| Alcohol abuse and dependence is becoming a serious public health problem worldwide. Alcoholic liver disease (ALD) is caused by long-term excessive drinking and is serious harmful to human health and life. With the rapid development of economic and increasing of living standards, the incidence of ALD increased too. In some areas, ALD has became the second major cause of liver injury. In-depth study of the pathogenesis of ALD and developing effective medicine are necessary. Primary cultured hepatocytes have an important role in experimental studies. The important foundation of this project is to obtain hepatocytes which are high yield, good dynamic and strong function through a series of improvements. Glutathione S-transferase Al (GSTAl) is a subtype of GSTs family. It is also an important part of the body antioxidant defense system. Catalyzed many xenobiotics with GSH, promote degradation and clear, protect the body. Our team has successful build acute alcoholic liver injury model in mice. On this basis, we have studied change of GSTA1in acute liver injury to provide a evidence for GSTAl as a marker for liver injury. The goal of this research is to establish alcoholic liver injury model in vitro and determine GSTAl change in primary cultured hepatocytes injury induced be ethanol to provide more evidence for GSTA1becoming an biomarker of acute liver injury. In this study, we studied protective effect of solanum nigrum on the basis of primary cultured hepatocytes injury model and acute liver injury model induced by ethanol in mice.This research used male KM mice as experimental animal. Establish an improved Seglen’s two-step in situ perfusion method to isolate mice hepatocytes. According to the determination of cell culture supernatants ALT, AST and cell MDA, SOD, GSH, choose the optimal exposure dose and time to build primary hepatocytes injury model induced by ethanol. Then, study the GSTA1change compared with ALT and AST through dose-effect and time-effect tests on the model and determine GSTA1through ELISA. Evaluate the protective effect of solanum nigrum on primary hepatocytes injury model and acute liver injury model induced by ethanol in mice by the level of ALT, AST, MDA, SOD, GSH, GSH-Px and liver histology analysis.The result shows:1. We successful obtained mice primary hepatocytes through improving the Seglen’s two-step in situ perfusion method. The viability of hepatocytes is more than85%and the hepatocytes number of each mouse is about (1.4×107)～(2.2×107). 2. The primary hepatocytes injury model induced by ethanol in mice can be set up through100mmol·L-1, for8h. The extent of hepatocytes damage is proportional to the ethanol concentration and reaction time. The level of cell culture supernatants ALT and AST has significant increased compare with control group (P<0.01). The level of cell MDA has significant increased compare with control group (P<0.01), SOD, GSH has significant decreased compare with control group (P<0.01).3. The change of cell culture supernatants GSTA1is proportional to the ethanol concentration and reaction time. In dose-effect test:from50mmol·L-1GSTA1has significant increased compare with control group (P<0.01), ALT and AST has significant increased at75mmol·L-1(P<0.05) and has significant increased at100mmol·L-1(P<0.01). In time-effect test:GSTA1has significant increased compare with control group (P<0.01) from2h compared with ALT and AST at6h has significant increased (P<0.05) and8h (P<0.01). It’s showed that GSTA1is more sensitive than ALT and AST.4. Effective of solanum nigrum on primary hepatocytes injury induced by ethanol showed:In the group which dose of Solanum nigrum is100μg·mL-1, ALT, AST, MDA has significant decreased compare with model group (P<0.05), GSTA1is significant decreased (P<0.01), MDA, SOD, GSH has significant increased compare with model group (P<0.05).5. Effective of solanum nigrum on acute hepatic injury induced by ethanol showed:At200mg·kg-1Solanum nigrum group, ALT, AST, GSTA1, MDA have significant decreased compare with model group (P<0.01), SOD, GSH, GSH-Px, have significant increased (P<0.01). At150mg·kg-1Solanum nigrum group, ALT, AST, GSTA1have significant decreased (P<0.01), MDA has significant decreased (P<0.05), SOD, GSH, GSH-Px, have significant increased (P<0.05).My research successfully isolated and cultured mice primary hepatocytes and replicated the primary hepatocytes injury model induced by ethanol. Dose-effect and time-effect tests showed GSTA1was more sensitive than ALT and AST in liver injury. So it could be a marker for liver injury. Solanum nigrum showed protective role in primary hepatocytes injury and acute liver injury in mice. It indicated that Solanum nigrum may have anti-oxidative effect. |
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