基于肠道菌群探讨醋炙对乳香治疗溃疡性结肠炎的增效机制

Exploring Synergistic Mechanism of Processing with Vinegar on Olibanum in Treatment of Ulcerative Colitis Based on Intestinal Flora

目的:基于肠道菌群角度探讨乳香醋炙前后对甘氨脱氧胆酸(GDCA)干预溃疡性结肠炎(UC)小鼠的改善作用及其醋炙增效机制。方法:30只C57BL/6J雄性小鼠随机分为正常组、模型组、GDCA组、乳香组(1.5 g·kg^(-1))、醋乳香组(1.5 g·kg^(-1)),每组6只。正常组自由饮水,其余组按周期自由摄入2%葡聚糖硫酸钠(DSS)建立UC小鼠模型,造模期间GDCA组、乳香组、醋乳香组腹腔注射GDCA(0.05 g·kg^(-1))进行干预;于造模后第13天起,乳香组、醋乳香组灌胃给予相应剂量药物,1次/d,连续给药36 d,期间记录小鼠体质量,评估疾病活动指数(DAI)。第48天,收集粪便进行16S r RNA和宏基因组测序分析肠道菌群变化。第49天取小鼠结肠,采用苏木素-伊红(HE)染色法观察小鼠结肠组织病变,酶联免疫吸附测定法(ELISA)检测小鼠血清中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6含量。采用Spearman相关性分析探讨差异菌种和炎症因子水平的相关性。结果:与正常组比较,模型组体质量显著降低(P<0.01),DAI评分明显升高(P<0.05),TNF-α、IL-1β和IL-6水平显著升高(P<0.01),结肠部分炎性细胞浸润;与模型组比较,GDCA组小鼠体质量显著降低、DAI显著升高,TNF-α、IL-1β和IL-6水平显著升高(P<0.01),结肠隐窝结构严重破坏,大量炎性细胞浸润且杯状细胞明显减少;与GDCA组比较,乳香组、醋乳香组体质量明显升高、DAI评分显著降低、TNF-α、IL-6和IL-1β水平明显降低(P<0.05,P<0.01),且醋乳香的调节作用明显优于乳香。Alpha多样性显示,GDCA干预后UC小鼠Chao1指数显著升高(P<0.01),Shannon指数明显降低(P<0.05);Beta多样性显示,5组菌群结构具有明显变化,乳香、醋乳香可一定程度回调GDCA干预后UC小鼠肠道菌群结构变化。菌群测序结果显示,与正常组比较,模型组厚壁菌门/拟杆菌门值明显升高(P<0.05),3个菌属和5个菌种相对丰度发生明显变化(P<0.05,P<0.01);与模型组比较,GDCA组厚壁菌门/拟杆菌门值明显升高(P<0.05),7个致病菌属和4个菌种相对丰度明显增加(P<0.05,P<0.01),3个有益菌属和Bacteroides_intestinalis相对丰度明显降低(P<0.05,P<0.01),乳香组和醋乳香组可回调厚壁菌门/拟杆菌门值、致病菌和有益菌相对丰度,且醋乳香组对厚壁菌门/拟杆菌门值、3个菌属和5个菌种相对丰度的回调作用明显优于乳香(P<0.05,P<0.01)。相关性分析显示,Bacteroides_intestinalis相对丰度与TNF-α、IL-6、IL-1β水平呈负相关;Prevotella_sp_CAG873、Bacteroides_sp_CAG927、Bacteroidales_bacterium_52_46、Bacteroidales_bacterium相对丰度与TNF-α、IL-6、IL-1β水平呈正相关。结论:GDCA可加重UC结肠炎症,乳香与醋乳香对于GDCA介导的UC均具有改善作用,以醋乳香作用更强,其作用机制可能与调节肠道有益菌和致病菌的丰度和结构、降低炎症因子水平有关。

