黄连水提物对脾胃湿热型胃溃疡大鼠的治疗作用机制

Mechanism of Rhizoma coptidis water extract in the treatment of the rats of gastric ulcer of spleen-stomach damp-heat type

关键词:  黄连水提物  脾胃湿热型胃溃疡  免疫  炎症因子  氧化应激  肠道菌群

Key words:  Rhizoma coptidis water extract  gastric ulcer of spleen-stomach damp-heat type  immunity  inflammatory factor  oxidative stress  intestinal flora

摘要:

[目的] 观察黄连水提物对于脾胃湿热型胃溃疡大鼠的治疗作用，探讨其可能的作用机制。[方法] 将24只大鼠随机分为对照组、模型组、黄连水提物组、盐酸雷尼替丁组，每组6只。除对照组外，构建脾胃湿热型胃溃疡大鼠模型。采用高温高湿环境、高脂饲料和56%乙醇联合诱导造模；给药组给予相应药物灌胃14 d，每日1次。实验期间，记录大鼠体质量、进食量、旷场运动等情况；测定大鼠空腹血糖、总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）、胃液中氢离子（H⁺）浓度、胃蛋白酶含量，苏木精-伊红（HE）染色观察大鼠胃组织病理变化，流式细胞术检测大鼠脾脏组织中CD4⁺、CD8⁺T细胞、CD4⁺/CD8⁺水平，实时荧光聚合酶链式反应（RT-PCR）法检测大鼠胃组织中白细胞介素-4（IL-4）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的表达情况，使用生化试剂盒检测大鼠血浆中超氧化物歧化酶（SOD）及胃组织中丙二醛（MDA）水平，采用16S rRNA基因测序技术检测各组大鼠盲肠内容物的肠道菌群变化。[结果] 与模型组比较，黄连水提物组大鼠体质量、摄食量、抓力及旷场运动总距离增加（P<0.05）；大鼠空腹血糖和血清中的TC、LDL-C水平降低（P<0.01或P<0.05）；胃液中H⁺浓度、胃蛋白酶水平和溃疡指数降低（P<0.01或P<0.05）；胃组织溃疡减轻，出血、充血情况好转，病理切片可见溃疡情况明显改善；脾脏组织中CD8⁺细胞数降低（P<0.05）；胃组织中抗炎因子IL-4 mRNA水平上升（P<0.05），促炎因子IL-6和TNF-α mRNA水平下降（P<0.01或P<0.05）；血浆中SOD活性增加（P<0.001），MDA含量降低（P<0.05）。肠道菌群测序结果显示Lachnoclostridium、乳杆菌属相对丰度降低，但阿克曼氏菌属、考拉杆菌属、肠球菌属等有益菌相对丰度增加。[结论] 黄连水提物对脾胃湿热型胃溃疡大鼠模型疗效显著，其机制可能与调控免疫、炎症、氧化应激及大鼠肠道菌群组成有关。

Abstract:

[Objective] To observe the therapeutic effect of Rhizoma coptidis water extract on rats with gastric ulcer of spleen-stomach damp-heat type，and to explore its possible mechanism of action. [Methods] Twenty-four rats were randomly divided into control group，model group，Rhizoma coptidis water extract group and ranitidine hydrochloride group，6 rats in each group. All groups of rats were used to establish the model of gastric ulcer of spleen-stomach damp-heat type except control group. The model was induced by high temperature and high humidity environment，high fat diet and 56% ethanol；14 days of drug intervention was given to rats of Rhizoma coptidis water extract group and ranitidine hydrochloride group. During the experimental period，the body weight，food intake and open field test were recorded；fasting blood glucose，TC，TG，LDL-C，H⁺ content and pepsin content of gastric tissue were measured；and HE staining was used to observe the pathological changes in rat’s gastric tissues；flow cytometry was applied to detect the levels of CD4⁺，CD8⁺ T cells，CD4⁺/CD8⁺ in rat spleen tissues；RT-PCR was used to detect the mRNA expression of interleukin-4（IL-4），interleukin-6（IL-6） and tumor necrosis factor（TNF-α） in rat gastric tissue；SOD activity in plasma and MDA level in gastric tissues of rats were detected using biochemical kits；the changes of intestinal flora in the cecum contents were detected using 16S rRNA gene sequencing technology. [Results] Compared with the model group，Rhizoma coptidis water extract significantly increased the body weight，food intake，and total distance of open field exercise of rats（P<0.05）；the blood glucose concentration and the levels of TC and LDL-C in serum were significantly decreased（P<0.01 or P<0.05）；H⁺ concentration，pepsin level，and ulcer index were decreased（P<0.01 or P<0.05）；ulcerations of gastric tissues were reduced，and the conditions of hemorrhage and congestion were significantly improved by the treatment of Rhizoma coptidis water extract. Rhizoma coptidis water extract significantly reduced the number of CD8⁺ cells in spleen tissues（P<0.05）；the level of anti-inflammatory factor IL-4 mRNA in gastric tissues was significantly increased（P<0.05），while the levels of pro-inflammatory factors IL-6 and TNF-α mRNA were significantly decreased（P<0.01 or P<0.05）；and the plasma activity of SOD increased（P<0.001），but MDA levels were significantly decreased（P<0.05）. The results of 16S rRNA gene sequencing showed that the relative abundance of Lachnoclostridium and Lactobacillus decreased，but the relative abundance of beneficial bacteria，such as Ackermannia，Phascolarctobacterium and Enterococcus increased. [Conclusion] Rhizoma coptidis water extract showed significant therapeutic effects in a rat model of gastric ulcer of damp-heat type in spleen and stomach，and the mechanism may be related to the regulation of blood glucose and lipids，immunity，inflammation，oxidative stress and the composition of intestinal flora.