Effects And Mechanism Of The Chikusetsusaponin Ⅳa On Protection Against Hepatocytic Inflammation Based On IRE1α-TLR4 Signaling Pathway

Objective In recent years the increasing prevalence of non-alcoholic steatohepatitis(NASH)has caused serious harm to human health in China.There is still a lack of effective intervention strategy for the NASH.Endoplasmic reticulum stress(ERS)sensitizes Toll-like receptor 4(TLR4)signal pathway to increase production of inflammatory cytokines that is the newly discovered key factor for the inflammatory diseases.Thus,the purpose of this study is as follows:(1)To build the inflammatory model by using of palmitic acid and thapsigargin to stimulate LO2 cells.(2)To investigate the molecular mechanism of endoplasmic reticulum stress(ERS)amplifying toll-like receptor 4(TLR4)signaling pathway.(3)To research the effects of the Chikusetsusaponin Ⅳa on protection against hepatocellular inflammation.Methods(1)The inflammatory model through the amplification of ERS signaling molecules to TLR4 signal pathway was built by using of palmitic acid and thapsigargin to stimulate LO2 cells,then different concentrations of TG,PA alone and combined were used to treat with LO2 cells for 24 hours,the activation situation of XBP-1 was detected by RT-PCR,the expression of TLR4 protein was detected by flow cytometry,the expression of IL-6 protein was detected by ELISA kits,According to these results,the optimum concentration of palmitic acid and thapsigargin used to build the inflammatory model through the amplification of ERS signaling molecules to TLR4 signal pathway was determined.(2)The inflammatory model was induced by PA and TG,using of inhibitor,siRNA,Chikusetsusaponin Ⅳa respective intervention.The activation situation of XBP-1was detected by RT-PCR;the expressions of IRE1α and p-IRE1α proteins were detected by Western Blotting;the expression of TLR4 protein was detected by flow cytometry;the nuclear translocation of NF-κB protein was detected by immunofluorescence;the expression of IL-6 protein was detected by ELISA kits;the possible targets of Chikusetsusaponin Ⅳa were predicted based on computer aided drug design.Results(1)Compared with the control group,the expression levels of p-IRE1α,TLR4 and IL-6 proteins in model group were significantly increased(p<0.05),the NF-κB protein shifted into nucleus in model group;The inflammatory model through the amplification of ERS signaling molecules to TLR4 signal pathway was successfully established by stimulating LO2 cells for 24 hours with the 25 μM concentration of palmitic acid and the 1 n M concentration of thapsigargin.(2)Compared with the model group,the expression levels of p-IRE1α,TLR4 and IL-6 proteins were significantly decreased(p<0.05)in Chikusetsusaponin Ⅳ a treatment group,Chikusetsusaponin Ⅳ a could prevent NF-κB protein shifted into nucleus.(3)The result based on computer aided drug design showed that Chikusetsusaponin Ⅳa could bind to IRE1α protein.(4)Compared with the model group,the expression levels of p-IRE1α,TLR4 and IL-6 proteins were significantly decreased(p<0.05)in siRNA or 4μ8C treatment group,siRNA or 4μ8C could prevent NF-κB protein shifted into nucleus.(5)Compared with the model group,the expression levels of TRAF2,IL-1,IL-6 and TNFα genes were significantly decreased(p<0.05)in 4μ8C treatment group,but the expression levels of IKK gene changed inconspicuously.Conclusion(1)The ERS signaling molecules can increase the release of inflammatory factors by amplifying the TLR4 signaling pathway.(2)ChikusetsusaponinⅣa has a good effect on improving NASH through regulation of ERS signaling molecules which amplifies TLR4 inflammatory signaling.