Emodin Exerts Neuroprotective Effects On Experimental Autoimmune Encephalomyelitis By Regulating The SIRT1/PGC-1α/NLRP3 Pathway

Objective: Multiple sclerosis(MS)is an autoimmune,demyelinating disease that occurs primarily in the central nervous system.It affects more than two million people worldwide,mainly young women.The typical pathological features of MS are focal demyelinating plaques,axonal damage and astrogliosis in the central nervous system.The clinical manifestations of MS patients are diverse and the etiology is not fully understood.Current treatments cannot cure MS,and commonly used therapeutic drugs,such as dimethyl fumarate,have been reported to have certain side effects.Inflammasomes are multimolecular signaling complexes that sense injury and stress signals and induce the maturation and secretion of pro-inflammatory cytokines.Among them,NLRP3 inflammasomes are the most widely studied inflammasomes.Studies have shown that NLRP3 inflammasomes are involved in a variety of immune inflammatory diseases and have also been reported in central nervous system diseases.Inflammasomes can mediate pyroptosis,which is a kind of programmed cell death and plays a crucial role in MS.Microglia,as important immune cells in the brain,also play a crucial role in MS.Emodin is a natural product derived from herbs such as rhubarb,which has anti-inflammatory,antioxidant and anti-tumor effects.Current studies have found that emodin has neuroprotective effects on central nervous system diseases.In this study,we used classical animal models of MS,experimental autoimmune encephalomyelitis(EAE)rat models,to investigate the neuroprotective effects of emodin on EAE and the potential mechanisms involved in this process.Methods:Ⅰ.Establish EAE rat models and microglial inflammation models to explore the neuroprotective effect of emodin on EAE rat models and its inhibition on microglial inflammation1.EAE rat models were established.From the day of modeling,emodin or vehicle was injected intraperitoneally every day until the day before sampling.The daily body weight and neurobehavioral scores of the rats were recorded;the degree of inflammatory cell infiltration in the brain and spinal cord was evaluated by HE staining;the degree of spinal cord demyelination was evaluated by LFB staining;q RT-PCR,NO assay and ELISA were used to evaluate the expression levels of inflammation-related factors;and the expression levels of Iba-1 and CD68 in the spinal cord were evaluated by immunohistochemical staining.2.Establish inflammation models of microglia.CCK8 assay was used to evaluate the cell viability;the expression level of inflammation-related factors was evaluated by NO detection and ELISA;the level of ROS was detected by fluorescence and flow cytometry experiments.Ⅱ.Investigate the effects of emodin on NLRP3 inflammasomes and pyroptosis in EAE rat models and microglial inflammation models1.In in vivo experiments,the protein expression levels of NLRP3,ASC and cleavedcaspase-1 were detected by Western blot;ELISA and q RT-PCR were used to assess the expression levels of IL-1β and IL-18;and the level of pyroptosis was evaluated by LDH assay and Western blot assay of GSDMD-N.2.In in vitro experiments,the protein expression levels of NLRP3,ASC and cleavedcaspase-1 were detected by Western blot;the expression levels of IL-1β and IL-18 were assessed by ELISA;and the level of pyroptosis was evaluated by LDH assay and Western blot assay of GSDMD-N;the degree of cell death was evaluated by Calcein-AM/PI staining.Ⅲ.Study on the mechanism of emodin affecting NLRP3 inflammasomes and pyroptosis1.Predict the potential target of emodin on MS through network pharmacology.2.Simulate the combination of emodin and potential target through molecular docking;the protein expression levels of potential target in vivo and in vitro were detected by Western blot.3.In in vitro experiments,microglia were transfected with si RNA to knock down potential target(fluorescence,q RT-PCR and Western blot for verification of transfection efficiency);target inhibitor was used to suppress protein expression of potential target(CCK8 assay and Western blot for verification of inhibition efficiency).The protein expression levels of potential target and its downstream molecule,NLRP3,ASC and cleaved-caspase-1 were detected by Western blot;the expression levels of IL-1β and IL-18 were assessed by ELISA;LDH assay and Western blot assay of GSDMD-N were used to evaluate the level of pyroptosis.4.In in vitro experiments,protein expression of downstream molecule of potential target was inhibited by using inhibitor(CCK8 assay and Western blot for verification of inhibition efficiency).The protein expression levels of potential target and its downstream molecule,NLRP3,ASC