摘要
目的:探究大黄酒蒸前后对便秘模型小鼠治疗的作用,以及潜在的作用机制.方法:将54只雄性ICR小鼠随机分为空白组、模型组、乳果糖组(1.5 mg·kg-1),生、熟大黄高、中、低剂量组(8、4、1 g·kg-1),除空白组外,其余各组灌胃盐酸洛哌丁胺(6mg·kg-1)构建便秘模型.造模2周后,各给药组灌胃相应剂量药液,空白组及模型组给予等体积生理盐水,1次/d,连续2周.给药结束后,收集粪便用于肠道菌群的16S rRNA测序;酶联免疫吸附测定法(ELISA)检测结肠组织中胃泌素(GAS)、胃动素(MTL)、白细胞介素-6(IL-6)、γ干扰素(IFN-γ)含量;苏木素-伊红(HE)染色观察结肠组织病理学变化;流式细胞仪检测外周血中CD4+、CD8+及调节性T细胞(Treg)比例变化.结果:与空白组比较,模型组24 h粪便粒数、质量及粪便含水率显著降低(P＜0.01),结肠可见黏膜上皮坏死脱落,固有层内局部肠腺变性、坏死、萎缩,坏死区见少量淋巴细胞散在浸润,GAS及MTL含量显著降低(P＜0.01),IL-6、IFN-γ含量明显升高(P＜0.05,P＜0.01),CD4+、CD8+及Treg比例显著下降(P＜0.01).与模型组比较,生、熟大黄高剂量组24 h粪便粒数、质量及粪便含水率明显升高(P＜0.05,P＜0.01),结肠病理损伤有不同程度缓解,GAS、MTL、IL-6、IFN-γ含量明显回调(P＜0.05,P＜0.01),CD4+、CD8+细胞比例显著上升(P＜0.01),Treg比例虽有升高趋势,但差异无统计学意义.肠道菌群结果表明,与空白组比较,模型组中扩增子序列变体(ASV)数及Alpha多样性降低,Beta多样性存在明显差异,乳杆菌属(Lactobacillus)相对丰度降低,芽孢杆菌属(Bacillus)及螺杆菌属(Helicobacter)相对丰度升高;与模型组比较,生、熟大黄高剂量组ASV数及Alpha多样性有所升高,Beta多样性有向空白小鼠转归的趋势,乳杆菌属相对丰度升高,芽孢杆菌属及螺杆菌属相对丰度降低.结论:生、熟大黄可能通过改善肠道菌群失调,促进免疫系统激活,抑制炎症因子释放以提升胃肠功能,这可能是其治疗便秘的潜在作用机制之一.
Abstract
Objective:To study on the different therapeutic effects and potential mechanisms of Rhei Radix et Rhizoma(RH)before and after steaming with wine on constipation model mice.Methods:Fifty-four male ICR mice were randomly divided into control group,model group,lactulose group(1.5 mg·kg-1),high,medium and low dose groups of RH and RH steaming with wine(PRH)(8,4,1 g·kg-1).Except for the control group,the constipation model was replicated by gavage of loperamide hydrochloride(6 mg·kg-1)in the other groups.After 2 weeks of modeling,each administration group was gavaged with the corresponding dose of drug solution,and the control and model groups were given an equal volume of normal saline,1 time/d for 2 consecutive weeks.After administration,the feces were collected for 16S rRNA sequencing,the levels of gastrin(GAS),motilin(MTL),interleukin-6(IL-6),γ-interferon(IFN-γ)in the colonic tissue were detected by enzyme-linked immunosorbent assay(ELISA),the histopathological changes of colon were observed by hematoxylin-eosin(HE)staining,flow cytometry was used to detect the proportion changes of CD4+,CD8+and regulatory T cell(Treg)in peripheral blood.Results:Compared with the control group,the model group showed significantly decrease in fecal number in 24 h,fecal quality and fecal water rate(P＜0.01),the colon was seen to have necrotic shedding of mucosal epithelium,localized intestinal glands in the lamina propria were degenerated,necrotic and atrophied,a few lymphocytes were seen to infiltrate in the necrotic area in a scattered manner,the contents of GAS and MTL,the proportions of CD4+,CD8+and Treg were significantly reduced(P＜0.01),the contents of IL-6 and IFN-γ were significantly elevated(P＜0.05,P＜0.01).Compared with the model group,the fecal number in 24 h,fecal quality and fecal water rate of high-dose groups of RH and PRH were significantly increased(P＜0.05,P＜0.01),the pathological damage of the colon was alleviated to varying degrees,the contents of GAS,MTL,IL-6 and IFN-γ were significantly regressed(P＜0.05,P＜0.01),and the proportions of CD4+and CD8+were significantly increased(P＜0.01),although the proportion of Treg showed an upward trend,there was no significant difference.In addition,the results of intestinal flora showed that the number of amplicon sequence variant(ASV)and Alpha diversity were decreased in the model group compared with the control group,and there was a significant difference in Beta diversity,with a decrease in the relative abundance of Lactobacillus and an increase in the relative abundances of Bacillus and Helicobacter.Compared with the model group,the ASV number and Alpha diversity were increased in the high-dose groups of RH and PRH,and there was a trend of regression of Beta diversity to the control group,the relative abundance of Lactobacillus increased,and the relative abundances of Bacillus and Helicobacter decreased.Conclusion:RH and PRH can improve dysbacteriosis,promote immune system activation,inhibit the release of inflammatory factors for enhancing the gastrointestinal function,which may be one of the potential mechanisms of their therapeutic effect on constipation.
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