Structure-activity Relationship Of Sesquiterpene Lactones As Hepatoprotective Agents Through LPS-TLR4 Signaling Pathway

Objective:Yun Muxiang（Saussurea lappa C.B.Clarke）and Zang Muxiang（Inula racemosa Hook.F）are a perennial herb of Compositae and their dried roots are always regarded as the medicinal parts.It exerts the anti-inflammatory effects and has been widely studied and applicated in clinic.Numerous evidences suggest that the anti-inflammatory mechanism of the major active ingredients（sesquiterpene lactones）involves in the following aspects:（1）The relevant proteins and genes of NF-κB,MAPKs,TRIF or STAT signaling pathways were absolutely inhibited;（2）The expressions of the inflammatory factors,such as TNF-α,PGE2,NO,IL-1,IL-6 and IL-8,were reduced as well as the suppression of the generation of cytokines.In this study,the activated ingredients were isolated and purified from Yun Muxiang and Zang Muxiang.Here,the hepatoprotecitve activity and structure-activity relationship of five sesquiterpene lactones including micheliolide（MCL）,dehydrocostus lactone（DL）,costunolide（CTL）,alantolactone（ATL）and isoalantolactone（IAL）were investigated.Methods and results:1.The roots of Yun Muxiang and Zang Muxiang were crushed,extracted with95%ethanol（solid-liquid rate=1:4）and refluxed at 60°C for 2h,repeated this procedure for 3 times.The extract liquid was collected and condensed at vacuum,and next up,the extractum was suspended with water and separately extracted with petroleum ether,ethyl acetate and n-butanol to obtain different active fractions.The fraction of petroleum ether extract was further purified by silica gel column and get monomers（DL,CTL,ATL and IAL）.These structures were identified by ¹H-NMR.The purities of DL,CTL,ATL and IAL were determined by HPLC（>95%purity）.MCL（>98%purity）was purchased from Chengdu Ruifens co.,LTD.2.The in vitro cell experiment was used RAW264.7 macrophages stimulated by lipopolysaccharide（LPS）to establish the classical inflammatory cell model.The phagocytic ability was detected by neutral red staining.The ROS level was measured by flow cytometry.The levels of pro-inflammatory cytokines including NO,IL-1βand TNF-αin the cell supernatant were analysized through enzyme linked immunosorbent assay（ELISA）,and the expressions of key proteins in the LPS-TLR4 signaling pathway were evaluated by western blot.TLR4 inhibitor was used to observe the expressions of TLR4 and its downstream proteins.The results found that five sesquiterpene lactones increased the phagocytosis of RAW264.7 macrophages,decreased the generation of ROS,NO,IL-1βand TNF-α,and inhibited the expression of key proteins（TLR4,p65,c-fos,c-jun,p-IRF3）in the LPS-TLR4 signaling pathway.Additionally,when cells were pretreated with TLR4 inhibitor,sesquiterpene lactones reducing the expression of TLR4 and its downstream proteins exhibited stronger.Thus,five sesquiterpene lactones blocked the interaction between TLR4 and LPS.3.The in vivo mice model with fulminant hepatic failure induced by LPS/D-Gal N was established sucessfully.The effects of five sesquiterpene lactones on regular biological indicators in mice serum and liver were respectively detected by Chemical kits and ELISA.The expression of proteins in LPS-mediated TLR4 signaling pathway was tested by western blot.The results indicated that five sesquiterpene lactones inhibited the levels of serum aspartate aminotransferase,alanine aminotransferase,nitric oxide and prostaglandin,suppressed the activities of nitric oxide synthase and cyclooxygenase,and improved the expression of catalase,superoxide dismutase and glutathione.Furthermore,five sesquiterpene lactones down-regulated the expression of TLR4,My D88,NF-κB,MAPKs and p-IRF3.4.By the establishment of structure-activity relationship,the antioxidative,anti-inflammatory and hepatoprotective activities of five sesquiterpene lactones were compared and found the results as follows:MCL<DL was relevant to the presence of C₄-C₁₅ and C₁₀-C₁₄ double bonds in DL,and C₄-OH in MCL.MCL,DL<CTL was probably due to the structure difference between the open ring[10+5]in CTL and the closed ring[5+7+5]in DL.IAL≈ATL indicated that there was no significance at double bond between an outer ring（C₅-C₆）in ATL and an inner ring(C₄-C₁₅)in IAL.MCL,DL,CTL<ATL and IAL meant that the anti-oxidative activities of the compounds with the structures of[5+7+5],[10+5]were less than that of the structure of[6+6+5].Therefore,the antioxidative,anti-inflammatory and hepatoprotective activities of the five sesquiterpene lactones were MCL<DL<CTL<ATL≈IAL.In summary,the extraction,separation and purification of sesquiterpene lactones were performed firstly,and then the purity and structures were determined and confirmed later.The purities of four sesquiterpene lactones including DL,CTL,IAL and ATL were greater than 95%.MCL was purchased from Chengdu Ruifens co.,LTD.The in vitro and in vivo results indicated that five sesquiterpene lactones alleviated LPS-induced inflammation and exerted the inhibitory effect on fulminant hepatic failure induced by LPS/D-Gal N in mice.Five sesquiterpene lactones reduced the levels of inflammation factors and the generation of ROS,improved antioxidative ability of the organism,alleviated the aggregation and infiltration of inflammatory factor in liver,as well as down-regulated LPS-TLR4-mediated signaling pathway.Structure-activity relationship showed that there was a close relation between the structures and activities of five sesquiterpene lactones,which provided an orientation for the further chemical modification of sesquiterpene lactones.