 Goat anti-mouse IgG (H+L)	1: 1000	Thermo Fisher Scientific	A-11001	AB_143165
secondary antibody, Alexa Fluor™ 488 Goat anti-rabbit IgG (H+L)	1: 1000	Thermo Fisher Scientific	A-11010	AB_2534069
secondary antibody, Alexa Fluor™ 546 Goat anti-mouse IgG (H+L)	1: 1000	Thermo Fisher Scientific	A-11003	AB_2534077
secondary antibody, Alexa Fluor™ 546 Goat anti-rabbit IgG (H+L)	1: 1000	Thermo Fisher Scientific	A-21244	AB_2534071
secondary antibody, Alexa Fluor™ 647	1: 1000	Thermo Fisher Scientific		AB_2535812

CD206: Cluster of differentiation 206; CD68: cluster of differentiation 68; cGAS: Cyclic GMP-AMP synthase; CHOP: CCAAT-enhancer-binding protein homologous protein; Iba1 : Ionized calcium-binding adapter molecule 1; MBP: myelin basic protein; NeuN: neuron-specific nuclear protein; NF : neurofilament.

For spinal cord tissue, the tissue sections were initially fixed with 4% PFA solution for 30 minutes and then treated with sodium citrate antigen retrieval solution (C1032, Solarbio) for 30 minutes at 90°C. Following antigen retrieval, the spinal cord sections were permeabilized and blocked with 5% bovine serum albumin (Sigma-Aldrich) in PBS containing 0.3% Triton X-100 (Solarbio) for 1 hour at RT. Subsequently, the tissue sections were incubated with primary antibodies (Additional Table 1) overnight at 4°C. The next day, the sections were washed three times with PBS and then incubated with the appropriate secondary antibodies (Additional Table 1) for 1 hour at RT. The sections were then stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000, Merck Millipore, Billerica, MA, USA) to visualize nuclei. All images were acquired and processed using identical settings on the Olympus SLIDEVIEW^TM VS200 microscope and Nikon A1 confocal microscope (Nikon, Tokyo, Japan) to capture single optical Z sections and the three-dimensional images. Two researchers independently evaluated the immunostaining positive ratios, fluorescence intensities, and the colocalization ratios using ImageJ software and Photoshop, and calculated the mean values of the results for statistical analysis.

To detect the proportion of amoeboid cells in cultured BV2 cells, we evaluated Iba1⁺ (a marker of microglia) BV2 cells with phalloidin-labeled hypertrophic bodies and multiple retracted cytoplasmic processes. CD86 (an M1 microglia marker) and CD206 (an M2 microglia marker) staining was performed in BV2 cells and primary cultured microglia, and the proportions of CD86-labeled and CD206-labeled BV2 cells were quantified.

On day 3 after SCI in mice, we detected the proportions of cGAS⁺ (a marker of cGAS-STING pathway activation) and p-STING⁺ (an activated form of STING) cells in total Iba1⁺ cells near the spinal cord lesion areas. On day 7 after SCI in mice, we analyzed the colocalization of CD86/CD68 (a marker of activated microglia) and CD206/CD68 signaling near spinal cord lesion areas. Neuron-specific nuclear protein (NeuN) staining was used to evaluate neurons. On day 28 after SCI mice, neurofilament (NF, a marker of axons) was used to detect the proportion of myelinated axons of total NF⁺ axonal puncta near lesion areas, and the average gray value intensities of myelin basic protein (MBP, a marker of myelination) and NF were measured within the epicenter of the mice spinal cords.

The ratios of cleaved caspase-3 (CC-3, a marker of apoptosis)/NeuN and CCAAT-enhancer-binding protein homologous protein (CHOP, a marker of ER stress activation)/NeuN staining in cultured N2a cells were quantified.

Quantitative reverse transcription-polymerase chain reaction

The extraction of total RNA was performed using the RNA-Quick Purification Kit (ES-RN001, YISHAN Biotechnology, Beijing, China) according to the manufacturer’s protocol. In brief, approximately 1 × 10⁶ BV2 cells or 80 mg of spinal cord tissue (approximately 0.3 mm above and below the injured segment [T8–T10]) were placed into 500 μL of lysis buffer and subsequently lysed using either an ultrasonic cell disruptor (Scientz, Ningbo, China) or a high-throughput tissue grinder (Scientz,), respectively. Then, the lysate was centrifuged at 12,000 × g at RT for 2 minutes, and the resulting supernatants were translocated into fresh 1.5 mL Eppendorf tubes. Then, an equal volume of anhydrous ethanol was added to the supernatants. The mixture was thoroughly mixed and then transferred to the spin columns. These spin columns were then centrifuged at 12,000 × g at RT for 1 minute, and then were supplemented with 500 μL of wash buffer and centrifugated at 12,000 × g at RT for 1 minute. Subsequently, the spin columns were placed into 1.5-mL RNase-free Eppendorf tubes, and their lids were left open to dry for 2 minutes. After that, 20–30 μL of elution buffer was added into the spin columns, which were then centrifugated at 12,000 × g at RT for 1 minute. Finally, total RNA was collected and Thermo Scientific^TM NanoDrop^TM One (OD 260/280 = 1.90–2.20) was used to quantify RNA concentration. Subsequently, 1 μg of RNA from each sample was reverse transcribed into cDNA using the cDNA DyNAmo Kit (Vazyme Biotech, R211-01/02). The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was conducted using the SYBR Green PCR master mix (Vazyme Biotech, Q511-02/03). The PCR real-time cycling parameters were set as follows: an initial step at 95°C for 15 minutes, followed by a cycling step comprising denaturation at 94°C for 15 seconds, annealing for 30 seconds, and a final extension at 72°C for a duration of 30 seconds × 39 cycles. Subsequently, a melting curve analysis was performed to confirm the specificity of the primers. β-Actin was used as the endogenous control for data normalization. Shanghai Sangon Biotech synthesized the primers (Additional Table 2) used in these experiments.

Additional Table 2.

Primer sequences for quantitative reverse transcription-polymerase chain reaction

Primer	Sequences	Annealing temperatures (°C)	Product size (bp)
IL-1β	(F): 5’-AAGGGCTGCTTCCAAACCTTTGAC-3’ (R): 5’-ATACTGCCTGCCTGAAGCTCTTGT-3’	65	100
IL-6	(F): 5’-ATCCAGTTGCCTTCTTGGGACTGA-3’ (R): 5’-TAAGCCTCCGACTTGTGAAGTGGT-3’	64	134
TNF-α	(F): 5’-TCTCATGCACCACCATCAAGGACT-3’ (R): 5’-ACCACTCTCCCTTTGCAGAACTCA-3’	64	92
CD206	(F): 5’-TCAGCTATTGGACGCGAGGCA-3’ (R): 5’-TCCGGGTTGCAAGTTGCCGT-3’	66	105
Arg-1	(F): 5’-TTATCGAGCGCCTTTCTCAA-3’ (R): 5’-TGGTCTCTCAGGTCATACTCTGT-3’	60	120
β-actin	(F): 5’-GGCACCACACCTTCTACAATG-3’ (R): 5’-GGGGTGTTGAAGGTCTCAAAC-3’	59	133

Arg-1 : Arginase-1; CD206: cluster of differentiation 206; F : forward; IL-6: interleukin-6; R: reverse; TNF-α: tumor necrosis factor-α.

