TGR5 protects against pSNL-induced mechanical allodynia by alleviating neuroinflammation in the injured nerves of male mice

TGR5 protects against pSNL-induced mechanical allodynia by alleviating neuroinflammation in the injured nerves of male mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TGR5 protects against pSNL-induced mechanical allodynia by alleviating neuroinflammation in the injured nerves of male mice Wen-Ge Shi, Yao Yao, Ya-Jing Liang, Jie Lei, Shi-Yang Feng, Yue Tian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3852075/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neuropathic pain is a pervasive medical challenge that currently lacks effective treatment solutions. Molecular changes occurring at the site of peripheral nerve damage contribute to the development of peripheral and central sensitization, which are critical components of neuropathic pain. This study aimed to investigate the role of the G protein-coupled bile acid receptor (GPBAR1, also known as TGR5) in the peripheral mechanisms underlying neuropathic pain induced by partial sciatic nerve ligation (pSNL) in male mice. TGR5 was upregulated in injured nerves and colocalized predominantly with macrophages. Peri-sciatic nerve administration of the TGR5-specific agonist INT-777 provided sustained relief from mechanical allodynia. Transcriptome sequencing revealed that pain relief was primarily attributable to reduced neuroinflammation. This finding was corroborated by a reduction in myeloid cells and proinflammatory mediators (including CCL3, CXCL9, IL-6, and TNF-α), accompanied by an increase in the percentage of anti-inflammatory M2 macrophages following INT-777 administration. Furthermore, myeloid cell-specific TGR5 knockdown in the sciatic nerve following pSNL exacerbated both mechanical allodynia and neuroinflammation. This is substantiated by data from the bulk RNA-seq and upregulated expression levels of inflammatory mediators (including CCL3, CCL2, IL-6, TNF-α and IL-1β), as well as increased monocytes/ macrophages in the injured nerve. Besides, the activation of microglia in the ipsilateral dorsal horn of spinal cord induced by pSNL altered when TGR5 in the sciatic nerve was manipulated. In summary, TGR5, present in injured nerves, plays a protective role and offers potential as a target for treating neuropathic pain. Neuropathic pain Mechanical allodynia Sciatic nerve TGR5 Macrophages Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or similar stimuli (Raja et al. 2020 ). Neuropathic pain occurs when damage or disease affects the somatosensory nervous system (Scholz et al. 2019 ). Neuropathic pain often becomes chronic and contributes significantly to the global disease burden, affecting 6.9–10% of the global population (van Hecke et al. 2014 , Bouhassira and Attal 2016 , Rice et al. 2016 ). The mechanisms underlying neuropathic pain are complex and vary between different states of the disease (Baron et al. 2010 ). At present, clinical treatments for neuropathic pain predominantly focus on drugs that regulate neural conduction. However, these drugs frequently lead to unavoidable side effects and less-than-ideal treatment efficacy (Bannister et al. 2020 ). Further research is imperative to identify treatment targets based on distinct pathological mechanisms. Neuropathic pain triggers neuroinflammation (Ji et al. 2016 ). Following peripheral nerve injury, immune cells infiltrate the injury site, secrete cytokines and chemokines, activate local immune cells, and attract more circulating leukocytes to both the injury site and along the neural pain pathway (Thacker et al. 2007 , Ellis and Bennett 2013 , McMahon et al. 2015 ). In the peripheral nervous system, macrophages participate in pain modulation by direct contacting with neurons (Iwai et al. 2021 , Tanaka et al. 2023 ) and releasing soluble mediators that bind to receptors expressed on other cells (Donnelly et al. 2020 , Domoto et al. 2021 , Msheik et al. 2022 ). However, clinical trial results for patients with neuropathic pain treated with neutralizing antibodies targeting proinflammatory cytokines are inconclusive (Williams et al. 2013 ). Therefore, a deeper understanding of the neuroimmune processes involved in the development and resolution of neuropathic pain is necessary for the development of effective treatment strategies. TGR5 is a G protein-coupled bile acid receptor (GPBAR1), which was initially reported to mediate the anti-inflammatory effects of bile acids on lipopolysaccharide (LPS)-treated monocytes and macrophages (Kawamata et al. 2003 ). The beneficial role of TGR5 activation in attenuating inflammation, including neuroinflammation induced by subarachnoid hemorrhage (Zuo et al. 2019 , Hu et al. 2021 ), sepsis (Jin et al. 2021 ), and intracerebroventricular injection of LPS (Wu et al. 2019 , Wu et al. 2019 ), have been discovered in several disease models (Perino et al. 2021 ). However, the role of TGR5 in pain modulation is not well understood and the conclusions of previous studies are paradoxical. Activation of TGR5 in sensory nerves, achieved through intra-plantar or intrathecal injection of bile acids leads to analgesia in response to mechanical paw stimulation (Alemi et al. 2013 ). In contrast, TGR5 activation wa shown to promot visceral hypersensitivity in a mouse model of irritable bowel syndrome (Castro et al. 2019 ). Given the aforementioned uncertainties, the role of TGR5 in the pathological processes of neuropathic pain remains unclear. Therefore, this study aimed to investigate the role of TGR5 in the peripheral mechanisms of mechanical allodynia in a mouse model of neuropathic pain induced by partial sciatic nerve ligation (pSNL). 2. Materials and methods 2.1 Animals Experiments were performed following the ethical principles outlined by the Animal Care and Use Committee of the Peking University Center of Health Science. We housed 6–8-week-old inbred male C57BL/6J (Vital River Laboratory Animal Technology Co. Ltd.) and Lyz2-Cre (Cat. NO. NM-KI-215037; Shanghai Model Organisms Center, Inc.) mice in a pathogen-free environment. 2.2 Neuropathic pain model Neuropathic pain was induced in mice through pSNL by modifying previously published methods (Seltzer et al. 1990 , Malmberg and Basbaum 1998 , Yao et al. 2016 ). Briefly, under 1% sodium pentobarbital anesthesia, the right sciatic nerve was exposed and carefully separated from the adjacent connective tissues near the trochanter. A 9–0 silk suture with a 3/8 curved mini-needle was used to ligate approximately half of the dorsal portion of the sciatic nerve. 2.3 Behavioral tests All tests were conducted during the light (rest) phase. The mice were habituated to the testing environment 2 days prior to testing. The investigator was blinded to the mouse groups. Mechanical allodynia was measured using a previously reported Von Frey test which we modified (Callahan et al. 2008 , Deuis et al. 2017 ). Von Frey filaments (Stoelting, Wood Dale) were used, beginning with a 0.16 g filament. The presence or absence of a positive withdrawal response (flicking, licking, or lifting) determined the choice of lower- or higher-weight filaments. Four additional responses were observed after the initial change. The 50% paw withdrawal threshold was calculated based on the recorded test results (Dixon 1980 , Chaplan et al. 1994 ). 2.4 Western blot analysis Under 1% sodium pentobarbital anesthesia, the mice underwent transcardial perfusion with chilled PBS. Approximately 1.5 cm of sciatic nerve tissue (including the injured site and proximal and distal parts) was dissected distal to the semitendinosus nerve branch. Proteins in the tissues were extracted using RIPA buffer (Applygen), and 1 mM phenylmethylsulfonyl fluoride and phosphatase inhibitors were added. The PVDF membranes, carrying proteins, were initially blocked using a 5% non-fat milk solution. Subsequently, they were incubated overnight at 4°C with the following primary antibodies: rabbit anti-TGR5 (1:1000; ab72608, Abcam), mouse anti-NLRP3 (1:1000; AG-20B-0014, AdipoGen), rabbit anti-ASC (1:1000; 67824S, CST), rabbit anti-Caspase-1 (1:1000; 24232S, CST), and rabbit anti-IL-1β (1:200; ab133357, Abcam). HRP-conjugated secondary antibodies (1:1000; Immunoway) were used to probe the blots, and enhanced chemiluminescence detection (Tanon) was used to detect the signals. Band intensities were quantified using ImageJ, and internal controls were used to calculate the relative protein expression. 2.5 Immunofluorescence staining Under 1% sodium pentobarbital anesthesia, the mice underwent transcardial perfusion with a pre-warmed 0.9% saline solution, followed by a chilled 4% paraformaldehyde solution. The ipsilateral sciatic nerves were dissected, fixed for 24 h, and subsequently subjected to overnight cryoprotection at 4°C in a 30% sucrose solution. After embedding in an Optimum Cutting Temperature Compound (OCT, Sakura), the nerves were sliced into 10 µm thick frozen sections. These sections, affixed to slides, were blocked with 10% donkey serum in PBS enriched with 0.3% Triton X-100 and incubated overnight at 4°C with the following primary antibodies: rabbit anti-TGR5 (1:500; ab72608, Abcam), mouse anti-PGP9.5 (1:1000, NB600-1160, Novus), rat anti-mouse F4/80 (1:500; MCA497, Biorad), mouse anti-NLRP3 (1:500; AG-20B-0014, AdipoGen), rabbit anti-ASC (1:500; 67824S, CST) and rabbit anti-IL-1β (1:200; ab133357, Abcam). Subsequently, the appropriate secondary antibodies (1:1000; Jackson ImmunoResearch) were used for incubation, including Cy3 donkey anti-rabbit IgG, AF488 donkey anti-rat IgG, AF488 donkey anti-mouse IgG, Cy3 donkey anti-mouse IgG, and AF647 donkey anti-rabbit IgG. Nuclear labeling was accomplished by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an Olympus microscope. To ensure reproducibility, a minimum of three fields per section were examined. Quantitative analyses were performed blindly using ImageJ Pro Plus software (Media Cybernetics Inc.). 2.6 Bulk RNA-seq and data analysis Total RNA was extracted from sciatic nerves using TRIzol reagent (Life Technologies) and assessed for quality, concentration, and chemical purity using spectrophotometry (NanoDrop). High-quality RNA libraries were generated and sequenced using the BGI system (Shenzhen, China). Subsequently, the sequencing data were analyzed using the Dr. Tom Multi-omics Data Mining System (available at https://biosys.bgi.com ), with significance levels adjusted using a stringent threshold (Q value ≤ 0.05). 2.7 RT-qPCR analysis A total of 400 ng RNA extracted from the sciatic nerve or bone marrow-derived macrophages (BMDMs) was reverse-transcribed. Subsequently, 1 µL of the resulting template cDNA was amplified in a 20 µL reaction volume, with a concentration of 0.5 µM for the specified PCR primer. Quantitative real-time PCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) with SYBR Premix Ex Taq II (Takara) following a previously described method (Liang et al. 2019 ). Specifc primers were employed for amplifcation, including Tgr5 (forward: 5′-ACTGGTCCTGCCTCCTTCTCC-3′, reverse: 5′- ACACTGCCATGTAGCGTTCCC-3′), Ccl3 (forward: 5′-TTGCTGTTCTTCTCTGTACCAT-3′, reverse: 5′- AATAGTCAACGATGAATTGGCG-3′), Ccl4 (forward: 5′-CTTGCTCGTGGCTGCCTTC-3′, reverse: 5′- TGCTGGTCTCATAGTAATCCATCAC-3′), Tnfa (forward: 5′-GCCTCTTCTCATTCCTGCTTGTGG-3′, reverse: 5′-GTGGTTTGTGAGTGTGAGGGTCTG-3′), Il1b (forward: 5′-TCGCAGCAGCACATCAACAAGAG-3, reverse: 5′-AGGTCCACGGGAAAGACACAGG-3′), Nlrp3 (forward: 5′-GCCGTCTACGTCTTCTTCCTTTCC-3′, reverse: 5′-CATCCGCAGCCAGTGAACAGAG-3′) and Gapdh (forward: 5′-AACTTTGGCATTGTGGAAGGGCTC-3′, reverse: 5′-TGGAAGAGTGGGAGTTGCTGTTGA-3′). 2.8 Flow cytometry assay Cells of sciatic nerves were isolated according to a previously described protocol (Liu et al. 2017 ). Part of the sciatic nerve was carefully dissected and transferred to 200 µL papain solution (1x HBSS/papain [15 U/mL]/DNAase [10 µg/mL]; 1x HBSS, Gibco; Papain, Roche; DNase, Sigma) on ice. After finely chopping the tissue samples into small pieces and digesting them at 37°C for 30 min, 400 µL of Solution A (1x HBSS/10% FBS/10 µg/mL DNase; FBS, Gibco) was added to terminate the digestion, followed by homogenization using a 1 mL syringe fitted sequentially with a 21 and 23 G needle. After homogenization, the solution was centrifuged at 12,000 rpm for 10 s. The cell pellets were washed with 1 mL of FACS buffer (1x HBSS/10% FBS) and resuspended in 100 µL of FACS buffer, to which 1 µL of rat anti-mouse CD16/CD32 clone 2.4G2 (BioLegend) was added. Following a 30-min incubation on ice, the following fluorescent antibodies were introduced into the incubation buffer and incubated on ice for another 30 min: PerCP-Cy5.5 rat anti-CD11b (550993, BD), PE/Cyanine7 anti-mouse F4/80 (123114, BioLegend), PE anti-mouse CD86 (12-0862-81, Thermo Fisher Scientific), and APC anti-mouse CD206 (141708, BioLegend). Subsequently, the cells were washed and resuspended in FACS buffer for flow cytometry. Cellular events were acquired using a Beckman Galios machine and the data were analyzed using FlowJo software. 2.9 Cytokine measurement Cytokines (IFN-γ, IL-10, CCL4 [MIP-1β], IFN-α, CXCL9 [MIG], CXCL10 [IP-10], TNF-α, IL-6, VEGF, IL-4, CCL3 [MIP-1α], and CCL2 [MCP-1]) in the sciatic nerve were measured using a LEGENDplex Tm MU Cytokine Release Syndrome Panel w/FP (741023, BioLegend), following the manufacturer's protocol and analyzed using a Beckman Galios flow cytometer. Sciatic nerve tissues were homogenized in 100 µL of cold PBS. The supernatant was collected after centrifugation at 12,000 × g for 10 min at 4°C. Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). For the analysis, 25 µL of sciatic nerve extract was used. The reported values in pg/mL were normalized to pg/mg of total protein, considering the protein concentration. 2.10 Lentivirus‑mediated shRNA construction The shRNA lentivirus for knockdown sequence identification was constructed by OBiO Technology (Shanghai, China) using the lentiviral vector, pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE. Three sequences targeting GPBAR1 mRNA were as follows: 5′-CCTACCTCTACCTGGAAGTTT-3’ (shGPBAR1-1), 5′-CTCTGTTATCGCTCATCTCAT-3′ (shGPBAR1-2), and 5′-TGCTTCTTCCTAAGCCTACTA-3’ (shGPBAR1-3). The sequence of the control shRNA was 5′-CCTAAGGTTAAGTCGCCCTCG-3′ ( referred to as shControl). The viral titers of shGPBAR1 and shControl reached 2.7 × 10 8 and 7.7 × 10 8 TU/mL, respectively. A Cre-dependent shRNA lentivirus for TGR5-specific knockdown was constructed by BrainVTA (Wuhan, China). The lentiviral vector sequence was rLV-CMV-DIO-EGFP-5'miR30-shRNA-3'miR30-WPRE. The sequence targeting GPBAR1 mRNA was 5′-CCTACCTCTACCTGGAAGTTT-3 and named shGPBAR1. The viral titers of shGPBAR1 and shControl reached 1 × 10 9 TU/mL. 2.11 BMDMs culture and treatments The isolation and incubation of bone marrow derived macrophages (BMDMs) were conducted following a well-established protocol with minor adjustments (Perino et al. 2014 ). Briefly, cells in the femur and tibia were flushed out of the bone using sterile HBSS. Following filtration through a 70-µm cell strainer, the cell solution was lysed using a lysis buffer (Solarbio) to remove the blood cells. After centrifugation, the cell pellets were suspended in 10 mL complete medium (RPMI 1640 medium/5 mM penicillin/streptomycin/10% FBS/30 ng/ml macrophage colony-stimulating factor (MCSF)) (RPMI 1640 medium, Gibco; penicillin/streptomycin, Gibco; MCSF, BioLegend) and plated in a 10-cm dish for incubation at 37°C in an atmosphere of 5% CO 2 . For lentivirus transfection, the BMDM incubated for 3 days was infected with a new complete medium containing lentiviruses at a multiplicity of infection of 100 for 12 h. After discarding the lentiviruses, the cells were cultured for an additional 72–96 h in a fresh complete medium and then prepared for western blotting, immunofluorescence staining, and RT-qPCR analysis. 