| Background: Diabetes mellitus is an independent risk factor for cardiovascular disease. Patients with diabetes are more likely to develop severe ischemic heart disease than non-diabetic patients. Diabetic neuropathy is one of the common complications of diabetes, which can affect the normal functions of sensory nerve, leading to a decrease of perception to external noxious stimuli and the absence of anginal pain in acute myocardial infarction patients, that is, silent myocardial ischemia(SMI). SMI often causes the patient miss the best time to rescue and increase the mortality of ischemic heart disease. TRPV1(transient receptor potential vanilloid 1, TRPV1) is distributed in terminals of cardiac sensory nerve. It could lead to the synthesize and release of sensory neuropeptides such as calcitonin gene-related peptide(CGRP) and substance P(SP), which participate in maintaining normal physiological function of heart, and play important protective roles in myocardial ischemia and reperfusion. And diabetic neuropathy could result in the decrease of sensory neuropeptide CGRP and SP. Therefore, we established the current study to investigate whether increased myocardial vulnerability is related to the loss of cardiac sensory neuropeptide induced by diabetic neuropathy.Objective:(1) To investigate whether diabetic neuropathy could lead to the sensory nerve impairment and the decrease of CGRP and SP synthesis in myocardium and dorsal root ganglion(DRG) innervating the heart.(2) To investigate the association of the diabetic neuropathy with increases of myocardial vulnerability and injury in diabetic animals.(3) Whether chronic stimulation of TRPV1 by dietary hot pepper could increase the synthesis of CGRP and SP, and improve myocardial ischemia-reperfusion injury in diabetic rats.Method:(1) The Male Sprague-Dawley rats(250-300 g) were randomly divided into normal control group(Control group), diabetic group(DM group) and diabetic hot pepper-diet group(DP group). Each group was divided into two groups: sham operation group(Sham group) and ischemia reperfusion group(IR group). Diabetes was induced in the animals of DM group and DP group with injection of STZ, while the animals in DP group were fed with the same diet supplemented with red pepper powder; Animals in IR group were subjected to 30 min of myocardial ischemia followed by 120 min reperfusion. The experiments were carried out at 8 weeks after injection of STZ;(2) The tail flick latency was measured to assess the changes of heat pain threshold in diabetic rats, and the changes of TRPV1 and CGRP/SP positive structures in spinal cord and DRG were observed by double immunofluorescence histochemical staining;(3) Double-label immunofluorescence and in situ hybridization were respectively used to observe the distribution of TRPV1 and CGRP/CGRPm RNA and SP/SPm RNA positive structures in myocardium. After myocardial ischemia reperfusion, the expression of TRPV1 and CGRP/SP in ischemic myocardium and DRG and the levels of CGRP and SP in serum were examined by using Western blot and ELISA; The expression of TRPV1 m RNA and CGRPm RNA/SPm RNA in ischemic myocardium and DRG were detected by using Rt-PCR;(4) The change of Left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), heart rate(HR) and maximal rate of rise/fall of left ventricle pressures(±d P/dtmax) during myocardial ischemia-reperfusion were detected to evaluate cardiac functions. The myocardial infarct size was determined by the Evans blue and TTC double staining; The c Tn I levels in serum and caspase-3 activity in myocardium were performed by ELISA and TUNEL staining was used to detect the apoptosis rate of myocardial cell. Ventricular premature beat, ventricular tachycardia and ventricular fibrillation were also recorded and analyzed during myocardial ischemia reperfusion.Result:(1) Compared with Control group, tail flick latency of DM group was significantly increased(p<0.05), and the proportion of TRPV1 and CGRP/SP co-expression positive neurons in DM group was significantly decreased(p<0.05), and the average optical density of TRPV1 and CGRP/SP co-expression positive structures in the superficial layers of spinal dorsal horn in DM group was significantly decreased(p<0.05). Compared with DM group, tail flick latency of DP group was significantly decreased(p<0.05), and the proportion of TRPV1 and CGRP/SP co-expression positive neurons in DP group was significantly