Effects Of Fatty Acids On Diabetic Neuropathy And Increasing Of Myocardial Vulnerability In Streptozocin-induced Diabetic Rats And The Potential Mechanism

Background: Diabetic peripheral neuropathy(DPN)and myocardial injury are common and serious complications of diabetes.Despite the global prevalence and severe complications of diabetes,the pathophysiological mechanisms of diabetic neuropathies have not been elucidated.Previous studies have focused on the effects of hyperglycemia on neurons and myocardial cells,while the effects of fatty acids on diabetic complications are often been overlooked.The transient receptor potential vanilloid subtype 1(TRPV1)is a non-select cation channel,which is activated by noxious heat,low p H,capsaicin,endogenous ligands and proinflammatory mediators.Activation of TRPV1 causes neuronal depolarization,which leads to pain and release of sensory neuropeptides such as CGRP and SP from peripheral and central nerve terminals.For the research in our lab showed that,the expression of TRPV1 were significantly decreased in dorsal root ganglia(DRG)and myocardium of diabetic rats,and that chronic activation of TRPV1 by pepper or capsaicin in dietary supplement could improve vulnerability to neuropathy and myocardial ischemia reperfusion injury in diabetic rats.Activation of TRPV1 and preservation of CGRP and SP exerted cardioprotection and hemodynamic regulation in ischemic preconditioning and postconditioning.Then,activation of TRPV1-CGFP/SP attenuated diabetic rats-induced neuropathy and cardiac ischemia/reperfusion dysfunction.At the same time,we found that some of the elevated fatty acids in diabetic patients have an inhibitory effect of TRPV1,whether these fatty acids have an damage effect on diabetic neuropathy and myocardial ischemia-reperfusion injury and whether the protective effect of TRPV1-CGRP axis couldbe inhibited by high concentration of fatty acids? The experiment mainly screened the increased fatty acids in diabetic patients reported in the literature and then verified the role on the TRPV1.The corresponding fatty acid was applied to DRG neurons of STZ-induced diabetic rats and myocardium for ischemia reperfusion in vitro to observe the growth of DRG neurons and influence of myocardial function.Finally,we will further explore the potential mechanism.Objective:(1)To detect the effects of different concentrations of oleic acid(OA)on the activity of TRPV1 channel on DRG neurons.(2)The effects of OA on DRG neurons of diabetic neuropathy rats was observed.Firstly,we observed the expression of TRPV1 and its downstream CGRP,SP,and NGF in different conditions.Meanwhile,the oxidative stress,mitochondrial function,apoptosis and neurite outgrowth of DRG neurons were detected.Then prototypical agonist of TRPV1 capsaicin(8-methyl-N-vanillyl-6-nonenamide)was used to detect whether the effects of OA on diabetic DRG neurons could be reversed.(3)Activation of TRPV1 lead to the release of CGRP,so whether CGRP may exert protective function on diabetic neuropathy rats? We used exogenous administration of CGRP or lentivirus-mediated overexpression of CGRP on diabetic rats DRG neurons,SP and NGF expressions were detected as well as diabetic DRG neurons apoptosis,mitochondrial membrane potential and production of reactive oxygen species(ROS),axonal growth were evaluated.(4)The changes of cardiac function were detected by using OA in vitro perfusion of normal rat hearts.Further administration of LY294002,an inhibitor of the PI3K/Akt signaling pathway,simply demonstrated the mechanism of TRPV1 on ischemia reperfusion injury.Methods:(1)Primary cultured DRG neurons from normal and diabetic SD rats were loaded with Fluo-4 molecular probes for calcium imaging.Effects of capsaicin and OA at different concentrations on TRPV1 activity were detected by the changes of intracellular Ca2+ concentration in DRG neurons.