Resveratrol Alleviates Temporomandibular Joint Inflammatory Pain By Recovering Disturbed Gut Microbiota

Pain caused by temporomandibular disorders(TMD),characterized by persistent dull pain,is one of the most common chronic oral and maxillofacial pains.TMD generally affects young people,and can seriously affects patients' daily life.There is currently no effective prevention and treatment method.Clinical data shows that 94%of TMD patients have the symptom of temporomandibular joint(TMJ)pain,which is mainly caused by TMJ inflammation.Previous research suggested that TMJ inflammation could activate microglia in spinal trigeminal nucleus caudalis(Sp5C).Activated microglia release large amount of pro-inflammatory cytokines,which further regulates synaptic plasticity of neurons and facilitates the transmission of pain signals.However,how this process could cause pain remains unknown.The gut microbiota is an essential microorganism for the regulation of the central inflammatory response and chronic pain by regulating the function of microglia,but the role of gut microbiota in TMJ inflammatory pain is currently unclear.Resveratrol as a natural bioactive polyphenolic compound,its powerful anti-inflammatory and analgesic effect makes it a potential drug for the treatment of joint inflammation and pathological pain.Recent studies have found that RSV can significantly improve gut microbiota dysbiosis,and relieves trigeminal neuralgia by suppressing the glial activation in the Sp5 C.However,the role of RSV in CFA-induced TMJ inflammatory pain and the role of gut microbiota in RSV regulating CFA-induced TMJ inflammatory pain is not clear.ObjectiveIn this study,intra-TMJ injection of CFA was used to construct a TMJ inflammatory pain model in mice.Through the observation of the changes of inflammatory indicators in the Sp5 C,gut microbiota and pain response in model mice before and after RSV and fecal microbiota transplantation(FMT)experiments,the mechanism of gut microbiota in RSV in relieving CFA-induced TMJ inflammatory pain was investigated.The study provides a theoretical basis for clinical treatment of inflammatory TMD pain.Part ?Influence of CFA-induced inflammatory TMJ pain on gut microbiota and the expression of pro-inflammatory factors in the Sp5C Methods1.Male C57 BL/6J wild-type(WT)mice and TNF? KO mice were used to construct TMJ inflammatory pain models,and von Frey filaments were used to test facial mechanical hysensitivity in each group of mice.2.Detection of neuroinflammation related indicators: q RT-PCR and Western blot were used quantitatively detect the expression levels of tumor necrosis factor alpha(TNF?)m RNA and protein in the Sp5C;immunofluorescence staining was applied to analyze the colocalization of TNF? with the neuron marker neuronal nuclei(Neu N),the astrocyte marker glial fibrillary acidic protein(GFAP)and the microglia marker ionized calcium-binding adapter molecule 1(Iba1),and morphology of microglia activation in the Sp5C;Evans blue perfusion was performed to examine blood-brain barrier(BBB)integrity in brainstem.3.Gut microbiota structure and metabolite detection: gas chromatography-mass spectrometry(GC-MS)analysis was used to measure the concentration of short-chain fatty acids(SCFAs)in the fecal samples of mice;Real-time fluorescent quantitative PCR was used to detect the number of SCFAs-producing gut bacteria in the fecal samples of mice;16S r DNA amplicon sequencing technology was used to detect bacterial gene diversity in the fecal samples of mice,and to analyze differences in the structure of gut microbiota between different mice groups.Results1.CFA induced inflammatory TMJ pain in WT mice.Bilateral injection of CFA robustly decreased head withdrawal threshold in both sides of trigeminal nerve V3branch-innervated facial skin area in WT mice;However,the genetic deletion of TNF? in the KO mice markedly diminished CFA-decreased head withdrawal threshold in both trigeminal nerve V3 branch-innervated skin areas on the same time points.2.CFA resulted in increased the expression of pro-inflammatory factor TNF?,Iba1-labeled activated microglia,massive co-labeled expression of TNF? and Iba1-positive microglia in the Sp5 C,and brainstem BBB injury.3.CFA caused a decrease in the microbial abundance and diversity in the gut of mice,in which the number of Bacteroidetes and Lachnospiraceae decreased significantly,and the gut microbiota that was