Activation Of TRPV1Prevents Atherosclerosis Vascular Dysfunction Induced By Disorders Of Glucose And Lipid Metabolism Through UCP2Up-regulation

Back Ground and Objectives:Atherosclerosis (AS) is still the leading cause of the cardiogenic sudden death and strokein many countries. Nowadays, therapeutic methods such as drugs, interventional therapies andsurgeries can not prevent the increase morbility and mortality of cardiovascular andcerebrovascular diseases. Several studies indicated dietary factor such as DASH diet,Mediterranean diet and Prudent diet can decreased the morbility and mortality ofcardiovascular and cerebrovascular diseases effectively. Epidemiology studies data from ourcountry showed that, the prevalence of morbility of cardiovascular and cerebrovasculardiseases in the south of China is much lower than in the north of China. Besides for the highersalt intake of the north China people, the cause of this difference may be also due to the spicyfood prevalence in south especially in the southwestern of China. Capsaicin is the majorgradient in Hot pepper, its target is Transient Receptor potential vanilloid1(TRPV1), whencapsaicin binding to the TRPV1, it can lead to rise calcium ion (Ca2+) level intracellular, thusmediate a series of pathologic physiology reaction. Our previous studies implicated thatdietary capsaicin exist cardiometabolic protection through TRPV1activation. Activation ofTRPV1in endotheliual cells (ECs) promote the Ca2+influx to the intracellular, then activatesthe cAMP/PKA/eNOS signal pathway thus increase the nitric oxide (NO) release fromvascular endothelium, improves the endothelium-dependent relaxation, hence lower the bloodpressure in Spontanously Hypertensive Rats (SHR). Activation of TRPV1in preadipocyte(3T3-L1) cells, inhibited the maturity of the3T3-L1thus prevents lipid formation and obesity.Recently, we found that activation of TRPV1by the specific agonists, capsaicin andresiniferatoxin (RTX), can dose-dependently increased cytosolic calcium and significantlyreduced the accumulation of lipids in VSMC from Wild type (WT) mice but not TRPV1knock out (TRPV1-/-) mice. Long-term activation of TRPV1significantly reduced lipidstorage and atherosclerotic lesions in the aortic sinus and in the thoracoabdominal aorta from AS mice treated with high-fat diet. These findings indicated that TRPV1activationameliorates high-fat diet-induced atherosclerosis. Chronic administration of capsaicin reducedthe high-salt diet-induced endothelial dysfunction and nocturnal hypertension in part bypreventing the generation of superoxide anions and NO reduction of mesenteric arteriesthrough vascular TRPV1activation. The above studies suggested that activation of TRPV1has protected effects against vascular dysfunction cause by oxidative stress. Lipid metabolismdisorders, chronic inflammation and oxidative stress are the major mechanisms of AS. Ourrecently study found that TRPV1activation by dietary capsaicin improves lipids metabolismthus prevents lipids accumulation. However, the effects of TRPV1activation on oxidativestress under the pathological state of AS is need further investigated.Under the cardiovascular risk factors, such as high cholesterol diet and high glucoselevel, reactive oxide species (ROS) generates from mitochondrial, excessive ROS can damagethe mitochondrial DNA, lead to abnormal mitochondrial membrane pore channels open,decrease mitochondrial membrane potential hence lead to the mitochondrial respiratory chaindysfunction and promote cell apoptosis. More important, excessive generates of ROS alsolead to endothelial nitric oxide synthase (eNOS) uncoupling. NO, which generate from L-arginine will bond to superoxide anion then generation more destructive products,peroxynitrite. Damage cells structure and relevant enzymes as well as impaired the NO/cGMPsignal pathway thus lead to the vascular dysfunction in AS.Recent years, researchers discoveried that uncoupling protein2(UCP2), a protein existed inthe mitochondrial membrane, can modulates the mitochondrial membrane potential, thusreducing the mitochondrial ROS generation. Some other studies found UCP2knock out(UCP2-/-) mice fed with high fat diet can develop AS significantly. Our previous study showedthat dietary capsaicin prevents non-alcoholic fatty liver through up-regulates UCP2expression in liver via TRPV1activation. Therefore, we propose the hypothesis that,activation of artery endothelial cells TRPV1by dietary capsaicin decreases the ROSproduction induced by the metabolism disorder of lipids and glucose, improves vasculardysfunction.This study will further clarify the important role of TRPV1in the process of vasculardysfunction in atherosclerosis, provide new therapy target for AS vascular dysfunction therapy,and also, provide theoretical basis on dietary factors intervention AS especially coronary atherosclerosis heart disease.Material and Methods:ApoE knock out (ApoE-/-) mice is a well model of AS, mice can formation ofhyperlipidemia and AS spontaneously. Leptin receptor deficient db/db mice existedhyperinsulinism, insulin resistant, high glucose levels and obesity that were similar to humantype2diabetes. It is also an important animal model for type2diabetes and related vasculardysfunction study. In order to verify our hypothesis, we use the TRPV1-/-，ApoE-/-and UCP2-/-mice generates the ApoE-/-/TRPV1-/-, ApoE-/-/UCP2-/-mice and then we use these mice forhigh fat induced atherosclerosis vascular dysfunction study. On the other hand, we useTRPV1-/-and WT mice, purchase the db/db mice and the matched WT mice to explore theeffects of dietary capsaicin on high glucose induced vascular dysfunction.We explore the effects of dietary capsaicin on AS mice coronary function by wiremyograph. Eight weeks old male ApoE-/-, ApoE-/-/TRPV1-/-and ApoE-/-/TRPV1-/-mice werefed with normal chow (ND), high fat diet (HF) and high fat diet