Mechanism of Dahuang Zhechongwan in Inhibiting Renal Fibrosis in Rats by Regulating Intestinal Flora Based on 16S rDNA Sequencing / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate the effect and mechanism of Dahuang Zhechongwan （DHZCW） on adenine-induced renal fibrosis in rats from the perspective of intestinal flora. MethodThirty-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. ResultCompared 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 of α-SMA， ColⅠ， and ColⅢ were 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 of α-SMA， ColⅠ， and ColⅢ were 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. ConclusionDHZCW can effectively improve renal function and inhibit renal fibrosis， and its mechanism of action may be related to the regulation of intestinal flora.