imbalanced returned to the basic level on the day 12 after CFA injection.CFA caused a reduction of SCFAs in the gut of mice.Part ?Effects of resveratrol on gut microbiota and the expression ofpro-inflammatory factors in the Sp5 C of mice with CFA-induced inflammatory TMJ pain Methods1.RSV(40 mg/kg,80 mg/kg)or DMSO(vehicle control)were intraperitoneally injected 1 h after intra-TMJ injection of CFA,once a day for 4 consecutive days;von Frey filaments were used to assess the analgesic effect of RSV on the facial mechanical hypersensitivity in each group of mice.2.Detection of neuroinflammation related indicators: q RT-PCR and Western blot were used to quantitatively detect the expression levels of TNF? m RNA and protein in the Sp5C;immunofluorescence staining was used to analyze the co-expression of TNF? and Iba1-positive microglia and microglia morphology in the Sp5C;Evans blue perfusion was performed to examine BBB integrity in brainstem.3.Gut microbiota structure and metabolite detection: GC-MS analysis was used to measure the concentration of SCFAs in the fecal samples of CFA-induced TMJ inflammatory pain mice before and after RSV treatment;Real-time fluorescent quantitative PCR method was used to detect the number of SCFAs-producing gut bacteria in CFA-induced TMJ inflammatory pain mice before and after RSV treatment.Results1.RSV inhibited CFA-induced inflammatory TMJ pain.RSV had no effect on the head withdrawal threshold in control mice,but it dose-dependently increased the CFA-decreased head withdrawal threshold in both sides of trigeminal nerve V3branch-innervated facial skin area compare to DMSO treatment.2.RSV blocked CFA-enhanced the expression of pro-inflammatory factor TNF?,microglia activation,co-labeled expression of TNF? and Iba1-positive microglia in the Sp5 C,and BBB leakage in brainstem.3.RSV reversed CFA-caused reduction of SCFAs and significantly recovered CFA-decreased SCFAs-producing Bacteroidetes and Lachnospiraceae in the mice gut.Part ? FMT attenuates CFA-induced inflammatory TMJ pain Methods1.FMT(fecal microbiota transplantation): collect fresh feces from mice with CFA+DMSO or CFA+RSV(40 mg/kg)treatment,and perform FMT(FMT1 and FMT2 respectively)1 h after intra-TMJ injection of CFA,once a day for 4consecutive days;use von Frey filaments to assess the analgesic effect of FMT on the facial mechanical hypersensitivity in CFA-induced TMJ inflammatory pain model.2.Gut microbiota structure and metabolite detection: GC-MS analysis was used to measure the concentration of SCFAs in the fecal samples of CFA-induced TMJ inflammatory pain mice before and after FMT treatment;Real-time fluorescent quantitative PCR method was used to detect the number of SCFAs-producing gut bacteria in the fecal samples of CFA-induced TMJ inflammatory pain mice before and after FMT treatment.3.Detection of neuroinflammation related indicators: Western blot was used to quantitatively detect the expression of TNF? protein in the Sp5C;immunofluorescence staining was used to analyze the co-expression of TNF? and Iba1-positive microglia in the Sp5C;Evans blue perfusion was performed to examine BBB integrity in brainstem.Results1.FMT with feces from RSV-treated mice attenuated CFA-induced inflammatory TMJ pain.The transplantation(FMT2)of fecal slurry from RSV-treated mice significantly increased head withdrawal threshold in the ipsilateral V3branch-innervated facial skin area compare with the transplantation(FMT1)of fecal slurry from DMSO-treated mice,while the FMT2 had no effect on the head withdrawal threshold in the contralateral V3 branch-innervated facial skin area.On the other head,the head withdrawal threshold of both ipsilateral and contralateral sides in mice with FMT1 were similar to those in the PBS-treated group.2.FMT with feces from RSV-treated mice reversed CFA-caused reduction of SCFAs and significantly recovered CFA-decreased SCFAs-producing Bacteroidetes and Lachnospiraceae in the mice gut.3.FMT with feces from RSV-treated mice inhibited CFA-enhanced microglial activation and the expression of pro-inflammatory factor TNF? in the Sp5 C,and blocked BBB leakage in brainstem.Conclusion1.CFA-induced inflammatory TMJ pain can cause gut microbiota imbalance;2.RSV can effectively exert analgesic effect on inflammatory TMJ pain and recover gut microbiota imbalance;3.The recovery of disturbed gut microbiota is an important mechanism for the analgesic effect of RSV.