| Objective: To investigate the protective effects of the water-soluble extract(AERW)and petroleum ether extract(AERP)of Xinjiang soft comfrey(Arnebia euchroma(Royle)Johnst,AER)on LPS/D-Gal N-induced acute liver injury(ALI)in mice using an in vivo pharmacodynamic assay.The monomeric compounds were screened for in vitro antiinflammatory effects and in vivo protection against inflammation-induced liver injury,providing a theoretical basis for the anti-inflammatory effects and regulation of inflammatory liver injury by the active ingredients of Comfrey.Methods: The protective effects of AERW and AERP on liver injury in mice were initially investigated through the analysis of serum indexes,NO content in liver tissues and liver histopathological sections,and in order to further investigate the effective anti-inflammatory components in Comfrey AERP and AERW,the MTT colorimetric method was used to investigate the main components of AERP,namely,purpureol(SK),levorin(L-SK),acetyl purpureol(A-SK),β-β-dimethylacryloylshikonin(D-SK)and two phenolic acid compounds,caffeic acid tetramer(CATE)and caffeic acid tetramer isomer(CATI),isolated from the AERW site,were used to investigate their toxic effects on Raw264.7 cells.The effect of L-SK,A-SK,D-SK,CATE and CATI on cellular NO production was investigated by Griess method,and the mechanism of anti-inflammatory effect of L-SK,CATE and CATI was further investigated.The distribution and fluorescence intensity of P-Erk1/2 and P-P65 proteins in the cells were measured by cellular immunofluorescence assay,and the protective effects of L-SK and CATE on LPS/D-Gal N-induced liver injury were further investigated.The survival rate and the changes of liver and spleen index in each group of mice were investigated.The serum indicators and NO,superoxide dismutase(SOD)and malondialdehyde(MDA)in liver tissue homogenates were measured.Results: The results showed that both AERW and AERP groups were able to reduce AST and ALT activities in serum and NO levels in liver tissues to different degrees,and liver lesions were improved to different degrees.The results of in vitro anti-inflammatory experiments showed that,by studying the NO inhibition rate of L-SK,A-SK,D-SK,CATE and CATI,it was found that L-SK,CATE and CATI had significant inhibition effect on NO,and all three could significantly inhibit LPS-induced INOS,COX2,IFN-β and pro-inflammatory factors IL-6 in RAW264.7 cells In addition,L-SK significantly inhibited INOS and COX2 protein expression as well as NF-κB signaling pathway protein expression,while CATE and CATI not only inhibited INOS,COX2 and NF-κB signaling pathway protein expression,but also significantly decreased MAPK pathway protein phosphorylation level,and their effects on P-Erk1/2 and P P-Erk1/2 and P-65 nuclear ectopics were also improved.The in vivo hepatoprotection results showed that both L-SK and CATE significantly improved the hepatic and splenic indices,significantly decreased AST,ALT and AKP levels in serum,significantly decreased NO and MDA levels in liver tissue homogenates,and increased SOD activity,which significantly improved the pathological liver tissue damage and alleviated the inflammatory infiltration of liver tissue in mice.The expression of inflammatory factor proteins and MAPK/NF-κB pathway proteins in liver tissues was significantly inhibited by CATE.Conclusion: Both Comfrey AERW and AERP have good hepatoprotective effects,in which the lipid-soluble component L-SK and the water-soluble compounds CATE and CATI have good anti-inflammatory effects in LPS-induced inflammation in RAW264.7 cells in vitro.L-SK inhibited the protein expression of phosphorylated P65 and IKKα/β in the NF-κB signaling pathway by inhibiting the protein expression of IKKα/β,CATE and CATI exerted antiinflammatory effects in vitro by inhibiting protein expression of MAPK/NF-κB signaling pathway to different degrees,while L-SK and CATE had protective effects against acute liver injury in mice,and their mechanisms of action may be related to the anti-inflammatory effects. |
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