益气健脾方对香烟烟雾暴露小鼠的肺保护作用及潜在机制

Protective Effect of Yiqi Jianpi Recipe on Lung Injury in Mice Exposed to Cigarette Smoke and Its Potential Mechanism

[目的]初步探讨益气健脾方对香烟烟雾(cigarette smoke,CS)暴露小鼠肺损伤的作用及潜在机制。[方法]60只C57BL/6小鼠随机分为正常组、模型组、预防组、治疗组和防治组。除正常组外,所有小鼠均接受为期4周的CS暴露,其中预防组、防治组分别在暴露初期接受4周及8周的益气健脾方干预,治疗组则于暴露结束后接受4周干预。记录各组小鼠一般情况,苏木精-伊红(hematoxylin-eosin,HE)染色观察肺组织病理学改变;吉姆萨染色观察肺泡灌洗液细胞分类并计数;原位末端转移酶标记(terminal-deoxynucleoitidyl transferase mediated nick end labeling,TUNEL)检测肺组织凋亡细胞DNA片段,免疫印迹检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X,Bax)、胱天蛋白酶-3(cysteine aspartate proteinase-3,caspase-3)、激活型caspase-3(cleaved-caspase-3)、多聚ADP核糖聚合酶1[poly(ADP-ribose)polymerase 1,PARP1]、激活型PARP1(cleaved-PARP1)蛋白表达。[结果]至第4周,模型组小鼠生存率仅为72.72%;至第8周,模型组小鼠体质量最低(P<0.05),肺组织内可见明显炎性细胞浸润及结构破坏。与模型组比较,益气健脾方干预的三组小鼠体质量增加,死亡率降低(P<0.05),肺部炎症浸润及组织损伤均有所减轻(P<0.05),其中以防治组改善最为显著(P<0.01)。与正常组比较,模型组小鼠细胞凋亡指数明显增加(P<0.01),中药干预后则显著减少(P<0.01)。与正常组比较,模型组小鼠肺组织Bcl-2表达减少,Bax表达增多,Bcl-2/Bax比值下调,并且caspase-3、PARP1蛋白及其活化水平均上调(P<0.01,P<0.05)。与模型组比较,经中药干预8周后,小鼠肺组织中Bcl-2表达增加,Bax表达减少,Bcl-2/Bax比值上调,而Bax、caspase-3、cleaved-caspase-3、cleaved-PARP1表达均降低(P<0.05,P<0.01)。[结论]益气健脾方可有效减轻CS暴露造成的小鼠肺组织损伤及炎症反应,其机制可能与抑制Bcl-2-Bax/caspase-3/PARP1细胞凋亡通路相关。

[Objective]To investigate the effect of Yiqi Jianpi Recipe(YQJPR)on lung injury in mice exposed to cigarette smoke(CS),and its potential mechanism.[Methods]Sixty C57BL/6 mice were randomly divided into normal group,model group(CS),prevention group(CS+P),treatment group(CS+T)and prevention and treatment group(CS+P+T).Except for normal group,all mice were exposed to CS for 4 weeks,CS+P group and CS+P+T group were subjected to YQJPR for 4 weeks and 8 weeks from CS exposure beginning,while CS+T group received YQJPR for 4 weeks after CS exposure.The general condition of mice in each group was recorded.Hematoxylin-eosin(HE)staining and Giemsa staining were respectively used to observe the lung histopathology and the classification and counts of inflammatory cells in alveolar lavage fluid.Terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)apoptosis assay was used to detect the DNA fragments of apoptotic cells in lung tissues,and Western blot was used to assess the protein expression of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X(Bax),cysteine aspartate proteinase-3(caspase-3),cleaved-caspase-3,poly(ADP-ribose)polymerase 1(PARP1)and cleaved-PARP1.[Results]At 4th week,the survival rate of mice in model group was only 72.72%;at 8th week,compared with other groups,the mice in model group had decreased body weights with increased mortalities(P<0.05).HE staining showed obvious inflammatory cells infiltrations and structural damages in lung tissue.Compared with model group,the mice in three groups dministrated with YQJPR had gained body weights and reduced mortalities(P<0.05).Meanwhile,their lung inflammatory infiltrations and tissue damages were alleviated(P<0.05).As expected,the pathological changes had most significant improvement in mice with 8-week YQJPR administration(P<0.01).TUNEL apoptosis assay showed that compared with normal group,the apoptotic index in model group was significantly increased(P<0.01),while in mice with YQJPR,all three groups had a lower apoptosis rate in lung(P<0.01).Western blot showed that compared with normal group,the expression level of Bax was increased and the expression level of Bcl-2 was decreased in model group.The ratio of Bcl-2 to Bax was also decreased,caspase-3,PARP1 and their activation proteins were totally upregulated(P<0.01,P<0.05).Intriguingly,compared with model group,8-week treatment of YQJPR could inhibit the expression of Bax,caspase-3,cleaved caspase-3,cleaved PARP1,whereas promote the expression of Bcl-2 and raise the ratio of Bcl-2 to Bax(P<0.05,P<0.01).[Conclusion]YQJPR could effectively protect the lung injury and inflammation from CS exposure in mice,the mechanism may relate to the inhibition of apoptosis pathway of Bcl-2-Bax/caspase-3/PARP1.