Western blotting

Cultured BV2 cells and N2a cells were collected and lysed in lysis buffer containing ice-cold RIPA buffer (Solarbio, R0010), 100 mM PMSF (protease inhibitor, Solarbio), 100 mM Na3VO4 and 100 mM NaF (phosphatase inhibitors, Solarbio). Subsequently, the lysates were sonicated using an ultrasonic cell disruptor (Scientz), followed by incubation at 4°C for 15 minutes and then centrifugation at 14,000 × g at 4°C for 15 minutes. The spinal cord sections (approximately 0.3 mm above and below the injured segments [T8–T10]) were lysed by a high-throughput tissue grinder (Scientz) in the lysis buffer and the lysate was placed at 4°C for 30 minutes and then centrifugated at 14,000 × g at 4°C for 30 minutes. After quantification with the BCA (Thermo Fisher Scientific, 23227) technique, proteins were diluted in 5× loading buffer and boiled at 100°C for 15 minutes. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Merck Millipore) activated by methanol (HUSHI, Shanghai, China). After blocking with 5% skim milk for 1 hour at RT, the immunoblots were washed three times in tris-buffered saline with Tween 20 (TBST) and then incubated with different primary antibodies (Additional Table 3) overnight at 4°C. Subsequently, the membranes were washed three times with TBST and then incubated with the secondary antibodies (Additional Table 3) for 1 hour at RT. Then, the membranes were washed in TBST three times, and the protein bands were visualized using the ECL detection kit (Biomedical Technology, Wuxi, China). The gray values of the bands were quantified using ImageJ software. To quantify the relative expression levels of CD206, CD86, cGAS, p-STING, p65, nuclear factor-κB inhibitory protein α (IκBα), B-cell lymphoma-2 (Bcl-2, a marker of apoptosis), Bcl-2-related X (Bax, a marker of apoptosis), eukaryotic initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4, a marker of ER stress activation), and CHOP, their gray values were normalized by their corresponding β-actin gray values. The gray values of p-p65 (a marker of NF-κB activation), p-IκBα, and p-eIF2α (a marker of ER stress activation) were normalized by those of p65, IκBα, and eIF2α, respectively.

Additional Table 3.

Antibodies used for western blotting

Antibody	Dilution	Supplier	Cat#	RRID
Mouse anti-IκBα	1: 1000	Cell Signaling Technology (Danvers, MA, USA)	4814	AB_390781
mouse anti-Bcl-2	1: 1000	Cell Signaling Technology	15071	AB_2744528
mouse anti-Bax	1: 1000	Cell Signaling Technology	89477	AB_2927544
mouse anti-β-actin	1: 10000	Sigma-Aldrich (St. Louis, MO, USA)	A5316	AB_476743
rabbit anti-CD86	1: 1000	Proteintech (Wuhan, China)	26903-1-AP	AB_2880677
rabbit anti-CD206	1: 1000	Proteintech	18704-1-AP	AB_10597232
rabbit anti-cGAS	1: 1500	Proteintech	26416-1-AP	AB_2880507
rabbit anti-p-STING	1: 1000	Cell Signaling Technology	50907	AB_2827656
rabbit anti-NF-κB p65	1: 1000	Cell Signaling Technology	8242	AB_10859369
rabbit anti-p-NF-κB p65	1: 1000	Cell Signaling Technology	3033	AB_331284
rabbit anti-p-IκBα	1: 1000	Cell Signaling Technology	2859	AB_561111
rabbit anti-p-eIF2α	1: 1000	Proteintech	28740-1-AP	AB_2881204
rabbit anti-eIF2α	1: 1000	Proteintech	11170-1-AP	AB_2096489
rabbit anti-ATF4	1: 1000	Proteintech	10835-1-AP	AB_2058600
rabbit anti-CHOP	1: 1000	Cell Signaling Technology	2895	AB_2089254
Goat anti-rabbit			A0545
HRP-conjugated secondary antibodies Goat anti-mouse	1: 10000	Sigma-Aldrich	A9044	AB_257896
HRP-conjugated secondary antibodies	1: 10000	Sigma-Aldrich		AB_258431

ATF4: Activating transcription factor 4; CD206: cluster of differentiation 206; CD86: cluster of differentiation 86; cGAS: cyclic GMP-AMP synthase; CHOP: CCAAT-enhancer-binding protein homologous protein; eIF2α: eukaryotic initiation factor 2 α; HRP: horseradish peroxidase; IκBα: nuclear factor-κB inhibitory protein α; p-eIF2α: phosphorylated eukaryotic initiation factor 2 α; p-IκBα: phosphorylated nuclear factor-κB inhibitory protein α; p-NF-κB p65: phosphorylated nuclear factor-κB p65.

Primary microglial culture

The primary microglial culture was performed as described previously (Lian et al., 2016). Newborn mouse pups (P0) were supplied by the Animal Center of the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China. Briefly, after the unconscious newborn mouse pups (P0) were euthanized by freezing with ice, the cortex and hippocampus were collected, chopped, and subsequently incubated in 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) at 37°C for 15 minutes, and dissociated into a single-cell suspension via fine scissors. The cells were plated on poly-D-lysine (PDL, 0.1 mg/mL, Merck Millipore)-coated culture flasks and cultured with DMEM + 10% FBS (Gibco) in a cell culture incubator with 95% air and 5% CO₂ at 37°C. After approximately 7 days, astrocytes formed successive cell layers at the bottom of the flasks and microglia grew above the astrocytic layer. Flasks were shaken at 250 r/min for 4 hours to collect floating cells in conditioned culture media (MM Microglia Medium, ScienCell, Carlsbad, CA, USA) to obtain purified primary cultured microglia. The cells were seeded into PDL-coated culture vessels and replenished with fresh culture medium after about 2 hours if the microglia had adhered to the bottom surface.

Animals

The animal experimental protocols used in this study were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (approval No. WYYY-AEC-2023-024) on March 13, 2023. C57BL/6 male mice (18–22 g, 6–8 weeks, specific-pathogen-free level) were obtained from the Animal Center of the First Affiliated Hospital of Wenzhou Medical University. The experimental animals were housed five per cage at a temperature of 18–22°C on a 12/12-hour day/night light cycle with unrestricted access to food and water. All experiments were conducted in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al., 2020). Mouse experiment analyses were carried out according to the experimental timeline as shown in Additional Figure 1 ^{(2.7MB, tif)} .

Spinal cord injury surgical procedures

A total of 200 C57BL/6 male mice were randomized into four equal groups (50 mice per group): Sham + DMSO (Solarbio), Sham + Tryp (HY-N6607, MedChemExpress, Monmouth Junction, NJ, USA; its chemical structure was shown in Additional Figure 2 ^{(341.8KB, tif)} ), SCI + DMSO (control), SCI + Tryp. All four groups were administered general anesthesia (20 mL/kg) via an intraperitoneal injection with avertin (2,2,2-tribromoethanol, Merck Millipore) in 0.9% saline solution. The clip-compressive SCI model was performed as described in previous studies (de Almeida et al., 2011). Briefly, a laminectomy at T8–T10 was performed using a surgical microscope (SMZ745, Nikon) and a mouse spinal cord adapter (68094, RWD, Shenzhen, China). The spinal cord was compressed by a vascular clip (30 g force, Kent Scientific, Torrington, CT, USA) for 15 seconds (Joshi and Fehlings, 2002; Marques et al., 2009). Extended and rotated legs and a sagging tail indicated the successful establishment of the clip-compressive SCI model. After being disinfected with a povidone-iodine solution and washed with saline, the muscles were meticulously sutured in a layered manner, and the skin was secured with staples. The mice were rehydrated with 2 mL of 0.9% NaCl (subcutaneous injection) and kept warm in an incubator. Upon awakening, the mice were transferred to clean cages in a warm room with accessible food and water. Only laminectomy was performed in the Sham group. The mice received the following treatments after the surgeries: DMSO-treated mice were administered a 5.0% sterile saline-diluted DMSO solution (at a dose of 0.2 mL/d for 14 days) by intragastric administration. Tryp-treated mice were intragastrically administered Tryp (concentration of 3 μg/mL, at 30 mg/kg per day for 14 days) dissolved in sterile saline containing 5.0% DMSO. All groups received their initial treatment at 3 hours post-surgery, followed by subsequent daily treatments at consistent intervals until the 14^th day after surgery. The bladders of mice in both the SCI + DMSO group and the SCI + Tryp group were emptied twice daily until normal urination was restored (Yao et al., 2018). The inflammatory responses and microglial signaling pathway in the spinal cord were analyzed at 3 and 7 days post-injury (dpi). Tissue repair was evaluated in mice at 7 and 14 dpi. To assess the therapeutic effectiveness of Tryp in the regeneration and remyelination of axons, and the functional recovery after SCI, immunostaining and behavioral analyses were conducted at multiple time points until 28 dpi. The experimental results were evaluated in a double-blind manner.

