| Objective:The aim of the study was to investigate the heat pain threshold of diabetic rats and the expression of CGRP(calcitonin gene related peptide) and SP(substance P)in myocardium and DRG(dorsal root ganglia) following myocardial ischemia reperfusion in diabetic rats. Methods:(1) Thirty two male Sprague-Dawley rats, weighing 180-200 g, were randomly divided into control group(C) and diabetic group(DM), 16 rats in each group. Rats in DM group were fed with high sugar-fat diet for 14 weeks and were given streptozotocin(STZ, 30mg/mg, i.p.) at the end of the 4th week, to set up type 2 diabetic rat model. The animals in control group were fed with standard laboratory diet.(2) Tail flick latency to thermal stimulation was measured weekly. At the end of 10 weeks after administration of STZ, diabetic rats(and rats in control group) were further divided into myocardial ischemia/reperfusion group(I/R group) and sham operation group(Sham group). The left anterior descending branch of coronary artery was occluded for 30 min followed by reperfusion for 120 min, establishing myocardial ischemia/reperfusion. The histological immunofluorescence assay and Enzyme-linked immunosorbent assay(ELISA) were carried out to evaluate the changes of the expressions of CGRP and SP in the dorsal root ganglia(T1-T5).(3) 10 weeks later,DM rats and Normal control rats were randomly divided into ischemia/reperfusion group(I/R group) and sham operation group(Sham group). The left anterior descending branch of coronary artery was occluded under anesthesia by ligation of the artery to establish myocardial ischemia reperfusion model. The histological immunofluorescence assay and Enzyme-linked immunosorbent assay(ELISA) were performed to evaluate the changes of the expressions of CGRP and SP in the ischemia myocardium and serum.(4) The aim of the study was to investigate the possible mechanism of diabetic myocardial ischemia and reperfusion injury. Enzyme-linked immunosorbent assay(ELISA) were carried out to evaluate the changes of the expressions of norepinephrine(NE) in the ischemia myocardium and serum. Western Blot were performed to evaluate the changes of the expressions of beta-1 adrenergic receptor. Result:(1) The body weight of diabetic group had a significant increasing trend during the first four weeks of high-fat diet compare with the normal control group, and the serum insulin levels increased significantly(P<0.05), sensitivity of insulin decreased significantly(P<0.05). After 10 weeks of injection of STZ, the body weight of diabetic animal decreased obviously(P<0.05). Fasting blood glucose in diabetic group were significantly increased compared with the control group(P<0.01). The terminal insulin content of DM group decreased significantly compared with the control group(P<0.05). While there was an increasing trend of serum triglyceride content in DM group compared with Control group, but the difference was not statistically significant.(2) The diabetic tail flick time was significantlt longer than normal control group(P<0.01). CGRP and SP positive neurons are rich in DRG, however both of them decreased significantly in diabetic rats(P<0.05),CGRP positive neurons decreased 46%,small neurons decreased 70%,SP positive neurons decreased 62%,small neurons decreased 59%. CGRP and SP positive neurons concentrate in the superficial layers of the spinal dorsal horn(first and second layer). The comparison between normal control ischemia reperfusion group(C-I/R) and normal control sham operation group(C-SHAM): CGRP content in DRG increased significantly(P=0.001). The comparison between diabetic ischemia reperfusion group(DM-I/R) and diabetic sham operation group(DM-SHAM): CGRP content in DRG increased significantly(P=0.001). The comparison between diabetic sham operation group(DM-SHAM) and normal control sham operation group(C-SHAM): CGRP content in DRG decreased significantly(P=0.001). The comparison between diabetic ischemia reperfusion group(DM-I/R) and normal control ischemia reperfusion group(C-I/R): CGRP and SP content in DRG decreased significantly(P<0.01).(3) Cardiac CGRP and SP positive nerve fibers mainly expressed in the epicardial and myocardial layers, especially around perivascular vascular endothelium. Both of them decreased obviously in diabetic rats too. The comparison between normal control ischemia reperfusion group(C-I/R) and normal control sham operation group(C-SHAM): CGRP and SP in left ischemic ventricular tissue and serum increased significantly(P<0.05). The comparison between diabetic ischemia reperfusion group(DM-I/R) and diabetic sham operation group(DM-SHAM): CGRP in left ischemic ventricular tissue and serum increased significantly(P<0.05). The comparison between diabetic sham operation group(DM-SHAM) and normal control sham operation group(C-SHAM): CGRP and SP in left ischemic ventricular tissue and serum decreased significantly(P<0.05). The comparison between diabetic ischemia reperfusion group(DM-I/R) and normal control ischemia reperfusion group(C-I/R): CGRP and SP in left ischemic ventricular tissue and serum decreased significantly(P<0.05).(4) The content of NE in ischemic myocardium of left ventricular: the Sham group had an increasing trend compare with I/R group in both DM group and Control group, but the difference was not statistically significant, there is no difference between DM group and Control group too. The content of NE in serum: there is no difference between DM group and Control group, however I/R group increased significantly compare with Sham group in both DM group and Control group(P<0.05). The expression of beta 1 receptor: the expression of C-SHAM increased significantly compare with the C-I/R(P<0.05). D-SHAM had an increasing trend compare with D-I/R, but the difference was not statistically significant. There is a decreasing trend in DM group compare with Control group, however the difference is also not statistically significant.Conclusion:(1) Application of high glucose and high fat diet, body weight of diabetic group rats increased significantly, and there was a certain degree increase of insulin content and decrease of insulin sensibility, after a low dose of streptozotocin intraperitoneal injection, blood glucose increased significantly, the symptom of diabetic group rats appeared similar with the pathological features of human type 2 diabetes.After 10 weeks of STZ injection,the insulin content was insufficient in DM rats, the triglyceride content rose up.(2) Diabetes causes sensory denervation and obvious reduction of expression of SP and CGRP in the sensory neuron innervating heart during myocardial ischemia/ reperfusion, indicating impairment of adaptive reactivity of neuro-endocrine function of cardiac sensory nerves.(3) The diabetic myocardium sensory nerve denervation too. The changes of CGRP and SP which decreased in DM rats may participate in the pathophysiology of diabetic myocardial ischemia reperfusion injury.(4) Diabetes influenced the expression of beta-1 adrenergic receptor,the biological significance need further research. |
PDF Full Text Request