2.12 Peri-sciatic nerve injection Perineural injections were administered according to a previously established protocol (Kiguchi et al. 2010 ). A 30-gauge needle attached to a micro-syringe was gently inserted into the scar site induced by pSNL procedure. The substances were administered to the region surrounding the sciatic nerve in the upper thigh of the hind limb. Successful injection and targeting of the sciatic nerve were confirmed using blue dye which was deposited in the nerve after injection. The TGR5-specific agonist, INT-777 (HY-15677, MCE), was dissolved in 10% DMSO to concentrations of 0.5 µg/µL, 5 µg/µL, and 50 µg/µL. INT-777 or 10% DMSO (4 µL) was administered daily by peri-sciatic nerve injection at the time of pSNL surgery and on days 1–6 after pSNL under isoflurane anesthesia. Cre-dependent shRNA lentivirus (shGPBAR1 or shControl) was perineurally injected at a final titer of 1 × 10 9 TU/mL, with a total volume of 4 µL per mouse, concurrent with pSNL surgery. Additional injections were administered on the first and second days following pSNL surgery, while the animals were under isoflurane anesthesia. 2.13 Data analysis Statistical analyses were performed using GraphPad Prism software version 9.0. Data are presented as mean ± SEM. The mean values of the two groups were compared using a two-tailed unpaired Student’s t-test. Data from multiple groups were compared using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Two-factor interaction experiments were conducted using two-way ANOVA with Šídák’s post hoc test. Significance levels were labeled as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. 3. Results 3.1 TGR5 increased in the sciatic nerve after pSNL, mainly colocalized with macrophages In the mouse model of neuropathic pain, pSNL-induced mechanical allodynia, indicated by the decreased paw withdrawal threshold, was detected on post-operative day (POD) 1 and persisted until POD14 (Fig. 1 A). TGR5 expression in the injured sciatic nerve was determined using western blot analysis (Fig. 1 B). The results indicated that the protein levels of TGR5 significantly and continuously increased from POD1, peaked on POD7, and remained elevated until POD14 (Fig. 1 C). To investigate the cellular localization of TGR5, F4/80 (a macrophage marker) and PGP9.5 (a nerve fiber marker) were double-stained with TGR5 in sciatic nerves from mice of sham group and pSNL group (Fig. 1 D and 1 E). Consistent with the data of western bolting, TGR5 increased significantly in injured nerves, especially in the site of injury, marked with pentagrams in immunofluorescent microphotographs. Furthermore, it seemed that TGR5 predominantly colocalized with F4/80 positive cells. Statistically, the colocalization ratio of TGR5 with F4/80, as indicated by the percentage of area, was significantly higher on PDO7 than that of TGR5 colocalized with PGP9.5. In the sham group, TGR5 colocalized with F4/80 and PGP9.5 at the equivalent level (Fig. S1 ). In summary, these results revealed the increase of TGR5 in sciatic nerves after pSNL, with the predominant location in macrophages. 3.2 Macrophages in the sciatic nerve altered dynamically after pSNL, and located closely with nerve fibers To confirm the involvement of macrophages in the pathological process induced by pSNL, the alterations of macrophages in sciatic nerves were assessed using flow cytometry (Fig. S2 ). The results indicated a significant increase in the percentage of CD11b + myeloid cells on POD1 and POD3 (Fig. 2 A). Compared with the sham group, the percentages of F4/80 + and CD11b + F4/80 + macrophages increased significantly on POD3 (Fig. 2 B and 2 C). Additionally, the population of CD86 + CD206- M1 macrophages increased on POD1, showed a relative decrease on POD3 and POD7, and eventually returned to the levels of the sham group by POD14 (Fig. 2 D). The percentage of CD86-CD206 + M2 macrophages followed a pattern opposite to that of M1 macrophages (Fig. 2 E). Consequently, the ratio of M1 macrophages to M2 macrophages significantly increased on POD1 (Fig. 2 F). Furthermore, immunofluorescent staining showed that macrophages in the sciatic nerve were located closely with PGP9.5 + nerve fibers (Fig. 2 G). In summary, macrophages increased and were closely associated with nerve fibers in sciatic nerves after pSNL, displaying proinflammatory characteristics. 3.3 Peri-sciatic nerve injection of TGR5 agonist INT-777 alleviated pSNL-induced mechanical allodynia Based on the robust alterations of TGR5 induced by pSNL as well as the predominant colocalization with macrophages, it was hypothesized that TGR5 involved in pSNL-induced mechanical allodynia. To confirm the hypothesis, the TGR5-specific agonist INT-777 was administered daily viaperi-sciatic nerve injection from day 0 to day 6, and 50% paw withdrawal thresholds were measured (Fig. 3 A). The results indicated that the activation of TGR5 by 50 µg/µL INT-777 alleviated mechanical allodynia on POD5 and POD7, while the similar effects were not observed when INT-777 was administered at concentrations of 0.5 µg/µL or 5 µg/µL (Fig. 3 B and 3 C). Moreover, the mitigating effect of INT-777 on mechanical allodynia continued after the cessation of drug administration until POD21 (Fig. 3 D). 3.4 pSNL-induced transcriptome alterations reversed by INT-777 administration were predominantly inflammation-related To illustrate the upstream mechanisms of mechanical allodynia alleviation induced by INT-777 treatment, transcriptional profilings of sciatic nerves from three groups (DMSO_Sham, DMSO_pSNL, and INT-777_pSNL) on POD3 were analyzed by bulk RNA-seq (Fig. 4 A). Principal component analysis (PCA) revealed distinct transcriptome characteristics among the three groups (Fig. 4 B). Specifically, 562 downregulated genes and 556 upregulated genes (cluster 1 and cluster 2 indicated in the Fig. S3 A) were detected in the DMSO_pSNL group compared to those in the DMSO_Sham group (definition of differentially expressed genes [DEGs]: | log2FC | ≥ 2 and Q value ≤ 0.05; Fig. 4 C). Compared to vehicle treatment, 156 DEGs were downregulated and 84 DEGs were upregulated by INT-777 treatment after pSNL (Fig. 4 C). These genes were indicated as cluster 3 and cluster 4 in the volcano map (Fig. S3 B). To understand the functions of DEGs, the biological processes engaged by four cluster genes were analyzed separately using Gene Ontology (GO) enrichment analysis. Specifically, DEGs in cluster 1 were mainly related to the biosynthetic and metabolic processes of sterol, steroid, and cholesterol (Fig. S3 C). DEGs in cluster 2 were mainly related to inflammatory response and the processes associated with cytokines and chemokines (Fig. S3 D). DEGs in cluster 3 were mainly related to immune response (Fig. S3 E). DEGs in cluster 4 were mainly related to the processes of sarcomere organization, muscle contraction, and organism development (Fig. S3 F). Notably, DEGs in the cluster 2 and cluster 3, which were genes upregulated after pSNL and genes downregulated after INT-777 treatment, were involved in similar biological processes. Specifically, there were 29 overlapped genes between the two clusters (Fig. 4 D and 4 E). GO enrichment analysis indicated that these genes involved in the immune system process, the regulation process of interleukin-1 beta (IL-1β) and tumor necrosis factor (TNF) production, cellular response process to interferon-gamma (IFN-γ), and chemotaxis (Fig. 4 F). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that these genes are associated with cytokine and cytokine receptor interaction pathway, Toll-like receptor signaling pathway, and NF-kappa B signaling pathway as well as other pathways (Fig. 4 G). Considering the opposite expression pattern induced by pSNL and INT-777 treatment, these 29 genes and related biological processes might hold the key to mediate the modulatory effects of TGR5 activation against pSNL. In summary, data of bulk RNA-seq provided an overview indicating that the transcriptome alterations in sciatic nerves induced by pSNL, further reversed by TGR5 activation, were closely associated with inflammatory response, particularly genes related to the production processes of pro-inflammatory cytokines and chemokines. The regulation on these inflammatory mediators may play a pivotal role in the mechanisms underlying the mechanical allodynia alleviation induced by INT-777 treatment. 3.5 pSNL-induced increase of inflammatory mediators and pro-inflammatory monocytes/macrophages in the sciatic nerve were partially reversed by INT-777 administration To verify the effect of INT-777 administration on cytokine/chemokine expression, sciatic nerves were subjected to cytometric bead arrays analysis using a kit containing biomarkers involved in cytokine release syndrome. The results indicated that CCL3, CXCL9, TNF-α, and IL-6 increased in injured nerves after pSNL, and the administration of INT-777 reversed their upregulation (Fig. 5 A- 5 D). Additionally, the expression levels of CCL2, CXCL10 and VEGF, which were altered by pSNL, remained unchanged following INT-777 treatment (Fig. 5 E- 5 G). Unexpectedly, no difference in the expression of CCL4, IL-4, IL-10, IFN-α, and IFN-γ was detected among three groups (Fig. 5 H- 5 L). To determine the effect of local TGR5 activation on monocytes/macrophages, sciatic nerves of mice treated with either DMSO or INT-777 for 7 days were analyzed by flow cytometry (Fig. S4). The data revealed a significant reduction in the percentages of CD11b + monocytes and CD11b + F4/80 + macrophages following INT-777 administration, although the percentage of F480 + cells was not significantly different between the two groups (Fig. 5 M– 5 O). While no significant differences were observed in the individual percentage of CD86 + CD206- M1 macrophages, INT-777 administration significantly increased the percentage of CD86-CD206 + M2 macrophages and decreased the ratio of M1 macrophages to M2 macrophages in the treated sciatic nerves (Fig. 5 P– 5 R). These results indicated that INT-777 administration significantly decreased pro-inflammatory mediators, reduced monocyte/macrophage and increased the prevalence of anti-inflammatory macrophages in the sciatic nerve. In summary, the local TGR5 activation resulted in reduced neuroinflammation. 3.6 Myeloid cell-specific TGR5 knockdown in the sciatic nerve exacerbated pSNL-induced mechanical allodynia Macrophages in the sciatic nerve include tissue-resident peripheral nerve macrophages and monocyte-derived infiltrating macrophages. In the state condition, resident macrophages exhibit slow renewal by the bone marrow-derived macrophages. Upon injury, macrophages in the sciatic nerve were reported to be predominantly circulating monocyte-derived macrophages (Ydens et al. 2020 ). Given the abovementioned results, to gain a further insight into the contribution of macrophages TGR5 in the injured sciatic nerve to pain perception, targeted knockdown of TGR5 in myeloid cells was performed in Lyz2-cre mice. This was achieved by peri-sciatic nerve injection of the LV-CMV-DIO-EGFP-shRNA virus carrying either the shGPBAR1 or shControl sequence. The most effective shRNA sequence among three different sequences was selected depending on its ability to reduce the mRNA expression of TGR5 in BMDMs (Fig. S5A). Finally, the sequence of shGPBAR1-1 was designated as shGPBAR1 and further used. The specific knockdown effect of the LV-CMV-DIO-EGFP-shRNA (shGPBAR1) virus was validated in BMDMs from Lyz2-cre mice using RT-qPCR (Fig. S5B), immunofluorescence staining (Fig. S5C and S5D), and western blotting (Fig. S5E and S5F). Considering that the increase of myeloid cells in the sciatic nerve was most significant in the first three days after pSNL, the peri-sciatic nerve injection of lentivirus was performed three times after surgery (Fig. 6 A). To evaluate the cell specificity of virus infection, sciatic nerves were subjected to flow cytometry analysis. The results indicated that most of the GFP labeled cells were CD11b positive (Fig. 6 B). In addition, the GFP labeled CD11b + cells accounted for about 20% of the total CD11b + cells (Fig. 6 C). The knockdown efficiency of TGR5 in the sciatic nerve was further confirmed using RT-qPCR (Fig. 6 D), western blotting (Fig. 6 E and 6 F) and immunofluorescence staining (Fig. 6 G and 6 H). In this circumstance, pSNL-induced mechanical allodynia was evaluated. Compared with the shControl virus, the application of the shGPBAR1 virus from day 0 to day 2 after pSNL induced exacerbated mechanical allodynia on POD7 (Fig. 6 I). In summary, these results indicated that TGR5 expressed on myeloid cells in the sciatic nerve plays a protective role against pSNL-induced mechanical allodynia. 