基于内质网应激信号通路探讨温肺化纤颗粒对肺纤维化模型小鼠肺损伤的影响

Effect of Wenfei Huaxian Granule(温肺化纤颗粒)on Lung Injury in Mice with Pulmonary Fibrosis based on Endoplasmic Reticulum Stress Signaling Pathway

目的探讨温肺化纤颗粒治疗肺纤维化的可能作用机制。方法将50只昆明小鼠随机分为空白对照组、模型组及温肺化纤颗粒低、中、高剂量组,每组10只。除空白对照组外,其余各组小鼠通过气管内滴注博来霉素构建肺纤维化模型。造模后,温肺化纤颗粒低、中、高剂量组小鼠分别给予温肺化纤颗粒混悬液2.925、5.85和11.7 g/(kg·d)灌胃,空白对照组和模型组给予生理盐水0.1 ml/10 g灌胃,连续28天。末次灌胃24h后采用HE和Masson染色观察小鼠肺组织病理损伤;Real-time PCR检测小鼠肺组织克老素(Klotho)、转化生长因子β1 (TGF-β1)、α平滑肌肌动蛋白(α-SMA)、葡萄糖调节蛋白78 (GRP78)、C/EBP同源蛋白(CHOP) mRNA表达;Western blot法检测小鼠肺组织Klotho、GRP78、CHOP、B淋巴细胞瘤2相关X (Bax)、B淋巴细胞瘤2 (Bcl-2)蛋白表达;免疫组化法观察肺组织TGF-β1、α-SMA蛋白表达。结果与空白对照组比较,模型组小鼠肺脏病理切片显示肺泡间隔增厚、病变区肺泡结构受到破坏,可见明显的蓝色胶原纤维沉积。肺组织中TGF-β1、α-SMA、GRP78、CHOP mRNA表达明显升高,Klotho mRNA表达明显降低(P<0.01),TGF-β1、α-SMA、GRP78、CHOP、Bax蛋白表达明显增加,Klotho、Bcl-2蛋白表达明显减少(P<0.01)。与模型组比较,温肺化纤颗粒各剂量组小鼠肺脏损伤减轻、肺泡间隔稍增厚、病变肺泡结构破坏减轻,小鼠肺脏中蓝色胶原纤维沉积减少,肺组织中TGF-β1、α-SMA、GRP78、CHOP mRNA表达明显降低,Klotho mRNA表达明显升高(P<0.01),温肺化纤颗粒中、高剂量组GRP78、CHOP、Bax蛋白表达明显降低,温肺化纤颗粒各剂量组Klotho、Bcl-2蛋白表达明显升高(P<0.01);与温肺化纤颗粒低、高剂量组比较,温肺化纤颗粒中剂量组蓝色胶原纤维沉积最少,α-SMA、GRP78、CHOP mRNA降低,Klotho mRNA表达升高,Bax、GRP78、CHOP蛋白表达降低,Bcl-2蛋白表达升高(P<0.05或P<0.01)。结论温肺化纤颗粒具有抑制肺纤维化的作用,其机制可能是通过抑制内质网应激介导的凋亡发生,从而减少上皮间质转化。

ObjectiveTo investigate the possible mechanism of Wenfei Huaxian Granule(温肺化纤颗粒,WHG)in treating pulmonary fibrosis(PF).MethodsFifty Kunming mice were randomly divided into blank con-trol group,model group,WHG low,medium and high dose groups,with 10 mice in each group.Except for theblank control group,bleomycin were instilled into the trachea of the mice to develop PF models.After modelling,thesuspension of WHG at the dose of 2.925 g/(kg·d),5.85 g/(kg·d)and 11.7 g/(kg·d)were given by gavage to themice in WHG low,medium and high dose groups,respectively,while the blank control group and the model groupwere given 0.1 m L/10 g of normal saline by gavage;the administration in all the groups lasted for 28 days.HE andMasson staining were used to detect the pathological changes of lung in mice 24 h after the last administration;realtime PCR was used to detect the Klotho,transforming growth factorβ1(TGF-β1),α-smooth muscle actin(α-SMA),glucose regulatory protein 78(GRP78)and C/EBP homologous protein(CHOP)m RNA expression;the ex-pressions of Klotho,GRP78,CHOP,B lymphoma 2 related X(Bax)and B lymphoma 2(Bcl-2)protein were detect-ed by Western blotting;TGF-β1 andα-SMA protein expression in the lung tissues were detected by immunohisto-chemistry.ResultsCompared to those in the blank group,the alveolar septum in the model group was thickened;the alveolar structure in the lesion area was damaged,and obvious deposition of blue collagen fibers was observed.The expression of TGF-β1,α-SMA,GRP78 and CHOP m RNA increased significantly(P<0.01),while Klothom RNA decreased significantly(P<0.01);the expression of TGF-β1,α-SMA,GRP78,CHOP and Bax protein in-creased significantly(P<0.01),while the expression of Klotho and Bcl-2 protein decreased significantly(P<0.01).Compared to those in the model group,the lung injury,alveolar septum thickening,alveolar structure dam-age and deposition of blue collagen fibers in the lung of mice in WHG low,medium and high dose groups were im-proved;the expression of TGF-β1,α-SMA,GRP78 and CHOP m RNA decreased significantly(P<0.01),whileKlotho m RNA expression increased significantly(P<0.01);the expression of GRP78,CHOP and Bax proteins inthe WHG medium and high dose groups decreased significantly(P<0.01),and the expression of Klotho and Bcl-2 proteins in all the dose groups of WHG increased significantly(P<0.01).When compared between different dosesof WHG,the medium dose group had the least blue collagen fiber deposition,the lowest expression ofα-SMA,GRP78,and CHOP m RNA,the highest expression of Klotho m RNA,the lowest expression of Bax,GRP78 and CHOPproteins,and the highest expression of Bcl-2 protein(P<0.05 or P<0.01).ConclusionWHG can inhibit thePF,and its mechanism maybe related to the inhibition of endoplasmic reticulum stress-induced apoptosis,thereby re-ducing epithelial-mesenchymal transition.