| Part 1 Establishing the model of ischemia-reperfusion injured diabetic mice heartBackground:Diabetes mellitus(DM)is associated with clustered risk factors for cardiovascular disease.Meanwhile,cardiovascular mortality in diabetic patients is rising.Over 60%of people with DM die from some form of heart diseases.Coronary artery disease(CAD)is the main cause of death.Notably,ischemia/reperfusion injury(I/R)plays an important role in the pathogenesis of CAD.Since the mechanisms contributing to diabetes and CAD remain largely unknown,we seek to investigate the issue using an I/R injured diabetic mice model and look for potential therapeutic strategies.Objectives:To establish a model of ischemia-reperfusion injured diabetic mice heart.Methods:(1)Mice were fed a high-fat diet for two weeks and injected with 110 mg/kg streptozotocin(STZ)intraperitoneally to establish a diabetic model.Control mice were fed a normal chow diet and were injected with citrate buffer only.(2)Sensory nerve conduction velocity in the mice was measured at 0 and 8 weeks after the injection of STZ.(3)Mice were subjected to a Langendorff-induced ischemia/reperfusion injury model and cardiac parameters were recorded.Results:(1)Eight weeks after STZ injection,the diabetic mice showed an average of 28.81±4.67 mmol/L of plasma glucose compared to 8.56±1.64 mmol/L in the normal controls(P<0.05).The high glucose level was maintained until the end of the study.(2)An impaired sensory nerve conduction velocity developed in the diabetic mice in comparison with the non-diabetic controls(22.22±3.43 vs.31.29±2.48 m/s,P<0.01),indicating the development of diabetic peripheral neuropathy.(3)Cardiac parameters including LVSP、±dp/dt、HR and CF were significantly declined in diabetic group after ischemia/reperfusion injury(P<0.05 compared with the controls).Conclusions:We established a mouse model of diabetic neuropathy.Meanwhile,Langendorff-perfused I/R injured diabetic mice heart was obtained for the following experiments.Part 2 Protective effects of NGF on ischemia/reperfusion injured diabetic mice heartBackground:Due to the presence of cardiac neuropathy,chest pain is usually mild and not typical in diabetic patients.Therefore,prompt treatment is usually unavailable and mortality increases.Nerve growth factor(NGF)is a kind of polypeptide which maintains the function of sympathetic and sensory nerves.Previous study has shown that NGF can improve the function of diabetic rat heart with autonomic neuropathy.However,it is still unknown whether NGF can improve the function of ischemia/reperfusion injured diabetic mice heart.Objectives:Exploring the potential role of NGF on ischemia/reperfusion injured diabetic mice heart.Methods:(1)Adenoviral vectors encoding NGF(Ad-NGF)was constructed using genetic engineering technology.(2)Assessment of NGF expression in diabetic mice heart by western blot analysis.(3)Gene delivery for the Ad-NGF to myocardium.(4)Observing the effects of NGF on Langendorff-perfused ischemia/reperfusion injured diabetic mice heart,cardiac parameters including LVSP,LVEDP,HR,±dp/dt and CF were recorded by the RM6240 signal acquisition system.(5)Sample fluids were collected during the reperfusion period.The level of lactate dehydrogenase(LDH)was measured.Results:(1)Western blot analysis revealed the expression of fusion proteins,indicating the successful construction of recombinant adenoviral vectors.(2)Decreased expression of NGF was observed in diabetic heart,as compared to normal control heart.Meanwhile,upregulation of NGF was observed after adenovirus mediated transgene delivery in normal and diabetic hearts.(3)The ischemia/reperfusion injured diabetic mice heart showed a decreased LVSP,±dp/dt,and CF when compared to the normal controls(P<0.05).Meanwhile,an increased LVEDP and LDH was observed in diabetic preparations(P<0.05).(4)Beneficial effects of Ad-NGF treatment were detected in the diabetic and normal mice.Increases in LVSP,±dp/dt,and CF,accompanied by a decrease in LVEDP and LDH(P<0.05),were observed in Ad-NGF treated diabetic and non-diabetic hearts.Conclusions:Ad-NGF could be effectively transferred to the myocardium and exert protective effects on the ischemia/reperfusion injured diabetic mice heart.Part 3 The role of TRPV1 in NGF induced cardiac protectionBackground:Transient receptor potential vanilloid type 1(TRPV1)is widely distributed in sensory nerve terminals and in the epicardium of the heart.Activation of peripheral TRPV1 has been shown to have cardioprotective effects after ischemia/reperfusion(I/R)injury.Nonetheless,the relationship between NGF and TRPV1 in diabetic mice heart following ischemia/reperfusion injury remains largely unknown.Objectives:To identify the involvement of TRPV1 receptor and downstream neurotransmitters calcitonin gene related peptide(CGRP)and subatance P(SP)during the process of NGF induced cardiac protection.Methods:(1)Measurement of the expression of TRPV1 receptor using western blot analysis.The level of CGRP and SP was assessed by an ELISA kit.(2)Gene delivery for the Ad-NGF to myocardium.(3)CGRP8-37(a selective CGRP antagonist),RP67580(SP antagonist),or capsaicin(exogenous TRPV1 activator)was added to the perfusion fluid 5 min before ischemia in the experimental groups.Cardiac parameters including LVSP,LVEDP,HR,±dp/dt and CF were recorded by the RM6240 signal acquisition system.(4)Sample fluids were collected during the ischemia/reperfusion period.The level of CGRP and SP was measured by an ELISA kit.Results:(1)Decreased expression of TRPV1 was observed in diabetic heart,as compared to normal controls(P<0.05).Meanwhile,the concentrations of CGRP and SP were significantly reduced in diabetic heart in comparison with normal heart(P<0.05).(2)In line with the up-regulation of TRPV1,CGRP was increased following Ad-NGF treatment in the diabetic heart(P<0.05).The change in SP after Ad-NGF administration was not significant(P>0.05).(3)Treatment with CGRP8-37 inhibited cardiac recovery by lowering the LVSP,±dp/dt,and CF compared with untreated hearts(P<0.05).Meanwhile,the LVEDP was increased by CGRP8-37 treatment(P<0.05).Low-dose capsaicin increased the LVSP,±dp/dt,CF and decreased LVEDP in diabetic heart(P<0.05).RP67580 exerted little effect on the cardiac performance of each group.Conclusions:It is reasonable to suggest that the NGF-induced upregulation of TRPV1,by increasing the synthesis and release of CGRP,leads to improved cardiac performance in I/R-injured diabetic heart. |
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