3.7 Myeloid cell-specific TGR5 knockdown induced significant upregulation of pro-inflammatory genes in the sciatic nerve While the contribution of myeloid cells to neuroinflammation is well reported, whether the regulation of neuroinflammation is the main effect of myeloid TGR5 manipulation remains unknown. To screen the transcriptomic alterations after myeloid cell-specific TGR5 knockdown, sciatic nerves obtained from lyz2-cre mice treated with shGPBAR1 or shControl virus were subjected to bulk RNA-seq analysis. Principal component analysis revealed distinct transcriptome characteristics between the two groups (Fig. 7 A). Volcano map of DEGs revealed that 5 genes were downregulated and 77 genes were upregulated in the shGPBAR1 group compared to those in the shControl group (Fig. 7 B). GO enrichment analysis indicated that the top 5 biological processes engaged by upregulated genes were immune response, neutrophil chemotaxis, inflammatory response, immune system process and chemotaxis. Specifically, several genes were involved in regulating NIK/NF kappaB signaling and cytokine production process, especially the production process of TNF, IL-6 and IL-1β (Fig. 7 C). The major pathways engaged by upregulated genes were further evaluated using KEGG enrichment analysis. Cytokine and cytokine receptor interaction as well as chemokine signaling are the major enriched pathways. Similarly, genes in the NF-kappa B, IL-17, NOD-like receptor and TNF signaling pathways were significantly enriched (Fig. 7 D). The expression of genes in the above enriched biological processes was indicated in the clustering heatmap (Fig. 7 E). Among these 24 DEGs, Tnf , Il1b and Nlrp3 were three key genes according to enrichment analysis. Besides, Ccl3 and Ccl4 were two of three DEGs that also belong to the 29 overlapped gene cluster regulated reversely by pSNL and INT-777 treatment. The gene expression of five genes was validated using RT-qPCR. Except for Nlrp3 , the expression of other four genes were significantly upregulated in the shGBAR1 group (Fig. 7 F- 7 J). For the five downregulated DEGS, GO enrichment analysis revealed that these genes involved in dendritic spine morphogenesis, negative regulation of excitatory postsynaptic potential and cellular response to nutrient levels (Fig. S6A). KEGG enrichment analysis implied that these genes were closely associated with axon guidance and neuroactive ligand-receptor interaction pathway (Fig. S6B). The expression of genes in the above enriched biological processes was indicated in the clustering heatmap (Fig. S6C). In summary, these results revealed that the neuroinflammation was the primary biological process influenced by myeloid TGR5 knockdown, especially the cytokine/chemokine signaling. 3.8 Myeloid cell-specific TGR5 knockdown upregulated pro-inflammatory mediators and increased proportion of monocytes/macrophages in the sciatic nerve To confirm the effects of myeloid cell-specific TGR5 knockdown on neuroinflammation-related alterations implied by the data of RNA-seq, the protein levels of inflammatory mediators in the sciatic nerve were assessed. Cytometric bead arrays analysis indicated that the expression of CCL2, CCL3, TNF-α and IL-6 were significantly upregulated in the shGPBAR1 group (Fig. 8 A- 8 D). The expression of CCL4 was not significantly different between the two groups, although the mRNA levels of Ccl4 was significantly increased in the shGPBAR1 group (Fig. S7A). The expression of CXCL9, CXCL10, and VEGF, which were upregulated by pSNL, were not altered by myeloid cell-specific TGR5 knockdown (Fig. S7B-S7D). Consistent with the effects of pSNL and TGR5 activation on the expression of IL-4, IL-10, IFN-α and IFN-γ, TGR5 knockdown did not regulate their protein levels (Fig. S7E-S7H). NLRP3-ASC inflammasome was reported to regulate the generation of IL-1β (Broz and Dixit 2016). Given the significant enrichment of DEGs to NLRP3 regulation and IL-1β production pathways, their protein expression was assessed using western blot analysis (Fig. 8 E). The results indicated that the expression of NLRP3 was not significantly different between shControl and shGPBAR1 groups, while the expressions of ASC, Caspase-1, and IL-1β were significantly upregulated by TGR5 knockdown (Fig. 8 F- 8 I). Furthermore, immunofluorescence staining indicated that NLRP3-ASC inflammasome and IL-1β colocalized with F480 + macrophage in the injured sciatic nerve (Fig. 8 J and 8 K). Based on the robust alterations of cytokines and chemokines induced by myeloid-cell specific TGR5 knockdown, monocytes/ macrophages in the sciatic nerve were evaluated by flow cytometry (Fig. 9 A). The data revealed a significant increase of CD11b + monocytes, F480 + macrophages and CD11b + F4/80 + macrophages in the shGPBAR1 group compared to those in the shControl group (Fig. 9 B- 8 D). However, no significant differences were observed in the individual percentage of CD86 + CD206- M1 macrophages, CD86-CD206 + M2 macrophages, and the ratio of M1 macrophages to M2 macrophages between the two groups (Fig. 9 E– 9 G). These results confirmed the substantial regulatory impact of myeloid TGR5 on the expression of pro-inflammatory mediators and the proportion of monocytes/ macrophages in the sciatic nerve. In summary, myeloid-cell specific TGR5 knockdown exaggerated neuroinflammation in the sciatic nerve after pSNL. 3.9 Activation of microglia in the dorsal horn of spinal cord induced by pSNL altered when TGR5 in the sciatic nerve was manipulated Peripheral neuroinflammation contributes to central sensitization which is the pivotal mechanism of neuropathic pain (Woolf and Salter 2000 ). To estimate the effects of TGR5 manipulations on central sensitization in the spinal cord level, the activation of microglia indicated with the Iba-1 positive area in the ipsilateral dorsal horn of spinal cord induced by pSNL were analyzed on POD7. Immunofluorescence staining indicated that the activation of microglia was decreased by peri-sciatic nerve INT-777 treatment (Fig. 10 A and 10 B) and was increased by myeloid cells-specific TGR5 knockdown in the sciatic nerve compared to their respective control groups (Fig. 10 C and 10 D). 4. Discussion In this study, we confirmed that TGR5 in injured nerves protect against pSNL-induced mechanical allodynia by modulating neuroinflammation. Specifically, the expression of TGR5 increased in injured nerves, and local activation of TGR5 alleviated mechanical allodynia by reversing the increase of pro-inflammatory cytokines/chemokines as well as the increase of monocytes/macrophages induced by pSNL. On the contrary, specific knockdown of TGR5 in myeloid cells aggravates mechanical allodynia by increasing the expression of pro-inflammatory mediators and the proportion of monocytes/macrophages. TGR5, a GPCR protein, is abundantly expressed in the gallbladder epithelium, and is minimally to moderately expressed in almost all other tissues and cell types (Vassileva et al. 2006 ). In the nervous system, TGR5 is found in both neurons and non-neuronal cells, including resident and infiltrating immune cells (Lieu et al. 2014 ). The distinct functions of TGR5 in neurons and non-neuronal cells was previously reported. For neurons, administration of the TGR5 agonist increases the intrinsic excitability of DRG neurons and activates colon-innervating DRG neurons separated from naïve mice (Alemi et al. 2013 , Castro et al. 2019 ). In non-neuronal cells, TGR5 activation mitigates LPS-induced inflammatory responses in both microglia and macrophages (Hogenauer et al. 2014 , Yanguas-Casas et al. 2017 ). Moreover, the known roles of TGR5 expressed in different anatomical region in pain modulation are also inconsistent. In the mouse model of paclitaxel-induced peripheral neuropathic pain, TGR5 expressed in the DRG neurons contribute to the upregulation of CCR5, and then mediates the deoxycholic acid induced mechanical allodynia (Zhong et al. 2023 ). In the mouse model of spared nerve injury (SNI)-induced neuropathic pain, intrathecal injection of TGR5 agonist alleviates mechanical allodynia by inhibiting the activation of glial cells and modulating the function of GABAA receptors in the spinal cord(Wu et al. 2023 ). In this study, TGR5 in the injured sciatic nerve was mainly expressed in the macrophages, and pSNL-induced mechanical allodynia was significantly alleviated by peri-sciatic administration of the TGR5-specific agonist INT-777. Underlying this phenotype, the molecular mechanism regulated by TGR5 activation is more consistent with a negative regulation effect on inflammation rather than a positive impact on neuron excitability. The inference was validated by bulk RNA-seq analysis of injured nerve tissues. The sequence data indicated that, among the entire transcriptome changes, the inflammatory response is the primary process engaged by DEGs which were regulated reversely by pSNL and TGR5 activation. In addition, pSNL-induced mechanical allodynia was further exaggerated when TGR5 was specifically knockdown in myeloid cells along with the transcriptomic alterations predominantly related to the inflammatory response. Taken together, although the specific role of TGR5 expressed by neuron fibers in pain modulation was not evaluated in this study, the present data proved that increased TGR5 in the injured sciatic nerve is a molecular target to modulate mechanical allodynia by regulating neuroinflammation. In the field of neuropathic pain, ample evidence has underscored the substantial role of neuroinflammation in driving both peripheral and central sensitization, thereby enhancing pain hypersensitivity (Grace et al. 2014 , Bethea and Fischer 2021 ). Recent findings indicate that specific subsets of immune cells contribute to the alleviation of neuropathic pain (Fiore et al. 2023 ). Among them, macrophages are a subset of cells which exhibit remarkable plasticity and adopt functionally distinct phenotypes (Mosser et al. 2020 ). The inflammatory mediators, which are highly expressed by pro-inflammatory M1 macrophages, such as IL-1β (Binshtok et al. 2008 ), IL-6 (Liu et al. 2019 ) and TNF-α (Jin and Gereau 2006 ) were reported to induce neuronal sensitization and circulating leukocytes recruitment into inflamed tissue (Wang et al. 2022 ). The antinociceptive properties of the mediators secreted by anti-inflammatory M2 macrophages, such as IL-10 (Kwilasz et al. 2019 , Niehaus et al. 2021 ) and IL-4 (Celik et al. 2020 , Labuz et al. 2021 ) have also been validated. In this study, we confirmed the dynamic changes of macrophages and their close proximity to neuron fibers in injured nerves. In the context of TGR5 manipulation, accompanied by the alterations of mechanical allodynia, pro-inflammatory mediators and the proportion of monocytes/macrophages in the sciatic nerve were significantly regulated, with no significant difference detected in the expression of anti-inflammatory mediators. Activation of microglia in the SDH is an important part of central sensitization and is known to contribute to the neuropathic pain (Inoue and Tsuda 2018 ). In the present study, manipulations of TGR5 in the sciatic nerve significantly altered the activation level of microglia. In summary, the high relevance between macrophage-mediated neuroinflammation and pSNL-induced mechanical allodynia was further confirmed in this study. Regarding the neuronal mechanisms that mediate the effect of TGR5-modulated neuroinflammation on pain perception, although five genes involved in dendritic spine morphogenesis and negative regulation of excitatory postsynaptic process were detected to be downregulated by TGR5 knockdown, the specific roles of these genes still need to be further investigated. Since its detection, the modulatory effects of TGR5 on the functions of monocytes and macrophages have been extensively studied. Specifically, the production of pro-inflammatory cytokines in macrophages, such as TNF-α, IL-6 and IL-1β, are reported to be inhibited by TGR5 activation via NF-κB pathway (Pols et al. 2011 , Yang et al. 2017 ), and to be exacerbated by TGR5 deficiency via stabilizing the β-catenin destruction complex (Rao et al. 2020 ). The production of anti-inflammatory cytokines, such as IL-4 and IL-10, is reported to be upregulated by macrophage TGR5 activation, but the controversy remains regarding whether the presence of TGR5 is indispensable for the polarization of anti-inflammatory M2 macrophages (Perino et al. 2014 , Zhao et al. 2022 ). The expressions of chemokines, such as CCL2, CCL3 and CCL4, are also regulated by TGR5. In the presence of LPS, TRG5 deficiency exaggerates the upregulation of chemokines, while the activation of TGR5 poses the opposite impact on macrophages via the mTOR-C/EBPβ pathway (Perino et al. 2014 ). In addition, the activation of NLRP3 inflammasome is inhibited by bile acids via TGR5-cAMP-PKA axis (Guo et al. 2016 ), and the deficiency of TGR5 exaggerates the inflammatory response of macrophages stimulated with palmitic acid by promoting the activation of NLRP3 inflammasome (Shi et al. 2021 ). In the present study, the increase of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, as well as chemokines CCL3 and CCL2 in sciatic nerves was exacerbated by myeloid-cell specific TGR5 knockdown. These results are consistent with the known effects of TGR5 on macrophages. Regarding the NLRP3 inflammasome, although the expression of NLRP3 was not altered, the expression of the adaptor protein ASC, which connects the inflammasome sensor molecule to caspase-1, as well as the expression of caspase-1, which initiates downstream responses, were significantly upregulated by TGR5 knockdown, indicating the further activation of NLRP3 inflammasome. As for the specific molecular pathways underlying the modulations of TGR5 on inflammatory mediators in macrophages, which located in the injured sciatic nerve, further research is needed to illustrate the intrinsic alterations of these cells in situ rather than the changes induced by mimicked inflammatory response in cell experiments. The endogenous ligands of TGR5 are primary bile acids synthesized from cholesterol and secondary bile acids metabolized by the gut microbiota (Collins et al. 2023 ). The majority of bile acids are generated and exist in the enterohepatic system and a minority of them spillover into the circulation (Perino et al. 2021 ). In the peripheral and central nervous system, the presence of bile acids is confirmed (Xing et al. 2023 ). In addition to being transported from circulation, bile acids are also synthesized locally in the brain and spinal cord, as evidenced by the detection of rate-limiting enzymes for synthesis (Hurley et al. 2022 ). In the present study, data of bulk RNA-seq indicated that genes involved in the biosynthetic and metabolic process of cholesterol were significantly regulated in the sciatic nerve after pSNL, implying the potential alterations of endogenous ligands of TGR5. However, whether the protein levels of bile acids in injured sciatic nerves changes and whether these ligands participate in the modulation of pathological processes of neuropathic pain need to be further studied. The primary limitation of this study is the use of only male mice. Given the well-documented sex differences in neuroinflammatory mechanisms (del Rivero et al. 2018 , Gregus et al. 2021 ), whether TGR5 plays the same regulatory role in neuroinflammation and mechanical allodynia in female mice remains unknown. In conclusion, this study established that TGR5, expressed by macrophages within injured nerves, is a protective factor against mechanical allodynia. This protective effect involves alleviating neuroinflammation by modulating the pSNL-induced expression of inflammatory mediators. Consequently, TGR5, which operates at the peripheral nerve injury site, has emerged as a promising molecular target for treating neuropathic pain. Declarations Ethical approval and consent to participate All animal procedures were performed in compliance with experimental guidelines approved by the Animal Care and Use Committee of the Peking University Center of Health Science. Declaration of interest The authors declare no competing interest. