半夏泻心汤对DSS诱导的溃疡性结肠炎小鼠肠道微生态及Th17/Treg细胞平衡的影响

Effect of Banxie Xiexin Decoction on Gut Microbiota and Th17/Treg Cell Balance in Mice with Ulcerative Colitis Induced by Dextran Sodium Sulfate

目的观察半夏泻心汤对饮用葡聚糖硫酸钠(DSS)诱导建立的溃疡性结肠炎(UC)模型小鼠Th17/Treg细胞平衡及肠道微生态的影响,从免疫失衡及菌群紊乱角度分析半夏泻心汤治疗UC的作用机制。方法将40只C57BL/6J雄性小鼠随机分为空白组、模型组、阳性药(美沙拉嗪)组及半夏泻心汤组,除空白组外的各小鼠采用2.5%DSS水溶液自由饮用7天诱导建立UC小鼠模型。自实验第8日起,分别采用灭菌水、美沙拉嗪水溶液、半夏泻心汤水溶液给上述各组别小鼠灌胃。采用HE染色观察结肠组织病理学变化,ELISA方法检测组织炎症因子含量,流式细胞术检测Th17及Treg细胞的相对数量,16S rRNA测序技术进一步检测模型小鼠粪便中菌群的变化。结果与空白组比较,模型组小鼠结肠损伤严重,血清炎症因子含量显著升高(P<0.01);与模型组比较,中药干预组小鼠结肠损伤减轻,血清TNF-α、IFN-γ含量显著下降(P<0.05)。较空白组小鼠相比,模型组小鼠Th17细胞百分比显著升高(P<0.01),而Treg细胞百分比显著降低(P<0.01)。经半夏泻心汤干预后,其小鼠Th17细胞数量均显著降低(P<0.05),Treg细胞数量则显著升高(P<0.05)。同时,半夏泻心汤可升高门水平上Firmicutes、属水平上Lachnospiraceae_NK4A136_group、unclassified_f_Lachnospiraceae、Prevotellaceae_UCG-001、Alistipes;降低门水平上Actinobacteriota,科水平上Bifidobacteriaceae、Atopobiaceae、Clostridiaceae,属水平上Clostridium_sensu_stricto_1、Coriobacteriaceae_UCG-002、Bifidobacterium的相对丰度。结论半夏泻心汤可使Treg细胞相对数量升高、Th17细胞相对数量降低,以维持Th17/Treg细胞动态平衡;同时可降低有害菌Clostridium_sensu_stricto_1、Coriobacteriaceae_UCG-002的相对丰度,升高产短链脂肪酸类菌Lachnospiraceae_NK4A136_group、Prevotellaceae_UCG-001、unclassified_f_Lachnospiraceae等菌属的相对丰度,调控肠道微生物结构及动态平衡,从而发挥治疗UC的作用。

Objective To observe the effect of Banxia Xiexin Decoction on Th17/Treg cell balance and intestinal microecology in DSS-UC(Model of ulcerative colitis induced by free consumption of Dextran sulfate sodium)mice,and to explore mechanism of treating ulcerative colitis from the perspective of immune imbalance and flora disorder.Methods Forty C57BL6/J mice were randomly divided into control group,model group,positive drug group and Banxia Xiexin Decoction group.Except for control group,each mouse was freely drunk with 2.5%DSS aqueous solution for7 days to induce UC model.On the 8th day,normal saline,mesalazine,and Banxia Xiexin Decoction aqueous solution were given for 14 days.Finally,HE staining was used to observe the histopathological changes of the colon,ELISA to detect the content of tissue inflammatory factors,flow cytometry to detect the relative number of Th17 and Treg cells,and16S rRNA sequencing technology to detect the changes of fecal intestinal flora.Results Compared with control group,the mice in model group had severe colon damage,and the contents of TNF-αand IFN-γlevels in serum were significantly increased(P<0.01).Compared with model group,the mice in Banxia Xiexin Decoction group had less colon damage.The serum levels of TNF-αand IFN-γdecreased significantly(P<0.05).Compared with control group,the percentage of Th17 cells in model group was significantly increased(P<0.01),while Treg cells was significantly reduced(P<0.01).After intervention of Banxia Xiexin Decoction,the number of Th17 cells decreased significantly(P<0.05),and Treg cells increased significantly(P<0.05).At the same time,Banxia Xiexin Decoction increased the richness of Firmicutes at the phylum level,and of Lachnospiraceae_NK4A136_group,unclassified_f_Lachnospiraceae,Prevotellaceae_UCG-001,Alistipes,unclassified_f_Lachnospiraceae,Prevotellaceae_UCG-001,Alistipes at the genus level;while reduced the richness of Actinobacteriota at the phylum level,of Bifidobacteriaceae,Atopobiaceae,Clostridiaceae at the family level,and of Bifidobacterium,Coriobacteriaceae_UCG-002,Clostridium_sensu_stricto_1 at the genus level.Conclusion Banxia Xiexin Decoction can maintain the balance of Th17/Treg cells by increasing the relative number of Treg cells and reducing the relative number of Th17 cells.At the same time,it can reduce the richness of harmful bacteria Coriobacteriaceae_UCG-002 and Clostridium_sensu_stricto_1,and increase the richness of bacteria such as Lachnospiraceae_NK4A136_group,unclassified_f_Lachnospiraceae,Prevotellaceae_UCG-001,which could produce short-chain fatty acids to regulate microbial structure and dynamic balance of the intestinal tract,thereby playing a role in the treatment of ulcerative colitis.