The Protective Effect Of Total C-21 Steroidal Glycosides From Baishouwu On Liver And Kidney Cells Based On TLR4 Signaling Pathway In Vitro

The root of Cynanchum auriculatum Royle ex Wight(Asclepiadaceae),known in a traditional Chinese medicine(TCM)as "baishouwu",has been widely used for protecting liver and kidney and prolonging life since ancient times.As the most important active components of baishouwu,the C-21 steroidal glycosides(TCSG),were proved to posess the effect of preventing and treating liver fibrosis and renal fibrosis in rats,while the mechanism was still largely unknown.Based on the pvevious research,this paper aimed to study the protective effect of TCSG on liver and kidney in vitro,as well as the molecular mechanism based on TLR4 signaling pathway.In the first chapter of this paper,the relationship between TLR4-MyD88-NF-κB signaling pathway with liver and renal fibrosis were reviewed.In the next chapter,three different methods of activation of human hepatic stellate cells(LX-2)or Epithelial-mesenchymal transition(EMT)of human renal tubular epithelial cells(HK-2)in vitro were evaluated.LX-2 or HK-2 cells were randomly divided into four groups:Control group,TGF-β1(10 ng/mL)group,LPS(10 μg/mL)group,and TGF-β1 with LPS group.After 24 hours of stimualtion in LX-2 or HK-2 cells,the expression of α-SMA was tested by immunofluorescence assay,the secretion of COLⅢ in cell supernatant was detected by radioimmunoassay,and the fibrosis related factors(TGF-β1,α-SMA,COLⅠ and COLⅢ)and inflammatory cytokines(TNF-α and IL-6)were detected by qRT-PCR.Western blot was applied to detect the expression levels of Vimentin,TGF-β1,α-SMA,IL-6 and the nucleus ratio of p-NF-κB/NF-κB in LX-2 or HK-2 cells.The results showed that the expressions ofα-SMA,TGF-β1,COLⅠ,COLⅢ,TNF-α and IL-6 of LX-2 or HK-2 cells were up-regulated by TGF-β1 and/or LPS,as well as the ratio of p-NF-κB/NF κB in nucleus(P<0.05～0.01).In addition,Vimentin protein expression in HK-2 cells was also greatly increased by TGF-β1 and/or LPS.The results also suggest that TGF-β1 and/or LPS may play an important role in promoting LX-2 activation or HK-2 transdifferentiation,and the combined stimulation was more significant.In third chapter,we studied the in vitro effect of TCSG on protecting liver and kidney function and the mechanism based on TLR4-MyD88-NF-κB signaling pathway.MTT assay was used to detect the effect of TCSG on cell viability.The protein expression of α-SMA was tested by immunofluorescence,the secretion of COLⅢ in the cell supernatant was detected by Radioimmunoassay,and TLR4-MyD88-NF-κB signaling pathway was studied by Western blot.MTT result indicated that there was no sigmificant effect on the cell viability when the concentration of TCSG was lower than 40 μg/mL and the treating time was less than 48 h.As a result,10,20 and 40 μg/mL of TCSG and 24 h treatment were used in drug intervention studies.Compared with the model group,TCSG greatly reduced the fluorescence intensity of α-SMA in LX-2 or HK-2 cells induced by TGF-β1 and LPS,decreased the secretion of COLⅢ in the cell supernatant,significantly depressed the mRNA expressions of TGF-β1,α-SMA,COLⅠ,COLⅢ,TNF-α and IL-6,and down regulated the protein expressions of α-SMA,TGF-β1,TLR4,MyD88,TRAF6,p-P38,p-JNK,p-ERK,IL-6,p-IκB-α and nuclear ratio of p-NF-κB/NF-κB(P<0.05-0.01).In addition,the expression of Vimentin was significantly decreased by TCSG(P<0.05～0.01)in HK-2 cells.The results showed that TCSG could significantly suppress the activation of LX-2 and the EMT of HK-2 cells in vitro,and its mechanism might be closely related to the regulation of TLR4-MyD88-NF-κB signaling pathway.In the fourth chapter,TLR4 inhibitor or transient transfection technique was used to observe the effect of silencing TLR4 gene on the activation of LX-2 cells and transdifferentiation of HK-2 cells induced by TGF-β1 and LPS in vitro.After treatment with TLR4 inhibitor(TAK242)or transient transfection,the protein and mRNA levels of TLR4 in LX-2 cells or HK-2 cells were significantly decreased,and so did the TGF-β1 and α-SMA expressions.The results showed that TLR4 played an important role in the activation of human hepatic stellate cells or Epithelial-mesenchymal transition(EMT)of human renal tubular epithelial cells in vitrl.The results suggested that the protective effect of TCSG on liver and kidney cells in vitro may be closely related to TLR4 signaling pathway,but there may be other mechanisms involved.