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding This study was supported by the National Natural Science Foundation of China (81870788, 82170979, 82371227, 82171226, 81974169, 82001192) and Natural Science Foundation of Beijing Municipality (7222105). Author Contribution Wen-Ge Shi, Kai-Yuan Fu, and Guo-Gang Xing were involved in experimental design and manuscript writing. Wen-Ge Shi performed the experiments and processed the data. Yao Yao, Jie Lei, Shi-Yang Fang, Ya-Jing Liang, Yue Tian, Zi-Xian Zhang, and Jie Cai provided technical support for experiments. 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"Blockade of CCR5 suppresses paclitaxel-induced peripheral neuropathic pain caused by increased deoxycholic acid." Cell Rep 42 (11): 113386. Zuo, G., T. Zhang, L. Huang, C. Araujo, J. Peng, Z. Travis, T. Okada, U. Ocak, G. Zhang, J. Tang, X. Lu and J. H. Zhang (2019). "Activation of TGR5 with INT-777 attenuates oxidative stress and neuronal apoptosis via cAMP/PKCepsilon/ALDH2 pathway after subarachnoid hemorrhage in rats." Free Radic Biol Med 143 : 441-453. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1supplementaryfigures17.docx Researchdata.xlsx UncroppedGelsandBlotsimagesinfiures.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3852075","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266419304,"identity":"58eecc66-e844-4065-9611-d01c094be008","order_by":0,"name":"Wen-Ge Shi","email":"","orcid":"","institution":"Peking University School and Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Wen-Ge","middleName":"","lastName":"Shi","suffix":""},{"id":266419305,"identity":"283d1ab5-11e2-4b39-8c96-dc440ab65021","order_by":1,"name":"Yao Yao","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Yao","suffix":""},{"id":266419306,"identity":"a1527fab-4e82-4860-9c9d-b8190a5b7b44","order_by":2,"name":"Ya-Jing Liang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ya-Jing","middleName":"","lastName":"Liang","suffix":""},{"id":266419307,"identity":"3b6614db-6018-4831-8b26-4b77e43031ea","order_by":3,"name":"Jie Lei","email":"","orcid":"","institution":"Peking University School and Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Lei","suffix":""},{"id":266419308,"identity":"99bb3718-27a1-4c33-93be-32e2fa9458a5","order_by":4,"name":"Shi-Yang Feng","email":"","orcid":"","institution":"Peking University School and Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Shi-Yang","middleName":"","lastName":"Feng","suffix":""},{"id":266419309,"identity":"0db3c53c-ea4a-4f6a-baf0-a34d1e2955e1","order_by":5,"name":"Yue Tian","email":"","orcid":"","institution":"Peking University, Peking University Health Science Center, Ministry of Education of China \u0026 National Health Commission of China","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Tian","suffix":""},{"id":266419310,"identity":"755c2d27-2657-482c-91cd-361b191173a0","order_by":6,"name":"Zi-Xian Zhang","email":"","orcid":"","institution":"Peking University, Peking University Health Science Center, Ministry of Education of China \u0026 National Health Commission of China","correspondingAuthor":false,"prefix":"","firstName":"Zi-Xian","middleName":"","lastName":"Zhang","suffix":""},{"id":266419311,"identity":"fe5cc8b2-903f-489d-8cf5-b69b7b66aca1","order_by":7,"name":"Jie Cai","email":"","orcid":"","institution":"Peking University, Peking University Health Science Center, Ministry of Education of China \u0026 National Health Commission of China","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Cai","suffix":""},{"id":266419312,"identity":"7ae8680e-4558-442f-a697-03290e30c613","order_by":8,"name":"Guo-Gang Xing","email":"","orcid":"","institution":"Peking University, Peking University Health Science Center, Ministry of Education of China \u0026 National Health Commission of China","correspondingAuthor":false,"prefix":"","firstName":"Guo-Gang","middleName":"","lastName":"Xing","suffix":""},{"id":266419313,"identity":"9e5e0b60-9793-4b32-b6c0-a1751a39033d","order_by":9,"name":"Kai-Yuan Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACCQY2BoYKBpsExgYQl41YLWcY0kjXcjgBwiVGi2R7WprEwbbzeczTzhgwfCg7zMA/uwG/FmmeZ8eAWm4XM87OMWCcce4wg8SdA/i1yEmkt0l/bLud2AjUwszbdpjBQCKBsBagLecgWv4So0VaIg3ksAMQLYzEaJHseZZsceBcMtAvaQUHe86l80jcIKBF4nia4Y0DZXZ5hrOTNz74UWYtxz+DgBYGhgQWCRBl2MDAcABI8xBSD9LC/AFEyROhdBSMglEwCkYoAAB4FkcdqwAueQAAAABJRU5ErkJggg==","orcid":"","institution":"Peking University School and Hospital of Stomatology","correspondingAuthor":true,"prefix":"","firstName":"Kai-Yuan","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2024-01-11 02:59:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3852075/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3852075/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49511419,"identity":"1e91c1c3-1362-4fb1-ac87-9cd44679cf06","added_by":"auto","created_at":"2024-01-12 06:25:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1831785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of TGR5 in the sciatic nerve after pSNL. \u003c/strong\u003e(A) Alterations in the 50% paw withdrawal threshold assessed through the Von Frey test after pSNL. n = 6–7 per group. ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 vs. sham group; two-way ANOVA, Šídák’s post hoc test. (B) Representative western blot bands at different time points. (C) Densitometric quantification of TGR5 in the ipsilateral (right) sciatic nerve following pSNL. n = 4 per group. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 vs. sham group; one-way ANOVA, Tukey’s post hoc test. Data are presented as the mean ± SEM. Representative microphotographs illustrating the co-immunofluorescence staining of TGR5 (red) with macrophages (F4/80, green) (D) or nerve fibers (PGP9.5, green) (E) in the sciatic nerve. Pentagrams indicated the site of injury. Nuclei were stained with DAPI (blue). Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/2a1e336cc7979a78bb971a39.png"},{"id":49511404,"identity":"c3f3a6dc-ee9f-4e5d-a085-935195e160f4","added_by":"auto","created_at":"2024-01-12 06:25:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":942969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations of macrophages in the sciatic nerve after pSNL.\u003c/strong\u003ePercentages of CD11b+ cells (A), F4/80+ cells (B), and CD11b+F4/80+ cells (C) in single cells, defined by FS-A and FS-W. Percentages of CD86+CD206- cells (D) and CD86-CD206+ cells (E) in CD11b+F4/80+ cells. (F) The ratio of CD86+CD206- M1 macrophages to CD86-CD206+ M2 macrophages. n = 4–5 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 vs. sham group; one-way ANOVA, Tukey’s post hoc test. Data are presented as the mean ± SEM. (G) Representative microphotographs illustrating the staining of macrophages (F4/80, green) and nerve fibers (PGP9.5, red) in the sciatic nerve on POD3. Nuclei were stained with DAPI (blue). Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/529de45fcb37450c01255abc.png"},{"id":49511406,"identity":"6af21b50-9e3b-41e4-bcfe-f496fee55dee","added_by":"auto","created_at":"2024-01-12 06:25:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":352321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of peri-sciatic nerve administration of the TGR5-specific agonist INT-777 on pSNL-induced mechanical allodynia. \u003c/strong\u003e(A) Schematic of experimental design. (B) Changes in the 50% paw withdrawal threshold evaluated by the Von Frey test with the peri-sciatic nerve administration of DMSO and INT-777 at concentrations of 0.5 μg/μL, 5 μg/μL, and 50 μg/μL. n = 5–7 per group. ****P \u0026lt; 0.0001 vs. DMSO group; two-way ANOVA, Šídák’s post hoc test. (C) Alterations in the 50% paw withdrawal threshold were monitored during the peri-sciatic nerve treatment phase, in which DMSO or INT-777 (50 μg/μL) was daily injected. n = 8–11 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. DMSO group; two-way ANOVA, Šídák’s post hoc test. (D) Alterations in the 50% paw withdrawal threshold were evaluated following the discontinuation of peri-sciatic nerve treatment. n = 8–10 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. DMSO group; two-way ANOVA, Šídák’s post hoc test. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/c6c2355a60864396f9ccaf4d.png"},{"id":49511405,"identity":"3b746f38-8a6d-4606-a976-bf79ef53762e","added_by":"auto","created_at":"2024-01-12 06:25:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":712812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome alterations in the sciatic nerve rescued by administration of INT-777 after pSNL. \u003c/strong\u003e(A) Schematic of experimental design. (B) PCA of sciatic nerves from the 3 groups. n = 3–4 per group. (C) Diagram illustrating DEGs in the DMSO_pSNL group compared to those in the DMSO_Sham group, and DEGs in the INT-777_pSNL group compared to those in the DMSO_pSNL group. (D) Venn diagram illustrating the overlapped genes between cluster 2 (upregulated DEGs in the DMSO_pSNL group compared to that in the DMSO_Sham group) and cluster 3 (downregulated DEGs in the INT-777_pSNL group compared to that in the DMSO_pSNL group). (E) Heatmap of 29 overlapped genes from the Venn diagram. (F) GO enriched biological processes of 29 overlapped DEGs. (G) KEGG enriched pathways of 29 overlapped DEGs.\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/608121186bb280f474c99db0.png"},{"id":49513028,"identity":"b6d2bf86-cf90-4eee-ae29-fe4fccfd8acf","added_by":"auto","created_at":"2024-01-12 06:49:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":823034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of administration of INT-777 on alterations in inflammatory mediators and macrophages induced by pSNL.\u003c/strong\u003e Protein levels of CCL3 (A), CXCL9 (B), TNF-α (C), IL-6 (D), CCL2 (E), CXCL10 (F), VEGF (G), CCL4 (H), IL-4 (I), IL-10 (J), IFN-α (K), and IFN-γ (L) in the sciatic nerve were measured via cytometric bead array analysis. n = 4–5 per group. The displayed data came from two independent repeated tests. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001; one-way ANOVA, Tukey’s post hoc test. Percentages of CD11b+ cells (M), F4/80+ cells (N), and CD11b+F4/80+ cells (O) in single cells, defined by FS-A and FS-W. Percentages of CD86+CD206- cells (P) and CD86-CD206+ cells (Q) in CD11b+F4/80+ cells. (R) The ratio of M1 macrophages to M2 macrophages. n = 5 per group. *P \u0026lt; 0.05; unpaired t-test. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/69f3308dbeaa88a1fa296c81.png"},{"id":49511454,"identity":"e5450e77-61e9-4175-9ee9-02dd9c108144","added_by":"auto","created_at":"2024-01-12 06:25:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":935952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of myeloid cell-specific TGR5 knockdown and the effect of TGR5 knockdown on mechanical allodynia.\u003c/strong\u003e (A) Schematic of experimental design. Representative flow cytometry pseudocolor dot plots indicating the percentage of CD11b+ cells in total GFP+ cells (B), and the percentage of GFP+ cells in total CD11b+ cells (C) in sciatic nerves extracted from mice 7 days after virus injection. (D) mRNA expression of TGR5 in the sciatic nerves of Lyz2-cre mice treated with shControl virus or shGPBAR1 virus. n = 6 per group. *P \u0026lt; 0.05; unpaired t-test. (E) Representative western blot bands and (F) densitometric quantification of TGR5 in the sciatic nerves of Lyz2-cre mice treated with shControl virus or shGPBAR1 virus after pSNL. n = 5 per group. ***P \u0026lt; 0.001; unpaired t-test. (G) Representative microphotographs of immunofluorescence staining of TGR5 in the sciatic nerves of Lyz2-cre mice treated with shControl virus or shGPBAR1 virus. Scale bar: 100 μm. (H) Analysis of the TGR5+ mean fluorescence density. n = 4 per group. *P \u0026lt; 0.05; unpaired t-test. (I) Comparisons of the 50% paw withdrawal threshold evaluated by the Von Frey test between the shControl and shGPBAR1 group on POD7. n = 10 per group. *P \u0026lt; 0.05; unpaired t-test. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/38f3f085fa672171f253afbb.png"},{"id":49512425,"identity":"228da0db-7ae9-434b-9459-cf7db6cca2ee","added_by":"auto","created_at":"2024-01-12 06:33:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":720126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome alterations in the sciatic nerve induced by myeloid cell-specific TGR5 knockdown after pSNL. \u003c/strong\u003e(A) PCA of sciatic nerves from the shControl group and shGPBAR1 group. (B) Volcano plots of DEGs in the shGPBAR1 group compared to that in the shControl group, which including 77 upregulated genes and 5 downregulated genes. GO enriched biological processes (C) and KEGG enriched pathways (D) of 77 upregulated genes. (E) Heat map of DEGs involved in the production of cytokines. Gene expression of\u003cem\u003e \u003c/em\u003eCcl3 (F), Ccl4 (G), Tnfα\u003cem\u003e \u003c/em\u003e(H),\u003cem\u003e \u003c/em\u003eIl1b\u003cem\u003e \u003c/em\u003e(I), and Nlrp\u003cem\u003e3\u003c/em\u003e (J) was validated through RT-qPCR. n = 6 per group. *P \u0026lt; 0.05; unpaired t-test. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/4a2e7c8212a42b129c38fa8a.png"},{"id":49512610,"identity":"bf204d1a-a679-4a55-a5ca-3e92af8a3191","added_by":"auto","created_at":"2024-01-12 06:41:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1594642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of myeloid cell-specific TGR5 knockdown on the expression of inflammatory mediators after pSNL.\u003c/strong\u003e Protein levels of CCL3 (A), CCL2 (B), TNF-α (C), and IL-6 (D) in the sciatic nerve were measured via cytometric bead array analysis. n = 6 per group. *P \u0026lt; 0.05; unpaired t-test. Representative western blot bands (E) and densitometric quantification of NLRP3 (F), ASC (G), Caspase-1 (H), and IL-1β (I) in sciatic nerves of Lyz2-cre mice treated with shControl virus or shGPBAR1 virus. n = 4 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01; unpaired t-test. Data are presented as the mean ± SEM. (J) Representative microphotographs showing immunofluorescence staining of F4/80 (green), NLRP3 (red), and ASC (cyan) in the sciatic nerve after pSNL. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (K) Representative microphotographs showing immunofluorescence staining of F4/80 (green) and IL-1β (red) in the sciatic nerve after pSNL. Nuclei were stained with DAPI (blue). Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"floatimage18.