Ac-SDKP通过抑制内质网应激对肺纤维化细胞凋亡的影响

Effect of Ac-SDKP ameliorates pulmonary fibrosis by inhibiting endoplasmic reticulum stress induced apoptosis

目的探讨体内、外肺间质纤维化模型中Ac-SDKP延缓肺纤维化发展的可能机制。方法体内实验:将24只健康雄性C57BL/6小鼠随机分为对照组(8只)、肺纤维化模型组(8只)、肺纤维化+Ac-SDKP治疗组(8只)。采用气管内注入博来霉素(5 mg/kg)法建立小鼠肺纤维化模型,对照组为气管内注入等体积0.9%氯化钠溶液。造模后肺纤维化+Ac-SDKP治疗组小鼠腹腔内埋入Ac-SDKP微量注射泵(Ac-SDKP 800μg·kg^(-1)·d^(-1),冰乙酸0.1 mol/L,卡托普利100 mg·kg^(-1)·d^(-1)),持续干预21 d。对照组及肺纤维化模型组小鼠均于造模完成后第21天处死,肺纤维化+Ac-SDKP治疗组小鼠于干预21 d后处死,取肺组织观察其病理学变化;按试剂盒说明测定羟脯氨酸含量测定;TUNEL法测定肺泡上皮细胞凋亡率;Western-blot、免疫组化法检测GRP78、CHOP蛋白及mRNA的表达水平。体外实验:培养A549细胞,经TGF-β干预24 h后收集细胞,RT-PCR检测GRP78、CHOP mRNA的表达水平。正态分布计量资料多组间比较釆用方差分析,两两比较采用SNK-q检验;非正态分布计量资料多组间比较采用kruskal-WallisH检验。结果(1)一般观察:对照组小鼠双肺表面及切面光滑,未见结节形成;肺纤维化模型组肺组织表面及切面可见散在小结节;肺纤维化+Ac-SDKP治疗组肺组织表面也可见小结节,但较肺纤维化模型组稀少。(2)HE及Masson染色:肺纤维化模型组出现明显的纤维化改变,肺纤维化+AC-SDKP治疗组肺纤维化程度有所减轻。(3)羟脯氨酸含量3组间羟脯氨酸含量差异有统计学意义(H-20.490,P<0.01),其中肺纤维化模型组、肺纤维化+Ac-SDKP治疗组明显高于对照组(q-0.517、0.167,P<0.05),肺纤维化+Ac-SDKP治疗组明显低于肺纤维化模型组(q-0.351,P<0.05)。(4)肺泡上皮细胞凋亡指数:3组间差异有统计学意义(F-57.720,P<0.01),其中肺纤维化模型组、肺纤维化+Ac-SDKP组明显高于对照组(q-8.471、3.639,均P<0.01),肺纤维化+Ac-SDKP组明显低于肺纤维化模型组(q-4.832,P<0.01)。(5)CHOP、GRP78蛋白水平表达:肺纤维化模型组、肺纤维化+Ac-SDKP治疗组较对照组明显下降(q-22.630、8.921,138.812、41.233;P<0.01),肺纤维化+Ac-SDKP治疗组较肺纤维化组明显升高(q-13.711、97.583,P<0.01)。(6)GRP78、CHOP mRNA表达量:TGF-β干预组较对照组明显升高(q-8.791、7.782,P<0.05);TGF-β+AC-SDKP干预组较单纯Ac-SDKP干预组明显升高(q-4.399、5.561,P<0.05)。结论 Ac-SDKP可能通过抑制内质网应激减缓肺泡上皮细胞凋亡,从而发挥其抗肺间质纤维化的作用。

Objective to discuss the possible mechanism of Ac-SDKP to delay the development of pulmonary fibrosis in the internal and external pulmonary fibrosis model.Methods In vivo experiment:24 healthy male C57BL/6 mice were randomly divided into control group(n-8),pulmonary fibrosis model group(n-8),pulmonary fibrosis+Ac-SDKP treatment group(n-8).A model of pulmonary fibrosis was established by using the endotracheal injection of boliomycin(5 mg/kg).Pulmonary fibrosis after building + Ac-SDKP treatment group mice abdominal cavity into Ac-SDKP trace injection pump(Ac-SDKP 800 μg·kg^(-1)·d^(-1),ice acetic acid 0.1 mol/L,captopril 100 mg·kg^(-1)·d^(-1)),continued intervention 21 d.The mice of control group and pulmonary fibrosis model group were executed on the 21 st day of the completion of the mold,the mice of pulmonary fibrosis+Ac-SDKP treatment group were executed after intervention 21 d,and the lung tissue was observed to observe its pathological changes.The determination of the content of hydroxyprosine in accordance with the experimental formulae;TUNEL method to measure apoptosis of alveolar epithelial cells;The Western-blot,immunohistochemical method detects the expression levels of GRP78,CHOP protein,and mRNA.In vitro experiments:cultured A549 cells,which collect cells after TGF-β intervention 24 h,and RT-PCR for the expression of Grp78 and CHOP mRNA.For the normal distribution data,the variance analysis was used to compare the differences between groups,and the SNK-q test was used to compare the differences between two groups;for the non normal distribution data,kruskal-Wallis H test was used to compare the differences between groups.Results(1) General observation:the control mice had a smooth surface and smooth surface,and no nodules were formed.Pulmonary fibrosis model group lung tissue surface and shard visible in small nodules;Pulmonary fibrosis+Ac-SDKP treatment group of lung tissues also have small nodules on the surface,but they are rare in the pulmonary fibrosis model.(2) HE and Masson dyeing:a significant fibrosis change in the pulmonary fibrosis model group,pulmonary fibrosis,and Ac-SDKP treatment group were reduced in pulmonary fibrosis.(3) Hydroxyproline content:hydroxyproline content differences between the three groups was statistically significant(H-20.490,P<0.01),among them,pulmonary fibrosis,pulmonary fibrosis model group+Ac-SDKP treatment group is significantly higher than control group(q-0.517,0.167,p<0.05),pulmonary fibrosis+Ac-SDKP treatment group significantly lower than the pulmonary fibrosis model group(q-0.351,P<0.05).(4) The alveolar epithelial cell apoptosis:the rate was statistically significant difference between the 3 groups(F-57.720,P<0.01),among them,the pulmonary fibrosis model group and pulmonary fibrosis model group+Ac-SDKP group is significantly higher than the control group(q-8.471,3.639,P<0.01),pulmonary fibrosis+Ac-SDKP group was lower than that in group pulmonary fibrosis model(q-4.832,P<0.01).(5) CHOP and Grp78 protein level expression:the pulmonary fibrosis model group and pulmonary fibrosis+Ac-SDKP treatment group significantly increased than the control group(q-22.630,8.921,and 138.812,41.233,P<0.05),the pulmonary fibrosis+Ac-SDKP treatment group significantly decreased in the pulmonary fibrosis group(q-13.711,97.583,P<0.05).(6) CHOP and Grp78 mRNA expression:TGF-β intervention group significantly increased than the control group(q-8.791,7.782,P<0.05),TGF-β+Ac-SDKP intervention group significantly increased than the Ac-SDKP intervention group(q-4.399,5.561,P<0.05).Conclusion Ac-SDKP may play a role in the anti-pulmonary fibrosis by suppressing the internal network stress to slow the apoptosis of alveolar epithelial cells.