(2)DRGs were dissected from STZ-induced diabetic rats.Effects of OA and capsaicin on diabetic rats DRG neurons were measured by production of ROS,changes of mitochondrial membrance potential(MMP),apoptosis(TUNEL)and neurite outgrowth(anti-?? tubulin antibody)as well as expression of TRPV1?CGRP?SP and NGF(western blot,ELISA and PCR).(3)Lentiviral vector mediated over-expression of CGRP or 10-8mol/L CGRP were used to verify its effects on diabetic DRG neurons.production of ROS,changes of MMP,apoptosis and neurite outgrowth(anti-?? tubulin antibody)as well as expression of CGRP?SP and NGF(western blot,ELISA and PCR).(4)The normal rat hearts were taken under general anesthesia and subjected to 30 min myocardial ischemia followed by 60 min reperfusion by Langendorff device in vitro.Isolated rat hearts were pre-treated with capsaicin,OA or inhibitor of PI3K/Akt LY294002,left ventricular end-diastolic pressure(LVEDP),left ventricular active systolic pressure(LVSP),left ventricular developed pressure LVDP(LVDP = LVSP-LVEDP),heart rate(HR),maximal rate of myocardial contraction(+dp/dtmax),maximal rate of myocardial relaxation during diastole(-dp/dtmax)and ventricular arrhythmia,including single,multiple ventricular premature,ventricular tachycardia and ventricular fibrillation were recorded to evaluate the effect of OA on cardiac function after I/R injury.Western blot was used to determine the expression of phosphorylated Akt in ischemic myocardium.Results:(1)For primary cultured of DRG neurons,we used PGP9.5 polychonal antibody to identify DRG neurons.It was found that DRG neurons grew well,and axons would grow after cells adhering to the wall.TRPV1 and CGRP,SP,NGF were co-expressed on DRG neurons.(2)DRG neurons were isolated and cultured in vitro from normal SD rats weighting(220-270g)and STZ-induced diabetic rats.Ca2+ imaging was performed after 24 h of culturing and 10-7,10-6,10-5mol/L capsaicin all can activate TRPV1 in a concentration-dependent manner(P<0.001)for normal DRG neurons.10-6 mol/L capsaicin was used to act on DRG neurons in normal and diabetic rats,respectively.The increase of Ca2+ concentration in DRG neurons in diabetic rats was less than that in normal DRG neurons,which also verified the decrease of TRPV1 expression in DRG of diabetic rats.5×10-8,5×10-7,5×10-6mol/L oleic acid(OA)and 10-6mol/L capsazepine(Cpz)were used to act on normal rat DRG neurons,respectively.5×10-6mol/L OA and Cpz had similar effects,and there was no significant difference in intracellular Ca2+ concentration(P > 0.05).Compared with Cpz,5×10-8,5×10-7mol/L of OA increased intracellular Ca2+ concentration(P < 0.001).Then,administration of 10-6mol/L Cap,the TRPV1 receptor was further activated and the intracellular Ca2+ concentration actived by Cpz and 5×10-6mol/L OA showed no significant change after Cap treatment(P > 0.05).Compared with Cpz,5×10-8,5×10-7mol/L of OA significantly increased intracellular Ca2+ concentration under Cap treatment(P < 0.001).It indicates that low concentration of OA like 5×10-8mol/L and could 5×10-7mol/L have a certain activation effect on TRPV1,while high concentration of OA 5×10-6mol/L like antagonist capsazepine and has an inhibitory effect on TRPV1 receptor.(3)The effects of OA on DRG neurons from STZ-induced diabetic rats.DRGs from diabetic rats exhibited a marked accumulation of ROS(P<0.01),increased apoptosis cells(P<0.01)and lactate dehydrogenase(LDH)in cell culture medium,loss of MMP(P<0.01),as well as impaired neuronal axon regeneration(P<0.01).TRPV1,CGRP,SP and NGF(protein and m RNA)in DRG neurons were significantly decreased(P<0.05).Whereas administration of 10-6mol/L capsaicin could promoted peripheral nerve regeneration(P<0.01)as well as inhibiting the ROS production(P<0.01)and improve MMP(P<0.01),which were associated with neurons apoptosis.Meanwhile,TRPV1,CGRP,SP and NGF expression were increased(P<0.05).An antagonist of TRPV1,apsazepine,can cancel the protective effect of capsaicine(P>0.05).5×10-6mol/L OA was used to treat diabetic DRG neurons,and there was no further increase in DRG injury(P>0.05).However,capsaicin showed no protective effect after capsaicin administration(P>0.05),and TRPV1,CGRP,SP and NGF expression were not increased(P>0.05).This indicates that 5×10-6mol/L OA plays a similar role as an antagonist and cancels the protective effect of capsaicin in activating TRPV1.