Studies On Protective Effects And Mechanisms Of Shen Kang Injection And Effective Substances On Chronic Renal Disease

【Background】 Chronic kidney disease(CKD) is associated with high mortality rate, and the prevalence is estimated to be 8–16% worldwide. It seriously endangers the health of patients, imposes enormous socioeconomic burdens on families and societies. Drugs for treatment of this disease are including diuretic, glucocorticoid, antihypertensive and immunosuppressive drugs. But these drugs have shortcomings, such as side effects and adverse reaction. Traditional Chinese medicine(TCM) has a long history for treatment of CKD. At present, Shen Kang injection(SKI), Shenkang pill and Astragalus-Angelica mixture are widely used in clinic. But due to the effective substance of these drugs is not clear, and the mechanism of action is unknown. In this study, we chose SKI as the research object. First, we determined the effect and mechanism of SKI using rat and cell model. Then according to the active ingredients have detected in SKI, we screened the most effective antioxidantand antifibrosis ingredients using in vitro and in vivo models to find the efficacy material, and explored the molecular mechanism for treatment of CKD.【Objectives】 1. To investigate the anti-fibrosis and anti-oxidation effects of SKI, and to research the potential mechanisms involved in the treatment of CKD. 2. To evaluate the anti-oxidative and anti-fibrosis effect of 10 traditional Chinese medicine monomers(TCMMs): rhein, emodin, chrysophanol, aloe-emodin, physcion, gallic acid, rosemarinic acid, protocatechuic aldehyde, danshensu and calycosin glycosides using in vitro and in vivo models, and find out the potential monomers for treating CKD. 3. To evaluate the effect and mechanism of rhein for treatment of CKD using 5/6Nx rats and HK-2 cell model.【Methods】 1. In experimental animal studies, CKD was established by 5/6 nephrectomy(5/6Nx). Serum creatinine(Scr) and blood urea nitrogen(BUN) were determined. Histopathologic tests were performed by HE and Masson trichrome stained. The protein expressions of fibronectin(FN), collagen Ι, α-smooth muscle actin(α-SMA) and transforming growth factor-β(TGF-β) and phosphorylation of Smad3 were measured in 5/6Nx rats. In HK-2 cells, the effects of TGF-β/Smad3 signaling pathway on renal fibrosis and oxidative injury were examined. 2. The anti-oxidative effect of the 10 TCMMs in SKI was examined by studying the inhibition of lipid peroxidation in rat liver microsomes. The anti-oxidative capacity was also evaluated by studying the free radical, and the levels of reactive oxygen species(ROS), malondialdehyde(MDA), superoxide dismutase(SOD), glutataione(GSH) and oxidized glutathione(GSSG) in HK-2 cells. Meanwhile the ability of anti-fibrosis was examined by measuring the expressions of transforming growth factor- beta(TGF-β) and connective tissue growing factor(CTGF).3. We measured renal interstitial pathologic injury, cell apoptosis and ultrastructural changes in 5/6Nx rats using HE, MASSON, TUNEL stain and electron microscope. And also measured the indication of renal fibrosis and oxidative by western blot. SIRT3 gene silencing of HK-2 cell model is established, and the expression protein of FOXO3α; oxidative stress related indicators: ROS, 8-OHd G, p47 phox and gp91phox; fibrosis related indicators: α-SMA, E-cadherin and TGF-β; and the expression of apoptosis related indicators were detected.【Results】 1. Treatment of 5/6Nx rats with SKI markedly reduced levels of Scr and BUN, alleviated the change of renal tissue morphology, decreased the expression of TGF-β/p-Smad3 signaling pathway and fibrosis associated signaling molecules, such as FN, collage Ι, α-SMA. Meanwhile, in HK-2 cells, after exposure to TGF-β and H2O2, the proteins expression of renal fibrosis and oxidative stress were significantly increased. Treatment with SKI, The generation of oxidative stress and fibrosis were reduced by inhibiting the production of TGF-β and p-Smad3. 2. The results demonstrated that these monomers of rhein, emodin, gallic acid, rosemarinic acid, protocatechuic aldehyde and danshensu were potent in inhibiting lipid peroxidation and scavenging of DPPH. Among them, the ability of decreased HK-2 cells oxidative stress and fibrosis of rhein, emodin and rosemarinic acid was better than the other 7 components. In addition, these three monomers could increase HK-2 cells viability and reduce the release of lactate dehydrogenase(LDH). 3. Rhein could improve renal tissue morphology and ultrastructural changes, reduce renal cell apoptosis, relieve renal interstitial fibrosis and oxidative damage in 5/6Nx. In HK-2 cells, rhein could improve cell oxidative stress, fibrosis, but does not reduce cell apoptosis by activating SIRT3/FOXO3α singnaling pathways.【Conclusions】 1. SKI inhibits renal fibrosis and oxidative stress through down-regulation of TGF-β/Smad3 signaling pathway. 2. Rhein, emodin and rosemarinic acid had the strong ability of anti-oxidative and anti-fibrosis, with the potential to be developed into vital candidate for treatment of CKD. 3. Rhein protected kidney against 5/6Nx- induced renal injury through inhibiting oxidative stress, renal fibrosis and apoptosis. Moreover, the anti-oxidative and anti-fibrotic effects of rhein were dependent on activation of SIRT3/FOXO3α signaling pathway.