Alleviation of the doxorubicin-induced nephrotoxicity by fasudil in vivo and in vitro.

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.

Fasudil Ameliorates Osteoporosis Following Myocardial Infarction by Regulating Cardiac Calcitonin Secretion.

We hypothesis that Rho kinase inhibitor fasudil ameliorates osteoporosis following myocardial infarction (MI) by regulating cardiac calcitonin secretion. A mice model of MI and cultured neonatal cardiomyocytes exposed to hypoxia and serum deprivation (H/SD), and fibroblasts exposed to TGF-ß were used, respectively. Cardiac function in vivo was assessed with echocardiography. Osteoporosis in vivo was assessed with X-ray and micro-CT. In vivo and in vitro studies used histological and immunohistochemical techniques, along with western blots. In mice post-MI, fasudil ameliorates the microstructure and bone metabolism of the lumbar, improved cardiac function, and attenuated myocardial fibrosis. In vitro, fasudil or αCGRP could effectively inhibit the proliferation of primary fibroblasts treated with TGF-ß. Moreover, fasudil ameliorates the cardiac calcitonin secretion induced by MI in vivo or by H/SD in vitro. Our findings suggest that fasudil improved MI-induced osteoporosis by promoting cardiac secreting calcitonin.

Fasudil Protects Against Adriamycin-induced Acute Heart Injury by Inhibiting Oxidative Stress, Apoptosis, and Cellular Senescence.

BACKGROUND: The clinical utility of Adriamycin (ADR) is limited due to its toxicity, particularly cardiotoxicity. Therefore, effective cardioprotective adjuvants to minimize ADR-induced acute cardiotoxicity are urgently needed. Our previous studies have demonstrated the protective roles of fasudil on tissue injury. Here, we further explore whether inhibition of Rho-kinase could alleviate the acute heart injury induced by ADR. METHODS: C57BL6 mice were randomly divided into the following four groups: ① ADR group; ② low-dose fasudil (ADR+L); ③ high-dose fasudil (ADR+H); and ④ control group (CON). Animals were injected i.p 20 mg/kg ADR once in group ①~③. Animals were injected i.p fasudil (2 or 10 mg/kg/day) daily for consecutive 6 days in groups ② and ③, respectively. Blood samples and heart tissues were collected for assays. H9C2 cells were treated with fasudil for 30 mins and then incubated with ADR for 24 hours. Cells were collected for immunohistochemistry and western blot study, respectively. RESULTS: In the mouse model, administration of fasudil significantly ameliorated ADR-induced cardiac damage, suppressed cell apoptosis and senescence, and ameliorated redox imbalance and DNA damage. In vitro, fasudil treatment ameliorated ADR-induced immunofluorescence reaction of 8-OHdG, decreased the expression of TUNEL cells and proteins of Bax, Caspase-3 and p53, and increased the expression of proteins of Bcl-2 and SIRT 1. CONCLUSION: Fasudil has a protective effect on ADR induced acute cardiotoxicity, which is partially attributed to its antioxidant, anti-senescence, and anti-apoptotic effects.

Overexpression of circRNA SNRK targets miR-103-3p to reduce apoptosis and promote cardiac repair through GSK3ß/ß-catenin pathway in rats with myocardial infarction.

