摘要
目的 研究转化生长因子-β1/smad3信号通路对小鼠肺纤维化中细胞凋亡的调控作用及机制. 方法 54只健康雄性C57BL/6小鼠随机分为正常对照组、肺纤维化模型组、肺纤维化+TGF-β1/smad3抑制剂干预组,每组18只.肺纤维化模型组、干预组经气管内注入博莱霉素3.5 mg/kg诱导肺纤维化模型,干预组于造模后的第3、4、5天经腹腔注射TGF-β1/smad3抑制剂SB431542(按4.2 mg/kg,用10%乙醇溶解),正常对照组给予等剂量生理盐水.造模后第7、14、28天各组随机处死6只小鼠,留取右肺上叶组织石蜡包埋切片行苏木素-伊红(HE)染色和马松三色(Masson)染色;右肺中下叶组织碱水解法测羟脯氨酸(HYP)含量;DNA断裂的原位末端标记(TUNEL)法观察细胞凋亡情况;左肺组织经蛋白印记法(Western blot)测定肺组织半胱氨酸天冬氨酸蛋白酶-3(caspase3)和磷酸化smad3 (p-smad3)的表达. 结果 正常对照组肺泡结构正常,而肺纤维化模型组第7天时肺泡呈炎性改变,第28天时发展为明显肺纤维化;随时间推移肺纤维化模型组羟脯氨酸含量、细胞凋亡指数呈上升趋势,各时间点的变化均明显高于正常对照组(P<0.05);肺纤维化模型组caspase3、p-smad3的含量较正常对照组各时间点均增加(均P<0.05);干预组肺泡炎、肺纤维化程度、羟脯氨酸含量、细胞凋亡指数及caspase3、p-smad3的表达较肺纤维化模型组相对应的时间点均减轻(均P<0.05). 结论 细胞通过TGF-β1/Smad3信号通路使细胞凋亡上调而促进了肺纤维化的发生,SB431542可以通过抑制该信号通路从而减轻肺纤维化.
Objective To explore the effect and mechanism of TGF beta1/smad3 signaling pathways on apoptosis in mouse pulmonary fibrosis.Methods Fifty-four healthy male C57BL/6 mice were randomly divided into three groups:normal control (n=18),pulmonary fibrosis model (n =18) and TGF-β1/smad3 inhibitor group (n=18).Six mice in each group were randomly killed on days 7,14 and 28.Hematoxyli^eosin and Masson staining were adopted to evaluate the severity of pulmonary inflammation and fibrosis.The content of hydroxyproline (Hyp) in the lung tissues was detected by alkaline hydrolysis technique.The apoptosis was observed by tunnel apoptosis assay kit.P-smad3 and caspase3 protein expressions were assessed via Western blot.Results Lung in model mice versus normal control showed alveolar inflammatory change in 7 days and significant pulmonary fibrosis in 28 days(P<0.05).Meanwhile,apoptosis index,hydroxyproline content,caspase3,and phosphorylated Smad3 were obviously higher in model mice than in control group (P < 0.05).Compared with model group,TGF-β1/smad3 inhibitor group showed that alveolitis and pulmonary fibrosis degree,hydroxyproline content,cell apoptosis index,the expressions of p-smad3 and caspase3 were decreased at same time point (P < 0.05).Conclusions TGF beta1/smad3 signaling pathways may participate the abnormal apoptosis during the development of pulmonary fibrosis,and TGF-β1/smad3 inhibitor SB431542 could inhibit this process.
出处
《中华老年医学杂志》
CAS
CSCD
北大核心
2014年第7期802-806,共5页
Chinese Journal of Geriatrics
基金
山西省自然科学基金资助(2012011037-5)
关键词
转化生长因子Β1
肺纤维化
细胞凋亡
Transforming growth factor betal
Pulmonary fibrosis
Apoptosis