Study On The Therapeutic Effect Of Lagotis Brachystachys Maxim On Chronic Alcoholic Liver Injury And Acute Gouty Arthritis Based On Inflammatory Signaling Pathway

Objection:In this study,we established rat models of chronic alcoholic liver injury and acute gouty arthritis to investigate the effects of Tibetan medicine Lagotis brachystachys Maxim total extract and different polar site on chronic alcoholic liver injury and acute gouty arthritis.Through the method of network pharmacology,this paper systematically and comprehensively analyzed the common molecular mechanism of L.brachystachys Maxim,search for its possible active ingredients and provide ideas for further research on its mechanism of action.Methods:（1）The rat model of chronic alcoholic liver injury was established by8 weeks alcoholic gavage to study the effects of Tibetan medicine L.brachystachys Maxim extracts(0.5g·kg^-1、1 g·kg^-1、2g·kg^-1)on Toll-like receptor（TLR）/myeloid differentiation factor 88（My D88）/nuclear factor kappa B（NF-κB）an NALP3 signaling pathways.The levels of serum aspartate aminotransferase（AST）,alanine aminotransfease（ALT）,totalcholesterol（TC）,Triglyceride（TG）,Interleukin-1β（IL-1β）were measured.the expression of TLR2,TLR4,My D88,NF-κB and NALP3 protein in liver were detected by Western blot;The pathological changes of liver tissue were observed by hematoxylin-eosin（HE）staining.（2）The rat model of chronic alcoholic liver injury was established by 8 weeks alcoholic gavage to study the effects of Tibetan medicine L.brachystachys Maxim different polar parts（Water part,30% ethanol part,50%ethanol part,95%ethanol part）on TLR/My D88/NF-κB and NALP3signaling pathways.The serum levels of AST and other indicators were measured as before,and the levels of Glutathione peroxidase（GSH）and superoxide dismutase （SOD）in liver homogenate were detected;the expression of TLR2,My D88,NF-κB and NALP3 protein in liver were detected by Western blot;The pathological changes of liver tissue were observed by HE staining.（3）The rat model of acute gouty arthritis was established by injecting sodium urate crystals（MSU）into the rat right hind ankle joint to study the effects of Tibetan medicine L.brachystachys Maxim extracts (0.8g·kg^-1、1.6 g·kg^-1、3.2g·kg^-1)on TLR/My D88/NF-κB and NALP3 signaling pathways.The joint swelling of the right hind ankle joint was measured by evolume measuring;serum tumor necrosis factorα（TNF-α）levels was detected;the expression of TLR2,TLR4,My D88,NF-κB and NALP3 protein in liver was detected by Western blot.The pathological changes of synovial joints were observed by HE staining.（4） The rat model of acute gouty arthritis was established by injecting sodium urate crystals（MSU）into the rat right hind ankle joint to study the effects of Tibetan medicine L.brachystachys Maxim different polar parts（Water part,30%ethanol part,50%ethanol part,95%ethanol part）on TLR/My D88/NF-κB and NALP3 signaling pathways.The joint swelling of the right hind ankle joint was measured by evolume measuring;serum TNF-αlevels was detected;the expression of TLR2,TLR4,My D88,NF-κB and NALP3 protein in liver was detected by Western blot.The pathological changes of synovial joints were observed by HE staining.（5）Using the network pharmacology method to predict the active ingredients,action targets and disease targets of L.brachystachys Maxim,Investigate the common mechanism of chronic alcoholic liver injury and acute gouty arthritis caused by"different diseases and same treatment".the active ingredients and their targets of L.brachystachys Maxim was obtained by TCMSP,Pharm Mapper Server,Swiss Target Prediction,and CTD database;The related targets of the two diseases were obtained through the Gene Cards,OMIM,and TTD databases.Cytoscape was used to draw the active ingredient-disease-common target network diagram and the common target protein interaction network.The GO process and KEGG pathway of short spike rabbit ear grass were enriched and analyzed through DAVID website.Result:（1）L.brachystachys Maxim extracts in each dose group can reduce serum AST,ALT,TC,TG,IL-1βlevels,which the high-dose effect is particularly significant;Western Blot results showed that the expression levels of TLR2,My D88,NF-κB and NALP3 in liver tissues were significantly down-regulated,and there was no significant effect on the protein expression of TLR4.Pathological sections showed that each dose group of L.brachystachys Maxim could improve the pathological changes of liver tissue to different degrees.（2）The 30%,50%and 95%ethanol part and the positive drug group of L.brachystachys Maxim significantly reduced serum ALT and AST levels;and the30%,50%ethanol part significantly reduced serum TG and IL-1βlevels;TC levels was no significant effect.30%,50%ethanol part can significantly reduce GSH levels in liver homogenates,50%ethanol part can significantly reduce SOD levels in liver homogenates and there was no significant difference among MDA groups;Western Blot results showed that 30%ethanol part can make the expression levels of My D88down-regulated,and 30%,50%ethanol part could significantly make the expression levels of TLR2,NF-κB and NALP3 down-regulated.Pathological section results showed that 30%and 50%ethanol parts improved the pathological changes of liver tissue in rats.（3）After 48 hours of modeling with MSU,the swelling rate of ankle joints in different dose groups of L.brachystachys Maxim and colchicine group significantly decreased;each dose group can significantly reduce the serum TNF-αlevel and no significant difference in IL-1βlevels among the groups;Western Blot results showed that the medium and high dose groups significantly reduced the expression levels of TLR2,TLR4,My D88,NF-κB and NALP3 in synovial tissue;pathological sections showed that H.breviflora could improve the synovium of rats Histopathological changes.（4）After 48 hours of modeling with MSU,the swelling rate of ankle joints in the colchicine group,the low-dose group with 30%ethanol,and the low and high dose groups with 50%ethanol were significantly reduced;Western Blot results showed that the colchicine group and 30%ethanol part could down-regulate TLR2,TLR4,My D88,NF-κB and NALP3 protein expression in synovial tissue.At the same time,the synovial pathological section showed that the 30%ethanol part could better improve the pathological changes of the synovial tissue in rats.（5）Network pharmacological results show:Seven effective compounds were screened from L.brachystachys Maxim,involving 5027 targets.From the database,4212 disease-related targets were searched,and 253 compound and disease-related targets were obtained by intersection.253 common targets exert their"simultaneous treatment"effect mainly by acting on 44 signal pathways,including adipocyte factor signal pathway,Toll-like receptor signal pathway,NOD-like receptor signal pathway,JAK-STAT signal transmission pathway,FcεRI signal pathway,PPAR signal pathway,T cell receptor signal pathway,etc.Conclusion:L.brachystachys Maxim has a certain protective effect on chronic alcoholic liver injury and acute gouty arthritis.and its treatment for both diseases is related to TLR/My D88/NF-κB and NALP3 signaling pathways.Among them,30%and 50%ethanol parts are the effective parts against chronic alcoholic liver injury,and 30%ethanol parts are the effective parts against gouty arthritis.At the same time,network pharmacological results also indicate that the drug effect of compounds in L.brachystachys Maxim may be related to Toll-like receptor signaling pathway and NOD-like receptor signaling pathway.