Objective:To explore the improving effects and its synergistic mechanism of Olibanum before and after processing with vinegar on glycodesoxycholic acid(GDCA)intervention in mice with ulcerative coliti(s UC)based on the perspective of intestinal flora.Method:C57BL/6J male mice were randomly divided into the normal group,model group,GDCA group,Olibanum group(1.5 g·kg^(-1))and vinegar-processed Olibanum(1.5 g·kg^(-1))group,with 6 mice in each group.Mice in the normal group drank water freely,and mice in the other groups were given 2%dextran sulfate sodium(DSS)periodically to establish a UC mouse model.During the modeling,GDCA group,Olibanum group and vinegar-processed Olibanum group were intervened by intraperitoneally injection of GDCA(0.05 g·kg^(-1)).From the 13th day after modeling,Olibanum group and vinegar-processed Olibanum group were given the corresponding doses of drugs by gavage,once a day,for 36 d.During this period,the body mass of mice was recorded and the disease activity index(DAI)was assessed.On day 48,faeces were collected for 16S rRNA and metagenomic sequencing to analyse changes in intestinal flora.On the 49th day,hematoxylin-eosion(HE)staining was used to observe the colon histological lesions,enzyme-linked immunosorbent assay(ELISA)was used to determine serum levels of tumour necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6,and Spearman correlation analysis was used to explore the correlation between differential bacterial species and inflammatory factor levels.Result:Compared with the normal group,the model group showed a significant decrease in body weigh(t P<0.01),a significant increase in DA(I P<0.05),and a significant increase in TNF-α,IL-1βand IL-6 levels(P<0.01),and there was partial infiltration of inflammatory cells in the colon.Compared with the model group,mice in the GDCA group showed a significant decrease in body weight,a significant increase in DAI and levels of TNF-α,IL-1βand IL-6(P<0.01),and severe disruption of colonic crypt structure,extensive infiltration of inflammatory cells,and a significant decrease in goblet cells.Compared with the GDCA group,both the Olibanum and vinegar-processed Olibanum groups showed a significant recovery in body weight,a significant decrease in DAI and levels of TNF-α,IL-6 and IL-1β(P<0.05,P<0.01),and the modulating effect of vinegar-processed Olibanum was significantly better than that of Olibanum.Alpha diversity showed that Chao1 index of UC mice significantly increased(P<0.01)and Shannon index decreased significantly(P<0.05)in UC mice after GDCA intervention.Beta diversity showed that the microbial community structure of the 5 groups had significant changes,Olibanum and vinegar-processed Olibanum could modulate the changes in the structure of the intestinal flora in UC mice after GDCA intervention.Microbial sequencing results indicated that,compared with the normal group,the Firmicutes/Bacteroidetes ratio in the model group was significantly highe(r P<0.05),and the relative abundance of 3 genera and 5 species of flora changed significantly(P<0.05,P<0.01).Compared with the model group,the Firmicutes/Bacteroidetes ratio in the GDCA group was significantly highe(r P<0.05),the relative abundance of 7 pathogenic bacterial genera and four species was significantly increased(P<0.05,P<0.01),and the relative abundance of three beneficial bacterial genera and Bacteroides_intestinalis was significantly decreased(P<0.05,P<0.01).Olibanum group and vinegar-processed Olibanum group could modulate the Firmicutes/Bacteroidetes ratio,the relative abundance of pathogenic bacteria and beneficial bacteria,and the vinegar-processed Olibanum group was significantly superior to Olibanum group in terms of modulating the Firmicutes/Bacteroidetes ratio,the relative abundance of the three genera and five species of bacteria(P<0.01,P<0.05).Correlation analysis showed that the relative abundance of Bacteroides_intestinalis was negatively correlated with the levels of TNF-α,IL-6 and IL-1β,the relative abundance of Prevotella_sp_CAG873,Bacteroides_sp_CAG927,Bacteroidales_bacterium_52_46 and Bacteroidales_bacterium was positively correlated with TNF-α,IL-6 and IL-1βlevels.Conclusion:GDCA can exacerbate UC colonic inflammation,and Olibanum and vinegar-processed Olibanum have an ameliorative effect on GDCA-mediated UC,with the vinegar-processed Olibanum showing a stronger ameliorative effect,the mechanism may be related to the regulation the abundance and structure of intestinal beneficial and pathogenic bacteria,and the reduction of inflammatory factor levels.