Mechanism of Modified Zhenwutang in Delaying Renal Interstitial Fibrosis in Chronic Renal Failure Based on Ang Ⅱ/AT1R/NOX4 Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo explore the underlying mechanism of modified Zhenwutang in delaying renal interstitial fibrosis in chronic renal failure （CRF） by observing the effects of modified Zhenwutang on the expression of angiotensin Ⅱ （Ang Ⅱ）， angiotensin Ⅱ type 1 receptor （AT1R）， nicotinamide adenine dinucleotide phosphate （NADPH） oxidase 4 （NOX4）， transforming growth factor-β1 （TGF-β1）， type I collagen （COL1A1）， and type Ⅲ collagen （COL3A1） in the serum and renal tissues of adenine-induced CRF rats. MethodFifty male SPF-grade SD rats were randomly divided into a normal group （n=10） and an experimental group （n=40） using a random number table. After one week of adaptive feeding， the experimental CRF model was established in rats by administering adenine at 150 mg·kg-1·d-1 orally. Three rats from each group were randomly selected to evaluate the model induction. After successful modeling， rats in the experimental group were randomly divided into a model group， low-， medium， and high-dose modified Zhenwutang groups， and a benazepril hydrochloride group， with six rats in each group. The rats were orally administered the corresponding drugs once daily for four weeks. At the end of the first week， 13th week， and 17th week of the experiment， 24 hour urinary protein quantification （24 h-UTP） was measured. At the end of the 17th week， the rats were euthanized， and blood samples were collected from the abdominal aorta for the measurement of total protein （TP）， albumin （ALB）， creatinine （Cr）， and blood urea nitrogen （BUN） in the serum. Enzyme-linked immunosorbent assay （ELISA） was used to measure the expression levels of serum Ang Ⅱ. Hematoxylin-eosin （HE） staining and Masson's trichrome staining were performed to observe the pathological changes in renal tissues. Immunohistochemistry （IHC） was performed to observe the expression of AT1R， NOX4， TGF-β1， COL1A1， and COL3A1. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to observe the mRNA expression levels of AT1R， NOX4， and TGF-β1. Western blot was conducted to measure the protein expression levels of AT1R， NOX4， and TGF-β1. Result① Compared with the normal group， the model group showed a significant increase in 24 h-UTP （P<0.01）. The levels of Cr and BUN in the model group were significantly higher （P<0.01）， while the levels of TP and ALB were significantly lower （P<0.01）. The serum Ang Ⅱ level in the model group was significantly elevated （P<0.01）. The model group exhibited widening of the renal glomerular mesangial space， necrotic glomeruli， increased interstitial width with extensive inflammatory cell infiltration， brownish precipitates blocking the renal tubular lumens， irregular renal tubules， and significant deposition of collagen fibers in the renal interstitium. Additionally， the collagen fibers around the renal vessels， outside the parietal layer of the renal sacs， glomerular basement membrane， and tubular basement membrane increased significantly. The expression of AT1R and NOX4 in the glomeruli and renal tubules of the model group was significantly enhanced， and TGF-β1 expression also significantly increased in the renal tubules. The expression of COL1A1 and COL3A1 in the renal interstitium significantly increased. The mRNA expression of AT1R and TGF-β1 in the model group significantly increased （P<0.01）， while NOX4 mRNA expression significantly decreased （P<0.01）. The protein expression of AT1R， NOX4， and TGF-β1 was significantly enhanced （P<0.01）. ② Compared with the model group， modified Zhenwutang significantly reduced 24h-UTP （P<0.01）， decreased levels of Cr and BUN （P<0.01）， increased levels of TP and ALB （P<0.01）， reduced serum Ang Ⅱ level （P<0.01）， alleviated renal pathological damage， reduced expression of AT1R， NOX4， TGF-β1， COL1A1， and COL3A1 in the glomeruli， renal tubules， and renal interstitium， reduced mRNA expression of AT1R and TGF-β1 （P<0.01）， increased NOX4 mRNA expression （P<0.01）， and weakened protein expression of AT1R， NOX4， and TGF-β1 （P<0.01）. The modified Zhenwutang groups showed a significant dose-effect trend. ConclusionModified Zhenwutang may delay renal interstitial fibrosis in CRF rats by reducing the expression of Ang Ⅱ， AT1R， NOX4， and TGF-β1 in the serum and renal tissues， thereby alleviating renal pathological damage， reducing proteinuria， protecting renal function， and delaying the progression of CRF. The modified Zhenwutang group exhibited a dose-effect trend.