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/e90c3d4a2ded4a0615ea6f23.png"},{"id":49512422,"identity":"0e54fbbd-991a-48a0-b905-a8ff41fede62","added_by":"auto","created_at":"2024-01-12 06:33:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1355722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of myeloid cell-specific TGR5 knockdown on alterations of macrophages after pSNL. \u003c/strong\u003e(A) Representative flow cytometry pseudo color dot plots for myeloid cell populations in the sciatic nerves of mice treated with shControl virus or shGPBAR1 virus. Percentages of CD11b+ cells (B), F4/80+ cells (C), and CD11b+F4/80+ cells (D) in single cells defined by FS-A and FS-W. Percentages of CD86+CD206- cells (E) and CD86-CD206+ cells (F) in CD11b+F4/80+ cells. (G) The ratio of M1 macrophages to M2 macrophages. n = 4 per group; *P \u0026lt; 0.05; unpaired t-test. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage19.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/723907392a9f3dda5d3dffb0.png"},{"id":49511452,"identity":"f6753184-4a7c-4b58-82f7-4d97158511c7","added_by":"auto","created_at":"2024-01-12 06:25:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":665112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of TGR5 manipulations on microglia activation in the spinal cord after pSNL. \u003c/strong\u003e(A) Representative microphotographs of immunofluorescence staining of Iba-1 in the ipsilateral dorsal horn of spinal cord of mice treated with DMSO or INT-777. Scale bar: 100 μm. (B) Analysis of the percentage of Iba-1+ area. n = 4 per group. **P \u0026lt; 0.01; unpaired t-test. (C) Representative microphotographs of immunofluorescence staining of Iba-1 in the ipsilateral dorsal horn of spinal cord of mice treated with shControl virus or shGPBAR1 virus. Scale bar: 100 μm. (H) Analysis of the percentage of Iba-1+ area. n = 4 per group. *P \u0026lt; 0.05; unpaired t-test. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage20.png","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/82eeed568ac64c5b68027728.png"},{"id":57626657,"identity":"8034c717-6dcf-491f-b2db-b9c179a4c542","added_by":"auto","created_at":"2024-06-03 14:05:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12588516,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/ecfd64fb-fc20-49ec-be95-7bb06341aaf1.pdf"},{"id":49512426,"identity":"ae43cc00-b053-4cd4-b8ac-7e5338dfe5c9","added_by":"auto","created_at":"2024-01-12 06:33:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4555382,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1supplementaryfigures17.docx","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/de9379001753182584b81f40.docx"},{"id":49511456,"identity":"8cf5c771-2340-4055-911a-c1c4c3335d33","added_by":"auto","created_at":"2024-01-12 06:25:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6519693,"visible":true,"origin":"","legend":"","description":"","filename":"Researchdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/f2e6de7bb5db62d6168fb061.xlsx"},{"id":49512427,"identity":"c317cea3-925c-4a00-890f-b82aae0a5fd0","added_by":"auto","created_at":"2024-01-12 06:33:48","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6329288,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedGelsandBlotsimagesinfiures.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3852075/v1/80e24be3bffb5226fd5fbc29.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"TGR5 protects against pSNL-induced mechanical allodynia by alleviating neuroinflammation in the injured nerves of male mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or similar stimuli (Raja et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Neuropathic pain occurs when damage or disease affects the somatosensory nervous system (Scholz et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Neuropathic pain often becomes chronic and contributes significantly to the global disease burden, affecting 6.9\u0026ndash;10% of the global population (van Hecke et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Bouhassira and Attal \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Rice et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The mechanisms underlying neuropathic pain are complex and vary between different states of the disease (Baron et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). At present, clinical treatments for neuropathic pain predominantly focus on drugs that regulate neural conduction. However, these drugs frequently lead to unavoidable side effects and less-than-ideal treatment efficacy (Bannister et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Further research is imperative to identify treatment targets based on distinct pathological mechanisms.\u003c/p\u003e \u003cp\u003eNeuropathic pain triggers neuroinflammation (Ji et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Following peripheral nerve injury, immune cells infiltrate the injury site, secrete cytokines and chemokines, activate local immune cells, and attract more circulating leukocytes to both the injury site and along the neural pain pathway (Thacker et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Ellis and Bennett \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, McMahon et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the peripheral nervous system, macrophages participate in pain modulation by direct contacting with neurons (Iwai et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Tanaka et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and releasing soluble mediators that bind to receptors expressed on other cells (Donnelly et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Domoto et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Msheik et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, clinical trial results for patients with neuropathic pain treated with neutralizing antibodies targeting proinflammatory cytokines are inconclusive (Williams et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, a deeper understanding of the neuroimmune processes involved in the development and resolution of neuropathic pain is necessary for the development of effective treatment strategies.\u003c/p\u003e \u003cp\u003eTGR5 is a G protein-coupled bile acid receptor (GPBAR1), which was initially reported to mediate the anti-inflammatory effects of bile acids on lipopolysaccharide (LPS)-treated monocytes and macrophages (Kawamata et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The beneficial role of TGR5 activation in attenuating inflammation, including neuroinflammation induced by subarachnoid hemorrhage (Zuo et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Hu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), sepsis (Jin et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and intracerebroventricular injection of LPS (Wu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), have been discovered in several disease models (Perino et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the role of TGR5 in pain modulation is not well understood and the conclusions of previous studies are paradoxical. Activation of TGR5 in sensory nerves, achieved through intra-plantar or intrathecal injection of bile acids leads to analgesia in response to mechanical paw stimulation (Alemi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In contrast, TGR5 activation wa shown to promot visceral hypersensitivity in a mouse model of irritable bowel syndrome (Castro et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the aforementioned uncertainties, the role of TGR5 in the pathological processes of neuropathic pain remains unclear. Therefore, this study aimed to investigate the role of TGR5 in the peripheral mechanisms of mechanical allodynia in a mouse model of neuropathic pain induced by partial sciatic nerve ligation (pSNL).\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003e Experiments were performed following the ethical principles outlined by the Animal Care and Use Committee of the Peking University Center of Health Science. We housed 6\u0026ndash;8-week-old inbred male C57BL/6J (Vital River Laboratory Animal Technology Co. Ltd.) and Lyz2-Cre (Cat. NO. NM-KI-215037; Shanghai Model Organisms Center, Inc.) mice in a pathogen-free environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Neuropathic pain model\u003c/h2\u003e \u003cp\u003eNeuropathic pain was induced in mice through pSNL by modifying previously published methods (Seltzer et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Malmberg and Basbaum \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Yao et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Briefly, under 1% sodium pentobarbital anesthesia, the right sciatic nerve was exposed and carefully separated from the adjacent connective tissues near the trochanter. A 9\u0026ndash;0 silk suture with a 3/8 curved mini-needle was used to ligate approximately half of the dorsal portion of the sciatic nerve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Behavioral tests\u003c/h2\u003e \u003cp\u003eAll tests were conducted during the light (rest) phase. The mice were habituated to the testing environment 2 days prior to testing. The investigator was blinded to the mouse groups. Mechanical allodynia was measured using a previously reported Von Frey test which we modified (Callahan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Deuis et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Von Frey filaments (Stoelting, Wood Dale) were used, beginning with a 0.16 g filament. The presence or absence of a positive withdrawal response (flicking, licking, or lifting) determined the choice of lower- or higher-weight filaments. Four additional responses were observed after the initial change. The 50% paw withdrawal threshold was calculated based on the recorded test results (Dixon \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1980\u003c/span\u003e, Chaplan et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blot analysis\u003c/h2\u003e \u003cp\u003eUnder 1% sodium pentobarbital anesthesia, the mice underwent transcardial perfusion with chilled PBS. Approximately 1.5 cm of sciatic nerve tissue (including the injured site and proximal and distal parts) was dissected distal to the semitendinosus nerve branch. Proteins in the tissues were extracted using RIPA buffer (Applygen), and 1 mM phenylmethylsulfonyl fluoride and phosphatase inhibitors were added. The PVDF membranes, carrying proteins, were initially blocked using a 5% non-fat milk solution. Subsequently, they were incubated overnight at 4\u0026deg;C with the following primary antibodies: rabbit anti-TGR5 (1:1000; ab72608, Abcam), mouse anti-NLRP3 (1:1000; AG-20B-0014, AdipoGen), rabbit anti-ASC (1:1000; 67824S, CST), rabbit anti-Caspase-1 (1:1000; 24232S, CST), and rabbit anti-IL-1β (1:200; ab133357, Abcam). HRP-conjugated secondary antibodies (1:1000; Immunoway) were used to probe the blots, and enhanced chemiluminescence detection (Tanon) was used to detect the signals. Band intensities were quantified using ImageJ, and internal controls were used to calculate the relative protein expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eUnder 1% sodium pentobarbital anesthesia, the mice underwent transcardial perfusion with a pre-warmed 0.9% saline solution, followed by a chilled 4% paraformaldehyde solution. The ipsilateral sciatic nerves were dissected, fixed for 24 h, and subsequently subjected to overnight cryoprotection at 4\u0026deg;C in a 30% sucrose solution. After embedding in an Optimum Cutting Temperature Compound (OCT, Sakura), the nerves were sliced into 10 \u0026micro;m thick frozen sections. These sections, affixed to slides, were blocked with 10% donkey serum in PBS enriched with 0.3% Triton X-100 and incubated overnight at 4\u0026deg;C with the following primary antibodies: rabbit anti-TGR5 (1:500; ab72608, Abcam), mouse anti-PGP9.5 (1:1000, NB600-1160, Novus), rat anti-mouse F4/80 (1:500; MCA497, Biorad), mouse anti-NLRP3 (1:500; AG-20B-0014, AdipoGen), rabbit anti-ASC (1:500; 67824S, CST) and rabbit anti-IL-1β (1:200; ab133357, Abcam). Subsequently, the appropriate secondary antibodies (1:1000; Jackson ImmunoResearch) were used for incubation, including Cy3 donkey anti-rabbit IgG, AF488 donkey anti-rat IgG, AF488 donkey anti-mouse IgG, Cy3 donkey anti-mouse IgG, and AF647 donkey anti-rabbit IgG. Nuclear labeling was accomplished by counterstaining with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI).\u003c/p\u003e \u003cp\u003eImages were acquired using an Olympus microscope. To ensure reproducibility, a minimum of three fields per section were examined. Quantitative analyses were performed blindly using ImageJ Pro Plus software (Media Cybernetics Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Bulk RNA-seq and data analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from sciatic nerves using TRIzol reagent (Life Technologies) and assessed for quality, concentration, and chemical purity using spectrophotometry (NanoDrop). High-quality RNA libraries were generated and sequenced using the BGI system (Shenzhen, China). Subsequently, the sequencing data were analyzed using the Dr. Tom Multi-omics Data Mining System (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biosys.bgi.com\u003c/span\u003e\u003cspan address=\"https://biosys.bgi.