Research On Liver Toxicity And Its Mechanism Induced By Cigarette Smoke In Rats

Objective: The model of passive smoking in rats with different exposure time and different exposure dose was constructed to evaluate the hepatotoxicity of cigarette smoke on rats and further explore its mechanism.Methods: 120 SD rats were randomly divided into 3 exposure time groups at 4,8 and 12 weeks according to the preliminary experimental results,with 40 animals in each group.Each time exposure group was divided into blank control group(0 cigarettes/day group),low dose group(10cigarettes/day group),medium dose group(20 cigarettes/day group)and high dose group(30 cigarettes/day group),10 animals in each dose group.Each dose group was poisoned once a day for 30 minutes,continuously for 4,8 and 12 weeks.When the poisoning is over,the changes of body weight,liver weight,liver viscera coefficient and liver histopathological structure were observed.The changes of liver tissue and serum transaminase and lipid metabolism of rats in each group were measured.The changes of MDA content,GSH-Px activity and SOD activity in rat liver were measured.mRNA expression levels of Bax,Bcl-2 and Caspase3 genes in mitochondrial mediated hepatocyte apoptosis were detected by RT-PCR.The expression levels of Bax,Bcl-2 and Cleaved-Caspase3 in the mitochondrial mediated hepatocyte apoptosis pathway were determined by Western-Blot and immunohistochemistry.Results:(1)Cigarette smoke caused the rats in the exposed group to have poor spirits,decreased voluntary activities,restlessness and other phenomena,and some of the rats had unsteady steps.The body weight of rats in the exposed group increased slowly compared with that of the blank control group,and the body weight of rats in the high-dose group was lower than that of the blank control group in each cycle,with statistically significant difference(All P<0.05).The liver weight of the rats exposed to different periods decreased compared with that of the blank control group,and the change of the rats exposed for 8 weeks wassignificant,with statistically significant difference(P < 0.05).(2)Pathological findings showed that at the initial stage of exposure,liver tissues in the exposed group showed focus-like necrosis,hepatic sinus and venous congestion,and expansion of the portal area.With the extension of exposure time,liver cells in the liver tissues were swollen and the cytoplasm was loosened.(3)Liver tissue exposed group AST and ALT transaminase level rise,the expression level of AST compared with blank control group increased obviously,the cycle of high dose group of ALT levels compared with blank control group increased obviously,the difference was statistically significant(All P<0.05).The serum TBIL of rats in each exposed group was slightly increased,but the difference was not statistically significant compared with the blank control group(All P > 0.05).Compared with the blank control group,serum TC in the exposed group showed an increasing trend compared with TG in the blank control group.There was a statistically significant difference in serum TC between the low-dose group at 8 weeks and the medium-dose group at 12 weeks(P<0.05),but no significant increase in serum TC in other groups(P>0.05).In addition to the low-dose group at 12 weeks,serum TG levels in other exposed groups showed statistically significant differences compared with the blank control group(All P < 0.05).(4)MDA content in liver tissues of rats in each exposed group was significantly higher than that of the blank control group(All P<0.05).SOD activity level decreased significantly compared with the blank control group(All P < 0.05).The activity level of GSH-Px decreased compared with that of the blank control group,which decreased significantly in the 8-week and 12-week groups(P < 0.05).(5)Bax gene showed an up-regulated trend in mRNA level,which was significantly up-regulated in the high-dose group at 4 weeks,8 weeks and 12 weeks compared with the blank control group,with statistically significant difference(All P<0.001).The mRNA level of Bcl-2gene showed a downward trend,which was significant in each exposed group compared with the blank control group(All P<0.05).The mRNA level of Cleaved-Caspase3 gene showed a tendency of up-regulation,which was significant in each exposed group compared with the blank control group(All P < 0.05).(6)Western-Blot and immunohistochemical analysis showed that Bax protein expression levels in the exposed group were significantly up-regulated,including those in the high-dose group of each cycle,with statistically significant differences(All P < 0.05).The expression level of Bcl-2 protein in the exposed group showed a downward trend,among which the 4-week histone protein was significantly down-regulated,and the difference was statistically significant(P<0.05).The protein expression level of Cleaved-Caspase3 in the exposedgroup was significantly up-regulated compared with that in the blank control group,and the difference was statistically significant(All P < 0.05).Conclusion:(1)Cigarette smoke exposure can inhibit the growth and development of rats,and cause inflammatory reactions in liver tissues.The bioenzyme activity and lipid metabolism in liver tissues change with the change of exposure period and dose,suggesting that cigarette smoke exposure can produce a certain degree of hepatotoxicity.(2)The accumulation of a lot of cigarette smoke,make the body produce oxidative stress of rats,eventually oxidation and anti-oxidation system imbalance,resulting in the phenomenon of apoptosis and Bax and Cleaved-Caspase3 in mRNA level and quantity of protein expression levels rise,the Bcl-2in the mRNA level and quantity of protein expression levels,cigarette smoke exposure is caused liver mitochondria mediated apoptosis phenomenon.Therefore,it can be concluded that rat liver may be one of the target organs of cigarette smoke toxicity,cigarette smoke exposure has hepatotoxicity in rats,and mitochondria mediated hepatocyte apoptosis may be one of the mechanisms of liver cell apoptosis in rats.