Spinal cord sections

On 3, 7, 14, and 28 dpi, three, six, six, and six mice per group, respectively, were anesthetized and perfused intracardially with 40 mL PBS, followed by 60 mL 4% PFA. Then, the spinal cords were immersed in 4% PFA for 24 hours and transferred to 30% sucrose for an additional 48 hours. Tissue located approximately 0.4 mm above and below the injured segment (T8–T10) was embedded in OCT, and then serial coronal sections (14 μm thick) and serial transverse sections (14 μm thick) were obtained using a cryostat (NX50, Thermo Fisher Scientific). Subsequently, the transverse and coronal sections measuring 150–300 μm from the epicenter of the spinal cord segment were obtained and then preserved at –20°C for staining analysis. The images were acquired using a digital slice scanner (SLIDEVIEW VS200, Olympus, Tokyo, Japan) and a Nikon A1 confocal microscope.

Behavioral analysis

The locomotor function of mice was analyzed by Basso Mouse Scale (BMS) and Footprint analysis on days 0, 1, 3, 7, 14, 21 and 28 after SCI.

Basso Mouse Scale scoring analysis

BMS scoring was conducted to evaluate locomotor function of hindlimbs in mice after SCI. All mice were evaluated by the same two observers who were blinded to the mice groupings. The observers assigned scores ranging from 0 to 9 in accordance with a previously established scoring system. This scoring system encompasses various aspects, including coordination, posterior ankle joint mobility, paw posture, trunk stability, and tail posture (Basso et al., 2006; Zhou et al., 2020a). Higher BMS scores indicate better locomotor function of mice.

Footprint analysis

To evaluate the locomotor ability of hindlimbs after surgery, the mice were directed to walk along a white paper-lined runway (Ma et al., 2001; Faulkner et al., 2004). Briefly, the mice were guided to walk along the runway (3 feet long, 3 inches wide) in a darkened cage and their forelimbs and hindlimbs were marked with red and blue nontoxic ink, respectively. The stride length and width were analyzed.

Hematoma area analysis

On 7 dpi, the SCI + DMSO and SCI + Tryp groups were anesthetized, and then perfused intracardially using 40 mL of 0.01 M PBS followed by 60 mL of 4% PFA. The spinal cords were removed and immersed in 4% PFA for 24 hours, and then transferred to 30% sucrose for another 48 hours. Images of ventral and dorsal spinal cord tissue of both groups were captured using a Nikon camera. The borders of hematomas in spinal cord tissue were labeled by red dashed lines, and the areas were measured by ImageJ software. The hematoma areas of dorsal and ventral spinal cord in 7 dpi SCI + Tryp mice were normalized by those of dorsal and ventral spinal cord in 7 dpi SCI + DMSO mice.

Hematoxylin-eosin staining

HE staining was performed as previously described (Liu et al., 2021a). After staining with hematoxylin (Solarbio) for 1 minute, the spinal cord sections were rinsed three times using double distilled water. Next, the slices were incubated in an acidic liquid alcohol differentiation solution for 30 seconds. Subsequently, they were stained with eosin (Solarbio) for 50 seconds, followed by sequential immersion in 95% ethanol and 100% ethanol, and cleared in xylene. Finally, the sections were covered using neutral resins. The borders of lesion areas in spinal cord tissue were labeled by black dashed lines, and the areas were measured using ImageJ software. The lesion areas of 7 dpi SCI + Tryp, 14 dpi SCI + DMSO, and 14 dpi SCI + Tryp mice were normalized by those of 7 dpi SCI + DMSO mice.

Oil Red O staining

The Oil Red O staining procedure was performed as described previously (Mehlem et al., 2013). In brief, spinal cord sections were air-dried, immersed in formalin for fixation, and then washed with running tap water for 1–10 minutes. Subsequently, sections were rinsed with 60% isopropanol and stained with a freshly prepared working solution of Oil Red O (Solarbio) for 15 minutes. Then, the samples were rinsed with a 60% isopropanol solution. The slices were then dipped into alum hematoxylin (Solarbio) five times, followed by thorough rinsing using distilled water. Finally, the specimens were mounted in aqueous glycerine jelly. All images were acquired and processed using identical settings on an Olympus SLIDEVIEW^TM VS200 microscope. The borders of Oil Red O+ areas in spinal cord tissue were labeled by black dashed lines, and the areas were measured using ImageJ software. The Oil Red O+ areas of 7 dpi SCI + Tryp, 14 dpi SCI + DMSO, and 14 dpi SCI + Tryp mice were normalized by those of 7 dpi SCI + DMSO mice.

Nissl staining

Nissl staining was performed as previously described (Wang et al., 2018). The spinal cord sections were incubated with a 0.1% solution of cresyl violet (Solarbio) for 3 minutes at RT. Afterwards, the slices were rinsed with double distilled water and then immersed in 95% ethanol. The specimens were then dehydrated in 100% ethanol and cleared in xylene, and finally mounted with neutral resins. For the spinal cord samples, the intermediate gray matter layers and ventral horn were analyzed. All images were captured and processed using identical settings on an Olympus SLIDEVIEW^TM VS200 microscope. The density of Nissl bodies (a marker of neurons) in the ventral horn was calculated using ImageJ. The numbers of neurons (indicated by Nissl staining) in the ventral horn of 14 dpi Sham + Tryp, 14 dpi SCI + DMSO, and 14 dpi SCI + Tryp mice were normalized by those of 14 dpi Sham + DMSO mice.

Conditional co-culture system

The conditional co-culture system of BV2 cells and N2a cells was used to assess the role of a microglia-derived inflammatory microenvironment on neuronal survival. In brief, after BV2 cells were treated with Ctrl (0.1% DMSO), LPS (1 μg/mL) + 0.1% DMSO, or LPS (1 μg/mL) + Tryp (10 μM) for 24 hours, the supernatants were discarded and 3 mL DMEM without FBS was added to the dishes. After 24 hours, the supernatants of BV2 cells were used to treat N2a cells as BV2 cell-derived conditional medium (CM) for 24 hours. Finally, immunofluorescence and western blotting analysis were performed on N2a cells as described above.

Statistical analysis

No statistical methods were used to predetermine sample sizes; however, our sample sizes were similar to those reported in a previous study (Fei et al., 2021). The data provided within this study originated from at least three separate and independent experiments. Statistical analyses were performed using either an unpaired two-tailed Student’s t-test or one-way/two-way analysis of variance with Tukey’s multiple comparisons test. Statistical significance was considered at P < 0.05.