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with significance levels adjusted using a stringent threshold (Q value\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RT-qPCR analysis\u003c/h2\u003e \u003cp\u003eA total of 400 ng RNA extracted from the sciatic nerve or bone marrow-derived macrophages (BMDMs) was reverse-transcribed. Subsequently, 1 \u0026micro;L of the resulting template cDNA was amplified in a 20 \u0026micro;L reaction volume, with a concentration of 0.5 \u0026micro;M for the specified PCR primer. Quantitative real-time PCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) with SYBR Premix Ex Taq II (Takara) following a previously described method (Liang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Specifc primers were employed for amplifcation, including Tgr5 (forward: 5\u0026prime;-ACTGGTCCTGCCTCCTTCTCC-3\u0026prime;, reverse: 5\u0026prime;- ACACTGCCATGTAGCGTTCCC-3\u0026prime;), Ccl3 (forward: 5\u0026prime;-TTGCTGTTCTTCTCTGTACCAT-3\u0026prime;, reverse: 5\u0026prime;- AATAGTCAACGATGAATTGGCG-3\u0026prime;), Ccl4 (forward: 5\u0026prime;-CTTGCTCGTGGCTGCCTTC-3\u0026prime;, reverse: 5\u0026prime;- TGCTGGTCTCATAGTAATCCATCAC-3\u0026prime;), Tnfa (forward: 5\u0026prime;-GCCTCTTCTCATTCCTGCTTGTGG-3\u0026prime;, reverse: 5\u0026prime;-GTGGTTTGTGAGTGTGAGGGTCTG-3\u0026prime;), Il1b (forward: 5\u0026prime;-TCGCAGCAGCACATCAACAAGAG-3, reverse: 5\u0026prime;-AGGTCCACGGGAAAGACACAGG-3\u0026prime;), Nlrp3 (forward: 5\u0026prime;-GCCGTCTACGTCTTCTTCCTTTCC-3\u0026prime;, reverse: 5\u0026prime;-CATCCGCAGCCAGTGAACAGAG-3\u0026prime;) and Gapdh (forward: 5\u0026prime;-AACTTTGGCATTGTGGAAGGGCTC-3\u0026prime;, reverse: 5\u0026prime;-TGGAAGAGTGGGAGTTGCTGTTGA-3\u0026prime;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Flow cytometry assay\u003c/h2\u003e \u003cp\u003eCells of sciatic nerves were isolated according to a previously described protocol (Liu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Part of the sciatic nerve was carefully dissected and transferred to 200 \u0026micro;L papain solution (1x HBSS/papain [15 U/mL]/DNAase [10 \u0026micro;g/mL]; 1x HBSS, Gibco; Papain, Roche; DNase, Sigma) on ice. After finely chopping the tissue samples into small pieces and digesting them at 37\u0026deg;C for 30 min, 400 \u0026micro;L of Solution A (1x HBSS/10% FBS/10 \u0026micro;g/mL DNase; FBS, Gibco) was added to terminate the digestion, followed by homogenization using a 1 mL syringe fitted sequentially with a 21 and 23 G needle. After homogenization, the solution was centrifuged at 12,000 rpm for 10 s. The cell pellets were washed with 1 mL of FACS buffer (1x HBSS/10% FBS) and resuspended in 100 \u0026micro;L of FACS buffer, to which 1 \u0026micro;L of rat anti-mouse CD16/CD32 clone 2.4G2 (BioLegend) was added. Following a 30-min incubation on ice, the following fluorescent antibodies were introduced into the incubation buffer and incubated on ice for another 30 min: PerCP-Cy5.5 rat anti-CD11b (550993, BD), PE/Cyanine7 anti-mouse F4/80 (123114, BioLegend), PE anti-mouse CD86 (12-0862-81, Thermo Fisher Scientific), and APC anti-mouse CD206 (141708, BioLegend). Subsequently, the cells were washed and resuspended in FACS buffer for flow cytometry. Cellular events were acquired using a Beckman Galios machine and the data were analyzed using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cytokine measurement\u003c/h2\u003e \u003cp\u003eCytokines (IFN-γ, IL-10, CCL4 [MIP-1β], IFN-α, CXCL9 [MIG], CXCL10 [IP-10], TNF-α, IL-6, VEGF, IL-4, CCL3 [MIP-1α], and CCL2 [MCP-1]) in the sciatic nerve were measured using a LEGENDplex\u003csup\u003eTm\u003c/sup\u003e MU Cytokine Release Syndrome Panel w/FP (741023, BioLegend), following the manufacturer's protocol and analyzed using a Beckman Galios flow cytometer. Sciatic nerve tissues were homogenized in 100 \u0026micro;L of cold PBS. The supernatant was collected after centrifugation at 12,000 \u0026times; g for 10 min at 4\u0026deg;C. Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). For the analysis, 25 \u0026micro;L of sciatic nerve extract was used. The reported values in pg/mL were normalized to pg/mg of total protein, considering the protein concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Lentivirus‑mediated shRNA construction\u003c/h2\u003e \u003cp\u003eThe shRNA lentivirus for knockdown sequence identification was constructed by OBiO Technology (Shanghai, China) using the lentiviral vector, pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE. Three sequences targeting GPBAR1 mRNA were as follows: 5\u0026prime;-CCTACCTCTACCTGGAAGTTT-3\u0026rsquo; (shGPBAR1-1), 5\u0026prime;-CTCTGTTATCGCTCATCTCAT-3\u0026prime; (shGPBAR1-2), and 5\u0026prime;-TGCTTCTTCCTAAGCCTACTA-3\u0026rsquo; (shGPBAR1-3). The sequence of the control shRNA was 5\u0026prime;-CCTAAGGTTAAGTCGCCCTCG-3\u0026prime; ( referred to as shControl). The viral titers of shGPBAR1 and shControl reached 2.7 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e and 7.7 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e TU/mL, respectively.\u003c/p\u003e \u003cp\u003eA Cre-dependent shRNA lentivirus for TGR5-specific knockdown was constructed by BrainVTA (Wuhan, China). The lentiviral vector sequence was rLV-CMV-DIO-EGFP-5'miR30-shRNA-3'miR30-WPRE. The sequence targeting GPBAR1 mRNA was 5\u0026prime;-CCTACCTCTACCTGGAAGTTT-3 and named shGPBAR1. The viral titers of shGPBAR1 and shControl reached 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e TU/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 BMDMs culture and treatments\u003c/h2\u003e \u003cp\u003eThe isolation and incubation of bone marrow derived macrophages (BMDMs) were conducted following a well-established protocol with minor adjustments (Perino et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Briefly, cells in the femur and tibia were flushed out of the bone using sterile HBSS. Following filtration through a 70-\u0026micro;m cell strainer, the cell solution was lysed using a lysis buffer (Solarbio) to remove the blood cells. After centrifugation, the cell pellets were suspended in 10 mL complete medium (RPMI 1640 medium/5 mM penicillin/streptomycin/10% FBS/30 ng/ml macrophage colony-stimulating factor (MCSF)) (RPMI 1640 medium, Gibco; penicillin/streptomycin, Gibco; MCSF, BioLegend) and plated in a 10-cm dish for incubation at 37\u0026deg;C in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFor lentivirus transfection, the BMDM incubated for 3 days was infected with a new complete medium containing lentiviruses at a multiplicity of infection of 100 for 12 h. After discarding the lentiviruses, the cells were cultured for an additional 72\u0026ndash;96 h in a fresh complete medium and then prepared for western blotting, immunofluorescence staining, and RT-qPCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Peri-sciatic nerve injection\u003c/h2\u003e \u003cp\u003ePerineural injections were administered according to a previously established protocol (Kiguchi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A 30-gauge needle attached to a micro-syringe was gently inserted into the scar site induced by pSNL procedure. The substances were administered to the region surrounding the sciatic nerve in the upper thigh of the hind limb. Successful injection and targeting of the sciatic nerve were confirmed using blue dye which was deposited in the nerve after injection.\u003c/p\u003e \u003cp\u003eThe TGR5-specific agonist, INT-777 (HY-15677, MCE), was dissolved in 10% DMSO to concentrations of 0.5 \u0026micro;g/\u0026micro;L, 5 \u0026micro;g/\u0026micro;L, and 50 \u0026micro;g/\u0026micro;L. INT-777 or 10% DMSO (4 \u0026micro;L) was administered daily by peri-sciatic nerve injection at the time of pSNL surgery and on days 1\u0026ndash;6 after pSNL under isoflurane anesthesia.\u003c/p\u003e \u003cp\u003eCre-dependent shRNA lentivirus (shGPBAR1 or shControl) was perineurally injected at a final titer of 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e TU/mL, with a total volume of 4 \u0026micro;L per mouse, concurrent with pSNL surgery. Additional injections were administered on the first and second days following pSNL surgery, while the animals were under isoflurane anesthesia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Data analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism software version 9.0. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The mean values of the two groups were compared using a two-tailed unpaired Student\u0026rsquo;s t-test. Data from multiple groups were compared using one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc test. Two-factor interaction experiments were conducted using two-way ANOVA with Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s post hoc test. Significance levels were labeled as follows: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 TGR5 increased in the sciatic nerve after pSNL, mainly colocalized with macrophages\u003c/h2\u003e\n \u003cp\u003eIn the mouse model of neuropathic pain, pSNL-induced mechanical allodynia, indicated by the decreased paw withdrawal threshold, was detected on post-operative day (POD) 1 and persisted until POD14 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). TGR5 expression in the injured sciatic nerve was determined using western blot analysis (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). The results indicated that the protein levels of TGR5 significantly and continuously increased from POD1, peaked on POD7, and remained elevated until POD14 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eTo investigate the cellular localization of TGR5, F4/80 (a macrophage marker) and PGP9.5 (a nerve fiber marker) were double-stained with TGR5 in sciatic nerves from mice of sham group and pSNL group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Consistent with the data of western bolting, TGR5 increased significantly in injured nerves, especially in the site of injury, marked with pentagrams in immunofluorescent microphotographs. Furthermore, it seemed that TGR5 predominantly colocalized with F4/80 positive cells. Statistically, the colocalization ratio of TGR5 with F4/80, as indicated by the percentage of area, was significantly higher on PDO7 than that of TGR5 colocalized with PGP9.5. In the sham group, TGR5 colocalized with F4/80 and PGP9.5 at the equivalent level (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). In summary, these results revealed the increase of TGR5 in sciatic nerves after pSNL, with the predominant location in macrophages.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Macrophages in the sciatic nerve altered dynamically after pSNL, and located closely with nerve fibers\u003c/h2\u003e\n \u003cp\u003eTo confirm the involvement of macrophages in the pathological process induced by pSNL, the alterations of macrophages in sciatic nerves were assessed using flow cytometry (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). The results indicated a significant increase in the percentage of CD11b\u0026thinsp;+\u0026thinsp;myeloid cells on POD1 and POD3 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Compared with the sham group, the percentages of F4/80\u0026thinsp;+\u0026thinsp;and CD11b\u0026thinsp;+\u0026thinsp;F4/80\u0026thinsp;+\u0026thinsp;macrophages increased significantly on POD3 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Additionally, the population of CD86\u0026thinsp;+\u0026thinsp;CD206- M1 macrophages increased on POD1, showed a relative decrease on POD3 and POD7, and eventually returned to the levels of the sham group by POD14 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). The percentage of CD86-CD206\u0026thinsp;+\u0026thinsp;M2 macrophages followed a pattern opposite to that of M1 macrophages (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Consequently, the ratio of M1 macrophages to M2 macrophages significantly increased on POD1 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). Furthermore, immunofluorescent staining showed that macrophages in the sciatic nerve were located closely with PGP9.5\u0026thinsp;+\u0026thinsp;nerve fibers (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). In summary, macrophages increased and were closely associated with nerve fibers in sciatic nerves after pSNL, displaying proinflammatory characteristics.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Peri-sciatic nerve injection of TGR5 agonist INT-777 alleviated pSNL-induced mechanical allodynia\u003c/h2\u003e\n \u003cp\u003eBased on the robust alterations of TGR5 induced by pSNL as well as the predominant colocalization with macrophages, it was hypothesized that TGR5 involved in pSNL-induced mechanical allodynia. To confirm the hypothesis, the TGR5-specific agonist INT-777 was administered daily viaperi-sciatic nerve injection from day 0 to day 6, and 50% paw withdrawal thresholds were measured (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results indicated that the activation of TGR5 by 50 \u0026micro;g/\u0026micro;L INT-777 alleviated mechanical allodynia on POD5 and POD7, while the similar effects were not observed when INT-777 was administered at concentrations of 0.5 \u0026micro;g/\u0026micro;L or 5 \u0026micro;g/\u0026micro;L (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, the mitigating effect of INT-777 on mechanical allodynia continued after the cessation of drug administration until POD21 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 pSNL-induced transcriptome alterations reversed by INT-777 administration were predominantly inflammation-related\u003c/h2\u003e\n \u003cp\u003eTo illustrate the upstream mechanisms of mechanical allodynia alleviation induced by INT-777 treatment, transcriptional profilings of sciatic nerves from three groups (DMSO_Sham, DMSO_pSNL, and INT-777_pSNL) on POD3 were analyzed by bulk RNA-seq (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Principal component analysis (PCA) revealed distinct transcriptome characteristics among the three groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Specifically, 562 downregulated genes and 556 upregulated genes (cluster 1 and cluster 2 indicated in the Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eA) were detected in the DMSO_pSNL group compared to those in the DMSO_Sham group (definition of differentially expressed genes [DEGs]: | log2FC | \u0026ge; 2 and Q value\u0026thinsp;\u0026le;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Compared to vehicle treatment, 156 DEGs were downregulated and 84 DEGs were upregulated by INT-777 treatment after pSNL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). These genes were indicated as cluster 3 and cluster 4 in the volcano map (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eTo understand the functions of DEGs, the biological processes engaged by four cluster genes were analyzed separately using Gene Ontology (GO) enrichment analysis. Specifically, DEGs in cluster 1 were mainly related to the biosynthetic and metabolic processes of sterol, steroid, and cholesterol (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eC). DEGs in cluster 2 were mainly related to inflammatory response and the processes associated with cytokines and chemokines (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eD). DEGs in cluster 3 were mainly related to immune response (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eE). DEGs in cluster 4 were mainly related to the processes of sarcomere organization, muscle contraction, and organism development (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eF). Notably, DEGs in the cluster 2 and cluster 3, which were genes upregulated after pSNL and genes downregulated after INT-777 treatment, were involved in similar biological processes. Specifically, there were 29 overlapped genes between the two clusters (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). GO enrichment analysis indicated that these genes involved in the immune system process, the regulation process of interleukin-1 beta (IL-1\u0026beta;) and tumor necrosis factor (TNF) production, cellular response process to interferon-gamma (IFN-\u0026gamma;), and chemotaxis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that these genes are associated with cytokine and cytokine receptor interaction pathway, Toll-like receptor signaling pathway, and NF-kappa B signaling pathway as well as other pathways (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). Considering the opposite expression pattern induced by pSNL and INT-777 treatment, these 29 genes and related biological processes might hold the key to mediate the modulatory effects of TGR5 activation against pSNL.