基于16S rDNA技术研究芍药甘草颗粒对肝郁脾虚证斑秃小鼠肠道菌群的影响

Effect of Shaoyao Gancao Granules on Intestinal Flora in Mice with Alopecia Areata and Syndrome of Liver Depression and Spleen Deficiency:A Study Based on 16S rDNA Technique

目的基于16S rDNA技术,从肠道菌群角度研究芍药甘草颗粒促进肝郁脾虚证斑秃(alopecia areata,AA)小鼠毛发再生的作用机制。方法将C57BL/6J小鼠随机分成空白组,模型组,芍药甘草颗粒高、中、低剂量组和阳性对照药组。除空白组外,其余各组均通过21 d慢性不可预知温和应激联合5%咪喹莫特乳膏方法构建肝郁脾虚证AA小鼠模型;在实验第8天进行药物干预:空白组、模型组给予生理盐水,芍药甘草颗粒高、中、低剂量组分别给予芍药甘草颗粒7.8、3.9、1.95 g/kg,阳性对照药组给予复方甘草酸苷片20 mg/kg,连续灌胃14 d。实验结束后,采用免疫组织化学法检测空白组、模型组小鼠皮损CD4^(+)、CD8^(+)T细胞表达情况,皮肤镜下观察小鼠皮损区毛发特征,各组采用悬尾实验和强迫游泳实验进行行为学测试,应用16S rDNA技术检测小鼠肠道菌群变化,通过PICRUSt2进行菌群功能预测,并对差异菌群与行为学测试进行相关性分析。结果模型小鼠皮损区的皮肤镜表现、免疫组织化学及行为学测试结果表明,芍药甘草颗粒可有效促进模型小鼠毛发再生,改善其宏观表征及行为学表现。与空白组比较,模型组小鼠肠道菌群厚壁菌门相对丰度显著降低,拟杆菌门相对丰度显著升高(P<0.05),Lactobacillus相对丰度显著降低(P<0.05),StaPhylococcus、Duncaniella及Paramuribaculum相对丰度显著升高(P<0.05);芍药甘草颗粒各剂量组对以上差异菌群的相对丰度均有显著的调节作用,可能涉及糖蛋白的生物合成与代谢、辅助因子和维生素代谢等多种代谢通路。强迫游泳实验与厚壁菌门相对丰度呈负相关,与Paramuribaculum相对丰度呈正相关。结论芍药甘草颗粒可有效促进肝郁脾虚证AA小鼠毛发再生,缓解其肝郁脾虚症状,其机制可能与调节肠道菌群构成,促进菌群平衡有关。

Objective To investigate the mechanism of action of Shaoyao Gancao Granules in promoting hair regenerationin mice with alopecia areata(AA)and syndrome of liver depression and spleen deficiency based on 16S rDNA technique from the perspective of intestinal flora.Methods C57BL/6J mice were randomly divided into blank group,model group,positive control drug group,and high-,middle-,and low-dose Shaoyao Gancao Granules groups.All mice except those in the blank group were treated with chronic unpredictable mild stress combined with 5%imiquimod cream for 21 days to establish a mouse model of AA with syndrome of liver depression and spleen deficiency.Drug intervention was initiated on day 8 of the experiment as follows:the mice in the blank group and the model group were given normal saline,those in the high-,middle-,and low-dose Shaoyao Gancao Granules groups were given Shaoyao Gancao Granules at a dose of 7.8、3.9、1.95 g/kg,respectively,and those in the positive control drug group were given compound glycyrrhizin tablets 20 mg/kg,by gavage for 14 consecutive days.After the experiment ended,immunohistochemistry was used to measure the expression levels of CD4^(+)and CD8^(+)T cells in skin lesion of the mice in the blank group and the model group;dermoscopy was used to observe the features of hair in the area of skin lesion;the tail suspension test(TST)and the forced swimming test(FST)were used for behavioral assessment;the 16S rDNA technique was used to measure the changes in intestinal flora,and PICRUSt2 was used to predict the function of intestinal flora;a correlation analysis was performed for differentially expressed flora and behavioral assessments.Results For the mice in the model group,dermoscopic findings of skin lesion,immunohistochemistry,and behavioral assessments showed that Shaoyao Gancao Granules effectively promoted hair regeneration and improved macroscopic representation and behavioral manifestation.Compared with the blank group,the model group had a significant reduction in the relative abundance of Firmicutes and a significant increase in the relative abundance of Bacteroidetes(P<0.05),as well as a significant reduction in Lactobacillus(P<0.05)and significant increases in StaPhylococcus,Duncaniella,and Paramuribaculum(P<0.05).Shaoyao Gancao Granules at different doses could significantly reverse the increases or reductions in the above differentially expressed flora,involving various metabolic pathways such as glycoprotein biosynthesis and metabolism and the metabolism of cofactors and vitamins.FST was negatively correlated with the relative abundance of Firmicutes and was positively correlated with Paramuribaculum.Conclusion For AA mice with syndrome of liver depression and spleen deficiency,Shaoyao Gancao Granules can effectively promote hair regeneration and alleviate the symptoms of liver depression and spleen deficiency,possibly by regulating the composition of intestinal flora and promote the balance of flora.