The Protective Effect And Mechanism Of N-acetyl-L-cysteine On Lung Injury Induced By Cigarette Smoking In Rats

Objectives:To observe whether cigarette smoking can cause oxidative stress and apoptosis of lung tissue in rats,moreover,to explore possible protective effect of N-acetyl-L-cysteine as well as related molecular mechanism.Methods:1.Thirty-two healthy male Wistar rats of SPF grade were randomly divided into four groups:control group,N-acetyl-L-cysteine group（NAC group）,cigarette smoking exposure group（CS group）,cigarette smoking exposure plus N-acetyl-L-cysteine group（CS+NAC group）.The rats of CS group and CS+NAC group were exposed to cigarette smoking for four weeks in a way of ten cigarettes every time,twice a day,five days a week.The rats of NAC group and CS+NAC group were administered intragastrically with NAC at a dose of 150mg·kg^-1·d^-1.After four weeks,all rats were anesthetized with 1%pentobarbital sodium(40mg·kg^-1),blood was drawn in rat hearts and serum was separated by centrifuging;lung tissues were snap-frozen in liquid nitrogen or fixed in 4%paraformaldehyde.2.The serum levels of malondialdehyde（MDA）and superoxide dismutase（SOD）were tested according to the protocol of ELISA kit.3.The expression of Bax,Bcl-2,Keap1,Nrf2 and HO-1 protein was detected by employing Western blotting method.4.The effects of CS and NAC on the morphology of lung tissue of rats were observed microscopically by HE staining.5.Immunohistochemistry was used to detect the expression of 8-OHd G and Caspase3 p12.6.Immunofluorescence double labeling method was used to detect the colocalization of SP-C with 8-Ohd G or Caspase3 p12.Results:1.NAC could improve weight gain in CS-induced rats.The weight gain in CS group obviously reduced compared with control group（P<0.05）.CS+NAC group produced a significant increase in animal weight gain,which was statistically significant since the second week（P<0.05）.2.NAC could ameliorate changes in lung tissue morphology caused by CS.Lung tissue of CS group was characterized by alveolar ectasia,edema,and inflammatory infiltration.Interestingly,it was significantly attenuated in CS+NAC group.3.NAC could improve CS-induced oxidative stress.Compared with control group,the MDA concentration in serum was significantly increased（P<0.05）in CS group,while the level of SOD was significantly decreased（P<0.05）in CS group;after treatment with NAC,the values of MDA SOD were both significantly restored（P<0.05）.In addition,the results of immunohistochemistry displayed that the expression of 8-OHd G in CS group was enhanced compared with control group,and it was decreased in CS+NAC group.4.NAC could improve CS-induced apoptosis.The expression of Bax protein in CS group was increased but not statistically significant（P>0.05）compared with control group,while the expression of Bcl2 protein was significantly decreased（P<0.05）.In CS+NAC group,the expression of Bax was significantly decreased（P<0.05）,and the expression of Bcl2 protein was significantly increased（P<0.05）compared with CS group.Moreover,the results of immunohistochemistry displayed the enhanced expression of Caspase3 p12 in CS group,and it was decreased in CS+NAC group.5.It was observed that 8-OHd G or Caspase3 p12 was co-located with SP-C by immunofluorescence double staining,which indicates the occurrence of oxidative stress and apoptosis in alveolar type II epithelial cells.6.The antagonistic effect of NAC on CS-induced oxidative stress and apoptosis may be related to Nrf2/HO-1.The expression of Nrf2 and HO-1 protein in CS group was significantly decreased（P<0.05）.In CS+NAC group,the expression of Nrf2 and HO-1 protein was both significantly increased compared with CS group（P<0.05）.Conclusions:1.CS can cause systemic（serum）and local（lung tissue）oxidative stress as well as apoptosis in rat lung tissue.2.NAC can protect against CS-induced oxidative stress and inhibit apoptosis.3.One of the protective pathway of NAC may be related to Nrf2/HO-1.