赛培新增产品文章：大鼠 IL-4、IL-13、IFN-γ ELISA试剂盒

发布时间：2022-09-16 17:00:12

益肺化痰汤减轻哮喘脾虚证大鼠气道高黏状态的作用与机制
翟建宾，赵宏达，赵臣亮，于士昌，程志鸿，赵亮* （河北省中医院，石家庄 050000） ［摘要］ 目的：探讨益肺化痰汤减轻哮喘脾虚证大鼠气道高黏状态的作用与机制。方法：将 55 只 8~9 周龄、清洁级 SD 大 鼠采用中医脾虚证复合西医哮喘动物建模方法制备哮喘脾虚证模型，造模成功后采用随机数字表法分为模型组、地塞米松组、 益肺化痰汤低、中、高剂量组，另取 11 只 SD 大鼠记为正常组。地塞米松组予 0.087 5 mg·kg-1醋酸地塞米松灌胃，益肺化痰汤 低、中、高剂量组分别予 0.8、1.6、3.2 g·kg-1益肺化痰汤流浸膏灌胃，模型组和正常组分别予 10 mL·kg-1蒸馏水灌胃。每日 1 次， 共 8 周。观察各组一般情况；酶联免疫吸附测定法（ELISA）检测各组大鼠支气管肺泡灌洗液（BALF）白细胞介素-4（IL-4）、白 细胞介素-13（IL-13）、γ 干扰素（IFN-γ）水平；苏木素-伊红（HE）染色观察大鼠肺组织病理改变；阿尔辛蓝-过碘酸雪夫（ABPAS）染色观察大鼠气道杯状细胞增生与黏液分泌状况；实时荧光定量聚合酶链式反应（Real-time PCR）检测大鼠肺组织转化 生长因子-β（1 TGF-β1）、Smad2、Smad3、黏蛋白 5AC（MUC5AC）、黏蛋白 5B（MUC5B）mRNA 表达；蛋白免疫印迹法（Western blot）检测大鼠肺组织 TGF-β1、Smad2、Smad3、MUC5AC、MUC5B 蛋白表达。结果：与正常组比较，模型组大鼠存在体质量下 降、肌肉消瘦、食量减少、饮水增多、肛温升高、倦怠少动及游泳耐力下降等脾虚证症状，同时伴随喘促、点头等呼吸困难症状。 与正常组比较，模型组大鼠 BALF 中 IL-4 和 IL-13 水平显著上升，IFN-γ 水平显著下降（P<0.01），气道黏膜层及黏膜下层见大量 炎性细胞浸润、气管平滑肌明显增厚、黏膜处存在大量上皮细胞增生、变形及脱落现象，肺组织病理评分显著上升（P<0.01），气 道可见大量杯状细胞增生、且形成大量黏液栓，气道阳性相对着色面积、TGF-β1、Smad2、Smad3、MUC5AC、MUC5B mRNA 和 蛋白质相对表达量显著上升（P<0.01）；与模型组比较，地塞米松组和益肺化痰汤各剂量组 BALF 中 IL-4、IL-13 水平下降，IFN-γ 水平上升（P<0.05，P<0.01），气道黏膜层及黏膜下层炎性细胞浸润逐渐减少、气管平滑肌增厚现象逐渐减少、黏膜处上皮细胞 增生、变形及脱落现象逐渐减少，肺组织病理评分显著下降（P<0.01），气道杯状细胞增生逐渐减轻，气道阳性相对着色面积、 TGF-β1、Smad2、Smad3、MUC5AC、MUC5B mRNA 和蛋白表达下降（P<0.05，P<0.01）；与地塞米松组比较，益肺化痰汤低剂量 组上述指标差异无统计学意义，益肺化痰汤中、高剂量组改善更明显（P<0.05，P<0.01）；上述指标改善益肺化痰汤各剂量组呈 剂量依赖性。结论：益肺化痰汤可减轻哮喘脾虚证大鼠气道高黏状态，可能与抑制 TGF-β1、Smad2、Smad3、MUC5AC、MUC5B 表达，下调 IL-4 和 IL-13 水平，上调 IFN-γ 水平相关。 ［关键词］ 益肺化痰汤；哮喘；脾虚证；气道黏液；转化生长因子-β1；黏蛋白 5AC；黏蛋白 5B ［中图分类号］ R285.5；R966；R562.2；R256.12 ［文献标识码］ ［文章编号］ 1005-9903（2022）16-0100-09 ［doi］ 10.13422/j.cnki.syfjx.20221694 [网络出版地址] https://kns.cnki.net/kcms/detail/11.3495.R.20220422.1909.004.html [网络出版日期] 2022-04-24 9:43
Effect of Yifei Huatan Decoction on Relieving Airway Hyperviscosity in Asthmatic Rats with Spleen Deficiency Syndrome and Its Mechanism
ZHAI Jianbin，ZHAO Hongda，ZHAO Chenliang，YU Shichang，CHENG Zhihong，ZHAO Liang* （Hebei Province Hospital of Traditional Chinese Medicine， Shijiazhuang 050000， China） ［Abstract］ Objective： To explore the effect of Yifei Huatan decoction on relieving airway hyperviscosity in asthmatic rats with spleen deficiency syndrome and its mechanism. Method： Fifty-five SPF level SD rats at 8-9 week of age were used to induce asthma with spleen deficiency syndrome by animal ［收稿日期］ 2022-02-16 ［基金项目］ 河北省中医药管理局科研计划项目（2018012） ［第一作者］ 翟建宾，硕士，从事心胸外科、微创胸外科医学研究，E-mail：zhaidoctor99@163. com ［通信作者］ * 赵亮，博士，副主任医师，从事心胸外科、微创胸外科医学研究，E-mail：zhaolcardiac@163. com ··100第 28 卷第 16 期 2022 年 8 月 中国实验方剂学杂志 Chinese Journal of Experimental Traditional Medical Formulae Vol. 28，No. 16 Aug. ，2022 modeling of traditional Chinese medicine combined with asthma of western medicine. After successful modeling，the rats were divided into model group，dexamethasone group，low，medium，and high-dose Yifei Huatan decoction groups by random number table method，and 11 clean SD rats at 8-9 week of age were recorded as a normal group. Rats in the dexamethasone group were given 0.087 5 mg kg-1 dexamethasone acetate by gavage. Rats in the low，medium，and high-dose Yifei Huatan decoction groups were given 0.8，1.6， 3.2 g kg-1 Yifei Huatan decoction liquid extract by gavage，respectively. Rats in the model group and the normal group were given 10 mL kg-1 distilled water. The medicine were given once per day for 8 w，and the general situation of each group was