\u003c/p\u003e\n \u003cp\u003eIn summary, data of bulk RNA-seq provided an overview indicating that the transcriptome alterations in sciatic nerves induced by pSNL, further reversed by TGR5 activation, were closely associated with inflammatory response, particularly genes related to the production processes of pro-inflammatory cytokines and chemokines. The regulation on these inflammatory mediators may play a pivotal role in the mechanisms underlying the mechanical allodynia alleviation induced by INT-777 treatment.\u003c/p\u003e\u003cspan\u003e\n \u003ch2\u003e3.5 pSNL-induced increase of inflammatory mediators and pro-inflammatory monocytes/macrophages in the sciatic nerve were partially reversed by INT-777 administration\u003c/h2\u003e\n \u003c/span\u003e\n \u003cp\u003eTo verify the effect of INT-777 administration on cytokine/chemokine expression, sciatic nerves were subjected to cytometric bead arrays analysis using a kit containing biomarkers involved in cytokine release syndrome. The results indicated that CCL3, CXCL9, TNF-\u0026alpha;, and IL-6 increased in injured nerves after pSNL, and the administration of INT-777 reversed their upregulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Additionally, the expression levels of CCL2, CXCL10 and VEGF, which were altered by pSNL, remained unchanged following INT-777 treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE-\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). Unexpectedly, no difference in the expression of CCL4, IL-4, IL-10, IFN-\u0026alpha;, and IFN-\u0026gamma; was detected among three groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH-\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eL).\u003c/p\u003e\n \u003cp\u003eTo determine the effect of local TGR5 activation on monocytes/macrophages, sciatic nerves of mice treated with either DMSO or INT-777 for 7 days were analyzed by flow cytometry (Fig. S4). The data revealed a significant reduction in the percentages of CD11b\u0026thinsp;+\u0026thinsp;monocytes and CD11b\u0026thinsp;+\u0026thinsp;F4/80\u0026thinsp;+\u0026thinsp;macrophages following INT-777 administration, although the percentage of F480\u0026thinsp;+\u0026thinsp;cells was not significantly different between the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eM\u0026ndash;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eO). While no significant differences were observed in the individual percentage of CD86\u0026thinsp;+\u0026thinsp;CD206- M1 macrophages, INT-777 administration significantly increased the percentage of CD86-CD206\u0026thinsp;+\u0026thinsp;M2 macrophages and decreased the ratio of M1 macrophages to M2 macrophages in the treated sciatic nerves (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eP\u0026ndash;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eR).\u003c/p\u003e\n \u003cp\u003eThese results indicated that INT-777 administration significantly decreased pro-inflammatory mediators, reduced monocyte/macrophage and increased the prevalence of anti-inflammatory macrophages in the sciatic nerve. In summary, the local TGR5 activation resulted in reduced neuroinflammation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Myeloid cell-specific TGR5 knockdown in the sciatic nerve exacerbated pSNL-induced mechanical allodynia\u003c/h2\u003e\n \u003cp\u003eMacrophages in the sciatic nerve include tissue-resident peripheral nerve macrophages and monocyte-derived infiltrating macrophages. In the state condition, resident macrophages exhibit slow renewal by the bone marrow-derived macrophages. Upon injury, macrophages in the sciatic nerve were reported to be predominantly circulating monocyte-derived macrophages (Ydens et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Given the abovementioned results, to gain a further insight into the contribution of macrophages TGR5 in the injured sciatic nerve to pain perception, targeted knockdown of TGR5 in myeloid cells was performed in Lyz2-cre mice. This was achieved by peri-sciatic nerve injection of the LV-CMV-DIO-EGFP-shRNA virus carrying either the shGPBAR1 or shControl sequence. The most effective shRNA sequence among three different sequences was selected depending on its ability to reduce the mRNA expression of TGR5 in BMDMs (Fig. S5A). Finally, the sequence of shGPBAR1-1 was designated as shGPBAR1 and further used. The specific knockdown effect of the LV-CMV-DIO-EGFP-shRNA (shGPBAR1) virus was validated in BMDMs from Lyz2-cre mice using RT-qPCR (Fig. S5B), immunofluorescence staining (Fig. S5C and S5D), and western blotting (Fig. S5E and S5F).\u003c/p\u003e\n \u003cp\u003eConsidering that the increase of myeloid cells in the sciatic nerve was most significant in the first three days after pSNL, the peri-sciatic nerve injection of lentivirus was performed three times after surgery (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). To evaluate the cell specificity of virus infection, sciatic nerves were subjected to flow cytometry analysis. The results indicated that most of the GFP labeled cells were CD11b positive (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, the GFP labeled CD11b\u0026thinsp;+\u0026thinsp;cells accounted for about 20% of the total CD11b\u0026thinsp;+\u0026thinsp;cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). The knockdown efficiency of TGR5 in the sciatic nerve was further confirmed using RT-qPCR (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD), western blotting (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF) and immunofluorescence staining (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH). In this circumstance, pSNL-induced mechanical allodynia was evaluated. Compared with the shControl virus, the application of the shGPBAR1 virus from day 0 to day 2 after pSNL induced exacerbated mechanical allodynia on POD7 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI). In summary, these results indicated that TGR5 expressed on myeloid cells in the sciatic nerve plays a protective role against pSNL-induced mechanical allodynia.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Myeloid cell-specific TGR5 knockdown induced significant upregulation of pro-inflammatory genes in the sciatic nerve\u003c/h2\u003e\n \u003cp\u003eWhile the contribution of myeloid cells to neuroinflammation is well reported, whether the regulation of neuroinflammation is the main effect of myeloid TGR5 manipulation remains unknown. To screen the transcriptomic alterations after myeloid cell-specific TGR5 knockdown, sciatic nerves obtained from lyz2-cre mice treated with shGPBAR1 or shControl virus were subjected to bulk RNA-seq analysis.\u003c/p\u003e\n \u003cp\u003ePrincipal component analysis revealed distinct transcriptome characteristics between the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Volcano map of DEGs revealed that 5 genes were downregulated and 77 genes were upregulated in the shGPBAR1 group compared to those in the shControl group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). GO enrichment analysis indicated that the top 5 biological processes engaged by upregulated genes were immune response, neutrophil chemotaxis, inflammatory response, immune system process and chemotaxis. Specifically, several genes were involved in regulating NIK/NF kappaB signaling and cytokine production process, especially the production process of TNF, IL-6 and IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). The major pathways engaged by upregulated genes were further evaluated using KEGG enrichment analysis. Cytokine and cytokine receptor interaction as well as chemokine signaling are the major enriched pathways. Similarly, genes in the NF-kappa B, IL-17, NOD-like receptor and TNF signaling pathways were significantly enriched (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). The expression of genes in the above enriched biological processes was indicated in the clustering heatmap (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE). Among these 24 DEGs, \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eNlrp3\u003c/em\u003e were three key genes according to enrichment analysis. Besides, \u003cem\u003eCcl3\u003c/em\u003e and \u003cem\u003eCcl4\u003c/em\u003e were two of three DEGs that also belong to the 29 overlapped gene cluster regulated reversely by pSNL and INT-777 treatment. The gene expression of five genes was validated using RT-qPCR. Except for \u003cem\u003eNlrp3\u003c/em\u003e, the expression of other four genes were significantly upregulated in the shGBAR1 group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF-\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eJ).\u003c/p\u003e\n \u003cp\u003eFor the five downregulated DEGS, GO enrichment analysis revealed that these genes involved in dendritic spine morphogenesis, negative regulation of excitatory postsynaptic potential and cellular response to nutrient levels (Fig. S6A). KEGG enrichment analysis implied that these genes were closely associated with axon guidance and neuroactive ligand-receptor interaction pathway (Fig. S6B). The expression of genes in the above enriched biological processes was indicated in the clustering heatmap (Fig. S6C). In summary, these results revealed that the neuroinflammation was the primary biological process influenced by myeloid TGR5 knockdown, especially the cytokine/chemokine signaling.\u003c/p\u003e\u003cspan\u003e\n \u003ch2\u003e3.8 Myeloid cell-specific TGR5 knockdown upregulated pro-inflammatory mediators and increased proportion of monocytes/macrophages in the sciatic nerve\u003c/h2\u003e\n \u003c/span\u003e\n \u003cp\u003eTo confirm the effects of myeloid cell-specific TGR5 knockdown on neuroinflammation-related alterations implied by the data of RNA-seq, the protein levels of inflammatory mediators in the sciatic nerve were assessed. Cytometric bead arrays analysis indicated that the expression of CCL2, CCL3, TNF-\u0026alpha; and IL-6 were significantly upregulated in the shGPBAR1 group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). The expression of CCL4 was not significantly different between the two groups, although the mRNA levels of Ccl4 was significantly increased in the shGPBAR1 group (Fig. S7A). The expression of CXCL9, CXCL10, and VEGF, which were upregulated by pSNL, were not altered by myeloid cell-specific TGR5 knockdown (Fig. S7B-S7D). Consistent with the effects of pSNL and TGR5 activation on the expression of IL-4, IL-10, IFN-\u0026alpha; and IFN-\u0026gamma;, TGR5 knockdown did not regulate their protein levels (Fig. S7E-S7H).\u003c/p\u003e\n \u003cp\u003eNLRP3-ASC inflammasome was reported to regulate the generation of IL-1\u0026beta; (Broz and Dixit 2016). Given the significant enrichment of DEGs to NLRP3 regulation and IL-1\u0026beta; production pathways, their protein expression was assessed using western blot analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE). The results indicated that the expression of NLRP3 was not significantly different between shControl and shGPBAR1 groups, while the expressions of ASC, Caspase-1, and IL-1\u0026beta; were significantly upregulated by TGR5 knockdown (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eF-\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eI). Furthermore, immunofluorescence staining indicated that NLRP3-ASC inflammasome and IL-1\u0026beta; colocalized with F480\u0026thinsp;+\u0026thinsp;macrophage in the injured sciatic nerve (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eJ and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eK).\u003c/p\u003e\n \u003cp\u003eBased on the robust alterations of cytokines and chemokines induced by myeloid-cell specific TGR5 knockdown, monocytes/ macrophages in the sciatic nerve were evaluated by flow cytometry (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA). The data revealed a significant increase of CD11b\u0026thinsp;+\u0026thinsp;monocytes, F480\u0026thinsp;+\u0026thinsp;macrophages and CD11b\u0026thinsp;+\u0026thinsp;F4/80\u0026thinsp;+\u0026thinsp;macrophages in the shGPBAR1 group compared to those in the shControl group (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB-\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). However, no significant differences were observed in the individual percentage of CD86\u0026thinsp;+\u0026thinsp;CD206- M1 macrophages, CD86-CD206\u0026thinsp;+\u0026thinsp;M2 macrophages, and the ratio of M1 macrophages to M2 macrophages between the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eE\u0026ndash;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eG).\u003c/p\u003e\n \u003cp\u003eThese results confirmed the substantial regulatory impact of myeloid TGR5 on the expression of pro-inflammatory mediators and the proportion of monocytes/ macrophages in the sciatic nerve. In summary, myeloid-cell specific TGR5 knockdown exaggerated neuroinflammation in the sciatic nerve after pSNL.\u003c/p\u003e\u003cspan\u003e\n \u003ch2\u003e3.9 Activation of microglia in the dorsal horn of spinal cord induced by pSNL altered when TGR5 in the sciatic nerve was manipulated\u003c/h2\u003e\n \u003c/span\u003e\n \u003cp\u003ePeripheral neuroinflammation contributes to central sensitization which is the pivotal mechanism of neuropathic pain (Woolf and Salter \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e). To estimate the effects of TGR5 manipulations on central sensitization in the spinal cord level, the activation of microglia indicated with the Iba-1 positive area in the ipsilateral dorsal horn of spinal cord induced by pSNL were analyzed on POD7. Immunofluorescence staining indicated that the activation of microglia was decreased by peri-sciatic nerve INT-777 treatment (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eB) and was increased by myeloid cells-specific TGR5 knockdown in the sciatic nerve compared to their respective control groups (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we confirmed that TGR5 in injured nerves protect against pSNL-induced mechanical allodynia by modulating neuroinflammation. Specifically, the expression of TGR5 increased in injured nerves, and local activation of TGR5 alleviated mechanical allodynia by reversing the increase of pro-inflammatory cytokines/chemokines as well as the increase of monocytes/macrophages induced by pSNL. On the contrary, specific knockdown of TGR5 in myeloid cells aggravates mechanical allodynia by increasing the expression of pro-inflammatory mediators and the proportion of monocytes/macrophages.