Results

Tryptanthrin inhibits microglia-derived inflammation through promoting polarization to M2 phenotype

To examine the effects of Tryp on microglia-derived inflammation and its potential mechanisms, the LPS-induced BV2 cell model was used to mimic the characteristics of microglia located in the spinal cord lesion area after SCI in vitro (Liu et al., 2021b). CCK-8 assay showed that Tryp was not cytotoxic at concentrations lower than 10.0 μM within 24 hours (Additional Figure 3 ^{(1.2MB, tif)} A). Additionally, incubation with LPS (1 μg/mL) did not cause cytotoxic effects on BV2 cells (Additional Figure 3 ^{(1.2MB, tif)} B). We next evaluated the phenotypes of BV2 cells based on their morphology by coimmunostaining of Iba1 and phalloidin, a marker of F-actin. As shown in Figure 1A and B, LPS significantly induced BV2 cells to transform into an amoeboid-like morphology, the M1-like phenotype. Tryp inhibited the morphological changes of LPS-induced BV2 cells in a dose-dependent manner. Furthermore, qRT-PCR analysis showed that the mRNA expression of M1 microglial markers (IL-6, IL-1β, and TNF-α) were significantly upregulated by LPS treatment, and were significantly suppressed by incubation with Tryp (Figure 1C–E). The mRNA levels of M2 microglial markers (CD206 and Arg-1) were significantly upregulated after Tryp treatment (Figure 1F and G), which indicated that Tryp promoted microglial polarization to the M2 phenotype. Immunostaining and western blot analyses of CD86 and CD206 further showed that Tryp promoted the shift of microglia from the M1 to M2 phenotype in a dose-dependent manner (Figure 1H–M). Moreover, we evaluated the protein levels of the apoptosis-related markers Bcl-2 and Bax (Murphy et al., 2000, Wei et al., 2001) in BV2 cells after Tryp treatment. As shown in Additional Figure 4 ^{(928.2KB, tif)} A–C, no significant differences were found in the expression of Bcl-2 or Bax between groups, suggesting that Tryp treatment did not induce the apoptosis of BV2 cells. Taken together, these results suggested that microglia-derived inflammation was inhibited by Tryp through modulating microglial polarization to the M2 phenotype rather than apoptosis.

Figure 1.

Tryp inhibits microglia-derived inflammation through shifting polarization to the M2 phenotype.

(A) Double immunostaining of phallotoxin (purple) and Iba1 (green) of BV2 cells incubated with different levels of Tryp (0, 2.5, 5.0, and 10.0 μM) with or without LPS for 24 hours. BV2 cells with hypertrophic bodies and multiple retracted cytoplasmic processes were identified as amoeboid cells (marked by white arrows). The areas marked by boxes are shown in the ‘Enlarge’ panels. Scale bars: 50 μm. (B) Quantitative analysis of the percentages of activated M1-like BV2 cells as shown in A (n = 12 individual samples of cells per group). (C–G) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (C), IL-1β (D), TNF-α (E), CD206 (F) and Arg-1 (G) gene expression levels in BV2 cells incubated with different levels of Tryp (0, 2.5, 5.0, 10.0 μM) with or without LPS for 24 hours (normalized to β-actin, n = 5 individual samples of cells per group). (H) Immunostaining of CD86 (CD86⁺ cells were marked by white arrows) and CD206 (CD206⁺ cells were marked by white arrows) in BV2 cells incubated with different levels of Tryp (0, 2.5, 5.0, 10.0 μM) with or without LPS for 24 hours. Scale bar, 50 μm. (I, J) Quantitative analysis of the percentages of CD86⁺ (I) and CD206⁺ (J) BV2 cells as shown in H (n = 6 individual samples of cells per group). (K) Western blot analysis of CD206 and CD86 in BV2 cells incubated with various concentrations of Tryp (0, 2.5, 5.0, 10.0 μM) with or without LPS for 24 hours. (L, M) Quantitative analysis of CD86 (L) and CD206 (M) protein levels shown in (K) (normalized to β-actin, n = 6 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; Iba1: ionized calcium-binding adapter molecule 1; IB: immunoblotting; IL-6: interleukin-6; LPS: lipopolysaccharide; ns: not significant; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Tryptanthrin promotes M2 polarization of microglia through inhibiting the cGAS/STING/NF-κB pathway

It was recently shown that LPS-induced cellular dyshomeostasis may lead to cytoplasmic dsDNA accumulation, which then activates the cGAS-STING pathway to shift microglia to the M1 phenotype (Li et al., 2022a). Thus, we next examined whether Tryp promoted microglia (BV2 cell) M2 polarization by inhibiting cGAS-STING signaling in vitro. Western blot analyses showed that cGAS and p-STING were significantly upregulated in BV2 cells treated with LPS + DMSO (LD-treated group), compared with those in the Ctrl group (P = 0.0372). The upregulation of cGAS and p-STING was significantly inhibited by Tryp treatment (LT-treated group), when compared with the LD-treated group (P = 0.0002; Figure 2A–C). Furthermore, the levels of both p-p65 and p-IκBα were significantly reduced in the LT-treated group compared with those in the LD-treated group (Figure 2D–H). These results suggested that the cGAS/STING/NF-κB pathway was inhibited in Tryp-treated BV2 cells.

Figure 2.

Tryp promotes the M2 polarization of microglia through the cGAS/STING/NF-κB pathway.

(A) Western blot analysis of cGAS and p-STING in BV2 cells incubated with Ctrl, LPS + DMSO (LD group) and LPS + Tryp (10.0 μM; LT group) for 1 hour. (B, C) Quantitative analysis of cGAS (B) and p-STING (C) protein levels as shown in A (normalized to β-actin, n = 8 individual samples of cells per group). (D) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells incubated with Ctrl, LD and LT for 1 hour. (E–H) Quantitative analysis of p-p65 (E, normalized to p65), p65 (F, normalized to β-actin), p-IκBα (G, normalized to IκBα), and IκBα (H, normalized to β-actin) protein levels shown in (D) (p-p65 and p65, n = 11 individual samples of cells per group; p-IκBα and IκBα, n = 8 individual samples of cells per group). (I) Western blot analysis of cGAS and p-STING in BV2 cells treated with Ctrl, LD, LT and LPS + Tryp + SR-717 (20.0 μM; LT + SR-717) for 1 hour. (J, K) Quantitative analysis of cGAS (J) and p-STING (K) protein levels shown in I (normalized to β-actin, n = 6 individual samples of cells per group). (L) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 1 hour. (M–P) Quantitative analysis of p-p65 (M, normalized to p65), p65 (N, normalized to β-actin), p-IκBα (O, normalized to IκBα) and IκBα (P, normalized to β-actin) protein levels shown in L (n = 6 individual samples of cells per group). (Q–U) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (Q), IL-1β (R), TNF-α (S), CD206 (T) and Arg-1 (U) mRNA expression levels within BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 24 hours (normalized to β-actin, n = 3 individual samples of cells per group). (V) Immunostaining of CD86 (CD86⁺ BV2 cells and CD86⁺ primary cultured microglia were both marked by white arrows) and CD206 (CD206⁺ BV2 cells and CD206⁺ primary cultured microglia were both marked by white arrows) in BV2 cells and primary cultured microglia treated with Ctrl, LD, LT and LT + SR-717 for 24 hours. Scale bar: 100 μm. (W, X) Quantitative analysis of the percentages of CD86⁺ and CD206⁺ BV2 cells (W; n = 6 individual samples of cells per group), and primary cultured microglia (X; n = 3 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; cGAS: cyclic GMP-AMP synthase; Ctrl: control; DMSO: dimethyl sulfoxide; IκBα: nuclear factor-κB inhibitory protein α; IL-6: interleukin-6; ns: not significant; LPS: lipopolysaccharide; p: phosphorylated; STING: stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

To confirm whether Tryp promoted microglial M2 polarization through inhibiting the cGAS-STING pathway, SR-717, an agonist of STING, was used to activate STING signaling (Chin et al., 2020). As shown in Additional Figure 5 ^{(255.7KB, tif)} , the effective concentration of SR-717 was approximately 20 μΜ, and this concentration was selected for the following experiments. Western blot analyses showed that cGAS, p-STING, p-p65, and p-IκBα were significantly upregulated in BV2 cells treated with LPS + Tryp and SR-717 (LT + SR-717 group; Figure 2I–P). The qRT-PCR (Figure 2Q–U) and immunostaining analyses (Figure 2V–W) further showed that SR-717 partially reversed Tryp-induced M2 polarization of BV2 cells. Consistent with the results of BV2 cells, immunostaining analyses of primary cultured microglia also showed that Tryp promoted M1-to-M2 polarization, and the effect of Tryp was partially reversed by SR-717 (Figure 2V and X). Taken together, these results suggested that Tryp promoted microglial polarization to the M2 subtype through inhibiting the cGAS/STING/NF-κB pathway.

