姜虎汤对结缔组织病相关间质性肺病模型小鼠免疫炎性损伤的影响

Effect of Jianghu Decoction on immune inflammatory injury in connective tissue disease associated interstitial lung disease model mice

[目的]探讨姜虎汤对博来霉素(BLM)诱导结缔组织病相关间质性肺病(CTD-ILD)模型小鼠肺间质病变的影响。[方法]采用SPF级雄性C57/BL6小鼠,经BLM(2 mg/kg)气管内滴注诱导间质性肺病模型,将造模成功的30只小鼠随机分为模型组6只、阳性药组6只、姜虎汤高、中、低剂量组各6只,另选6只未造模小鼠作为正常对照组。正常对照组和模型组予蒸馏水10 mL/(kg·d),阳性药组予泼尼松7.5 mg/(kg·d),姜虎汤高、中、低剂量组分别予姜虎汤26.66、13.33、6.67 g/(kg·d)剂量灌胃给药,其中姜虎汤高剂量组每日分2次灌胃给药。干预4周后处死小鼠,采集肺组织标本。以苏木精-伊红染色法(HE)观察肺组织病理改变;蛋白免疫印迹(Western Blot)技术检测肺组织高迁移率族蛋白B1(HMGB1)、转化生长因子β1(TGF-β1)、核因子-κB p65(NF-κB p65)、单磷酸腺苷(AMP)活化蛋白激酶α1(AMPKα1)、磷酸化AMPKα1(pAMPKα1)蛋白表达水平。[结果]模型组小鼠较正常对照组有明显病理改变(P<0.01),主要表现在肺组织炎症细胞浸润、间质纤维化;阳性药组肺组织炎症病变较模型组有改善(P<0.05);姜虎汤中、低组小鼠肺组织炎症细胞浸润较模型组有改善(P<0.05),姜虎汤高、中、低剂量组小鼠肺组织间质纤维化较模型组有改善(P<0.05)。Western Blot结果显示,与正常组比较,模型组小鼠肺组织中HMGB1、NF-κB p65蛋白表达水平显著升高(P<0.01),TGF-β1蛋白表达水平明显升高(P<0.05),AMPKα1、pAMPKα1蛋白表达水平明显降低(P<0.05,P<0.01)。与模型组比较,姜虎汤中、低剂量组小鼠肺组织中HMGB1、NF-κB p65蛋白表达水平明显降低(P<0.05),姜虎汤高剂量组小鼠肺组织TGF-β1蛋白表达水平明显降低(P<0.05),阳性药组小鼠肺组织中AMPKα1蛋白表达水平明显上调(P<0.05),姜虎汤高剂量组小鼠肺组织中pAMPKα1蛋白表达水平明显上调(P<0.05);与阳性药组比较,姜虎汤高剂量组小鼠肺组织中TGF-β1蛋白表达水平明显下调(P<0.05);姜虎汤各剂量组间比较,姜虎汤高剂量组TGF-β1蛋白表达水平与姜虎汤中剂量组差异明显(P<0.05)。[结论]姜虎汤中、低剂量组在改善小鼠肺组织炎症病理损伤,及抑制免疫炎性因子高表达的作用更具优势;姜虎汤高剂量组在改善小鼠肺组织间质纤维化、抑制纤维化生成因子高表达及上调免疫炎性抑制因子的作用更强。其分子机制可能与激活AMPK,从而抑制HMGB1/NF-κB/TGF-β1免疫炎性信号通路相关。

[Objective]To investigate the effect of Jianghu Decoction on pulmonary interstitial lesions in bleomycin(BLM)-induced connective tissue disease associated interstitial lung disease,CTD-ILD model mice.[Methods]The mice model of ILD was established in SPF male C57BL/6J mice by intratracheal dripping of BLM(2 mg/kg).The 30 mice with successful modeling were randomly divided into model group,positive drug group,high,medium and low dose groups of Jianghu Decoction,6 mice in each group.Six male C57BL/6J mice were set as a normal group.Mice in the normal group and the model group were given deionized water[10 mL/(kg·d)].Mice in the positive group was given prednisone[7.5 mg/(kg·d)].Mice in the Jianghu Decoction groups were given Jianghu Decoction 26.66,13.33 and 6.67 g/(kg·d),respectively.All mice in each group were given intragastric administration once a day,but the Jianghu Decoction high dose group was given twice a day.Lung tissue samples were collected after 4 weeks of continuous intervention.Hematoxylin-eosin(HE)staining method was used to observe the pathological morphology of the lung tissue in each group of mice.The protein expression levels of high mobility group box1(HMGB1),transforming growth factor beta1(TGF-β1),nuclear factor kappa-Bp65(NF-κB p65),Adenosine monophosphate AMP-activated protein kinaseα1(AMPKα1),pAMPKα1 in lung tissue were detected by Western Blot.[Results]Compared with the normal control group,the mice in the model group had obvious pathological changes(P<0.01),mainly manifested as inflammatory cell infiltration and interstitial fibrosis in lung tissue.The inflammatory lesions of lung tissue in the positive drug group were improved compared with the model group(P<0.05).The infiltration of inflammatory cells and interstitial fibrosis in the lung tissue of mice in each treatment group of Jianghu Decoction were improved compared with the model group(P<0.05).Western Blot results showed that compared with the normal group;the expression levels of HMGB1 and NF-κB p65 protein in the lung tissue of the model group were significantly increased(P<0.01);the expression level of TGF-β1 protein was significantly increased(P<0.05),and the expression levels of AMPKα1 and pAMPKα1 protein were significantly decreased(P<0.05,P<0.01).Compared with the model group,the expression levels of HMGB1 and NF-κB p65 protein in the lung tissue of mice in the middle and low dose groups of Jianghu Decoction were significantly decreased(P<0.05).The expression level of TGF-β1 protein in the lung tissue of mice in the high dose group of Jianghu Decoction was significantly decreased(P<0.05).The expression level of AMPKα1 protein in the lung tissue of mice in the positive drug group was significantly up-regulated(P<0.05),and the expression level of pAMPKα1 protein in the lung tissue of mice in the high dose group of Jianghu Decoction was significantly up-regulated(P<0.05).Compared with the positive drug group,the expression level of TGF-β1 protein in the lung tissue of mice in the high-dose Jianghu Decoction group was significantly down-regulated(P<0.05).The expression level of TGF-β1 protein in the high dose group of Jianghu Decoction was significantly different from that in the middle dose group of Jianghu Decoction(P<0.05).[Conclusion]The middle and low dose groups of Jianghu Decoction have more advantages in improving the inflammatory pathological injury of lung tissue in mice and inhibiting the high expression of immune inflammatory factors.The high-dose group of Jianghu Decoction has a stronger effect on improving the interstitial fibrosis of lung tissue in mice,inhibiting the high expression of fibrogenic factors and up-regulating the immune inflammatory inhibitory factors.The molecular mechanism may be related to the activation of AMPK,thereby inhibiting HMGB1/NF-κB/TGF-β1 immune inflammatory signaling pathway.