Study On The Effect Of Abstract Of Periplaneta Americana On Apoptosis And PI3K/AKT/p70^s6K Signal Pathway Of Rat Hepatic Stellate Cells

Object:The study focusing on the important cells of hepatic fibrosis-hepatic stellate cells (HSC), studied apoptosis and apoptosis related proteins Bcl-2, Bax, Caspase-3 mRNA expression after the Abstract of Periplaneta americana (APA) disposed rat hepatic stellate cells in vitro (HSC-T6) and investigate whether the mechanism of APA anti-fibrosis related to PI3K/Akt/p70S6K signaling pathway, providing a theoretical basis for the Abstract of Periplaneta americana treatment to liver fibrosis.Methods:1. Rat HSC strain (HSC-T6) were incubated with different concentrations (2mg/mL,4mg/mL,6 mg/mL) of APA for 24h and 48h, and setting blank control group and LY294002 group, HSC apoptosis rate was assayed by flow cytometry;2. The cultivation technique of HSC-T6 cells in vitro was applied and HSC-T6 cells were disposed by APA (2,4,6mg/ml) for 24h, setting blank control group and LY294002 group, Real-time quantitative nucleic acid amplification detection system (RT-real time Q-PCR) testing the mRNA expression levels of PI3K, Akt, p70s6k, a-SMA, Bcl-2, Bax, Caspase-3 of HSC-T6 cells.Then Application HSC-T6 cells cultured in vitro and setting blank control group and LY294002 group, different concentrations APA (2,4,6mg/ml) intervention HSC-T6 cells for 24h, Real-time quantitative nucleic acid amplification detection system assayed the mRNA expression level of Ⅰ、 Ⅲ collagen in HSC-T6 cells.3. Application techniques of HSC-T6 cells cultured in vitro, setting blank control group、LY294002 group、LY294002+APA4mg/mL group and different concentrations of APA(2,4,6mg/ml) intervention HSC-T6 cells for 24h, Western blotting assayed the expression levels of phosphorylated AKT and p70S6K protein of activated HSC-T6 cells.Results:1. The flow results indicated that:different concentrations (2 mg/mL,4 mg/mL,6 mg /mL) of APA intervention HSC-T6 cells 24 or 48 hours, it could promote apoptosis of HSC-T6 cells,when the concentration was greater than or equal to 4mg/mL could obviously promote apoptosis of HSC-T6 cells.2.RT-QPCR results indicated that:different concentrations of APA intervention cultured HSC-T6 cell for 24h and RT-QPCR assyed the mRNA expression of PI3K, Akt, P70S6K and a-SMA, Bcl-2, Bax, Caspase-3, type I collagen and type III collagen. The results showed that the concentration 4mg/mL and 6mg/mL of APA reduced the expression of signal transduction molecule PI3K mRNA; inhibition the upstream of the pathway, concentration of 6mg/mL reduced the mRNA expression of Akt, weakening the activity of downstream pathway; concentration of 6mg/mL down-regulation of the P70S6K gene mRNA expression and inhibited the downstream target phosphorylation level; three concentrations (2,4,6mg/mL) reduced the mRNA gene activation of a-SMA; weakening the ability to muscle fibrosis cells with a concentration dependent manner; the concentrations of 4mg/mL and 6mg/mL reduced the mRNA expression of Bcl-2; three concentrations (2,4,6mg/mL) up-regulated the mRNA expression of apoptosis gene Bax and Caspase-3, there was a dose-response relationship to apoptosis. Three concentrations (2,4,6mg/ml) of APA were cut the mRNA expression of type Ⅰ, Ⅲ collagen and reduced the quantity of ECM, delayed the progression of liver fibrosis. (P< 0.05), of which PI3K, Bcl-2, P70S6K, caspase-3 (P< 0.01).3. Western blotting results showed that:after different intervention of HSC-T6 for 24 hours, compared with the blank control group, except 2mg/mL and 4mg/mL of APA group,other experimental group the phosphorylated expression of Akt and P70S6K were significantly decreased, and there is a dose-response relationship. High concentration of APA (6mg/mL) group has the most obvious effect.Between the 2mg/mL and APA4 mg/mL of APA group the phosphorylation expression of Akt and P70S6K higher compared with LY294002 group;APA (6mg/ml) p-p70s6k expression was reduced compared with LY294002; APA (6mg/ml) p-Akt expression was higher compared with LY294002. The expression of p-AKT and p-p70S6K in the LY294002+APA4mg/mL group was significantly decreased compared with APA4mg/mL group.Conclusion:1. The Abstract of Periplaneta Americana (APA) can promote HSC apoptosis, the mechanism is down-regulated Bcl-2/Bax ratio and increased the expression of Caspase-3 with a concentration-dependent manner.2. The Abstract of Periplaneta Americana can inhibit the expression of α-SMA、 collagen Ⅰ and Ⅲ, thereby reducing extracellular matrix formation when hepatic fibrosis, which was in a concentration-dependent manner.3.The Abstract of Periplaneta Americana anti hepatic fibrosis by blocking PI3K/Akt/ P70S6k transduction pathways, down-regulating the gene expression of PI3K, Akt, p70s6k and the protein synthesis of phosphorylated Akt、P70S6K.