observed. The levels of interleukin-4（IL-4），IL-13，and interferon- γ（IFN- γ）in bronchoalveolar lavage fluid（BALF）were determined by enzyme-linked immunosorbent assay（ELISA）. The pathological changes in lung tissues of rats were observed by hematoxylin-eosin（HE）staining. Alcian blueperiodic acid Schiff（AB-PAS）staining was used to detect the hyperplasia of airway goblet cells and mucus secretion in rats. The mRNA expressions of transforming growth factor- β1（TGF- β1），Smad2，Smad3，mucin 5AC（MUC5AC），and mucin 5B（MUC5B）in the lung tissues of rats were detected by real-time quantitative polymerase chain reaction（Real-time PCR）. The protein expressions of TGF- β1，Smad2，Smad3，MUC5AC， and MUC5B in the lung tissues of rats were detected by Western blot. Result：As compared with the normal group，rats in the model group showed the symptoms of spleen deficiency syndrome，such as decreased body weight， muscle emaciation， decreased food intake， increased water intake， increased anal temperature， tiredness，and decreased swimming endurance，accompanied by dyspnea symptoms such as wheezing and nodding. As compared with the normal group，IL-4 and IL-13 levels in the BALF of the model group were significantly increased（P<0.01），while the IFN- γ level was significantly decreased（P<0.01）. In the model group，a large number of inflammatory cells were observed in the mucosa and submucosa of the airway，and the smooth muscle of the trachea was significantly thickened. The hyperplasia，deformation，and exfoliation of various epithelial cells were observed in the mucosa，and the pathological scores of lung tissue increased significantly（P<0.01）in the model group. A large number of goblet cells were observed in the airway with the formation of plenty of mucous thrombus in the model group，and the positive relative staining area of airway， and mRNA and protein expressions of TGF- β1，Smad2，Smad3，MUC5AC，and MUC5B were significantly increased（P<0.01）. As compared with the model group，IL-4 and IL-13 levels in BALF of the dexamethasone group and the Yifei Huatan decoction groups decreased，while the IFN-γ level increased. The inflammatory cell infiltration in airway mucosa and submucosa，the thickening of tracheal smooth muscle，the hyperplasia， deformation，and exfoliation of epithelial cells in mucosa were gradually decreased，and the pathological scores of lung tissues decreased significantly（P<0.01）in the dexamethasone group and the Yifei Huatan decoction groups. Goblet cell proliferation gradually decreased，and the positive relative staining area of airway，and mRNA and protein relative expressions of TGF- β1，Smad2，Smad3，MUC5AC，and MUC5B decreased with statistically significant difference（P<0.05，P<0.01）. There was no significant difference in the above indexes in the dexamethasone group and the Yifei Huatan decoction low-dose group. The above indexes were dosedependent in the low，medium，and high-dose Yifei Huatan decoction groups. Conclusion： Yifei Huatan decoction reduces airway hyperviscosity in asthmatic rats with spleen deficiency syndrome，which may be related to the inhibition of TGF-β1，Smad2，Smad3，MUC5AC，and MUC5B expressions，down-regulation of IL-4 and IL-13 levels，and up-regulation of IFN-γ level. ［Keywords］ Yifei Huatan decoction；asthma；spleen deficiency syndrome；airway mucus；transforming growth factor-β1；mucin 5AC；mucin 5B

益肺化痰汤减轻哮喘脾虚证大鼠气道高黏状态的作用与机制_翟建宾.pdf