加味半夏泻心汤发酵液对幽门螺旋杆菌相关性胃炎小鼠核因子-κB信号通路及肠道菌群的影响

A study on the effects of modified Banxia Xiexin Decoction fermentation broth on NF-κb signaling pathway and intestinal flora of helicobacter pylori infection-associated gastritis mice

关键词:  半夏泻心汤  幽门螺旋杆菌  植物乳杆菌  中药发酵  抑菌

Key words:  Banxia Xiexin Decoction  helicobacter pylori  lactobacillus plantarum  traditional Chinese medicine fermentation  bacteriostatic inhibition

摘要:

[目的] 探讨加味半夏泻心汤发酵液对幽门螺旋杆菌相关性胃炎小鼠NF-κB信号通路及肠道菌群的影响。[方法] 取40只SPF级8周龄BALB/c小鼠，随机分出34只，建立脾胃湿热型幽门螺旋杆菌相关性胃炎小鼠模型，未建模的6只小鼠作为空白对照组。造模后随机抽取4只小鼠检测其胃黏膜病理变化，将造模成功的小鼠随机分为模型组、加味半夏泻心汤发酵组、加味半夏泻心汤组、益生菌组、西药三联组，每组6只。按照分组分别给予对应药物灌胃治疗。给药过程中观察小鼠一般情况，包括小鼠毛发、饮食、大小便、精神状态、活动情况等；采用苏木素-伊红（HE）染色及吉姆萨染色观察胃黏膜病理学改变及幽门螺旋杆菌定植情况；采用ELISA检测小鼠血清中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）促炎因子表达水平；运用免疫组织化学法及qPCR法检测胃组织中NF-κB p65、IκB-α的蛋白和mRNA水平；采用qPCR法对四种肠道菌进行检测，比较各组小鼠肠道菌群变化。[结果] 给药过程中各治疗组小鼠一般情况均有所好转，其中加味半夏泻心汤发酵组小鼠饮食与饮水量明显增加，活动量增多，精神状态明显改善；吉姆萨染色结果显示：与模型组相比，各治疗组对幽门螺旋杆菌均有一定的抑制作用，其中西药三联组抑菌作用最强，幽门螺旋杆菌定植数量最少。HE染色结果显示：与模型组相比，各治疗组小鼠胃黏膜组织病理情况好转，其中加味半夏泻心汤发酵组与加味半夏泻心汤组效果最为明显，其炎性渗出物明显减少，上皮细胞排列规律；在炎症因子和NF-κB p65、IκB-α mRNA和蛋白含量检测方面，加味半夏泻心汤发酵组与加味半夏泻心汤组、益生菌组和西药三联组相比TNF-α、IL-6、IL-1β含量降低（P<0.05），NF-κB p65 mRNA和蛋白表达下调（P<0.01），IκB-α mRNA和蛋白表达上调，其中加味半夏泻心汤发酵组IκB-α mRNA水平与加味半夏泻心汤组差异无统计学意义（P>0.05）；qPCR法检测四种肠道菌结果显示，加味半夏泻心汤发酵组与其他治疗组相比，乳杆菌、双歧杆菌上调，肠球菌、肠杆菌下调（P<0.01）。[结论] 加味半夏泻心汤发酵液能够抑制幽门螺旋杆菌相关性胃炎小鼠胃黏膜幽门螺旋杆菌的定植，并且通过影响胃黏膜NF-κB p65、IκB-α蛋白表达，降低血清中促炎相关因子的表达，抑制炎症反应，并且可以调节肠道菌群，促进乳杆菌、双歧杆菌等益生菌增殖，抑制有害菌群生长。加味半夏泻心汤经植物乳杆菌发酵后能够显著增强其抗炎和调节肠道菌群的作用，但在抑菌和修复胃黏膜方面没有明显的提升。

Abstract:

[Objective] To investigate the effects of Modified Banxia Xiexin Decoction fermentation broth on NF-κB signaling pathway and intestinal flora of Hp-associated gastritis mice. [Methods] Forty SPF-grade 8-week-old BALB/c mice were taken，and 34 mice were randomly assigned to establish a mouse model of Helicobacter pylori-associated gastritis with damp-heat in the spleen and stomach，and 6 mice without modelling were used as a blank control group. As a result of the successful modeling of Hp-associated gastritis mice，they were randomly divided into the following groups：the model group the Modified Banxia Xiexin Decoction fermentation group，the Modified Banxia Xiexin Decoction group，the probiotic group（lactobacillus plantarum），and the western medicine group（triple drug）. We fed them the appropriate medicines through gastric gavage based on their groupings. During the dosing period，mice were observed for hair，diet，urine，feces and mental status. HE staining and Giemsa staining were used to observe pathological changes in gastric mucosa and Hp colonization. ELISA was used to detect levels of TNF-α，IL-1β and IL-6 inflammatory factors in mice serum.A combination of immunohistochemistry and qPCR was used to detect the protein and mRNA levels of NF-κB p65 and IκB-α in gastric tissues.By using QPCR，four types of intestinal bacteria were detected and differences in intestinal flora were compared between mice groups. [Results] The general conditions of the mice in all treatment groups improved during the administration of the drug，among which the dietary water intake and activity of the mice in the Modified Banxia Xiexin Decoction fermentation group increased significantly，and their mental conditions improved. The results of Giemsa staining showed that all treatment groups had a certain inhibitory effect on Hp colonization compared with the model group. Among them，the western medicine group had the strongest inhibitory effect and the lowest number of Hp colonization. HE staining results showed that compared with the model group，histopathological conditions of the gastric mucosa of mice improved in the treatment groups was improved，among which the effect was most obvious in the Modified Banxia Xiexin Decoction fermentation group and the Modified Banxia Xiexin Decoction group，with a significant reduction in inflammatory exudates and regular arrangement of epithelial cells. Regarding the detection of inflammatory factors and NF-κB p65，IκBα mRNA and protein levels，the Modified Banxia Xiexin Decoction fermentation group had lower levels of TNF-α，IL-6 and IL-1β（P<0.05），down-regulated expression of NF-κB p65 mRNA and protein（P<0.01），and up-regulated expression of IκBα mRNA and protein when compared to groups B，P and W. Among them，the Modified Banxia Xiexin Decoction fermentation group IκBα mRNA levels were not statistically different from the Modified Banxia Xiexin Decoction group（P>0.05）. The results of four kinds of intestinal bacteria detected by qPCR method showed that lactobacillus and Bifidobacterium were up-regulated and Enterococcus and Enterobacteriaceae were down-regulated in the Modified Banxia Xiexin Decoction fermentation group compared with the model group and other treatment groups（P<0.05）. [Conclusion] Modified Banxia Xiexin Decoction fermentation broth can inhibit the colonization of Hp in the gastric mucosa of Hp-associated gastritis model mice，and inhibit the inflammatory response by affecting the expression of NF-κB p65 and IκBα in the gastric mucosa，reduced the expression of pro-inflammatory factors in the serum，and improved the intestinal flora，promoted the proliferation of probiotics such as Lactobacillus and Bifidobacterium，and inhibited the harmful flora. Modified Banxia Xiexin Decoction fermented by Lactobacillus plantarum was able to significantly enhance its anti-inflammatory and intestinal flora-regulating effects，but there was no significant enhancement in bacteriostasis and gastric mucosa repair.