\u003c/p\u003e \u003cp\u003eTGR5, a GPCR protein, is abundantly expressed in the gallbladder epithelium, and is minimally to moderately expressed in almost all other tissues and cell types (Vassileva et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In the nervous system, TGR5 is found in both neurons and non-neuronal cells, including resident and infiltrating immune cells (Lieu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The distinct functions of TGR5 in neurons and non-neuronal cells was previously reported. For neurons, administration of the TGR5 agonist increases the intrinsic excitability of DRG neurons and activates colon-innervating DRG neurons separated from na\u0026iuml;ve mice (Alemi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Castro et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In non-neuronal cells, TGR5 activation mitigates LPS-induced inflammatory responses in both microglia and macrophages (Hogenauer et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Yanguas-Casas et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, the known roles of TGR5 expressed in different anatomical region in pain modulation are also inconsistent. In the mouse model of paclitaxel-induced peripheral neuropathic pain, TGR5 expressed in the DRG neurons contribute to the upregulation of CCR5, and then mediates the deoxycholic acid induced mechanical allodynia (Zhong et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the mouse model of spared nerve injury (SNI)-induced neuropathic pain, intrathecal injection of TGR5 agonist alleviates mechanical allodynia by inhibiting the activation of glial cells and modulating the function of GABAA receptors in the spinal cord(Wu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, TGR5 in the injured sciatic nerve was mainly expressed in the macrophages, and pSNL-induced mechanical allodynia was significantly alleviated by peri-sciatic administration of the TGR5-specific agonist INT-777. Underlying this phenotype, the molecular mechanism regulated by TGR5 activation is more consistent with a negative regulation effect on inflammation rather than a positive impact on neuron excitability. The inference was validated by bulk RNA-seq analysis of injured nerve tissues. The sequence data indicated that, among the entire transcriptome changes, the inflammatory response is the primary process engaged by DEGs which were regulated reversely by pSNL and TGR5 activation. In addition, pSNL-induced mechanical allodynia was further exaggerated when TGR5 was specifically knockdown in myeloid cells along with the transcriptomic alterations predominantly related to the inflammatory response. Taken together, although the specific role of TGR5 expressed by neuron fibers in pain modulation was not evaluated in this study, the present data proved that increased TGR5 in the injured sciatic nerve is a molecular target to modulate mechanical allodynia by regulating neuroinflammation.\u003c/p\u003e \u003cp\u003eIn the field of neuropathic pain, ample evidence has underscored the substantial role of neuroinflammation in driving both peripheral and central sensitization, thereby enhancing pain hypersensitivity (Grace et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Bethea and Fischer \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recent findings indicate that specific subsets of immune cells contribute to the alleviation of neuropathic pain (Fiore et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among them, macrophages are a subset of cells which exhibit remarkable plasticity and adopt functionally distinct phenotypes (Mosser et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The inflammatory mediators, which are highly expressed by pro-inflammatory M1 macrophages, such as IL-1β (Binshtok et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), IL-6 (Liu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and TNF-α (Jin and Gereau \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) were reported to induce neuronal sensitization and circulating leukocytes recruitment into inflamed tissue (Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The antinociceptive properties of the mediators secreted by anti-inflammatory M2 macrophages, such as IL-10 (Kwilasz et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Niehaus et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and IL-4 (Celik et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Labuz et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have also been validated. In this study, we confirmed the dynamic changes of macrophages and their close proximity to neuron fibers in injured nerves. In the context of TGR5 manipulation, accompanied by the alterations of mechanical allodynia, pro-inflammatory mediators and the proportion of monocytes/macrophages in the sciatic nerve were significantly regulated, with no significant difference detected in the expression of anti-inflammatory mediators. Activation of microglia in the SDH is an important part of central sensitization and is known to contribute to the neuropathic pain (Inoue and Tsuda \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, manipulations of TGR5 in the sciatic nerve significantly altered the activation level of microglia. In summary, the high relevance between macrophage-mediated neuroinflammation and pSNL-induced mechanical allodynia was further confirmed in this study. Regarding the neuronal mechanisms that mediate the effect of TGR5-modulated neuroinflammation on pain perception, although five genes involved in dendritic spine morphogenesis and negative regulation of excitatory postsynaptic process were detected to be downregulated by TGR5 knockdown, the specific roles of these genes still need to be further investigated.\u003c/p\u003e \u003cp\u003eSince its detection, the modulatory effects of TGR5 on the functions of monocytes and macrophages have been extensively studied. Specifically, the production of pro-inflammatory cytokines in macrophages, such as TNF-α, IL-6 and IL-1β, are reported to be inhibited by TGR5 activation via NF-κB pathway (Pols et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and to be exacerbated by TGR5 deficiency via stabilizing the β-catenin destruction complex (Rao et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The production of anti-inflammatory cytokines, such as IL-4 and IL-10, is reported to be upregulated by macrophage TGR5 activation, but the controversy remains regarding whether the presence of TGR5 is indispensable for the polarization of anti-inflammatory M2 macrophages (Perino et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Zhao et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The expressions of chemokines, such as CCL2, CCL3 and CCL4, are also regulated by TGR5. In the presence of LPS, TRG5 deficiency exaggerates the upregulation of chemokines, while the activation of TGR5 poses the opposite impact on macrophages via the mTOR-C/EBPβ pathway (Perino et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, the activation of NLRP3 inflammasome is inhibited by bile acids via TGR5-cAMP-PKA axis (Guo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and the deficiency of TGR5 exaggerates the inflammatory response of macrophages stimulated with palmitic acid by promoting the activation of NLRP3 inflammasome (Shi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, the increase of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, as well as chemokines CCL3 and CCL2 in sciatic nerves was exacerbated by myeloid-cell specific TGR5 knockdown. These results are consistent with the known effects of TGR5 on macrophages. Regarding the NLRP3 inflammasome, although the expression of NLRP3 was not altered, the expression of the adaptor protein ASC, which connects the inflammasome sensor molecule to caspase-1, as well as the expression of caspase-1, which initiates downstream responses, were significantly upregulated by TGR5 knockdown, indicating the further activation of NLRP3 inflammasome. As for the specific molecular pathways underlying the modulations of TGR5 on inflammatory mediators in macrophages, which located in the injured sciatic nerve, further research is needed to illustrate the intrinsic alterations of these cells in situ rather than the changes induced by mimicked inflammatory response in cell experiments.\u003c/p\u003e \u003cp\u003eThe endogenous ligands of TGR5 are primary bile acids synthesized from cholesterol and secondary bile acids metabolized by the gut microbiota (Collins et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The majority of bile acids are generated and exist in the enterohepatic system and a minority of them spillover into the circulation (Perino et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the peripheral and central nervous system, the presence of bile acids is confirmed (Xing et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition to being transported from circulation, bile acids are also synthesized locally in the brain and spinal cord, as evidenced by the detection of rate-limiting enzymes for synthesis (Hurley et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, data of bulk RNA-seq indicated that genes involved in the biosynthetic and metabolic process of cholesterol were significantly regulated in the sciatic nerve after pSNL, implying the potential alterations of endogenous ligands of TGR5. However, whether the protein levels of bile acids in injured sciatic nerves changes and whether these ligands participate in the modulation of pathological processes of neuropathic pain need to be further studied.\u003c/p\u003e \u003cp\u003eThe primary limitation of this study is the use of only male mice. Given the well-documented sex differences in neuroinflammatory mechanisms (del Rivero et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Gregus et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), whether TGR5 plays the same regulatory role in neuroinflammation and mechanical allodynia in female mice remains unknown.\u003c/p\u003e \u003cp\u003eIn conclusion, this study established that TGR5, expressed by macrophages within injured nerves, is a protective factor against mechanical allodynia. This protective effect involves alleviating neuroinflammation by modulating the pSNL-induced expression of inflammatory mediators. Consequently, TGR5, which operates at the peripheral nerve injury site, has emerged as a promising molecular target for treating neuropathic pain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were performed in compliance with experimental guidelines approved by the Animal Care and Use Committee of the Peking University Center of Health Science.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (81870788, 82170979, 82371227, 82171226, 81974169, 82001192) and Natural Science Foundation of Beijing Municipality (7222105).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWen-Ge Shi, Kai-Yuan Fu, and Guo-Gang Xing were involved in experimental design and manuscript writing. Wen-Ge Shi performed the experiments and processed the data. Yao Yao, Jie Lei, Shi-Yang Fang, Ya-Jing Liang, Yue Tian, Zi-Xian Zhang, and Jie Cai provided technical support for experiments. All authors contributed to and endorsed the submission of this manuscript and take responsibility for it.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlemi, F., E. Kwon, D. P. Poole, T. Lieu, V. Lyo, F. Cattaruzza, F. Cevikbas, M. Steinhoff, R. Nassini, S. Materazzi, R. Guerrero-Alba, E. Valdez-Morales, G. S. Cottrell, K. Schoonjans, P. Geppetti, S. J. Vanner, N. W. Bunnett and C. U. Corvera (2013). \u0026quot;The TGR5 receptor mediates bile acid-induced itch and analgesia.\u0026quot; \u003cu\u003eJ Clin Invest\u003c/u\u003e \u003cstrong\u003e123\u003c/strong\u003e(4): 1513-1530.\u003c/li\u003e\n \u003cli\u003eBannister, K., J. Sachau, R. Baron and A. H. 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Zhang (2019). \u0026quot;Activation of TGR5 with INT-777 attenuates oxidative stress and neuronal apoptosis via cAMP/PKCepsilon/ALDH2 pathway after subarachnoid hemorrhage in rats.\u0026quot; \u003cu\u003eFree Radic Biol Med\u003c/u\u003e \u003cstrong\u003e143\u003c/strong\u003e: 441-453.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neuropathic pain, Mechanical allodynia, Sciatic nerve, TGR5, Macrophages, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-3852075/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3852075/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeuropathic pain is a pervasive medical challenge that currently lacks effective treatment solutions. Molecular changes occurring at the site of peripheral nerve damage contribute to the development of peripheral and central sensitization, which are critical components of neuropathic pain. This study aimed to investigate the role of the G protein-coupled bile acid receptor (GPBAR1, also known as TGR5) in the peripheral mechanisms underlying neuropathic pain induced by partial sciatic nerve ligation (pSNL) in male mice. TGR5 was upregulated in injured nerves and colocalized predominantly with macrophages. Peri-sciatic nerve administration of the TGR5-specific agonist INT-777 provided sustained relief from mechanical allodynia. Transcriptome sequencing revealed that pain relief was primarily attributable to reduced neuroinflammation. This finding was corroborated by a reduction in myeloid cells and proinflammatory mediators (including CCL3, CXCL9, IL-6, and TNF-α), accompanied by an increase in the percentage of anti-inflammatory M2 macrophages following INT-777 administration. Furthermore, myeloid cell-specific TGR5 knockdown in the sciatic nerve following pSNL exacerbated both mechanical allodynia and neuroinflammation. This is substantiated by data from the bulk RNA-seq and upregulated expression levels of inflammatory mediators (including CCL3, CCL2, IL-6, TNF-α and IL-1β), as well as increased monocytes/ macrophages in the injured nerve. Besides, the activation of microglia in the ipsilateral dorsal horn of spinal cord induced by pSNL altered when TGR5 in the sciatic nerve was manipulated. In summary, TGR5, present in injured nerves, plays a protective role and offers potential as a target for treating neuropathic pain.\u003c/p\u003e","manuscriptTitle":"TGR5 protects against pSNL-induced mechanical allodynia by alleviating neuroinflammation in the injured nerves of male mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-12 06:25:43","doi":"10.21203/rs.3.rs-3852075/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e9d922c-ed01-48bd-9cdf-edbe2ce8843b","owner":[],"postedDate":"January 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-03T13:56:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-12 06:25:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3852075","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3852075","identity":"rs-3852075","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}