The aims of this study were to identify whether H3 relaxin protected against vascular injury induced by diabetes in vivo and potential mechanism. Our results demonstrated that vascular fibrosis, NLRP3 inflammasome activation and ERS were involved in the vascular complications of diabetes and that H3 relaxin improved vascular injury by inhibiting fibrosis, ERS and NLRP3 inflammasome activation.
In higher primates, relaxin family peptides contains seven members, which are relaxin-1, 2, 3 and insulin-like peptides (INSL) 3, 4, 5, 6. However, in rats, there are six members in relaxin family peptides, including relaxin-1 (similar to human relaxin-2), relaxin-3 and INSL-3, 4, 5, 6. The relaxin family peptides receptors contains RXFP1, 2, 3, 4, natural ligand of human relaxin-2 (rats relaxin-1) is RXFP1 which plays protective effects in the cardiovascular disease. Recent studies reported that relaxin-3 can inhibit myocardial injury by binding to RXFP1 in rats with myocardial infarction, although RXFP3, a natural receptor of relaxin-3, is mainly located in the brain. Later, study found that in rat atrial and ventricular cells, there existed relaxin-3 mRNA which expression was up-regulated in the myocardium after isoproterenol administration. Our recent study reported that H3 relaxin improved cardiac injury by regulating the activation of NLRP3 inflammasome in diabetic cardiomyopathy[7, 8]. This study focus on whether H3 relaxin inhibited diabetic vascular injury, and we choose the dose of 0.2 or 2ug/kg/day relaxin-3 treatment for diabetic rats as zhang et al. described previously. Interestingly, we found both doses of H3 relaxin were effective to a similar degree, and we will choose the very low dose H3 relaxin in future experiment. This study limitations contained that we did not test plasma relaxin-1 and relaxin-3 expression, so we can not clarify the issue that why both high and low dose H3 relaxin had similar protective effects in vascular injury in type 1 diabetes rats. We will aviod this question in future.
We found that the body weight increased and the glucose level decreased after H3 relaxin injection in diabetic rats as reported in our previous study, and which maybe a mechanism of that H3 relaxin inhibited vascular fibrosis, need to be verificated. Unfortunately, we did not detected the level of insulin, we will avoid this issue in future. In addition, we found that Relaxin-1, 3 and RXFP1, 3 mRNA were significantly up-regulated after STZ in the aortas of diabetic rats according to real-time PCR. The reason in increased expression of RXFP1 and RXFP3 in aortas of diabetic rats may be due to increased expression of endogous relaxin-1/3, however, these results clarified that endogenous relaxin-1/3 and receptors participated in the mechanism of vascular injury in diabetic rats.
The mechanism of vascular complications of diabetes contains endothelial dysfunction, oxidative stress and arterial remodelling. Oxidative stress is a determining mechanism in vascular complication of diabetes. Increased plasma TNF-a and IL-6 are associated with vascular dysfunction in patients with type 2 diabetes. NF-κB activity increased, TNF-a and intercellular adhesion molecule (ICAM) upregulated in vascular tissues of type 2 diabetic rats. Aljwaid H et al. found that compared with control, MDA levels were higher in patients with diabetes, which was related with higher levels of oxidized ascorbate. These studies clarfied that oxidative stress was involved in the mechanism of vascular dysfunction in diabetes. Recent data indicate that H3 relaxin protects against ischaemic injury by reducing the hypoxia-induced production of ROS. In our study, we found that plasma MDA and TNF-a levels were increased in rats with diabetes, and H3 relaxin inhibited vascular dysfunction by regulating MDA and TNF-a levels in rats with diabetes.
The expression of ERS markers were increased in kidney from patients with diabetes, were involved in the pathogenesis of diabetic nephropathy, however, CHOP knockout improved kidney injury in diabetic mice. In addition, ER stress activated inflammatory factors and mediated increased vascular permeability in retina of diabetes. In a recent study, we found that H3 relaxin inhibited the ERS mediated apoptosis of myocardial cells induced by high glucose. In our study, we found that compared with the controls, the expression of CHOP and GRP78 increased in the aortas of diabetic rats, which were inhibited by H3 relaxin administration, indicated that H3 relaxin inhibited ERS-induced vascular injury.
High glucose-induced NLRP3 inflammasome activation prompted vascular complications in diabetes. In vivo, NLRP3 inflammasome was activated in the aortas of rats after a high glucose diet, and inhibited by rutin administration. Aberrantly, in atherosclerotic pig aortas, the expression of NLPR3, ASC and IL-1β were increased, along with NF-κB activation and endothelial dysfunction. In patients with coronary heart disease, increased NLRP3 expression in aortas was positively associated the severity of coronary artery disease. In addition, our findings are consistent; compared with the controls, the expression and activation of the NLRP3 inflammasome were increased in the aortas of rats with diabetes, and were inhibited by H3 relaxin.
In conclusion, H3 relaxin is a potential candidate for treating diabetes-associated vascular complications by inhibiting fibrosis, the activation of ERS and NLRP3 inflammasome.