Targeting cGAS/STING/NF-κB pathway by tryptanthrin shifts microglia from M1 to M2 phenotype after spinal cord injury

To investigate whether targeting the cGAS/STING/NF-κB pathway by Tryp would polarize microglia to the M2 phenotype in vivo, we established the clip-compressive SCI model (Additional Figure 6 ^{(485.7KB, tif)} ). As resident innate immune cells in spinal cord, microglia play a vital role in the development of secondary inflammation after SCI (Devanney et al., 2020). As shown in Figure 3A and B, the expression levels of cGAS and p-STING were significantly decreased in Tryp-treated mice at 3 dpi compared with those in control-treated mice. Moreover, the coimmunostaining analyses of cGAS/Iba1 (Figure 3C and D) and p-STING/Iba1 (Figure 3E and F) showed that the microglial cGAS-STING pathway was inhibited in spinal cord of Tryp-treated mice at 3 dpi. Subsequently, we assessed the expression of p-p65, p65, p-IκBα, and IκBα at 7 dpi and found a significant inhibition of NF-κB activation in the Tryp-treated group (Figure 3G and H).

Figure 3.

Tryp shifts microglia from M1 to M2 phenotype after spinal cord injury by targeting the cGAS/STING/NF-κB pathway.

(A) Western blot analysis of cGAS and p-STING in spinal cord treated with Tryp at 3 dpi. (B) Quantitative analysis of cGAS and p-STING protein levels shown in A (normalized to β-actin, n = 6 mice per group). (C, E) Confocal laser scanning microscopy double immunostaining images (60× magnification) of cGAS (purple) and Iba1 (green) (C), and p-STING (purple) and Iba1 (green) (E) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 3 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. cGAS⁺Iba1⁺ cells and p-STING⁺Iba1⁺ cells were marked by white arrowheads. Scale bars, 50 μm (left) and 20 μm (enlarge). (D, F) Quantitative analysis of the percentages of cGAS⁺Iba1⁺ cells in Iba1⁺ cells (D, n = 3 mice per group), and p-STING⁺Iba1⁺ cells in Iba1⁺ cells (F, n = 3 mice per group). (G) Western blot analysis of p-p65, p65, p-IκBα and IκBα in spinal cord treated with Tryp at 7 dpi. (H) Quantitative analysis of p-p65 (normalized to p65), p65 (normalized to β-actin), p-IκBα (normalized to IκBα) and IκBα (normalized to β-actin) protein levels shown in G (n = 10 mice per group). (I, K) Confocal laser scanning microscopy double immunostaining images (60× magnification) of CD86 (red) and CD68 (green) (I), and CD206 (red) and CD68 (green) (K) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 7 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. CD86⁺CD68⁺ cells and CD206⁺CD68⁺ cells were marked by white arrowheads. Scale bars: 50 μm (left) and 20μm (enlarge). (J, L) Quantitative analysis of the percentages of CD68⁺/DAPI (left) and CD86⁺CD68⁺/CD68⁺ (right) (J, n = 6 mice per group), and CD68⁺/DAPI (left) and CD206⁺CD68⁺/CD68⁺ (right) (L, n = 6 mice per group). Data were analyzed by one-way analysis of variance with Tukey’s multiple comparisons test (left) and unpaired two-tailed Student’s t-test (right). (M) Western blot analysis of CD206 and CD86 expression in spinal cord treated with Tryp at 7 dpi. (N, O) Quantitative analysis of CD206 (N) and CD86 (O) protein levels shown in M (normalized to β-actin, n = 9 mice per group). (P–T) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (P), IL-1β (Q), TNF-α (R), CD206 (S) and Arg-1 (T) gene expression levels in spinal cords of Sham + DMSO, Sham + Tryp, SCI + DMSO and SCI + Tryp mice at 7 dpi (normalized to β-actin, n = 6 mice per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; cGAS: cyclic GMP-AMP synthase; DMSO: dimethyl sulfoxide; dpi: days post-injury; Iba1: ionized calcium-binding adapter molecule 1; IL-6: interleukin-6; ns: not significant; NF-κB: nuclear factor-κB; ns: not significant; p: phosphorylated; p-STING: phosphorylated stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

To further evaluate the role of Tryp in regulating the polarization and activation of microglia after SCI, CD86 and CD206 were each coimmunostained with CD68, which is highly expressed in activated microglia, near the spinal cord lesion area. As shown in Figure 3I–L, compared with those in the Sham group, the percentages of CD68⁺ cells were remarkably increased in the spinal cord of both control-treated and Tryp-treated mice at 7 dpi. There were no significant differences between control-treated and Tryp-treated mice, which indicated that Tryp did not suppress microglial activation at the early stage after SCI. The ratio of activated M1 microglia (CD68⁺CD86⁺ cells) was significantly reduced and the ratio of activated M2 microglia (CD68⁺CD206⁺ cells) was substantially increased in Tryp-treated mice compared with those in control-treated mice at 7 dpi (Figure 3I–L). Moreover, consistent with the immunostaining results, western blot analyses of the protein levels of CD206 and CD86 in lesion sites at 7 dpi (Figure 3M–O) showed that microglia were significantly shifted to the M2 phenotype in Tryp-treated mice compared with control-treated mice. Furthermore, qRT-PCR analysis of IL-6, IL-1β, TNF-α, CD206, and Arg-1 showed that a proinflammatory response was dominant at 7 dpi, and that Tryp promoted an anti-inflammatory response (Figure 3P–T). These results suggested that inhibition of the cGAS/STING/NF-κB pathway by Tryp promoted microglial polarization to the M2 subtype and suppressed microglia-derived neuroinflammation after SCI.

Tryptanthrin promotes functional recovery and tissue repair after spinal cord injury

To examine the effects of Tryp on functional recovery after SCI, footprint analysis and BMS scoring were performed. As shown in Figure 4A, the BMS score was significantly increased in Tryp-treated mice compared with that in control-treated mice (14 dpi, P = 0.0179; 21 dpi, P = 0.0179; 28 dpi, P = 0.0011). Footprint analysis showed that the stride length was significantly improved in Tryp-treated mice compared with that in control-treated mice at 14 dpi, whereas no significant difference was detected in the stride width between groups (Figure 4B–D). After SCI in mice, spinal cord lesions are detrimental to the reconstruction of neural networks across the epicenter and to the functional recovery of hindlimbs (Huang et al., 2021). Thus, we evaluated the histological and morphological changes in spinal cord tissue after SCI. Compared with that in control-treated mice, the hematoma area was decreased in both dorsal (P = 0.0002) and ventral (P = 0.0003) spinal cord in Tryp-treated mice at 7 dpi (Figure 4E and F). HE staining further showed that the lesion size in Tryp-treated mice was significantly decreased at both 7 and 14 dpi (7 dpi, P < 0.0001; 14 dpi, P = 0.0045) compared with those in control-treated mice (Figure 4G and H). Additionally, Oil Red O staining analyses showed that on 7 and 14 dpi, the diffusion of lipid-rich debris, which indicated the extent of inflammatory infiltration (Zhou et al., 2020b), was restricted in spinal cord tissue of Tryp-treated mice but not in control-treated mice (Figure 4I and J). These results suggested that Tryp promoted functional recovery and tissue repair after SCI in mice.

