柴胡皂苷D对克雷伯杆菌肺炎大鼠肺组织损伤和纤维化及免疫功能的调节作用

The regulating effect of saikosaponin D on lung tissue injury,fibrosis and immune function in klebsiella pneumoniae rats

目的研究柴胡皂苷D(saikosaponin D,SD)对克雷伯杆菌肺炎大鼠肺组织损伤、纤维化及免疫功能的影响。方法采用气管穿刺注入感染法制作克雷伯杆菌性重症肺炎模型。20只建模成功的大鼠随机分成肺炎模型组(pneumonia)和模型加药组(pneumonia+SD);另取20只SD健康大鼠随机分为健康对照组(control)和柴胡皂苷D单独处理组(SD);柴胡皂苷D单独处理组和模型加药组大鼠腹腔注射柴胡皂苷D,注射剂量为50 mg·kg-1,持续一周。苏木伊红(hematoxylin and eosin,HE)染色和马松(masson)染色观察肺组织损伤和纤维化情况;原位末端标记法(terminal-deoxynucleoitidyl transferase mediated nick end labeling,TUNEL)染色观察肺组织细胞凋亡情况。蛋白质印迹检测切割形式的半胱氨酸天冬氨酸蛋白酶3(cleaved cysteinyl aspartate-specific proteinase-3,cl-caspase-3)、cl-caspase-9、Toll样受体4(toll-like receptor 4,TLR4)、转化生长因子β激活激酶-1(transforming growth factorβactivated kinase-1,TAK1)和核因子-κB(nuclear factor-kappa B,NF-κB)P65的磷酸化情况。酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测白介素-6(interluekin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、诱导型一氧化氮(inducible nitric oxide,iNOS)和IL-10的含量。结果模型组与对照组相比较,大鼠肺组织损伤和纤维化严重;肺组织细胞凋亡数目明显增加(P<0.01);cl-caspase-3和cl-caspase-9的表达显著上调(P<0.01);IL-6、TNF-α和iNOS的含量显著上升,IL-10的表达没有显著变化;TLR4、磷酸化TAK1(phosphorylated TAK1,p-TAK1)和磷酸化P65(phosphorylated P65,p-P65)的表达显著上调,NF-κB P65总的表达没有明显变化。模型加药组与模型组相比较,大鼠肺组织损伤和纤维化明显减轻;肺组织细胞凋亡数目显著降低(P<0.01);cl-caspase-3和cl-caspase-9的表达显著下调(P<0.01);IL-6、TNF-α和iNOS的含量显著下降,IL-10的表达没有显著变化;TLR4、p-TAK1和p-P65的表达显著下调,NF-κB P65总的表达没有明显变化。结论柴胡皂苷D调节克雷伯杆菌肺炎大鼠免疫功能,缓解大鼠肺组织损伤和纤维化。

Objective To explore the effect of the saikosaponin D on lung tissue injury,fibrosis and immune function in klebsiella pneumoniae rats.Methods The rats′model of Klebsiella pneumoniae was established by injecting infection through trachea puncture.Twenty rats were randomly divided into pneumonia model group and model addition group,and 20 SD healthy rats were randomly divided into healthy control group and saikosaponin D alone treatment group(SD).The rats were injected with Radix Bupleuri saponins D,50 mg·kg-1 BW,in the single treatment group and model addition group,and last for a week.H&E staining and MASSON staining were used in the lung tissue injury and fibrosis;The apoptosis of lung tissue cells was observed by TUNEL staining.The expression level of caspase-3,caspase-9,TLR4,p-TAK1 and phosphorylated NF-κB P65 were detected by Western blot.The concentrations of IL-6,TNF-alpha,iNOS and IL-10 were detected by ELISA.SPSS18.0 was used for statistical analysis.Results Compared with the control group,lung tissue injury and fibrosis were more serious in rats.The number of apoptosis in lung tissue was significantly increased(P<0.01).The expressions of caspase-3 and caspase-9 were significantly increased(P<0.01);The concentrations of IL-6,TNF-alpha and iNOS increased significantly,and the expressions of IL-10 did not change significantly.The expressions of TLR4,p-TAK1 and p-P65 were increased significantly(P<0.01,and there was no significant change in the total expression of NF-κB P65.Compared with the model group,the lung tissue injury and fibrosis of rats were significantly reduced.The number of apoptosis of lung tissue was significantly decreased(P<0.01).The expressions of cl-caspase-3 and cl-caspase-9 decreased significantly(P<0.01);The concentrations of IL-6,TNF-alpha and iNOS decreased significantly(P<0.01,and the expression of IL-10 did not change significantly.The expression of TLR4,p-TAK1 and p-P65 was significantly down-regulated(P<0.01),and there was no significant change in the total expression of NF-κB P65.Conclusion Saikosaponin D regulates the immune function and alleviates lung tissue injury and fibrosis in rats.