(4)The effects of CGRP on DRG neurons from STZ-induced diabetic rats.Exogenous administration of 10-8mol/L CGRP in diabetic DRG neurons can improve the damage to DRG neurons caused by diabetic diseases,so as to reducing cell damage(P<0.05),reducing ROS production(P<0.01),increasing MMP(P<0.05),reduce apoptotic cells rate(P<0.01)and increase neurite outgrowth(P<0.01).Meanwhile,10-7mol/L antagonist CGRP8-37 can cancel the protective effect of CGRP(P>0.05).Then whether the protective role of CGRP depends on SP or NGF,we used Lentiviral vector mediated over-expression of CGRP in diabetic rats DRG neurons.The demage of diabetic DRG neurons were decreased(P<0.01)and neurite outgrowth were increased(P<0.01).At the same time,the expression of SP and NGF are detected,and the m RNA and protein expression of SP and NGF are not significantly increased(P>0.05).(5)The effects of OA ischemic preconditioning on ischemia/reperfusion(I/R)induced cardiac dysfunctions and ventricular arrhythmia in isolated normal rats heart.Isolated normal rats hearts were pre-treated with 10-6mol/L capsaicin,LVDP?HR and ±dp/dtmax were significantly increased(P<0.01),LVEDP was decreased(P<0.01).Whereas,5×10-6mol/L OA pre-treated on isolated hearts,LVDP,HR and ± dp/dtmax decreased significantly(P<0.05),while LVEDP increased(P<0.05),further administratin of capsaicin could inhibit the effects of OA on cardiac function(P>0.05).After I/R injury,capsaicin could significantly ameliorated the cardiac function damage after I/R,as LVDP,HR and ±dp/dtmax improved(P<0.01)and LVEDP decreased(P<0.01).Meanwhile,capsaicin can improve the damage of OA to cardiac function(P<0.05)The PI3 K antagonist LY294002 was given and capsaicin was found to have on protective effects(P>0.05).During I/R period,capsaicin induced more ventricular premature(P<0.05),the number of ventricular tachycardia and ventricular fibrillation had no significant difference(P>0.05).For independent roles of OA,the average number of ventricular premature(P<0.01),tachycardia and fibrillation(P<0.05)were increased.OA was given in advance followed by capsaicin,the average number of ventricular premature(P<0.01),ventricular tachycardia(P<0.05)and ventricular fibrillation(P<0.05)were all decreased.Compared with administration with capsaicin,LY294002 could decreased the ventricular premature(P<0.05).The expression of p-Akt in the myocardial ischemia area was detected.P-Akt expression was increased after the action of capsaicin(P<0.01).Both OA and LY294002 inhibited the increase of p-Akt induced by capsaicin(P<0.01).It indicated that OA can aggravate I/R injury by inhibiting TRPV1 and inhibiting PI3K/Akt signaling pathway.Conclusion:(1)The effect of OA on TRPV1 activity depending on concentration.Low concentration can mildly activate TRPV1 while high concentration can inhibit TRPV1.(2)For DRG neurons from diabetic neuropathy rats,the expression of TRPV1 and sensory neuropeptides CGRP,SP and NGF are reduced.Exogenous capsaicin can improve the oxidative stress injury of diabetic DRG and promote axonal regeneration.Exogenous administration of large doses of OA did not aggravate the injury of diabetic DRG neurons,but weaken the protective effect after the activation of TRPV1.(3)CGRP can protect DRG neurons from diabetic neuropathy injury without depending on SP and NGF.(4)In normal rats isolated heart ischemia reperfusion injury,OA can worsen the injury and inhibit the protection of capsaicin activate TRPV1 against I/R injury,which may be achieved through the PI3K/Akt signaling pathway.Large doses of oleic acid can inhibit the protective effect of TPRV1-CGRP axis in diabetic rat neuropathy and cardiac ischemia-reperfusion injury,which may be achieved by inhibiting the PI3K/Akt signaling pathway.