基于Ghrelin/NF-κB探讨银莱汤治疗胃肠积热合并肺炎的机制研究

Exploring the intervention mechanism of Yinlai Decoction on gastrointestinal heat retention syndrome and pneumonia based on Ghrelin/NF-κB signaling pathway

目的基于胃饥饿素(Ghrelin)/核转录因子κB(nuclear factor kappa-B,NF-κB)信号通路探讨银莱汤治疗胃肠积热合并肺炎幼龄大鼠的作用机制。方法36只SPF级4周龄雄性大鼠随机分为6组,分别为正常组、模型组、银莱汤组、胃饥饿素组、胃饥饿素受体拮抗剂组、胃饥饿素受体拮抗剂合银莱汤组,每组6只。除正常组外,其余各组均采用高热量饮食联合脂多糖(lipopolysaccharide,LPS)雾化的方式制备胃肠积热合并肺炎模型,并分别给予银莱汤、胃饥饿素或胃饥饿素受体拮抗剂处理。每日监测大鼠进食量、体质量、肛温等指标;采用苏木精—伊红(hematoxylin-eosin,HE)染色观察肺组织病理学改变;酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)检测各组大鼠血清中白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)炎症因子及Ghrelin水平,分析Ghrelin与各炎症因子水平的相关性;蛋白免疫印迹法(Western blot,WB)检测肺组织中细胞核因子-κB p65(nuclear factor-κB p65,NF-κB p65)、κBα磷酸化抑制剂(phosphorylated inhibitor of κBα,p-IκBα)、κBα抑制剂(inhibitor of κBα,IκBα)蛋白的表达。结果与正常组相比,模型组大鼠血清中炎症因子IL-1β、TNF-α、IL-6水平显著升高,Ghrelin水平显著降低(P<0.01),肺组织中NF-κB p65、p-IκBα相对表达显著升高,IκBα相对表达显著降低(P<0.01)。与模型组相比,银莱汤组、胃饥饿素组大鼠血清中炎症因子水平显著降低,Ghrelin水平显著升高(P<0.01),肺组织中NF-κB p65、p-IκBα相对表达显著降低,IκBα相对表达显著升高(P<0.01);胃饥饿素受体拮抗剂组大鼠血清中炎症因子水平显著升高,Ghrelin水平显著降低(P<0.01),肺组织中NF-κB p65相对表达明显升高(P<0.05),p-IκBα相对表达有升高趋势(P>0.05),IκBα相对表达显著降低(P<0.01)。与银莱汤组相比,胃饥饿素受体拮抗剂合银莱汤组大鼠血清中炎症因子水平显著升高,Ghrelin水平显著降低(P<0.01),肺组织中NF-κB p65、p-IκBα相对表达显著升高,IκBα相对表达显著降低(P<0.01)。血清中Ghrelin与IL-1β水平呈负相关(r=-0.958,P<0.01);Ghrelin与TNF-α水平呈负相关(r=-0.975,P<0.01);Ghrelin与IL-6水平呈负相关(r=-0.962,P<0.01)。结论银莱汤可通过提高胃肠积热合并肺炎幼龄大鼠体内Ghrelin水平,进而抑制NF-κB炎症信号通路激活,从而起到治疗作用。

Objective Based on Ghrelin/NF-κB signaling pathway to explore the mechanism of Yinlai decoction in the treatment of gastrointestinal heat retention syndrome and pneumonia in young rats.Methods Thirty six SPF 4-week old male rats were randomly divided into 6 groups:the normal group,the model group,the Yinlai decoction group,the Ghrelin group,the Ghrelin receptor antagonist group and the Ghrelin receptor antagonist combined Yinlai decoction group,with 6 rats in each group.In addition to the normal group,the other groups were treated with Yinlai decoction,Ghrelin or Ghrelin receptor antagonist respectively to prepare the model of gastrointestinal heat retention syndrome and pneumonia by high calorie diet combined with LPS atomization.The food intake,body mass and anal temperature of rats were monitored daily;the method of HE staining was used to observe the pathological changes of lung tissues;the method of ELISA kits to detect changes in the levels of IL-1β,TNF-α,IL-6 inflammatory factors and Ghrelin in serum of rats in each group;the expression of NF-κB p65,p-IκBα,IκBα proteins in lung tissues were detected by Western blot.Results Compared with the normal group,the levels of inflammatory factors IL-1β,TNF-α,IL-6 in the model group were increased significantly,Ghrelin was decreased significantly(P<0.01);the expression of NF-κB p65,p-IκBα was increased significantly,IκBα was decreased significantly(P<0.01).Compared with the model group,the levels of inflammatory factors in the Yinlai Decoction group and the Ghrelin group were decreased significantly,the levels of Ghrelin were increased significantly(P<0.01),and the expression of NF-κB p65,p-IκBα was decreased significantly,IκBαwas increased significantly(P<0.01);in the ghrelin receptor antagonist group,the levels of inflammatory factors were increased significantly,and Ghrelin was decreased significantly(P<0.01),the expression of NF-κB p65 was increased significantly(P<0.05),p-IκBα showed an upward trend(P>0.05),IκBαwas decreased significantly(P<0.01).Compared with the Yinlai decoction group,the levels of inflammatory factors in the combination of Ghrelin receptor antagonist and Yinlai decoction group were increased significantly,the level of Ghrelin was decreased significantly(P<0.01),and the expression of NF-κB p65,p-IκBα was increased significantly,IκBαwas decreased significantly(P<0.01).The level of Ghrelin and IL-1β was negatively correlated(r=-0.958,P<0.01);the level of Ghrelin and TNF-α was negatively correlated(r=-0.975,P<0.01);the level of Ghrelin and IL-6 was negatively correlated(r=-0.962,P<0.01).Conclusion Yinlai decoction can inhibit NF-κB signaling pathway by increasing the level of Ghrelin in young rats with gastrointestinal heat retention syndrome and pneumonia to play a therapeutic role.