Paeoniflorin Protect Diabetes Mice Against Myocardial Ischemic Injury And Its Associated Mechanisms

Background:Diabetes mellitus (DM) has become a global problem that threatens human health, Diabetes mellitus is characterized by increased plasma glucose levels and is often accompanied by several complications including neuropathy and increases cardiovascular events. Transient receptor potential vanilloid 1 (TRPV1) is a non-selective positive ion channel that is mainly expressed in sensory neurons. Pharmacological studies have shown that TRPV1 exerts an important cardioprotective effect. TRPV1 could activate the CaMK and then increase the CREB phosphorylation, which could increase the CGRP expression. CGRP is one of the cardioprotective neuropeptide:positive inotropic and potent vasodilatory effect. Paeoniflorin(PF), a monoterpene glycoside, exhibits various pharmacological activities including anti-inflammatory, antioxidant and immunoregulatory activities. It is reported that Paeoniflorin plays an essential role in neuroprotection and ischemic heart disease.Aim:Previous researched indicated that Paeoniflorin protect the mice from myocardial ischemia injury, however the mechanism is still unknown. In this study, we aimed to investigate the hypothesis that PF protects mice with diabetes mellitus from myocardial ischemic injury, and investigate its associated mechanisms.Methods:1) Male C57BL/6J and age-matched TRPV1 gene knockout mice were subjected to intraperitoneal injection with 150mg/kg of streptozotocin (STZ) in a fasting state. Then the mice were administrated with high-fat diet. Blood glucose concentration>16.7 mmol/l were up to the criteria for experimental DM mice at three days and eight weeks post-STZ injection.2) The diabetes mellitus mice were given 140 mg/kg/day of PF for 14 days by the intragastric route; the control group was treated with saline. After 14 days of PF treatment in mice, KN-93(300 μg/kg), a selective CaMKII inhibitor, was administered directly into the left ventricle. The animals were divided into six groups:(1) WTDM group:diabetes mellitus (DM) in wild type mice; (2) PF-WTDM:WTDM treated with 140 mg/kg of PF; (4) PF-WTDM-KN-93 group:KN-93 was used after PF administration; (5) TRPV1-/-DM:TRPV1-/-mice was used for the DM model; (6) PF-TRPV1-/-DM:TRPV1-/-DM treated with 140 mg/kg of PF; (7) PF-TRPV1-/-DM-KN-93 group:KN-93 was used after PF administration.3) The acute myocardial infarction animal model was induced by the permanent ligation of the left coronary artery. Twenty-four hours after coronary artery ligation, the heart was then isolated from the chest, the blood was collected into a test tube and stored at -20℃.4) Myocardial infarct size was measured with Evans blue and TTC staining, myocardial enzyme was measured using commercial kits, Radioimmunoassay was used to determine the circulating CGRP concentration, haematoxylin and eosin (H&E) and Massion’s trichrome staining were using to evaluate myocardial fibrosis. Western blot analyzed the expression of associated molecules (TRPV1, CaMKII, CREB, p-CREB) and compared among different experimental groups.Results:1) Before and after given paeoniflorin treatment, the weight and blood glucose were measured, the data has no significant difference in each group.2) In wild type DM mice, compared with the group treated with saline, PF significantly decreased myocardial infarct size, reduced the release of myocardial enzymes in serum, increased the activity of CGRP in serum, and alleviated the degree of fibrosis after myocardial infarction. Meanwhile, PF also increase the expression ofTRPV1.3) In TRPV1-/-DM mice, PF also decreased infarct size and myocardial enzyme levels compared with the group treated with saline. However, knockout of TRPV1 gene reduced the effect of PF on both myocardial infarct size and serum myocardial enzymes concentration. TRPV1-/-hearts released less CGRP, compared with hearts in the PF-WTDM group. PF reduced the interstitial fibrotic area in both WTDM and TRPV1-/-DM hearts, but the area in PF-WTDM was less than PF-TRPV1-/-DM group.4) In wild type DM mice, treated with PF could not alter the expression of CaMKII. KN-93 significantly alleviated the cardioprotection of PF, including increasing myocardial infarct size and the concentration levels of circulating myocardial enzymes; meanwhile, when pretreated with KN-93, the release of CGRP in serum significantly decreased and the myocardial fibrosis markedly increased. However, in TRPV1-/-DM mice, KN-93 could not change the myocardial infarction size, circulating myocardial enzymes, the activity of CGRP and myocardial fibrosis.5) Analysis the expression of CREB and CREB phosphorylation, Paeoniflorin significantly increases the phosphorylation of CREB and this effect could be alleviated with KN-93 in wild type DM mice. In TRPV1-/-DM mice, the expression CREB phosphorylation was low and PF could not change the CREB phosphorylation.Conclusion:Taken together, these findings demonstrate that PF protects diabetic mice against MI at least partially via the TRPV1/CaMK/CREB/CGRP signaling pathway.