银莱汤对胃肠积热合并肺炎大鼠5-HT水平及Th17/Treg细胞平衡的影响

Effects of Yinlai Decoction on 5-HT and Th17/Treg cell balance in rats with gastrointestinal heat retention syndrome and pneumonia

目的探究银莱汤对胃肠积热合并肺炎大鼠5-羟色胺(5-hydroxytryptamine,5-HT)水平,以及辅助性T细胞17(T helper cell 17,Th17)与调节性T细胞(regulatory T cell,Treg)细胞平衡的影响,阐明银莱汤治疗胃肠积热合并肺炎的相关机制。方法将30只4周龄SPF级大鼠随机分为正常组、胃肠积热组、肺炎组、胃肠积热合并肺炎组、银莱汤组治疗组,每组6只。正常组给予大鼠维持饲料、纯水灌胃、纯水雾化;胃肠积热组给予高热量饲料配合牛奶灌胃制备胃肠积热模型;肺炎组采用脂多糖(lipopolysaccharide,LPS)(0.5 mg/mL)雾化吸入制备肺炎模型;胃肠积热合并肺炎组以特制高热量饲料联合LPS雾化制备胃肠积热合并肺炎模型;银莱汤治疗组在胃肠积热合并肺炎模型的基础上给予银莱汤(每100g体重按照0.56 g/mL)灌胃。每日监测大鼠进食量、体质量、肛温等指标。采用HE染色观察肺组织病理学改变;酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测各组大鼠血清中5-HT及白细胞介素(interleukin,IL)-6、IL-1β、IL-17A、IL-10、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)等细胞因子水平;蛋白免疫印迹法(Western blot,WB)检测肠系膜淋巴结(mesenteric lymph nodes,MLNs)中Th17细胞、Treg细胞特异性转录因子Rorγt和Foxp3的表达水平;RT-PCR和WB检测结肠组织色氨酸羟化酶(tryptophan hydroxylase,Tph)1的表达。结果与肺炎组相比,胃肠积热合并肺炎组大鼠肺部炎性损伤明显;血清5-HT、IL-10水平降低,IL-17A水平升高,TNF-α、IL-1β、IL-6等促炎因子水平升高;结肠Tph1的mRNA及蛋白表达水平降低;MLNs中Rorγt蛋白表达升高、Foxp3蛋白表达降低。与胃肠积热合并肺炎组相比,银莱汤治疗组大鼠肺部炎性损伤减轻,血清5-HT水平、IL-10水平升高,IL-17A水平降低,TNF-α、IL-1β、IL-6等促炎因子水平降低;结肠Tph1 mRNA及蛋白表达水平升高;MLNs中Rorγt蛋白表达降低、Foxp3蛋白表达升高。结论胃肠积热合并肺炎可导致大鼠机体炎性因子水平升高、5-HT水平降低以及Th17/Treg细胞失衡;银莱汤可以通过调节胃肠积热合并肺炎大鼠5-HT水平,调节Th17/Treg细胞平衡,进而起到治疗作用。

Objective To explore the effects of Yinlai Decoction on 5-HT and the balance of Th17/Treg cells in rats with gastrointestinal heat retention syndrome and pneumonia,and to explore the mechanism of Yinlai Decoction in the treatment of gastrointestinal heat retention syndrome and pneumonia.Methods Thirty SPF rats were randomly divided into the normal group(N),the gastrointestinal heat retention syndrome group(G),the pneumonia group(P),the gastrointestinal heat retention syndrome combined with pneumonia group(GP)and the treatment group(T),6 rats in each group.The G group,the GP group and the T group were treated with high-calorie diet and milk for the intragastric administration group and the P group were treated with normal diet.The P group,the GP group and the T group were treated with LPS atomization to prepare a model of pneumonia,and the treatment group was given Yinlai Decoction(0.5 g/mL/100 g)by gavage.The method of HE staining was used to observe the inflammatory damage of lung tissue;the method of ELISA was used to detect serum levels of 5-HT,TNF-α,IL-1β,IL-6,IL-17A,IL-10 and other cytokines in each group of rats;the expression of RORγt and Foxp3 mRNA in Mesenteric Lymph Node tissues and the expression of Tph1 in colon tissues were detected by Western Blot.The expression of Tph1 mRNA in colon tissues were detected by RT-PCR.Results Compared with the N group,the G group,the P group,the inflammatory injury in the lungs of rats in the GP group was significantly severe,and the levels of serum,TNF-α,IL-1β,IL-6 were significantly increased(P<0.01);and the level of serum was significantly decreased(P<0.01);the expression of IL-17A in peripheral blood of the GP group rats was increased(P<0.01)while the expression of IL-10 was decreased(P<0.01).The expression of RORγt in MLNs tissues were increased(P<0.01),while the expression of Foxp3 was decreased(P<0.01).When treated with Yinlai Decoction,the results above get significantly relief.Conclusion Yinlai Decoction can effectively relieve the inflammatory injury of lung tissues in rats with gastrointestinal fever and pneumonia,and its mechanism of action maybe related to the regulation of 5-HT level and Th17/Treg cell balance.