Figure 4.

Tryp promotes functional recovery and tissue repair after SCI.

(A) Quantitative analysis of motor function measured via BMS scoring analysis at different stages in mice treated with Tryp after SCI (n = 8 mice per group). Data were analyzed by two-way analysis of variance with Tukey’s multiple comparisons test. (B) Representative images of footprint analysis of Sham + DMSO, Sham + Tryp, SCI + DMSO and SCI + Tryp mice at 14 dpi. Scale bar: 10 mm. (C, D) Quantitative footprint analysis of stride length (C) and stride width (D) at different time points after SCI (n = 8 mice per group). (E) Typical images showing the hematoma area (indicated by red dashed lines) of dorsal and ventral spinal cord in control-treated and Tryp-treated mice at 7 dpi. (F) Quantitative analysis of the relative hematoma areas shown in E (normalized to SCI + DMSO group, n = 6 mice per group). (G) Typical hematoxylin-eosin staining images showing the lesion area (labeled by black dashed lines) in spinal cords at 7 and 14 dpi. Scale bar: 500 μm. (H) Quantitative analysis of the relative lesioned areas shown in G (normalized to 7 dpi SCI + DMSO group, n = 10 sections of 5 mice per group). (I) Typical Oil Red O staining images showing the Oil Red O⁺ area (indicated by black dashed lines) in spinal cords at 7 and 14 dpi. Scale bar: 500 μm. (J) Quantitative analysis of the relative Oil Red O⁺ areas shown in I (normalized to 7 dpi SCI + DMSO group, n = 10 sections of 5 mice per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data were from at least three separate and independent studies. BMS: Basso Mouse Scale; DMSO: dimethyl sulfoxide; dpi: days post-injury; SCI: spinal cord injury; Tryp: tryptanthrin.

Tryptanthrin inhibits neuronal loss after spinal cord injury

Recent studies have shown that the locomotor functional recovery after SCI is strongly correlated with the retention of ventral motor neurons (VMNs) and the regeneration of axons within and nearby the spinal cord lesion area (Chen and Shifman, 2019, Huang et al., 2021). Moreover, some studies have shown that oligodendrocyte-derived myelin sheaths support the recovery of axon conduction after SCI (Llorens-Bobadilla et al., 2020). Both Nissl staining (P = 0.0122; Figure 5A and B) and NeuN immunostaining (P = 0.0090; Figure 5C and D) showed Tryp significantly reduced the injury-induced loss of VMNs compared with control-treated mice. Moreover, western blot analysis of Bcl-2 and Bax showed that compared with that in control-treated mice, Bcl-2 expression was significantly downregulated, whereas Bax expression was not changed significantly (Figure 5E–G). These results suggested that Tryp inhibited the apoptosis of VMNs.

Figure 5.

Tryp inhibits neuronal loss after spinal cord injury.

(A) Typical Nissl staining images showing the retention of VMNs in spinal cord of Sham + DMSO, Sham + Tryp, SCI + DMSO, and SCI + Tryp mice at 14 dpi. The areas marked by boxes were shown in the ‘Enlarge’ panels. Scale bars: 500 μm and 200 μm (enlarge). (B) Quantitative analysis of the relative density of VMNs shown in A (normalized to SCI + DMSO group, n = 10 sections of 5 mice per group). (C) Immunostaining of NeuN (green) in spinal cord of Sham + DMSO, Sham + Tryp, SCI + DMSO and SCI + Tryp mice at 14 dpi. The areas marked by boxes were shown in the ‘Enlarge’ panels. Scale bars: 500 μm and 200 μm (enlarge). (D) Quantitative analysis of NeuN⁺ cell density shown in C (n = 15 sections of 5 mice per group). (E) Western blot analysis of the expression of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated protein X (Bax) in spinal cord of Sham + DMSO, Sham + Tryp, SCI + DMSO and SCI + Tryp mice at 14 dpi. (F, G) Quantitative analysis of Bcl-2 (F) and Bax (G) protein levels shown in (E) (normalized to β-actin, n = 15 mice per group). (H, J) Confocal laser scanning microscopy double immunostaining images (left 40× and right 60× magnification) of MBP (red) and NF (green) (H), and MBP (red) and NF (green) (J) in spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 28 dpi. Higher magnification panels show single optical Z sections of areas marked by boxes. MBP⁺NF⁺ puncta were marked by white arrowheads. The dotted lines referred to the boundaries of the spinal cord lesion area. Scale bars, 100 μm and 20 μm (enlarge). (I) Quantitative analysis of the percentages of MBP⁺NF⁺/NF⁺ shown in H (n = 6 mice per group). (K, L) Quantitative analysis of the relative intensity of NF (K) (normalized to SCI + DMSO group, n = 6 mice per group) and MBP (L) (normalized to SCI + DMSO group, n = 6 mice per group) shown in J. Data were analyzed by one-way analysis of variance with Tukey’s multiple comparisons test in B, D, F, G, and I. Data of the relative intensity of NF (K) and MBP (L) were analyzed by unpaired two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were presented as mean ± SEM. The data were from at least three separate and independent studies. DMSO: Dimethyl sulfoxide; MBP: myelin basic protein; NeuN: neuron-specific nuclear protein; NF: neurofilament; ns: not significant; dpi: days post-injury; SCI: spinal cord injury; Tryp: tryptanthrin; VMNs: ventral motor neurons.

We also defined each NF⁺ axonal punctum colocalized with MBP as a myelinated axon, and counted the ratio of MBP⁺NF⁺ axon puncta to total NF⁺ axon puncta. The loss of myelin sheaths was significantly inhibited in the spinal cord lesion area in the Tryp-treated mice compared with control-treated mice (Figure 5H and I). As shown in Figure 5J–L, the fluorescence intensities of both NF and MBP in the epicenter of injured spinal cord in the Tryp-treated mice were higher than those in the control-treated mice. These results suggested that Tryp alleviated the loss of VMNs in the lesion area after SCI.

Microglia treated with tryptanthrin suppresses endoplasmic reticulum stress-related neuronal apoptosis

We next investigated whether the improvement of microglia-derived inflammatory microenvironment induced by Tryp promoted neuronal survival after SCI (Figure 6A). Coimmunostaining analysis of CC-3 and NeuN showed that LT-treated BV2 cell-derived conditional medium (LT-CM) inhibited the apoptosis of N2a cells, compared with that in the LD-CM (LD-treated BV2 cell-derived CM) group (P = 0.0057; Figure 6B and C). As expected, LT-CM increased Bcl-2 expression and downregulated Bax expression in N2a cells (Figure 6D–F) compared with the LD-CM group. To further investigate the potential mechanisms of the neuroprotective effect of Tryp-treated BV2 cells on N2a cells, we evaluated the activation of ER stress, which has been demonstrated to cause apoptosis, especially in neurons (Penas et al., 2007). Western blot analysis of p-eIF2α (P = 0.0013), eIF2α, ATF4 (P = 0.0004), and CHOP (P = 0.0110) showed that LT-CM treatment alleviated ER stress in N2a cells compared with LD-CM treatment (Figure 6G–K). Double immunostaining analysis of CHOP and NeuN also showed that LT-CM treatment inhibited ER stress in N2a cells (Figure 6L and M). Taken together, these results suggested that Tryp pretreatment of microglia reduced the ER stress-related apoptosis of neurons.

Figure 6.

Microglia treated with tryptanthrin suppresses neuronal apoptosis through ER stress–related signaling.

