Exploring Mechanism of Regulating Mitochondrial Autophagy and Inhibiting Hepatic Stellate Cell Activation by Ruangan Huaxian Granules(柔肝化纤颗粒)Based on miR-135a/FOXO1/PINK1 Pathway
Objective Based on miR-135a/FOXO1/PINK1 pathway,the effect and mechanism of Ruangan Huaxian Granules(柔 肝 化纤颗粒)on inhibiting activation and proliferation of hepatic stellate cells by regulating mitochondrial autophagy.Meth-ods HSC-T6 was divided into blank group,H2O2 group,miR-135a-NC group,miR-135a-mimic group,miR-135a inhibi-tor NC+H2O2 group,miR-135a-inhibitor+H2O2 group,drug-containing serum(Ruangan Huaxian Granules)control group,H2O2+drug-containing serum control group,H2O2+drug containing serum group,H2O2+drug-containing serum+miR-135a-NC group and H2O2+drug-containing serum+miR-135a-mimic group.The expressions of MiR-135a,recombinant forkhead box protein O1(FOXO1),PTEN induced putative kinase 1(PINK1),Parkin,human microtubule-associated protein light chain 3 Ⅱ(LC3 Ⅱ),Smad2,p-Smad2 and transforming growth factor beta 1(TGF-β1),nuclear factor kappa-B(NF-KB)p65,p-NF-κB p65,α-smooth muscle actin(α-SMA),type Ⅰ collagen,type Ⅲ collagen and tumor necrosis factor-α(TNF-α),mitochondrial membrane potential and reactive oxygen species(ROS)production were detected by Real time PCR,Western Blot,ELISA and flow cytometry,respectively.Results Administration of H2O2 and over-expression of miR-135a sig-nificantly up-regulated the expressions of HSC-T6 miR-135a,α-SMA,type Ⅰ collagen,type Ⅲ collagen,p-Smad2,TGF-β1,p-NF-κB p65,TNF-α and ROS production(P＜0.01),down-regulate the expressions of FOXO1,PINK1,Parkin and LC3 Ⅱ and mitochondrial membrane potential(P＜0.01).Administration of Ruogan Huaxian Granules and inhibition of miR-135a expression can significantly down-regulate the expressions of HSC-T6 miR-135a,α-SMA,type 1 collagen,type Ⅲcollagen,p-Smad2,TGF-β1,p-NF κB p65 and TNF-α and ROS production(P＜0.01),up-regulate the expressions of FOXO1,PINK1,Parkin and LC3 Ⅱ and mitochondrial membrane potential(P＜0.01).Simultaneous over-expression of miR-135a with Ruogan Huaxian Granules can inhibit the effect of Ruogan Huaxian Granules on HSC-T6(P＜0.01).Conclusions-Mitochondrial autophagy can inhibit HSC-T6 activation,and its mechanism is related to the inhibition of ROS production by mi-tochondrial autophagy,which in turn inhibits TGF-β1/Smad2 pathway and inflammatory response.Rougan Huaxian Granules can also inhibit the activation of HSC-T6,and its mechanism is related to the inhibition of miR-135a expression by Ruogan Huaxian Granules,activation of the FOXO1/PINK1 pathway and promotion of mitochondrial autophagy.
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