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

LIANG Jingtao; WANG Yao; HE Xiaoyan; LI Xin; HUANG Jing; GU Zhengzheng; XIAO Jingyi; WU Lijuan

doi:10.13422/j.cnki.syfjx.20231236

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

更新时间：2023-10-20

- Mechanism of Dahuang Zhechongwan in Inhibiting Renal Fibrosis in Rats by Regulating Intestinal Flora Based on 16S rDNA Sequencing
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 22, Pages: 37-46(2023)
- 作者机构：
  
  1.成都中医药大学附属医院，成都 610072
  2.成都中医药大学公共卫生学院，成都 610075
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20231236
  CLC： R2-0;R33;R569.2;R289
- Published：20 November 2023，
  
  Published Online：01 September 2023，
  
  Received：13 July 2023，
- 稿件说明：
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梁静涛,王尧,何晓艳等.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[J].中国实验方剂学杂志,2023,29(22):37-46.

LIANG Jingtao,WANG Yao,HE Xiaoyan,et al.Mechanism of Dahuang Zhechongwan in Inhibiting Renal Fibrosis in Rats by Regulating Intestinal Flora Based on 16S rDNA Sequencing[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(22):37-46.
梁静涛,王尧,何晓艳等.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[J].中国实验方剂学杂志,2023,29(22):37-46. DOI： 10.13422/j.cnki.syfjx.20231236.

LIANG Jingtao,WANG Yao,HE Xiaoyan,et al.Mechanism of Dahuang Zhechongwan in Inhibiting Renal Fibrosis in Rats by Regulating Intestinal Flora Based on 16S rDNA Sequencing[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(22):37-46. DOI： 10.13422/j.cnki.syfjx.20231236.

摘要

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

-平滑肌肌动蛋白（

-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（

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（

0.01）。与空白组比较，模型组肾组织中出现明显纤维化改变，

-SMA、ColⅠ、ColⅢ蛋白含量也显著升高（

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

-SMA、ColⅠ、ColⅢ蛋白含量显著降低（

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。

Abstract

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 proteins α-smooth muscle actin （α-SMA）， type Ⅰ collagen （ColⅠ）， and type Ⅲ collagen （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 （

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 （

0.01）， indicating that DHZCW intervention significantly improved renal function. In the model group， renal tissues exhibited significant fibrotic changes， and the protein levels of α-SMA， ColⅠ， and ColⅢ were significantly increased （

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 of

-SMA， ColⅠ， and ColⅢ were significantly decreased in the high-， medium-， and low-dose DHZCW groups， as well as the pirfenidone group （

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.

关键词

大黄䗪虫丸肾纤维化16S rDNA肠道菌群

Keywords

Dahuang Zhechongwanrenal fibrosis16S rDNAintestinal flora

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