(A) Schematic of BV2 and N2a cell treatments and the conditional co-culture system. (B) Double immunostaining of CC-3 (green) and NeuN (red) in N2a cells incubated with conditional medium (CM) of BV2 cells treated by Ctrl (Ctrl-CM), LPS + DMSO (LD-CM) and LPS + Tryp (LT-CM). CC-3⁺NeuN⁺ cells were marked by white arrows. Scale bar: 100 μm. (C) Percentages of CC-3⁺NeuN⁺ cells shown in B (n = 6 individual samples of cells per group). (D) Western blot analysis of Bcl-2 and Bax expression in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. (E, F) Quantitative analysis of Bcl-2 (E) and Bax (F) protein levels shown in D (normalized to β-actin, n = 6 individual samples of cells per group). (G) Western blot analysis of p-eIF2α, eIF2α, ATF4 and CHOP in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. (H–K) Quantitative analysis of p-eIF2α (H, normalized to eIF2α), eIF2α (I, normalized to β-actin), ATF4 (J, normalized to β-actin) and CHOP (K, normalized to β-actin) protein levels shown in G (n = 6 individual samples of cells per group). (L) Double immunostaining of CHOP (green) and NeuN (red) in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. The areas marked by boxes were shown in the ‘Enlarge’ panels. Scale bars: 50 μm and 20 μm (enlarge). (M) Percentages of CHOP⁺NeuN⁺ cells according to L (n = 6 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data were from at least three separate and independent studies. ATF4: Activating transcription factor 4; CC-3: cleaved caspase-3; CHOP: CCAAT-enhancer-binding protein homologous protein; DMSO: dimethyl sulfoxide; eIF2α: eukaryotic initiation factor 2α; ER: endoplasmic reticulum; LPS: lipopolysaccharide; NeuN: neuron-specific nuclear protein; ns: not significant; p: phosphorylated; Tryp: tryptanthrin.

Discussion

The results of the present study suggest that Tryp promoted microglial polarization to the M2 subtype, reduced ER stress-related neuronal apoptosis induced by microglia-derived neuroinflammation, and facilitated locomotor functional recovery after SCI. On the basis of these findings, we established a working model of the biological functions of Tryp in the functional recovery of SCI (Figure 7). In this model, Tryp suppresses microglia-derived neuroinflammation, attenuates injury-induced neuronal loss, and promotes functional recovery. Mechanistically, Tryp inhibits the activation of NF-κB signaling through inhibiting the cGAS-STING axis, and thereby shifts microglia from the M1 to M2 phenotype, which results in a reduction in neuroinflammation and eventually promotes functional recovery after SCI.

Figure 7.

Working model of tryptanthrin in microglial polarization after SCI.

Tryp promotes microglial polarization to the M2 phenotype by targeting the cGAS/STING/NF-κB axis, which inhibits neuroinflammation and neuronal loss, and eventually facilitates functional recovery after SCI. cGAS: Cyclic GMP-AMP synthase; ER: endoplasmic reticulum; IκB-α: nuclear factor-κB inhibitory protein α; NF-κB: nuclear factor-κB; SCI: spinal cord injury; STING: phosphorylated stimulator of interferon genes.

After SCI, injury-activated M1 microglia secrete various cytokines, including IL-6, IL-1β, and TNF-α, to trigger a proinflammatory response, which leads to axonal degeneration and demyelination, neuronal loss, and inhibition of reactive astrogliosis and glial scarring, and ultimately impairs locomotor recovery. For the past few years, an increasing number of studies have explored various methods to stably and effectively regulate microglial polarization after SCI to promote functional recovery (Chen et al., 2018; Fan et al., 2019, 2020; Gaojian et al., 2020; Kobashi et al., 2020). Previous studies have shown that Tryp inhibits not only the activation of some inflammatory cytokines in peripheral disorders (such as cyclooxygenase-2, 5-lipoxygenase, prostaglandin, and leukotriene) but also BV2 cell-derived inflammation in vitro (Kwon et al., 2017; Danz et al., 2002a, b). On the basis of these findings, we investigated whether Tryp regulates microglial polarization after SCI. The immunostaining and western blotting analyses of microglial polarization showed that Tryp reversed injury-induced microglia M1 polarization, thereby suppressing the secondary proinflammatory response at the early stage after SCI.

In the present study, the expression levels of IL-6, IL-1β, TNF-α, and CD86 were significantly upregulated in injured spinal cord in mice at 7 dpi, indicating that SCI triggered the proinflammatory response by inducing microglia to polarize toward the M1 phenotype. The expression of CD86 and these cytokines was inhibited by Tryp, whereas those of CD206 and Arg-1 were upregulated. Moreover, VMN retention, axonal regeneration and remyelination, and locomotor function recovery were enhanced after administration of Tryp in mice. These results indicate that Tryp may have neuroprotective effects through shifting M1/M2 polarization. Consistent with the results in vivo, the data from BV2 cells in vitro showed that incubation with Tryp significantly suppressed LPS-induced M1 polarization and inhibited the mRNA expression of microglia-derived proinflammatory cytokines, including IL-6, IL-1β, and TNF-α. Furthermore, at the maximum incubation concentration with non-toxic side effects, Tryp effectively modulated microglial polarization toward the M2 subtype, which possesses anti-inflammatory and neuroprotective functions. Nevertheless, the molecular mechanisms by which Tryp regulated microglial polarization remain unclear.

Recent studies indicated that SCI and LPS induced cellular dyshomeostasis and accumulation of cytoplasmic dsDNA, which augmented the activation of cGAS-STING signaling and ultimately promoted microglial differentiation toward the M1 phenotype (Hu et al., 2022; Li et al., 2022a). In addition to the interferon regulatory factor 3-mediated interferon response, TBK1-dependent and TBK1-independent NF-κB-mediated inflammatory responses are downstream of the cGAS-STING axis. However, we did not investigate whether Tryp’s effect on the cGAS/STING/NF-κB axis was dependent on TBK1 activity, which is a limitation of the present study and an issue worthy of study in the future. NF-κB is a vital transcription factor to mediate M1 microglia polarization and inflammatory response in the progression of neuroinflammatory diseases (Wang et al., 2014). Interestingly, we found that Tryp significantly suppressed LPS-induced activation of cGAS/STING/NF-κB signaling, thereby shifting the polarization of microglia toward the M2 subtype in vitro and in vivo. Additionally, we found that some cGAS⁺ puncta and p-STING⁺ puncta were present in the areas where Iba1 was absent, which suggested that SCI might induce the upregulation of both cGAS and p-STING not only in microglia but also in other cells, possibly astrocytes. In a previous study, Toll-like receptor 9, another important dsDNA receptor, was activated by CpG-rich DNA in microglia, and thereby promoted the microglia-derived inflammatory response and ultimately contributed to neurodegeneration and dysfunction in the CNS (Tauber et al., 2009). This process was suppressed by the physiological activity of glucocorticoid receptors in microglia (Maatouk et al., 2018). Our study did not address this important aspect, and it should be investigated in the future. Furthermore, SR-717, an agonist of STING (Chin et al., 2020), reversed the inhibitory effects of Tryp on the cGAS/STING/NF-κB axis, and shifted microglia toward the M1 phenotype. In summary, our results suggest that Tryp shifted microglia from the M1 to M2 phenotype and alleviated the microglia-derived proinflammatory response induced by SCI and LPS through targeting the cGAS/STING/NF-κB pathway.

