OBJECTIVE To investigate the effect of Shenshuai capsule on the Keap1/Nrf2/HO-1 antioxidant stress pathway and its renal protective role in spontaneously hypertensive rat(SHR).

METHODS Twelve 16-week-old WKY rats and 72 SHRs were acclimatized for 1 week. The WKY rats served as the blank group. The 72 SHRs were randomly divided into the model group, valsartan group(0.0072 g·kg⁻¹), Haikun Shenxi capsule group(0.1188g·kg⁻¹), and Shenshuai capsule groups at high, medium, and low doses(0.2160, 0.1080, 0.0540 g·kg⁻¹). Rats in the blank and model groups were intragastric administered an equivalent volume of 0.5% carboxymethylcellulose sodium solution. During the drug intervention period, the general condition of rats in each group was observed. Systolic blood pressure(SBP) and diastolic blood pressure(DBP) were monitored at specific time points before administration and every 2 weeks after administration. After 8 consecutive weeks of drug intervention, urinary albumin(ALB) and creatinine(Cr) were measured using a fully automated biochemical analyzer, and the ALB/Cr was calculated. Serum levels of malondialdehyde(MDA), NADPH oxidase 4(NOX4), and glutathione(GSH) activity were detected using ELISA. Renal histopathological changes were observed via HE staining. The protein levels of Keap1, Nrf2, and HO-1 in renal tissue were detected by Western blotying. The mRNA levels of Keap1, Nrf2, and HO-1 were determined by quantitative real-time PCR(qPCR). The cellular localization and distribution of Keap1, Nrf2, and HO-1 proteins were assessed by immunofluorescence.

RESULTS Compared with the blank group, the model group showed increased SBP and DBP, elevated urinary ALB/Cr levels, increased serum MDA and NOX4, decreased serum GSH levels, and structural damage to the kidneys characterized by focal glomerulosclerosis, turbid swelling of renal tubules, and significant renal interstitial fibrosis. In renal tissue, the protein and mRNA expression levels of Keap1 were increased, and its immunofluorescence intensity was enhanced; the protein and mRNA expression levels, and immunofluorescence intensity of Nrf2 and HO-1 were decreased. the levels of SBP, DBP and urinary ALB/Cr were significantly decreased in each treatment group(P<0.05). The valsartan group, the high and low dose Shenshuai capsule groups, and the Haikun Shenxi capsule group exhibited decreased serum MDA and NOX4 levels, and the high dose Shenshuai capsule group showed increased serum GSH activity(P<0.05), renal tissue damage was alleviated. Western blotting results showed that Keap1 expression was significantly inhibited in the valsartan group, Haikun Shenxi capsule group, and medium dose Shenshuai capsule group(P<0.05). Nrf2 protein levels were significantly upregulated in the valsartan group and the medium and low dose Shenshuai capsule groups(P<0.05). HO-1 protein levels were significantly upregulated in the valsartan group and the high and low dose Shenshuai capsule groups(P<0.05). Results of qPCR revealed that all treatment groups significantly downregulated the mRNA expression levels of Keap1, and upregulated the mRNA expression of Nrf2 as well as its downstream target gene HO-1(P<0.05 or P<0.01). Immunofluorescence results suggested that compared with the model group, Keap1 protein expression was significantly reduced in all treatment groups(P<0.05 or P<0.01). HO-1 protein expression was significantly increased in the valsartan group and different dose groups of Shenshuai capsule(P<0.05). Nrf2 protein expression was significantly increased in the valsartan group, Haikun Shenxi capsule group, and the high and medium dose Shenshuai capsule groups(P<0.05 or P<0.01). Furthermore, immunofluorescence localization observation revealed that drug intervention promoted the translocation and nuclear accumulation of Nrf2 from the cytoplasm.

CONCLUSION Shenshuai capsule may regulate the Keap1/Nrf2/HO-1 antioxidant stress pathway, inhibit the expression of Keap1, promote the nuclear translocation of Nrf2, and upregulate HO-1 expression. It can alleviate oxidative stress and reduce the urinary ALB/Cr, exert antioxidant and renal fibrosis-inhibiting effects, thereby further protecting renal tissues in SHRs.