plus capsaicin (HC,0.01%capsaicin inside). At the end of24weeks, the vasorelaxation of coronary induced by capsaicin,acetylcholine (Ach) and nitroglycerin (NTG) were studied. H&E stain, oil red stain andMasson stain were used to explore the effects of dietary capsaicin on the degree of coronaryartery stenosis. Using bioelectric amplifier, we observe the change of electrocardiogram (ECG)in mice. After intraperitoneal injection of isoprenaline (1.5mg/kg), we using dynamicelectrocardiogram to observe the ischemic ECG changes and the ST segement duration timein mice in order to assess the effects of dietary capsaicin to resist myocardial ischemia inmice.We also use small animal ultrasound to observe the effects of dietary intervention on thecardiac structure and function in AS mice, including cardiac ventricular septal thickness andsystolic function, diastolic function in left heart and aortic blood flow, coronary blood flowvelocity, etc. Fluorescence staining and western blotting to analysis the effects of long-termdietary capsaicin on ROS and Nitrotyrosine levels of aortic from AS mice. Observe theinfluence of long-term dietary on survival time of AS mice, and make the survival curve,calculating the average survival time.In vitro study, we cultured the pig iliac endothelial cells (PIEC), and treated withox-LDL, then using dihydroethidium (DHE) probe and immunobloting to observe the effects of capsaicin in ox-LDL induced ROS level and nitrotyrosine increase. Finally, we explore thechanges of TRPV1, eNOS, p-eNOS, PKA, p-PKA, UCP2in AS mice aortic tissue by westernblot and the effects of dietary capsaicin on these changes.In high glucose level induced vascular dysfunction, we first observe the effects ofTRPV1activation on blood glucose level. TRPV1-/-and WT mice were treat with dietarycapsaicin (0.01%) for24weeks, body weight, fasting blood glucose level, IntraperitonealGlucose Tolerance Testing (IPGTT), dynamic blood glucose levels were observed. Then weobserve the effects of dietary capsaicin on db/db mice body weight, fasting blood glucoselevels, intraperitoneal insulin-tolerance test (IPITT) and islet fuction and islet morphologywere observed by immunofluorescence and H&E stain. Myograph analysis the mesenteric andaortic arteries vasorelaxation in db/db mice and the WT mice treated with capsaicin or not.Use immunofluorescence to study the ROS and NO levels in mice mesenteric arteries. We useimmunoblotting analysis the expressions of TRPV1, eNOS, p-eNOS, PKA, p-PKA, UCP2inmice arteries tissues.Results:Capsaicin induced vasorelaxation in coronary arteries was impaired in high fat diettreated AS mice, and chronic dietary capsaicin improves the capsaicin induced vasorelaxationin ApoE-/-mice coronary arteries but not in TRPV1or UCP2knock out AS mice coronaryarteries. We also found Ach induced vasorelaxation was impaired in high fat diet treated mice,and chronic dietary capsaicin improves the vasorelaxation induced by Ach also in TRPV1andUCP2dependent manner. ECG studies found that there existed ST segment depression in highfat diet treated AS mice and24weeks dietary capsaicin improves the ST depression inTRPV1and UCP2dependent manner.Ultrasonic results indicated that, high fat diet caused the cardiac remodeling in AS micetreated with high fat diet. Dietary capsaicin decreased the interventricular septal thickness,improves the heart diastolic function but not the systolic function in TRPV1and UCP2dependent manner. TRPV1or UCP2knock out decreased the survival time of AS mice treatedwith high fat diet, and long time dietary capsaicin can prolong the span life time in ApoE-/-mice treated with high fat diet.Ox-LDL induced a significant increase superoxide anion and nitrotyrosine levels inPIECs, and capsaicin decrease the ROS and peroxynitrite level in a dose dependet manner, but this effects was blocked by TRPV1antagnist, PKA antagonist and UCP2inhibitorrespectively.In animal study, dietary capsaicin decreased the ROS production and increased the NOlevels in AS mice aortic treated with high fat diet. Western blot results indicated that, TRPV1expression decreased in high fat diet treat ApoE-/-mice aortic, and dietary capsaicin restorethe TRPV1expression in UCP2dependent manner. Dietary capsaicin also increased thep-eNOS, UCP2levels and decreased the nitrotyrosine levels in ApoE-/-mice vascular treatedwith high fat diet.TRPV1activation decreased the blood glucose level both in db/db mice and the WTmice. Dietary capsaicin also improves db/db mice insulin sensitivity and restores isletfunction.14weeks dietary capsaicin improves db/db mice arteries endothelium-dependentvasorelaxation but not the endothelium-independent vasorelaxation. Western blottingindicated dietary capsaicin increases TRPV1, p-eNOS, p-PKA and UCP2expression in db/dbmice arteries, decreases P22phoxin mice arteries.Conclusions:Dietary capsaicin restores capsaicin induced vasorelaxation, improves coronary arteriesendothelium-dependent vasorelaxation and decreases artherosclerosis stenosis hence improvesthe ischemic ECG changes in high fat treat AS mice in TRPV1and UCP2dependent manner.Long term dietary capsaicin improves cardiac remodeling and vascular velocity in AS micethus prolong the life span in AS mice also in UCP2dependent manner through TRPV1activation.Capsaicin prevents ox-LDL induced ROS and nitrotyrosine increase via PKA/UCP2pathway through TRPV1activation. Chronic dietary capsaicin increase TRPV1, p-eNOS,UCP2and p-PKA expression and decreased nitrotyrosine in AS mice.TRPV1activation decrease blood glucose levels, improves insulin sensitivity and restoreislet morphology in db/db mcie. Chornic dietary capsaicin decreases the ROS production andincreases NO level thus improves db/db mice endothelium-dependent vasorelaxation, thismay be due to the increases expression of TRPV1, p-eNOS, PKA and UCP2, decreases theexpression of P22phoxin mice arteries.