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賽培新增產品文章：大鼠 IL-4、IL-13、IFN-γ ELISA試劑盒

發(fā)布時間：2022-09-16 17:00:12

益肺化痰湯減輕哮喘脾虛證大鼠氣道高黏狀態(tài)的作用與機制
翟建賓，趙宏達，趙臣亮，于士昌，程志鴻，趙亮* （河北省中醫(yī)院，石家莊 050000） ［摘要］ 目的：探討益肺化痰湯減輕哮喘脾虛證大鼠氣道高黏狀態(tài)的作用與機制。方法：將 55 只 8~9 周齡、清潔級 SD 大 鼠采用中醫(yī)脾虛證復合西醫(yī)哮喘動物建模方法制備哮喘脾虛證模型，造模成功后采用隨機數字表法分為模型組、地塞米松組、 益肺化痰湯低、中、高劑量組，另取 11 只 SD 大鼠記為正常組。地塞米松組予 0.087 5 mg·kg-1醋酸地塞米松灌胃，益肺化痰湯 低、中、高劑量組分別予 0.8、1.6、3.2 g·kg-1益肺化痰湯流浸膏灌胃，模型組和正常組分別予 10 mL·kg-1蒸餾水灌胃。每日 1 次， 共 8 周。觀察各組一般情況；酶聯免疫吸附測定法（ELISA）檢測各組大鼠支氣管肺泡灌洗液（BALF）白細胞介素-4（IL-4）、白 細胞介素-13（IL-13）、γ 干擾素（IFN-γ）水平；蘇木素-伊紅（HE）染色觀察大鼠肺組織病理改變；阿爾辛藍-過碘酸雪夫（ABPAS）染色觀察大鼠氣道杯狀細胞增生與黏液分泌狀況；實時熒光定量聚合酶鏈式反應（Real-time PCR）檢測大鼠肺組織轉化 生長因子-β（1 TGF-β1）、Smad2、Smad3、黏蛋白 5AC（MUC5AC）、黏蛋白 5B（MUC5B）mRNA 表達；蛋白免疫印跡法（Western blot）檢測大鼠肺組織 TGF-β1、Smad2、Smad3、MUC5AC、MUC5B 蛋白表達。結果：與正常組比較，模型組大鼠存在體質量下 降、肌肉消瘦、食量減少、飲水增多、肛溫升高、倦怠少動及游泳耐力下降等脾虛證癥狀，同時伴隨喘促、點頭等呼吸困難癥狀。 與正常組比較，模型組大鼠 BALF 中 IL-4 和 IL-13 水平顯著上升，IFN-γ 水平顯著下降（P<0.01），氣道黏膜層及黏膜下層見大量 炎性細胞浸潤、氣管平滑肌明顯增厚、黏膜處存在大量上皮細胞增生、變形及脫落現象，肺組織病理評分顯著上升（P<0.01），氣 道可見大量杯狀細胞增生、且形成大量黏液栓，氣道陽性相對著色面積、TGF-β1、Smad2、Smad3、MUC5AC、MUC5B mRNA 和 蛋白質相對表達量顯著上升（P<0.01）；與模型組比較，地塞米松組和益肺化痰湯各劑量組 BALF 中 IL-4、IL-13 水平下降，IFN-γ 水平上升（P<0.05，P<0.01），氣道黏膜層及黏膜下層炎性細胞浸潤逐漸減少、氣管平滑肌增厚現象逐漸減少、黏膜處上皮細胞 增生、變形及脫落現象逐漸減少，肺組織病理評分顯著下降（P<0.01），氣道杯狀細胞增生逐漸減輕，氣道陽性相對著色面積、 TGF-β1、Smad2、Smad3、MUC5AC、MUC5B mRNA 和蛋白表達下降（P<0.05，P<0.01）；與地塞米松組比較，益肺化痰湯低劑量 組上述指標差異無統(tǒng)計學意義，益肺化痰湯中、高劑量組改善更明顯（P<0.05，P<0.01）；上述指標改善益肺化痰湯各劑量組呈 劑量依賴性。結論：益肺化痰湯可減輕哮喘脾虛證大鼠氣道高黏狀態(tài)，可能與抑制 TGF-β1、Smad2、Smad3、MUC5AC、MUC5B 表達，下調 IL-4 和 IL-13 水平，上調 IFN-γ 水平相關。 ［關鍵詞］ 益肺化痰湯；哮喘；脾虛證；氣道黏液；轉化生長因子-β1；黏蛋白 5AC；黏蛋白 5B ［中圖分類號］ R285.5；R966；R562.2；R256.12 ［文獻標識碼］ ［文章編號］ 1005-9903（2022）16-0100-09 ［doi］ 10.13422/j.cnki.syfjx.20221694 [網絡出版地址] https://kns.cnki.net/kcms/detail/11.3495.R.20220422.1909.004.html [網絡出版日期] 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 ［基金項目］ 河北省中醫(yī)藥管理局科研計劃項目（2018012） ［第一作者］ 翟建賓，碩士，從事心胸外科、微創(chuàng)胸外科醫(yī)學研究，E-mail：zhaidoctor99@163. com ［通信作者］ * 趙亮，博士，副主任醫(yī)師，從事心胸外科、微創(chuàng)胸外科醫(yī)學研究，E-mail：zhaolcardiac@163. com ··100第 28 卷第 16 期 2022 年 8 月 中國實驗方劑學雜志 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

益肺化痰湯減輕哮喘脾虛證大鼠氣道高黏狀態(tài)的作用與機制_翟建賓.pdf