基于16S rDNA测序探讨大黄䗪虫丸调控肠道菌群抑制大鼠肾纤维化的作用机制

Mechanism of Dahuang Zhechongwan in Inhibiting Renal Fibrosis in Rats by Regulating Intestinal Flora Based on 16S rDNA Sequencing

目的:从肠道菌群角度研究大黄䗪虫丸(DHZCW)对腺嘌呤诱导的肾纤维化大鼠的作用及机制。方法:将36只SD大鼠随机分为6组:空白组、模型组、大黄䗪虫丸高、中、低剂量组(0.168、0.084、0.042 g·kg^(-1))、吡非尼酮组(200 mg·kg^(-1)),每组6只。除空白组外,其余各组用腺嘌呤混悬液250 mg·kg^(-1)灌胃造模28 d,再进行药物干预4周,在代谢笼中收集大鼠尿液,测定肾功能指标尿素氮(BUN)、尿素(Urea)、尿肌酐(Crea)、胱抑素C(Cys C)、24小时尿蛋白(24 h TP)。收集肾脏样本并对其苏木素-伊红(HE)染色、马松(Masson)染色,观察各组大鼠肾组织病理变化,采用蛋白免疫印迹法(Western blot)检测关键效应蛋白α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(ColⅠ)、Ⅲ型胶原(ColⅢ)蛋白的表达量。收集大鼠粪便运用高通量测序16S rDNA技术对大鼠菌群进行物种差异性分析。结果:与空白组比较,模型组BUN、Urea、Crea、Cys C、24 h TP含量显著升高(P<0.01),与模型组比较,DHZCW高、中、低剂量组和吡非尼酮组BUN、Urea、Crea、Cys C、24 h TP均显著降低(P<0.01)。与空白组比较,模型组肾组织中出现明显纤维化改变,α-SMA、ColⅠ、ColⅢ蛋白含量也显著升高(P<0.01);与模型组比较,DHZCW高剂量组和吡非尼酮组组织结构基本正常,未见明显病理损伤改变,DHZCW中、低剂量组可见纤维化改变,低剂量组较为明显;DHZCW高、中、低剂量组和吡非尼酮组中的α-SMA、ColⅠ、ColⅢ蛋白含量显著降低(P<0.01),说明DHZCW能有效减少胶原异常沉积而抑制肾纤维化。肠道菌群来看,门水平上,与空白组比较,模型组中厚壁菌门(Firmicutes)的丰度明显增加,拟杆菌门(Bacteroidetes)丰度减少,二者比例明显失调。科水平上,模型组降低了毛螺菌科(Lachnospiraceae)、前蹄菌科(Prevotellaceae)、未分类拟杆菌科(Bacteroidota_unclassified),增加了瘤胃球菌科(Ruminococcaceae)、乳杆菌科(Lactobacillaceae)、颤螺旋菌科(Oscillospiraceae)菌群的丰度。属水平上,模型组的未分类厚壁菌(Firmicutes_unclassified)、未分类拟杆菌属(Bacteroidota_unclassified)、普雷沃氏菌属_UCG-001(Prevotellaceae_UCG-001)等菌群丰度显著降低,UCG-005、梭状芽孢杆菌(Clostridia_UCG-014_unclassified)等菌群丰度增加。与模型组比较,DHZCW能降低潜在致病菌而升高有益菌丰度,调节肠道菌群。结论:DHZCW能有效改善肾功能和抑制肾纤维化,其作用机制可能与调节肠道菌群有关。

Objective:To investigate the effect and mechanism of Dahuang Zhechongwan(DHZCW)on adenine-induced renal fibrosis in rats from the perspective of intestinal flora.Method:Thirty-six SD rats were randomly divided into a blank group,a model group,and high-,medium-and low-dose DHZCW groups(0.168,0.084,0.042 g·kg^(-1)),and a pirfenidone group(200 mg·kg^(-1)),with 6 rats in each group.Except for those in the blank group,rats in other groups were treated with adenine suspension(250 mg·kg^(-1))by gavage for 28 days for renal fibrosis model induction.Subsequently,they received drug intervention for 4 weeks.Urine samples were collected from rats in metabolic cages,and renal function indicators including blood urea nitrogen(BUN),urea,creatinine(Crea),cystatin C(Cys C),and 24-hour urine protein(24 h TP)were measured.Kidney samples were collected and subjected to hematoxylin-eosin(HE)staining and Masson's trichrome staining to observe the pathological changes in rat renal tissues.Western blot was used to detect the expression levels of key effector proteinsα-smooth muscle actin(α-SMA),typeⅠcollagen(ColⅠ),and typeⅢcollagen(ColⅢ)in the kidneys.High-throughput sequencing of 16S rDNA was used to analyze the species diversity of rat intestinal flora.Result:Compared with the blank group,the model group showed increased BUN,urea,Crea,Cys C,and 24 h TP levels(P<0.01).Compared with the model group,the high-,medium-,and low-dose DHZCW groups,as well as the pirfenidone group,showed significant reductions in BUN,urea,Crea,Cys C,and 24 h TP levels(P<0.01),indicating that DHZCW intervention significantly improved renal function.In the model group,renal tissues exhibited significant fibrotic changes,and the protein levels ofα-SMA,ColⅠ,and ColⅢwere significantly increased(P<0.01)compared to those in the blank group.Compared with the model group,the high-dose DHZCW group and the pirfenidone group had relatively normal tissue structure,with no significant pathological damage observed.However,fibrotic changes were observed in the medium-and low-dose DHZCW groups,with the changes being more significant in the low-dose group.The protein levels ofα-SMA,ColⅠ,and ColⅢwere significantly decreased in the high-,medium-,and low-dose DHZCW groups,as well as the pirfenidone group(P<0.01),indicating that DHZCW effectively reduced abnormal collagen deposition and inhibited renal fibrosis.From the perspective of intestinal flora,at the phylum level,compared with the blank group,the model group showed a significant increase in the abundance of Firmicutes and a decrease in Bacteroidetes,leading to a significant imbalance in their ratio.At the family level,the model group decreased the abundance of Lachnospiraceae,Prevotellaceae,and Bacteroidota_unclassified,and increased the abundance of Ruminococcaceae,Lactobacillaceae,and Oscillospiraceae.At the genus level,the model group showed significantly reduced abundance of Firmicutes_unclassified,Bacteroidota_unclassified,and Prevotellaceae_UCG-001,etc.,and increased abundance of UCG-005,Clostridia_UCG-014_unclassified,etc.Compared with the model group,DHZCW effectively reduced the abundance of potential pathogenic bacteria and increased the abundance of beneficial bacteria,regulating the intestinal flora.Conclusion:DHZCW can effectively improve renal function and inhibit renal fibrosis,and its mechanism of action may be related to the regulation of intestinal flora.