Research On The Inhibitory Effect And Mechanism Of Isovitexin On HSH-T6 Cells Based On PI3k/Akt And TLR4/NF-κB Signaling Pathways

Objective:The inhibitory effect of Isovitexin,an active component of coriander monomer,on HSC-T6 was studied by targeting on PI3K/Akt and TLR4/NF-κB signaling pathways.Methods:HSC-T6 cells were divided into six groups including normal control group,model group,positive control group(LY294002),high-,medium-,low-dosages of isovitexin treated group.HSC-T6 cells were pre-treated with PDGF-BB and then incubated with corresponding drugs for 24 h.1.The effect of Isovitexin on proliferation in HSC-T6 cellsThe toxicity of Isovitexin on HSC-T6 cells was detected by cck-8 method,and then the effect of Isovitexin on the proliferation and activation of HSC-T6 cells was detected.In addition,the effects of Isovitexin on ALT and AST release were detected with the kits.2.The effect of Isovitexin on flammation in HSC-T6 cellsTo evaluate the effect of Isovitexin in the inflammatory response,the production of inflammatory cytokines IL-1β and TNF-α in HSC-T6 cells were detectedby ELISA.3.The effect of Isovitexin on apoptosis in HSC-T6 cellsThe effect of Isovitexin on the apoptosis morphology of HSC-T6 cells was observed by AO/EB.The effect of Isovitexin on the apoptosis rate of HSC-T6 cells was detected by flow cytometry.In addition,the effect of Isovitexin on HSC-T6 apoptosis-related proteins such as Bax,Bcl-2,Caspase-3,Caspase-8and Caspase-9 were detected by Western blot.4.The effect of Isovitexin on collagen deposition in HSC-T6 cellsThe protein expression of α-SMA、Collagen-I、Collagen-III were detected by Western blot and the protein expression metal matrix enzymes such as MMP-2 and TIMP-1were also detected.5.The effect of Isovitexin on the PI3K/Akt signaling pathwayTo evaluate the effect of Isovitexin on the PI3K/Akt signaling pathway,the expressions of PI3K/Akt pathway related proteins Akt,PI3 K,m-TOR and P70S6 K and their phosphorylation were detected by Western blot.The m RNA expressions of Akt,PI3 K were analyzed by RT-q PCR.6.The effect of Isovitexin on the TLR4/ NF-κB signaling pathwayTo investigate the potential protective effect of Isovitexin on hsc-t6 cells,the expressions of TLR4/NF-κB pathway related proteins p-NF-κBp65,NF-κBp65,TLR4 and My D88 were detected by Western blot and the m RNA expressions of NF-κB p65 was analyzed by RT-q PCR.Results:1.Isovitexin effectively inhibited the proliferationThe result of CCK-8 showed that the IC50 of Isovitexin on HSC-T6 cells was 180.65 μmol/L.The result of cell proliferation experiment showed that PDGF-BB stimulation obviously promoted the proliferation of HSC-T6 cells(P<0.05).The proliferation of HSC-T6 cells was inhibited after intervened by Isovitexin and the inhibitory effect increased with the increase of drug concentration;the results of ALT and AST showed that a large amount of ALT and AST were released in the model group and Isovitexin treatment could significantly inhibit the release of ALT and AST.2.Isovitexin inhibited the inflammation of HSC-T6 cellsELISA results showed that PDGF-BB stimulation could promote the release of IL-1β and TNF-α in HSC-T6 cells while Isovitexin effectively inhibited the release of these inflammatory cytokines.It was suggested that Isovitexin reduced the production of inflammatory cytokines,thus inhibiting the further occurrence of inflammatory response.3.Isovitexin obviously promoted the apoptosis of HSC-T6 cellsAO/EB results showed that PDGF-BB stimulation had no significant effect on the apoptosis of HSC-T6 cells.The apoptosis of activated HSC-T6 cells were obviously promoted in Isovitexin treatment groups and with the increase of the concentration of administration,the effect of promoting apoptosis is also significantly enhanced(P<0.05);flow cytometry detection of apoptosis rate also showed the similar results;western blot experiments showed that Isovitexin can significantly increase the expression level of BAX and inhibit the expression of Bcl-2(P<0.05).These results suggested that Isovitexin may play a role in preventing liver fibrosis by regulating the expression of apoptosis-related proteins,inhibiting the activation of HSC-T6 cells and promoting apoptosis.4.Isovitexin inhibited the formation and deposition of collagenWestern blot showed that the expressions of α-SMA,Collagen-I and Collagen-III were increased significantly in PDGF-BB group(P<0.05),while the expressions of α-SMA,Collagen-I,and Collagen-Ⅲ were decreased significantly in the Isovitexin-treated groups.In addition,the results of Western blot showed that the protein level of TIMP-1 was significantly increased in the PDGF-BB group(P<0.05),but the expression of TIMP-1 was significantly inhibited and the expression of MMP-2 was increased after Isovitexin treatment(P<0.05).It was suggested that Isovitexin inhibited the production of collagen in HSC-T6 cells and reduced excessive deposition of extracellular matrix,which was the pharmacological basis for the treatment of liver fibrosis.5.Isovitexin significantly inhibited the expressions of the PI3K/Akt signaling pathwayThe results of western blot showed that compared with the normal control group,the phosphorylated expressions of Akt,PI3 K,m-TOR and P70S6 K in the PDGF-BB group were significantly increased(P<0.05),and after the intervention of Isovitexin,the expressions of these proteins were significantly decreased(P<0.05).In addition,RT-q PCR results also showed that Akt and PI3 K m RNA were significantly increased in the PDGF-BB group,and Isovitexin intervention can significantly inhibit the expression of these genes(P<0.05).It was suggested that the role of Isovitexin in inhibiting the activation of HSC-T6 cells may be related to the inhibition of the PI3K/Akt signaling pathway.6.Isovitexin significantly inhibited the expressions of the TLR4/NF-κB signaling pathwayThe results of western blot showed that compared with the normal control group,the phosphorylation levels of NF-κBp65 and the expression levels of TLR4 and My D88 in the PDGF-BB group were significantly increased(P<0.05),while the expression levels of these proteins were significantly down-regulated after Isovitexin intervention(P<0.05).In addition,RT-q PCR results also showed that NF-κBp65 m RNA was significantly increased in the PDGF-BB group,and Isovitexin intervention can significantly inhibit its gene expression(P<0.05).It was suggested that the role of Isovitexin in inhibiting HSC-T6 cell activation may be related to the inhibition of the TLR4/NF-κB signaling pathway.Conclusion:Isovitexin can significantly inhibit the proliferation and activation of HSC-T6 cells,induce apoptosis,inhibit collagen production and excessive deposition of extracellular matrix,and its potential mechanism may be related to the regulation of apoptosis-related proteins and the inhibition of PI3K/Akt and TLR4/NF-κB signaling pathways.