Results from our current study indicate that in rats with hyperlipidemia, either metformin or atorvastatin treatment is favorable for reducing CRP level, increasing NO production and decreasing Rho kinase activity. These cardio-protective effects are further enhanced by combined therapy with metformin and atorvastatin. Notably, metformin has no effects on lipid profile change, indicating that these benefits derived from metformin therapy may be associated with other mechanisms such as declining Rho kinase activity in the setting of hyperlipidemia since linear regression analyses show that Rho kinase activity is positively correlated with CRP level while negatively correlated with NO production in the metformin group.
Accordingly, the benefits of statins therapy can be categorized into two respects in terms of lipid-lowering efficacy and pleiotropic effects. Notably, statins’ pleiotropy is largely associated with its potent effect on inhibiting small GTP-binding proteins isoprenylation. With isoprenylation, small GTP-binding proteins such as Rho can play multiple and complex roles on endothelium, leukocyte and fibroblast. Under pathological setting such as hyperlipidemia and diabetes mellitus, Rho isoprenylation and its effector Rho kinase (ROCK) over-activation contribute detrimental effects on vascular system such as promoting inflammatory cells adhesion and infiltration, impairing endothelial function, increasing reactive oxidative species generation, destabilizing eNOS mRNA and decreasing NO production. Findings from our current study further corroborated previous reports that in the setting of hyperlipidemia, statins treatment not only could improve dyslipidemia, but also concomitantly decreased Rho kinase activity which consequently reduced CRP level and enhanced NO production[8, 21].
Interestingly and importantly, our current study also preliminarily suggested that in the setting of hyperlipidemia, 50 mg/Kg body weight per day of metformin administration was beneficial for NO production increment and CRP level reduction which was consistent to previous studies[14, 22, 23]. Nevertheless, the mechanisms associated with these benefits are still incompletely clear. In light of our current study, we speculated that the benefits derived from metformin therapy of rats with hyperlipidemia might be partially associated with its effects on inhibiting Rho kinase activity. Nevertheless, since metformin had no effects on cholesterol biosynthesis as revealed in our study, inhibiting Rho kinase activity of metformin might not be related to its inhibition of isoprenylation of small GTP-binding proteins. Rather, on the basis of previous studies[14, 24, 25], we considered that some alternative mechanisms might be associated with diminishment of Rho kinase activity with metformin therapy. First of all, basic researches suggest that cardio-protective effects of metformin are partially mediated by AMP-activated protein kinase (AMPK) activation which is responsible for eNOS up-regulation and NO production increase[14, 26]. Accordingly[27, 28], Rho kinas activated is partially dependent on reactive oxidative species, and up-regulation of eNOS expression and NO production ameliorate oxidative stress therefore inhibiting Rho kinase activation. Secondly, since high blood glucose is a potential stimulus for Rho kinase activation, and Rho isoprenylation and Rho kinase over-activation contribute to insulin resistance which reciprocally induces high blood glucose[25, 29], therefore we considered that it was possible that metformin declined Rho kinase activity by means of improving glucose metabolism. In our current study, we observed that rats with high-fat and high-cholesterol diet administration for 6 weeks, fasting blood glucose was somewhat increased in the hyperlipidemia groups than that of the sham group, indicating that hyperlipidemia per se might potentially compromise glucose metabolism. Nevertheless, with 4 weeks of metformin therapy, fasting blood glucose levels in the metformin and combined groups were reduced in comparison to the control and atorvastatin groups, suggesting that metformin therapy was favorable for improving glucose metabolism despite in non-diabetes condition. Although all the hyperlipidemia groups were not qualified to the criteria of diabetes mellitus, we considered that increased fasting blood glucose might be associated with Rho kinase activity enhancement, and in the metformin and combined groups, Rho kinase activity was a little bit lower than that of the atorvastatin group which might directly suggest that fasting blood glucose improvement with metformin therapy was favorable for declining Rho kinase activity.
Importantly, favorable effects in terms of CRP reduction, NO production, and Rho kinase activity decrease were more robust in the combined therapy groups, suggesting that atorvastatin and metformin might have synergistic protective effects. In light of previous reports and our current findings[23, 30–32], we considered that these benefits might derive from anti-inflammation and lipid-modification of atorvastatin as well as anti-inflammation and glucose-metabolism improvement of metformin which concomitantly leaded to Rho kinase activity diminishment.
No liver enzymes and creatinine kinase elevation were observed with four weeks of metformin and atorvastatin therapy, suggesting the high safety profile of current combined therapeutic strategy.