Effects Of Autophagy On Lung Injury Induced By Different Doses Of Na Hs In Rats

In recent years,NaHS as H2 S exogenous donor,has been applied to a variety of pathological models,such as heart and lung ischemia-reperfusion model,rat diabetes model,traumatic hemorrhagic shock model.The toxicity of Na HS has seriously affected its application in scientific research.The lungs act as a major target organ for Na HS toxicity,which is susceptible to NaHS toxic damage.In this study,the lungs were used as the study subjects.Three commonly used doses were selected.To study the injury degree of Na HS on lungs and its possible reasons,the histopathological changes of lungs were observed,and the levels of ROS,MDA and MPO were detected.The level of autophagy in lung injury induced by Na HS was determined by detecting the expression of Beclin-1 protein and the ratio of LC3-?/LC3-I.To study the effects of autophagy on Na HS-induced lung injury in rats,autophagy inhibitor 3-MA was used.The aim of this study is to screen out the clinically common NaHS safe dose range in lungs and provide a theoretical basis and clinical guidance for related experiments using Na HS in lungs.In this study,150 healthy Wistar rats were randomly divided into 7 groups: blank control group,Na HS ? group(Na HS 1 mg/kg,ip),Na HS ? group(Na HS 3 mg/kg,ip),Na HS ? group(3-MA 30 mg/kg+Na HS 5 mg/kg,ip),3-MA+Na HS ? group(3-MA 30 mg/kg+Na HS 1 mg/kg,ip),3-MA+Na HS ? group(3-MA 30 mg/kg+Na HS 3 mg/kg,ip),3-MA+Na HS ? group(3-MA 30mg/kg+NaHS 5 mg/kg,ip).Rats in control group was 6.Rats in the remaining groups was 24.Rats in the control group were sacrificed at 2 h after the start of the experiment.Six rats were sacrificed at 2 h,6 h,12 h and 24 h after Na HS injection in the remaining groups,respectively.The histopathological changes of lung tissue,the ratio of wet to dry,ROS,MDA,MPO detection,as well as autophagy-related protein Beclin-1,LC3 protein detection and immunohistochemistry were observed.The results showed that the changes of lung injury were observed before autophagy inhibition in three dose groups.And the pathological changes in three dose groups were more serious than those in the control group.The pathological changes in Na HS ? group and Na HS ? group were more serious than those in Na HS ? group.Aftre autophagy inhibition,the pathological changes in3-MA+Na HS ? group were more serious than those in Na HS ? group,but the pathological changes in 3-MA+Na HS ? group and 3-MA+Na HS ? group were less than those before autophagy inhibition.Compared with the control group,the ratio of wet to dry in Na HS ? group and Na HS ? group were both increased significantly at each time piont(P<0.01),but that inNa HS ? group only was increased significantly at 2 h(P<0.05).After autophagy inhibition,the ratio of wet to dry in 3 inhibitor groups were both increased significantly at each time piont(P<0.01).Whether autophagy was inhibited,the ROS levels in lungs were higher than the control group in all test groups(P<0.05).Compared with the control group,the levels of MDA in the Na HS ? group and Na HS ? group were significantly higher than those in the control group before autophagy inhibition(P<0.01),while that in Na HS ? group was significantly higher at 2 h.After autophagy inhibition,the levels of MDA in 3-MA+Na HS ? group and 3-MA+Na HS ?group were significantly higher than those in the control group at each time point(P<0.01),while3-MA+Na HS ? group was significantly increased at 2 h and 6 h(P<0.01).Before autophagy inhibition,the MPO activity in three dose groups increased significantly at 12 h,24 h(P<0.05).After autophagy inhibition,the MPO activity in 3-MA + Na HS ? group was significantly higher than that in the control group at 2 h,6 h and 12 h(P<0.01).The MPO activity in 3-MA+Na HS ?and 3-MA+Na HS ? group group was significantly higher than that in the control group at each time piont(P<0.05).Before 3-MA pretreatment,the expression of Beclin-1 protein in the Na HS ?group was significantly higher than that in the control group(P<0.01),and that in the NaHS ? and Na HS ? group was significantly higher than that in the control group(P<0.01).After 3-MA pretreatment,3-MA pretreatment did not significantly inhibit the expression of Beclin-1 protein in each group.Before 3-MA pretreatment,the ratio of LC3-?/LC3-? in the Na HS ? group was significantly higher than that in the control group at 6 h(P<0.01),and the ratio in Na HS ? group and Na HS ? group were higher than that in the control group at each time point(P<0.01).After3-MA pretreatment,the ratio of LC3-?/LC3-? in lung decreased significantly compared with the control group(P<0.05).The immunohistochemistry results in lung showed that 3-MA inhibited the transformation of LC3-? to LC3-?,and autophagy was successfully inhibited.Based on these results,it can be concluded that:(1)1 mg/kg,3 mg/kg and 5 mg/kg Na HS can i lung injury.These injuries are mainly caused by Na HS-oxidative stress and inflammatory response.(2)Autophagy has a protective effect in lung injury caused by 1 mg/kg Na HS,and lung injury may be aggravated by autophagy in the 3 mg/kg,5 mg/kg Na HS group.(3)It is recommended that the dose of Na HS in lung-related trials is not more than 1 mg/kg.