牛珀至宝微丸对内毒素致急性肺损伤纤维化大鼠辅助性T细胞的影响

Effects of Niupo Zhibao pellet on helper T cell expression in rats with endotoxin-induced acute pulmonary fibrosis

目的：观察牛珀至宝微丸对内毒素致急性肺损伤纤维化（RPF）大鼠辅助性T细胞（Th细胞）的影响。方法将224只健康Wistar大鼠按随机数字表法分为对照组、模型组、地塞米松组（地米组）和牛珀至宝微丸组（牛丸组），每组56只。采用内毒素“三次打击”造成RPF模型。于制模前3 d和制模后7 d牛丸组给予牛珀至宝微丸500 mg/kg灌胃，每日2次，共用10 d；地米组腹腔注射地塞米松3.0 mg/kg，每日1次，连用7 d；对照组给予等体积蒸馏水灌胃。于给药后1、3、7、9、14、21、28 d 7个时间点取血和支气管肺泡灌洗液（BALF），用酶联免疫吸附试验（ELISA）测定血清及BALF中γ-干扰素（IFN-γ）、白细胞介素-4（IL-4）含量；然后取肺组织用苏木素-伊红（HE）染色及Van Gieson法染色，光镜下观察肺组织形态学变化。结果 HE染色显示，模型组肺间质增厚，肺泡腔内大量炎性渗出，腔内微血栓形成；地米组肺间质增厚，炎性渗出减少明显；牛丸组渗出较地米组稍增多，肺间质基本正常。Van Gieson法染色显示：模型组胶原纤维表达明显；地米组胶原纤维表达减少不明显；牛丸组胶原纤维表达显著减少。ELISA试验显示：模型组BALF和血清中IFN-γ显著升高，地米组7 d时最低，14 d上升至治疗前水平，21 d逐渐下降；牛丸组IFN-γ持续处于高水平后缓慢回落，给药后7 d起各时间点BALF和血清中IFN-γ水平即明显高于模型组和地米组〔BALF（ng/L）：140.47±4.22比149.23±8.35、90.67±6.65；血清（ng/L）：140.47±4.15比100.43±11.05、99.35±7.85，P＜0.05或P＜0.01〕。各组BALF和血清中IL-4显著升高，7 d时最高，以后逐渐降低，给药后28 d牛丸组显著低于模型组和地米组〔BALF（ng/L）：6.60±1.05比7.20±1.25、8.55±1.05，血清（ng/L）：6.75±1.05比7.21±1.25、8.25±1.15，P＜0.05或P＜0.01〕。结论牛珀至宝微丸通过促进IFN-γ分泌、抑制IL-4分泌，调整Th细胞失衡，以达到抗炎、减轻肺损伤、抑制肺纤维化的作用。

Objective To observe the effects of Niupo Zhibao pellet, a compound traditional Chinese herbal medicine,on helper T cell（Th cell）expression in rats with endotoxin-induced rapid pulmonary fibrosis （RPF）. Methods Two hundred and twenty-four Wistar rats were randomly divided into four groups：normal control,model,dexamethasone（DXM）and Niupo Zhibao pellet（NW）groups（each n=56）. By using endotoxin three-hit regimen,the RPF model was established. Three days before and 7 days after the establishment of models in NW group,they were administered with the pellet by intragastric feeding,50 mg/100 g of distilled water twice a day,a total of 10 days medication. Rats in DXM group received DXM intraperitoneal injection,3.0 mg/kg once a day for consecutive 7 days. Rats in normal control group were administered with the same volume of distilled water by intragastric administration. On 1,3,7,9,14,21,28 days after the administration,blood and bronchoalveolar lavage fluid（BALF）specimens were collected. The contents of γ-interferon（IFN-γ）and interleukin-4（IL-4） in the serum and BALF were investigated by enzyme-linked immunosorbent assays（ELISA）. The lung tissue was stained by hematoxylin-eosin（HE）and Van Gieson respectively. Morphological changes in lung tissue were observed under light microscope. Results HE staining showed that the pulmonary interstitial tissues of rats in model group were thickened,there was a large amount of inflammatory exudates,and micro thrombi were seem in alveolar space. The pulmonary interstitial tissues in rats of DXM group were thickened too,but the inflammatory exudate in alveolar space was much less. Compared to rats in DXM group,the rats in NW group had slightly more inflammatory exudate and their pulmonary interstitial tissues were basically normal. Van Gieson staining showed that the expression of collagen fiber in model group was obvious,that of DXM group less than the former one but not significant,while that in the NW group was reduced markedly. ELISA assays demonstrated that the levels of IFN-γin BALF and serum in model group were increased significantly,those in DXM group were lowered to the minimum on the 7th day,raised to the levels before treatment on the 14th day,and gradually declined on the 21st day. In the NW group,the IFN-γwas consistently at a high level,and then gradually declined at a slow rate. After 7 days of drug administration,the IFN-γ levels in BALF and serum at various time points in NW group were obviously higher than those in model and DXM groups〔BALF（ng/L）：140.47±4.22 vs. 149.23±8.35,90.67±6.65;serum（ng/L）：140.47±4.15 vs. 100.43±11.05,99.35±7.85,P〈0.05 or P〈0.01〕. The levels of IL-4 in BALF and serum in each group increased significantly,reached to their maximum on the 7th day,and then gradually decreased. After the drug administration for 28 days,those levels in NW group were obviously lower than those in model and DXM group〔BALF（ng/L）：6.60±1.05 vs. 7.20±1.25,8.55±1.05,serum（ng/L）：6.75±1.05 vs. 7.21±1.25,8.25±1.15,P〈0.05 or P〈0.01〕. Conclusion Niupo Zhibao pellet can suppress inflammation,ameliorate injury of lungs and inhibit lung fibrosis by promoting IFN-γsecretion,restraining IL-4 secretion and adjusting the imbalance of Th cells.