Previous studies indicated that neuroinflammation was linked to neuronal apoptosis in neurodegenerative diseases (Gelders et al., 2018; Mangalmurti and Lukens, 2022). Neuroinflammation can lead to activation of ER stress, calcium efflux, and the ubiquitin–proteasome system, and eventually induce cell death (Gupta et al., 2021). Furthermore, CHOP, the kernel transcription factor of ER stress, can suppress the protein expression of Bcl-2, an anti-apoptotic protein of the Bcl family, to increase the level of CC-3 and lead to cell apoptosis. Therefore, to further explore the molecular mechanisms of microglia-mediated neuronal apoptosis, we evaluated the activation of the eIF2α/ATF4/CHOP axis. Our results showed that the ER stress was markedly inhibited in LT-CM-treated N2a cells, in which LD-CM-induced apoptosis was also significantly alleviated. However, the present study did not investigate the intercellular molecules in the crosstalk between microglia and neurons, which could be microglia-derived cytokines. This issue should also be studied in the future.

Previous studies (De La Torre et al., 1975; Bulama et al., 2022) showed that treatment with DMSO, a vehicle for Tryp administration in this study, improved functional recovery in dogs following spinal cord contusion injury and in rats with traumatic brain injury, compared with no treatment. To eliminate the potential therapeutic efficacy of DMSO itself, we compared the inflammatory cytokines mRNA expression and the number of Iba1⁺ cells in the spinal cords of saline-treated and 5% DMSO-treated mice at 7 dpi; no significant differences were found between inflammatory cytokines (Additional Figure 7 ^{(4.8MB, tif)} F–J) or between the quantity of Iba1⁺ cells in the spinal cord lesion area (Additional Figure 7 ^{(4.8MB, tif)} K–L). In addition, the CCK-8 assay and qRT-PCR analysis of inflammatory cytokines (Additional Figure 7 ^{(4.8MB, tif)} A–E) in BV2 cells showed that 0.1% DMSO did not have cytotoxic or additional effects on inflammation, indicating that 0.1% DMSO did not have the potential effects reported previously (Yuan et al., 2014) in our in vitro model.

This study had some limitations. We focused on the microglial response to Tryp treatment after SCI, and we cannot exclude the possibility that the cGAS/STING/NF-κB axis in astrocytes and other cells participates in the repair of injury after SCI. We are very interested in using mice with specific ablation of microglial cGAS for further research. In addition, sexual dimorphism has been reported in microglial activation and neuroinflammation in the CNS diseases (Villa et al., 2018; Li et al., 2022b). Female mice have better prognosis of functional recovery than male mice after SCI (Li et al., 2022b). Therefore, to streamline the evaluation of Tryp’s effects after SCI, we used only male mice in the present study. Future studies should investigate the efficacy of Tryp in female mice after SCI to investigate whether the pharmacological inactivation of the microglial cGAS/STING/NF-κB axis is independent of sex.

In conclusion, the present study showed that Tryp targets the cGAS/STING/NF-κB axis to promote microglia polarization to the M2 subtype, and thereby inhibits microglia-derived neuroinflammation and neuronal loss following SCI. The findings suggest that modulating M1/M2 microglial polarization via the cGAS/STING/NF-κB pathway is a prospective therapeutic target for SCI and other neuroinflammatory diseases.

Additional files:

Additional Table 1: Antibodies used for immunostaining.

Additional Table 2: Primer sequences for quantitative reverse transcription-polymerase chain reaction.

Additional Table 3: Antibodies used for western blotting.

Additional Figure 1 ^{(2.7MB, tif)} : Experimental timeline.

Additional Figure 1

Experimental timeline.

Additional Figure 2 ^{(341.8KB, tif)} : The chemical structure of tryptanthrin.

Additional Figure 2

The chemical structure of tryptanthrin.

Additional Figure 3 ^{(1.2MB, tif)} : Tryptanthrin has no cytotoxic effect at concentrations below 10 μM over a 24-hour period.

Additional Figure 3

Tryptanthrin has no cytotoxic effect at concentrations below 10 μM over a 24-hour period.

(A) Cell Counting Kit-8 assay of the effects of tryptanthrin on cell viability of BV2 cells treated with various concentrations of tryptanthrin (0-50 μM) for different durations (3, 6, 12, 24 h) (n = 5 per group). (B) Cell Counting Kit-8 assays of the effects of tryptanthrin on cell viability of BV2 cells treated with various concentrations of tryptanthrin (0-50.0 μM) in the absence or presence of LPS for 24 hours (n = 5 per group). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P< 0.0001. Data were mean ± SEM. LPS: Lipopolysaccharide; ns: not significant.

Additional Figure 4 ^{(928.2KB, tif)} : Tryptanthrin has no significant effects on the apoptosis of BV2 cells.

Additional Figure 4

Tryptanthrin has no significant effects on the apoptosis of BV2 cells.

(A) Western blot analysis of the expression of Bcl-2 and Bax in BV2 cells treated with various concentrations of Tryp (0, 2.5, 5.0, and 10.0 μM) with or without LPS for 24 hours. (B, C) Quantitative analysis of Bcl-2 (B) and Bax (C) protein levels as shown in (A) (normalized to β-actin, n = 6 per group). Data were mean ± SEM. Bcl-2: B-cell lymphoma-2; Bax: Bcl-2-related X; LPS: lipopolysaccharide; ns: not significant; Tryp: tryptanthrin.

Additional Figure 5 ^{(255.7KB, tif)} : SR-717 activates cGAS-STING pathway in BV2 cells.

Additional Figure 5

SR-717 activates cGAS-STING pathway in BV2 cells.

Western blot analysis of the expression of cGAS and p-STING in BV2 cells treated with various concentrations of SR-717 (0, 10, 20, 50, 100, and 200 μM) for 1 hour. cGAS: Cyclic GMP-AMP synthase; SR-717: an agonist of STING; STING: stimulator of interferon genes.

Additional Figure 6 ^{(485.7KB, tif)} : The typical images of clip-compressive spinal cord injury mouse model.

Additional Figure 6

The typical images of clip-compressive spinal cord injury mouse model.

Additional Figure 7 ^{(4.8MB, tif)} : 0.1% and 5% DMSO have no significant effects on the inflammation in vitro and in vivo respectively.

Additional Figure 7

0.1% and 5% DMSO have no significant effects on the inflammation in vitro and in vivo respectively.

(A-E) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (A), IL-1β (B), TNF-α (C), CD206 (D) and Arg-1 (E) gene expression levels in BV2 cells incubated with LPS (1 μg/mL) and LPS (1 μg/mL) + DMSO (Dimethyl sulfoxide) (0.1%) for 24 hours (normalized to β-actin, n = 4 individual samples of cells per group). (F-J) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (F), IL-1β (G), TNF-α (H), CD206 (I), and Arg-1 (J) gene expression levels in spinal cords of SCI + Saline and SCI + DMSO (5%) mice at 7 dpi after SCI (normalized to β-actin, n = 4 mice per group). (K) Immunostaining of Iba1 (purple) in the vicinity of spinal-cord gaps of SCI + Saline and SCI + DMSO mice at 7 dpi after SCI. Scale bar: 50 μm. (L) Quantitative analysis of relative number of Iba1+ cells as shown in (K) (normalized to SCI + Saline mice, n = 4 mice per group). Data were mean ± SEM. Arg-1: Arginase-1; CD206: cluster of differentiation 206; DMSO: dimethyl sulfoxide; Iba1: ionized calcium-binding adapter molecule 1; IL-6: interleukin-6; LPS: lipopolysaccharide; ns: not significant; SCI: spinal cord injury; TNF-α: tumor necrosis factor-α.

Funding Statement

Funding: This work was supported by the National Natural Science Foundation of China, Nos. 82071387 (to HT), 81971172 (to YW); the Natural Science Foundation of Zhejiang Province, China, No. LY22H090012 (to HT); and the Basic Research Project of Wenzhou City, China, No. Y20220923 (to MZ).

Data availability statement:

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