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This study investigated the renal protection effect and mechanism of Zuogui Jiangtang Yishen Prescription(ZGJTYSF) for db/db mice with diabetic kidney disease(DKD), focusing on the semaphorin 3a(Sema3a)/vascular endothelial growth factor A(VEGFA)/vascular endothelial growth factor receptor 2(VEGFR2) pathway. A DKD mouse model was established using 50 db/db mice(6 weeks, males) and 10 age-matched male db/m mice after 8 weeks on a regular diet. The db/db mice were randomly divided into 5 groups: the model group, the western medicine(dapagliflozin, 0.001 3 g·kg~(-1)) group, and the low-(6.4 g·kg~(-1)), medium-(12.8 g·kg~(-1)), and high-dose(25.6 g·kg~(-1)) ZGJTYSF groups, with 10 mice in each group. Another 10 db/m mice were set as the normal group. For the normal and model groups, the mice were administrated an equal amounts of saline by gavage. After intervention for 8 consecutive weeks, fasting blood glucose(FBG), urinary albumin-to-creatinine ratio(UACR), and liver and kidney function changes were detected in mice. Histopathological changes in kidney tissue of each group were observed by hematoxylin-eosin(HE), periodic acid-Schiff(PAS) staining, Masson staining, and transmission electron microscopy(TEM). Quantitative real-time polymerase chain reaction(real-time PCR) was used to detect the expression levels of glomerular endothelial cell marker(CD34), Sema3a, VEGFA, and VEGFR2 mRNA in renal tissues of each group, and Western blot was applied to measure the expression levels of CD34, Sema3a, VEGFA, and VEGFR2 proteins in renal tissues of each group. The results showed that compared with the normal group, mice in the model group had significantly higher FBG(P<0.05), significantly higher UACR(P<0.05), and significantly higher CD34, Sema3a, VEGFA, VEGFR2 mRNA expression and protein expression(P<0.05). The model group also showed an increased glomerular volume, fused podocyte protrusions, swollen endothelial cells, narrowed and damaged capillary lumen, increased stroma, and thickened glomerular basement membrane. After intervention, ZGJTYSF groups reduced FBG and UACR in DKD mice compared with the model group, which was positively correlated with the administration dosage, and the difference was statistically significant at the 8th week of administration(P<0.05). Compared with the model group, ZGJTYSF groups and the western medicine group improved glomerular and endothelial cells and podocyte injury. They could reduce CD34, Sema3a, VEGFA, VEGFR2 mRNA expression in kidney tissues, and the expression of CD34 and Sema3a mRNA was significantly reduced(P<0.05). They could also reduce the expression of CD34, Sema3a, VEGFA, and VEGFR2 proteins, and VEGFA proteins expression was significantly reduced in the dapagliflozin group and the medium-dose group of ZGJTYSF(P<0.05), Sema3a and VEGFR2 proteins expression were significantly reduced in the low-dose group of ZGJTYSF(P<0.05). The study revealed that ZGJTYSF can delay the progression of DKD by inhibiting the Sema3a/VEGFA/VEGFR2 pathway, and its mechanism, besides improving glucose metabolism in mice, may be related to the improvement of podocyte and endothelial cell injury.