Ischemic cardiomyopathy seriously endangers human health leading to a poor prognosis. Acute myocardial infarction (AMI) is the primary etiology, and the pathophysiological process concludes with the death of cardiomyocytes caused by acute and persistent ischemia and hypoxia in the coronary arteries. We identified a circRNA (circSNRK) which was downregulated in rats with myocardial infarction (MI), however, the role it plays in the MI environment is still unclear. This study contained experiments to investigate the role of circSNRK in the regulation of cardiac survival and explore the mechanisms underlying circSNRK functions. Quantitative real-time PCR (qRT-PCR) was performed to determine the circSNRK expression patterns in hearts. Gain-of-function assays were also conducted in vitro and in vivo to determine the role of circSNRK in cardiac repair. qRT-PCR, western blot, and luciferase reporter assays were used to study circRNA interactions with micro RNAs (miRNAs). Overexpression of circSNRK in cardiomyocytes reduced apoptosis and increased proliferation. Adeno associated virus 9 (AAV9) mediated myocardium overexpression of circSNRK in post MI hearts reduced cardiomyocyte apoptosis, promoted cardiomyocyte proliferation, enhanced angiogenesis, and improved cardiac functions. Overall, upregulation of circSNRK promotes cardiac survival and functional recovery after MI. Mechanistically, circSNRK regulates cardiomyocyte apoptosis and proliferation by acting as a miR-103-3p sponge and inducing increased expression of SNRK which can bind GSK3ß to regulate its phosphorylated activity. And thus circSNRK may be a promising therapeutic target for improving clinical prognosis after MI.

Rho Kinase Inhibition by Fasudil Attenuates Adriamycin-Induced Chronic Heart Injury.

Adriamycin (ADR)-induced chronic heart injury (CHI) is a serious complication of chemotherapy. The present study was designed to assess the ability of fasudil, a Rho kinase inhibitor, to prevent ADR-induced CHI. Forty male 6-week-old C57BL6 mice were randomly divided into the following four groups: (1) control group, (2) CHI induced by adriamycin (ADR group), (3) CHI plus low dose fasudil (ADR + L group), and (4) CHI plus high dose fasudil (ADR + H group). Animals from groups 2-4 received ADR (2.5 mg/kg, i.p.) once a week for 8 weeks, and the control group received saline. Meanwhile, the animals in groups 3-4 received 2 mg/kg/day or 10 mg/kg/day fasudil, respectively. After measurement of cardiac functions, blood samples were collected for biochemical assays. The hearts were excised for histological, immunohistochemistry and western blot study, respectively. Adriamycin produced evident cardiac damage revealed by cardiac functions changes: decreased left ventricular fractional shortening (FS), left ventricular ejection fraction (EF), increased left ventricular volume, cardiac injury marker changes (increased creatine kinase, lactate dehydrogenase), antioxidant enzymes activity changes (decreased superoxide dismutase), and lipid peroxidation (elevated malondialdehyde) to the control group. Fasudil treatment notably ameliorated ADR-induced cardiac damage, restored heart function, suppressed cell apoptosis and senescence, ameliorated redox imbalance, and DNA damage. Fasudil has a protective effect on ADR-induced chronic heart injury, which partially attributed to its antioxidant, anti-apoptotic effects of inhibiting the RhoA/Rho kinase (ROCK) signaling pathway.

Application of protein typing in molecular epidemiological investigation of nosocomial infection outbreak of aminoglycoside-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosan has emerged as an important pathogen elated to serious infections and nosocomial outbreaks worldwide. This study was conducted to understand the prevalence of aminoglycoside (AMG)-resistant P. aeruginosa in our hospital and to provide a scientific basis for control measures against nosocomial infections. Eighty-two strains of P. aeruginosa were isolated from clinical departments and divided into AMG-resistant strains and AMG-sensitive strains based on susceptibility test results. AMG-resistant strains were typed by drug resistance gene typing (DRGT) and protein typing. Five kinds of aminoglycoside-modifying enzyme (AME) genes were detected in the AMG-resistant group. AMG-resistant P. aeruginosa strains were classified into three types and six subtypes by DRGT. Four protein peaks, namely, 9900.02, 7600.04, 9101.25 and 10,372.87 Da, were significantly and differentially expressed between the two groups. AMG-resistant P. aeruginosa strains were also categorised into three types and six subtypes at the distance level of 10 by protein typing. AMG-resistant P. aeruginosa was cloned spread in our hospital; the timely implementation of nosocomial infection prevention and control strategies were needed in preventing outbreaks and epidemic of AMG-resistant P. aeruginosa. SELDI-TOF MS technology can be used for bacterial typing, which provides a new method of clinical epidemiological survey and nosocomial infection control.