Chengyu Xiang1, Yi Yan2, Dingguo Zhang3. 1. Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nangjing, China. 2. Department of Cardiology, Jiangyin People's Hospital, Jiangyin, China. 3. Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nangjing, China. Electronic address: zhdg0223@126.com.
Abstract
OBJECTIVE: Treatment with the chemotherapeutic agent, doxorubicin (DOX), is limited by side effects. We have previously demonstrated that fasudil, a Rho/ROCK inhibitor, has antioxidant, anti-inflammatory and anti-apoptotic effects in contrast-induced acute kidney injury model. The present study to investigated the possible protective effect of fasudil, on DOX-induced nephrotoxicity. MATERIALS AND METHOD: In vivo: Forty male C57BL/6 male mice were randomly divided into 4 groups: Control group, DOX treatment group (DOX group), DOX + low dose fasudil (DOX + L group), DOX + high dose fasudil (DOX + H group). Mice in 2-4 groups received DOX (2.5 mg/kg, i.p.) once a week for 8 weeks. The 3 and 4 group were given 2 mg/kg/d or 10 mg/kg/d fasudil before DOX injection. respectively. Meanwhile, the control group received saline. At the end of week eight, blood samples were collected for biochemical testing. The kidneys were removed for histological, immunohistochemical, Western blot, quantitative real-time PCR (qRT-PCR), and molecular detection. In vitro: NRK-52E cells were treated with 40 uM fasudil for 12 h, then incubated with 1 uM DOX for 24 h. Cells then collected for qRT-PCR and Western blot. RESULTS: In vivo, fasudil treatment ameliorated DOX-induced immunofluorescence reaction of DNA damage-related factors (8-OHdG), decreased the expression of Bax, Caspase-3, p16, p21 and p53, and increased the expression of protein of Bcl-2, Bmi-1 and Sirt-1. In the mouse model, administration of fasudil significantly ameliorated DOX-induced kidney damage, suppressed cell apoptosis and senescence, ameliorated redox imbalance and DNA damage. At the same time, DOX produced obvious kidney damage revealed by kidney functions changes: increased serum creatinine (SCr) and blood urea nitrogen (BUN) concentrations. In addition, kidney tissue staining in the DOX group showed abnormal structure and fibroproliferative disorders. And DOX could promote the oxidation and senescence of kidney cells, leading to increased expression of 8-OHdG and senescence and apoptosis-related factors. On the contrary, fasudil treatment can effectively inhibit redox imbalance and DNA damage caused by DOX, and inhibit cell senescence and apoptosis. Fasudil can inhibit excessive activation of Rho/ROCK signaling pathway, thereby improving kidney tissue fibrosis and recovery kidney function. CONCLUSION: Fasudil has a protective effect on DOX-induced nephrotoxicity in mice and NRK-52E cells, which can inhibit oxidative stress and DNA damage, inhibit apoptosis, and delays cell senescence by inhibiting RhoA/Rho kinase (ROCK) signaling pathway.
OBJECTIVE: Treatment with the chemotherapeutic agent, doxorubicin (DOX), is limited by side effects. We have previously demonstrated that fasudil, a Rho/ROCK inhibitor, has antioxidant, anti-inflammatory and anti-apoptotic effects in contrast-induced acute kidney injury model. The present study to investigated the possible protective effect of fasudil, on DOX-induced nephrotoxicity. MATERIALS AND METHOD: In vivo: Forty male C57BL/6 male mice were randomly divided into 4 groups: Control group, DOX treatment group (DOX group), DOX + low dose fasudil (DOX + L group), DOX + high dose fasudil (DOX + H group). Mice in 2-4 groups received DOX (2.5 mg/kg, i.p.) once a week for 8 weeks. The 3 and 4 group were given 2 mg/kg/d or 10 mg/kg/d fasudil before DOX injection. respectively. Meanwhile, the control group received saline. At the end of week eight, blood samples were collected for biochemical testing. The kidneys were removed for histological, immunohistochemical, Western blot, quantitative real-time PCR (qRT-PCR), and molecular detection. In vitro: NRK-52E cells were treated with 40 uM fasudil for 12 h, then incubated with 1 uM DOX for 24 h. Cells then collected for qRT-PCR and Western blot. RESULTS: In vivo, fasudil treatment ameliorated DOX-induced immunofluorescence reaction of DNA damage-related factors (8-OHdG), decreased the expression of Bax, Caspase-3, p16, p21 and p53, and increased the expression of protein of Bcl-2, Bmi-1 and Sirt-1. In the mouse model, administration of fasudil significantly ameliorated DOX-induced kidney damage, suppressed cell apoptosis and senescence, ameliorated redox imbalance and DNA damage. At the same time, DOX produced obvious kidney damage revealed by kidney functions changes: increased serum creatinine (SCr) and blood ureanitrogen (BUN) concentrations. In addition, kidney tissue staining in the DOX group showed abnormal structure and fibroproliferative disorders. And DOX could promote the oxidation and senescence of kidney cells, leading to increased expression of 8-OHdG and senescence and apoptosis-related factors. On the contrary, fasudil treatment can effectively inhibit redox imbalance and DNA damage caused by DOX, and inhibit cell senescence and apoptosis. Fasudil can inhibit excessive activation of Rho/ROCK signaling pathway, thereby improving kidney tissue fibrosis and recovery kidney function. CONCLUSION:Fasudil has a protective effect on DOX-induced nephrotoxicity in mice and NRK-52E cells, which can inhibit oxidative stress and DNA damage, inhibit apoptosis, and delays cell senescence by inhibiting RhoA/Rho kinase (ROCK) signaling pathway.
Authors: Nonhlakanipho F Sangweni; Derick van Vuuren; Lawrence Mabasa; Kwazi Gabuza; Barbara Huisamen; Sharnay Naidoo; Reenen Barry; Rabia Johnson Journal: Front Cardiovasc Med Date: 2022-06-22