increased(p<0.05), and the average optical density of TRPV1 and CGRP/SP co-expression positive structures in the superficial layers of spinal dorsal horn in DP group was significantly increased(p<0.05);(2) Co-expressions of TRPV1 and CGRP/SP were observed in myocardium, mainly in the myocardial endothelial cells and epicardium. Endothelial cells were further confirmed by in situ hybridization assay showing the co-expression of TRPV1 and CGRPm RNA/SPm RNA. Compared with Control group, LVSP, HR and +d P/dtmax in DM group were significantly reduced(p<0.05), while LVEDP and-d P/dtmax in DM group were significantly raised(p<0.05); the expression of TRPV1, CGRP and SP in myocardium and DRG and the levels of CGRP and SP in serum of DM group were significantly decreased(p<0.05). Compared with DM group, LVSP, HR and +d P/dtmax in DP group were significantly increased(p<0.05), while LVEDP and-d P/dtmaxin DP group were significantly decreased(p<0.05); the expression of TRPV1, CGRP and SP in myocardium and DRG and the levels of CGRP and SP in serum of DP group were significantly increased(p<0.05);(3) Compared with sham group, the contents of CGRP and SP in myocardium were greatly raised in IR group(p<0.05), and the expression of TRPV1 of C-IR and DP-IR group were significantly increased in myocardium(p<0.01), and the expression of TRPV1, CGRP and SP in DRG and the levels of CGRP and SP in serum of IR group were significantly increased(p<0.05). Compared with C-IR group, the expression of TRPV1, CGRP and SP in DRG and the levels of CGRP and SP in serum of DM-IR group were significantly decreased(p<0.01). Compared with DM-IR group, the expression of TRPV1, CGRP and SP in DRG and the levels of CGRP and SP in serum of D P-IR group were significantly increased(p<0.05);(4) Compared with sham group, the expressions of CGRPm RNA and SPm RNA of IR group were greatly raised in DRG(p<0.05), and the expression of TRPV1 of C-IR and DP-IR group were significantly increased in myocardium(p<0.05), and there was no significant increase of TRPV1 m RNA expression in myocardium and DRG of IR group(p>0.05). Compared with C-IR group, the expressions of TRPV1 m RNA, CGRPm RNA and SPm RNA were significantly reduced in myocardium and DRG of DM-IR group(p<0.05). Compared with DM-IR group, the expressions of TRPV1 m RNA, CGRPm RNA and SPm RNA were significantly increased in myocardium and DRG of DP-IR group(p<0.05);(5) Compared with sham group, the levels of c Tn I in serum, myocardial apoptosis rate and caspase-3 activity of IR group were significantly increased(p<0.01). Compared with C-IR, myocardial infarct size, the levels of c Tn I in serum, myocardial apoptosis rate and caspase-3 activity of DM-IR group were significantly increased(p<0.01). Compared with DM-IR group, myocardial infarct size, the levels of c Tn I in serum, myocardial apoptosis rate and caspase-3 activity of DP-IR group were significantly decreased(p<0.05);(6) During myocardial ischemia reperfusion, compared with C-IR group, DM-IR significantly reduced LVSP and +dp/dtmax(p<0.01) and enhanced LEVDP and-dp/dtmax(p<0.01), and DP-IR significantly reduced +dp/dtmax(p<0.01) and enhanced LEVDP and-dp/dtmax(p<0.01). Compared with DM-IR, DP-IR significantly increased LVSP and +dp/dtmax(p<0.01) and decreased LEVDP and-dp/dtmax(p<0.01);(7) Compared with C-IR, the number of ventricular premature of DM-IR group was significantly increased(p<0.01) and the duration of arrhythmias was brought forward, and there was a significant increase in incidence of malignant arrhythmia and incidence of ventricular fibrillation(p<0.05). Compared with DM-IR, the number of ventricular premature of DP-IR group was significantly reduced(p<0.01) and the duration of arrhythmias was late, and there was a significant decrease in incidence of malignant arrhythmia and incidence of ventricular fibrillation(p<0.05).Conclusions: Diabetes could lead to sensory nerve degeneration, heat pain threshold elevation, myocardial endogenous neuropeptide CGRP and SP reduction, cardiac dysfunctions and aggravation of myocardial ischemia reperfusion injury. Chronic uptake of hot pepper could significantly improve sensory nerve degeneration, cardiac dysfunctions, arrhythmia and alleviate myocardial ischemia reperfusion injury in DM animals which may be associated with the synthesis and release of endogenous sensory neuropeptide CGRP and SP following TRPV1 activation. |
PDF Full Text Request