Study On The Mechanism Of Abstract Of Periplaneta Americana On Anti-hepatic Fibrosis In Vitro

Objective:To study the effcets of Abstract of Periplaneta Americana (APA) on the rat hepatic stellate cells (HSC-T6) proliferation, apoptosis, cell cycle and expression of a-smooth muscle actin (a-SMA), I and III type collagen in vitro. To explore the mechanism of Anti-hepatic fibrosis with extracellular signal-regulated kinase1/2(ERK1/2) signaling pathway of HSC cells controll by APA, providing a experimental evidence for further treatment of liver fibrosis.Methods:1Rat HSC line (HSC-T6) and rat hepatocyte line (BRL-3A) were incubated with different concentrations (0.25mg/mL,0.5mg/mL,lmg/mL,2mg/mL,4mg/mL,6mg/mL,8mg/mL,]0mg/mL.16mg/mL) of APA and or without APA for24h,48h.72h, Cells proliferation was examined by MTT. And then to "determine the optimum concentration of APA for inhibiting proliferation of rat HSC.2Cell apoptosis rate and cell cycle of HSC-T6were analyzed using Annexin V-FITC/PI double-1abled kit and propduim kit by flow cytometry, and determination of expression of a-SMA,Ⅰ and Ⅲ type collagen by cellular immune fluorescent chemical method.3The cultured HSC-T6were divided into four experimental groups:Control group. PDGF stimulation group, PDGF+APA group, PDGF+PD98059group, after24hours, the ERK1/2, p-ERK1/2expression levels were detected by western blot.Results:1MTT assay indicated:different concentrations of APA intervention HSC-T6cell, proliferation in the experimental group were lower than the control group, the difference was statistically significant (P<0.01). and with the increase of drug concentrations and the extension of time, cell proliferation inhibition rate gradually increased in a dose and time dependent. IC50of APA in HSC-T6cells24h is8.086mg/ml,48h is3.749mg/ml and72h is2.410mg/ml. For the BRL-3A, Low concentrations of APA without toxic effects, when the concentration of APA is greater than the4mg/mL, it can inhibit the proliferation of BRL-3A cells, compared with the control group, the difference was statistically significant (P<0.01), and it showed that with the increase of drug concentration, the toxic effects of APA on BRL-3A cells increased. When the concentration of APA is greater than the4mg/mL, for BRL-3A cells, the toxicity is increased, the optimum concentration of APA for inhibiting proliferation of rat HSC is4mg/mL2The flow results indicated that different concentrations of APA (concentration of2mg/mL,4mg/mL,6mg/mL) intervention HSC-T6cells for24hours can promote apoptosis of HSC-T6cells, apoptosis rate was dose dependence, compared with the control group, the difference was statistically significant (P<0.05). Flow also indicated: With the increase of drug concentration, the cell percentage of G0/G1was increased and cell percentage of S was decreased. While the G2/M phase cells had no effect.3Cellular immune fluorescent chemical methods indicated:HSC-T6cells were incubated without APA, the expression of α-SMA, and Ⅰ, Ⅲ collagen was strongly positive. With a concentration of4mg/mL of the APA intervention for24hours, the expression of α-SMA, and Ⅰ, Ⅲ type collagen was decreased.4The results of western blotting assay indicated:PDGF increased obviously p-ERK1/2activity, and the expression of p-ERK1/2decreased relatively by APA and PD98059, between the two groups, APA group was significantly lower than PD98059group.Conclusion:1The Abstract of Periplaneta Americana can significantly inhibit the proliferation of HSC-T6in a dose-dependent and time-dependent manner;4mg/mL is the optimum concentration of Abstract of Periplaneta Americana for inhibiting.2The Abstract of Periplaneta Americana can promoted apoptosis of hepatic stellate cells, and the cell cycle from G1to S was arrest.3The Abstract of Periplaneta Americana can inhibit the expression of a-SMA, and can inhibit the secretion of type Ⅰ, Ⅲ collagen.4The Abstract of Periplaneta Americana could inhibit the proliferation and the expression of p-ERK1/2of HSC-T6induced by PDGF, The mechanism of Anti-hepatic fibrosis was related to the ERK1/2pathway.