and cleaved-caspase-1 were detected by Western blot;the expression levels of IL-1β and IL-18 were detected by ELISA;LDH assay and GSDMDN Western blot assay were used to evaluate the level of pyroptosis.Results:1.Compared with the normal control group rats,the weight of the disease model group rats decreased significantly,and the neurobehavioral score increased significantly;the degree of inflammatory cell infiltration and demyelination in spinal cord increased significantly;the degree of inflammatory cell infiltration in brain increased significantly;the expression level of inflammatory cytokines in spinal cord tissue and serum increased significantly;the expression of Iba-1 and CD68 increased in spinal cord tissue.Compared with the disease model group,these indicators have been significantly improved in the emodin treatment group.Moreover,compared with the normal control group,there were no significant changes in these indicators in the emodin group.2.Compared with microglia of control group,the expression of pro-inflammatory factors increased,the expression of anti-inflammatory factors decreased,and the level of ROS elevated in microglial inflammation models group.Emodin can significantly inhibit the expression of pro-inflammatory factors,increase the expression of anti-inflammatory factors,and decrease the level of ROS.3.Compared with the normal control group rats,the protein expression levels of NLRP3,ASC,cleaved-caspase-1 and GSDMD-N were apparently elevated in the spinal cord tissues of the disease model group rats;the m RNA expressions of IL-1β and IL-18 were significantly increased in the spinal cord tissues;and the serum levels of IL-1β,IL-18 and LDH were significantly increased.In the emodin treatment group,the expression levels of these indicators were significantly reduced.Compared with the normal control group,these indicators were not significantly changed in the emodin group.4.The protein expression levels of NLRP3,ASC,cleaved-caspase-1 and GSDMD-N were significantly increased in the microglial inflammation models group compared with microglia of control group;the expression of IL-1β,IL-18 and LDH were significantly increased in the cell culture supernatant;the degree of cell death increased.Emodin treatment significantly inhibited the increase of these indicators.5.Combining the results of network pharmacology and the available literature,SIRT1 may be a potential target of emodin to affect MS,and PGC-1α is a possible downstream of SIRT1;the molecular docking results indicate that the binding between emodin and SIRT1 is highly probable;the results of Western blot showed that in in vivo and vitro model groups,the expression of SIRT1 and PGC-1α decreased,while their expression could be increased after emodin treatment.6.In in vitro experiments,the use of SIRT1 si RNA transfection and SIRT1 inhibitor(EX527)significantly inhibited the expression of intracellular SIRT1.Compared with the treatment group,the expression levels of intracellular SIRT1 and PGC-1α were significantly reduced after transfection,and the expression of relevant indicators of NLRP3 inflammasomes and pyroptosis significantly increased in the cells of transfected group,suggesting that emodin regulates NLRP3 inflammasome activation and pyroptosis through SIRT1.However,the effect of the inhibitor group on the relevant indicators was not significant.7.In in vitro experiments,the use of PGC-1α inhibitor(SR-18292)significantly suppressed the intracellular PGC-1α expression level.Compared with the treatment group,the expression of PGC-1α was significantly reduced in the cells of the inhibitor group,but the expression level of SIRT1 was not significantly changed,which,combined with the previous results,suggests that PGC-1α may be a downstream molecule of SIRT1;in addition,the expression of relevant indicators of NLRP3 inflammasomes and pyroptosis was significantly increased in the cells of the inhibitor group,suggesting that PGC-1α is involved in the regulation of emodin on NLRP3 inflammasomes and pyroptosis.Therefore,emodin may regulate NLRP3 inflammasomes and pyroptosis through the SIRT1/PGC-1αpathway.Conclusions:1.Emodin could improve the clinical symptoms,inhibit the degree of inflammatory cell infiltration and demyelination in the spinal cord,inhibit the degree of inflammatory cell infiltration in the brain,reduce the level of inflammation and thus exert neuroprotective effects in the EAE rat models;emodin could inhibit the level of inflammation in the microglial inflammation models.2.In EAE rat models and microglial inflammation models,emodin can inhibit the level of NLRP3 inflammasomes activation and pyroptosis.3.Emodin regulates NLRP3 inflammasomes activation and pyroptosis via SIRT1/PGC-1αpathway.