温肺化纤汤对系统性硬化症相关间质性肺病模型小鼠肺组织纤维化及内质网应激的影响

The Effects of Wenfei Huaxian Decoction(温肺化纤汤)on Pulmonary Fibrosis and Endoplasmic Reticulum Stress in Systemic Sclerosis-Associated Interstitial Lung Disease Model Mice

目的探讨温肺化纤汤治疗系统性硬化症相关间质性肺病(SSc-ILD)肺纤维化的作用机制。方法将60只C3H/He雌鼠随机分为对照组、模型组、吡非尼酮组及温肺化纤汤低、中、高剂量组。除对照组外,其余各组小鼠予博来霉素溶液0.04 mg/d背部皮下连续注射28天建立SSc-ILD小鼠模型。造模成功后,吡非尼酮组予吡非尼酮胶囊300 mg/(kg·d)灌胃,温肺化纤汤低、中、高剂量组分别给予温肺化纤汤7.81、15.62、31.24 g/(kg·d)灌胃,对照组及模型组予生理盐水0.1 ml/10 g灌胃,共21天。干预结束后采用HE染色和Masson染色观察小鼠皮肤及肺组织的病理变化;检测小鼠皮肤及肺组织的羟脯氨酸含量;Western blot法测定小鼠肺组织中的内质网应激相关蛋白葡萄糖调节蛋白78(BIP)和C/EBP同源蛋白(CHOP)及核因子κB(NF-κB)通路中p65的蛋白表达水平;ELISA测定小鼠血清中的γ干扰素(IFN-γ)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的表达水平。结果HE染色、Masson染色结果显示,与对照组比较,模型组小鼠皮肤组织表皮棘层肥厚,胶原纤维明显增粗、膨大,数量增多,炎性细胞明显增多;肺组织出现明显的炎性细胞反应,大量胶原纤维增生,并伴有炎性细胞浸润。与模型组比较,吡非尼酮组和温肺化纤汤各剂量组皮肤组织和肺组织胶原纤维增生明显减少,炎性细胞浸润减少,且吡非尼酮组和温肺化纤汤中、高剂量组效果基本相当。与对照组比较,模型组小鼠皮肤组织和肺组织羟脯氨酸含量,血清IFN-γ、IL-6、TNF-α含量,肺组织BIP和CHOP蛋白表达水平均升高(P<0.05)。与模型组比较,吡非尼酮组和温肺化纤汤低、中剂量组皮肤组织羟脯氨酸含量降低,温肺化纤汤中剂量组肺组织羟脯氨酸含量下降,吡非尼酮组和温肺化纤汤高剂量组小鼠血清IFN-γ、IL-6、TNF-α含量及肺组织BIP、CHOP和p65蛋白表达水平降低(P<0.05)。温肺化纤汤中剂量组肺组织羟脯氨酸含量明显低于温肺化纤汤低、高剂量组,温肺化纤汤低、中剂量组小鼠血清IFN-γ、IL-6、TNF-α含量高于温肺化纤汤高剂量组,温肺化纤汤高剂量组BIP蛋白表达水平明显低于温肺化纤汤低、中剂量组(P<0.05)。结论温肺化纤汤可改善SSc-ILD模型小鼠皮肤组织及肺组织纤维化,其作用机制可能是通过抗炎、抑制NF-κB通路,进而抑制内质网应激,最终阻抑纤维化进程。

Objective To investigate the possible mechanism of Wenfei Huaxian Decoction(温肺化纤汤)in treat⁃ment of pulmonary fibrosis in systemic sclerosis-associated interstitial lung disease(SSc-ILD).Methods Sixty C3H/He female rats were randomly divided into a control group,a model group,a pirfenidone group,and low-,medium-,and high-dose Wenfei Huaxian Decoction groups.The SSc-ILD model mice was established by subcutaneous injection of bleomycin solution 0.04 mg/d into the back of mice for 28 days in all groups but the control group.After successful modelling,the pirfenidone group was given pirfenidone capsule 300 mg/(kg·d)by gavage,the low-,medium-and high-dose Wenfei Huaxian Decoction groups were given Wenfei Huaxian Decoction 7.81,15.62,and 31.24 g/(kg·d)by gavage,respectively,and the control group as well as the model group were given normal saline 0.1 ml/10 g by gavage,for a total of 21 days.At the end of the intervention,HE staining and Masson staining were used to observe the pathological changes in the skin and lung tissues;the hydroxyproline content of the skin and lung tissues was detected;the protein expression levels of endoplasmic reticulum stress-related proteins glucose-regulated protein 78(BIP)and C/EBP homologous protein(CHOP)as well as those of nuclear factor kappa B(NF-κB)pathway p65 were mea⁃sured by western blot;ELISA was performed to determine the expression levels of interferon gamma(IFN-γ),interleu⁃kin 6(IL-6)and tumour necrosis factor alpha(TNF-α)in serum of rats.Results The results of HE and Masson stain⁃ing indicated that compared with the control group,the dermis significantly thickened,the number of collagen fibers significantly enlarged,and the number of inflammatory cells significantly increased in the model group;the lung tissue showed a marked inflammatory cellular response with massive collagen fibre proliferation with inflammatory cell infiltration.Compared with the model group,the skin tissue and lung tissue collagen fibre proliferation significantly reduced and inflammatory cell infiltration reduced in the pirfenidone group and all dose groups of Wenfei Huaxian Decoction,and the effects of pirfenidone group and Wenfei Huaxian Decoction medium-and high-dose groups were basically comparable.Compared with the model group,the content of hydroxyproline in skin and lung tissue,the serum level of IFN-γ,IL-6 and TNF-α,and the expression levels of BIP and CHOP protein in lung tissue increased in model group(P<0.05).Compared with model group,the content of hydroxyproline in skin tissue of pirfenidone group,low-and medium-dose Wenfei Huaxian Decoction groups decreased,and the content of hydroxyproline in lung tissue of medium-dose Wenfei Huaxian Decoction group decreased.The serum level of IFN-γ,IL-6,TNF-αand the expres⁃sion levels of BIP,CHOP and p65 protein in lung tissue of rats in pirfenidone group and high-dose Wenfei Huaxian Decoction group decreased(P<0.05).The content of hydroxyproline in lung tissue of medium-dose Wenfei Huaxian Decoction group was significantly lower than that of low-dose and high-dose Wenfei Huaxian Decoction group,and the serum level of IFN-γ,IL-6,TNF-αin low-and medium-dose Wenfei Huaxian Decoction group were higher than those in high-dose Wenfei Huaxian Decoction group.The expression level of BIP protein in high-dose group was significantly lower than that in low-and medium-dose Wenfei Huaxian Decoction groups(P<0.05).Conclusion Wenfei Huaxian Decoction can improve the skin and lung fibrosis of SSc-ILD rats,which may act through anti-inflammation,inhibition of NF-κB pathway,and then inhibition of endoplasmic reticulum stress,which ultimately blocked the fibrotic process.