The Effect Of Tetramethylpyrazine On Hepatic Stellate Cells Activation In The Insulin Resistance State

Objective:Hepatic fibrosis is a common liver disease and effective drugs for this disease are still lacking in the clinic. Activation of hepatic stellate cells (HSCs) is the core event in the pathogenesis of hepatic fibrosis. Inhibition of HSC activation and blockade of the downstream signal transduction are rational approaches for developing antifibrotics. Metabolism syndrome especially insulin resistance may promote liver fibrogenesis and make it hard to reverse the pathogenesis. We previously have found that the natural product tetramethylpyrazine.(TMP) had antifibrotic activity. The present study was aimed at investigating the effects of TMP on HSC activation under the circumstance of insulin resistance, and elucidating the underling molecular mechanisms. To this end, We first established the insulin resistance model of HSC,and assessed glucose and insulin on HSC proliferation and fibrogenicbehaviors.Then we observed the effects of Tetramethylpyrazine on the changes in expression of extracellular matrix components.Then we focused on the insulin signaling mediated by insulin receptor (InsR) involving PI3K/AKT and MAPK pathways. These investigations will provide novel insights into TMP inhibition of HSC activation via modulation of insulin signal transduction, and experimental basis for developing TMP as a promising antifibrotic targeting HSC.Methods:This in vitro study mainly used HSCs cultured with high glucose and insulinand employed MTT, Western blot and Real-time PCR assays to determine:1) TMP effects on HSC proliferation, morphology and fibrogenicbehaviors, as well as expression of a-SMA, CTGF, fibronectin and pro-collAl; 2) TMP effects on protein and mRNA expression of ECM components including procollagen, fibronectin, a-SAM, MMP-2,-9 and TIMP-1; 3) TMP effects on expression of InsR and key effectors in PI3K/AKT and MAPK signaling pathways.Results:1) The glucose and insulin can promote the proliferation of the HSC with the concentration of 15mM、0.001 mU/ml (P<0.01); high glucose and insulin also changed HSC morphology, and meanwhile promoted HSC migration and adhesion(P<0.05). Key effectors implicated in HSC activation including a-SMA, CTGF, fibronectin and pro-collA1 were also upregualted under insulin resistance.2) Tetramethylpyrazine can inhibit the proliferation of HSC obviously,with the increased of drag concentration, the potential efficacy on inhibition of HSC-T6 proliferation shows an dose-dependent manner; Western blotting and Real-time PCR results showed that tetramethylpyrazine can dose-dependent inhibit a-SMA, CTGF, fibronectin and Pro-collA1 protein and mRNA expression of HSC, while tetramethylpyrazine also can promte MMP-2 protein and mRNA expression, while reducing MMP-9 and TIMP-1 expression;3) Tetramethylpyrazine can reduce the protein expression of InsR in a dose-dependent manner, and the effect reach strongest when the concentration was 30μM; tetramethylpyrazine can significantly inhibit the phosphorylation of PI3K, Akt, ERK, inhibit protein expression of P38 weakly, while the JNK protein expression has no significant difference.Conclusion:We found that high glucose and insulin played a critical role in HSC activation, and significantly induced HSC proliferation and changed HSC morphology, and meanwhile promoted HSC migration and adhesion. Key effectors implicated in HSC activation including a-SMA, CTGF, fibronectin and pro-collA1 were also upregualted under insulin resistance. Tetramethylpyrazine could inhibit HSC activation and reduce expression of a-SMA, CTGF, fibronectin and pro-collA1, which might be attributed to upregulation of MMP-2 and downregulation of MMP-9/TIMP-1. Furthermore, tetramethylpyrazine could suppress InsR expression and significantly reduce expression of key effectors in the downstream PI3K/AKT and MAPK pathways.