| Along with improvement of living standard and change of lifestyle in modern society,the proportion of obese population in world is rising rapidly.The World Health Organization(WHO)has announced that,more than 2.1billion people worldwide will be overweight or obese by 2013,in which United States accounts 13%,and China and India together account 15% of total.Obesity and its related diseases have been noticed by public and society more and more.Metabolic syndrome(MS),which is characterized by obesity,hyperlipidemia,atherosclerosis,hypertension,insulin resistance and diabetes mellitus,has become a group of diseases that are harmful to health of people.The earliest abnormalities in vascular function are endothelial dysfunction,which mainly display the higher reactivity to the vasoconstrictor but the lower reactivity to the vasodilatation substances.The cause of endothelial dysfunction is induced by hyperactivity of renin-angiotensin system,hypoxia/reoxygenation injury,ischemia/reperfusion injury,insulin resistance,inflammation/immune response,oxidative stress,endoplasmic reticulum stress(ERS),and so on.Endoplasmic reticulum(ER)is the main site of protein synthesis,folding and maturation,and the homeostasis of ER is the premise for its function.The destruction of protein folding,capture,and degradation results in the accumulation of unfolded or misfolded proteins in ER,which is defined as ERS.High-fat diet-induced protein synthesis is so fast that beyond protein folding capacity of ER,leading to accumulation of unfolded and misfolded protein,which is called as ERS.ERS can activate inflammatory response,promote insulin resistance in liver and adipose tissue,resulting in vascular endothelium dysfunction and atherosclerosis.Autophagy is an important lysosome-related cell processes which could degradate and recycle proteins and organelles.There is a functional coupling between ERS and autophagy.ERS acts as a stimulator for autophagy in order to remove misfunctional or damaged organelles,limit ERS and protect living body for survival in adverse environment.So autophagy acts as one of the important mechanisms for protein homeostasis and antioxidation.Autophagy flux is the dynamic process of autophagy and can be used to evaluate autophagy function.There are two key parts in autophagy,including formation of autophagosomes and its degradation in the lysosome.Defects or disorders of autophagy may exacerbate oxidative stress,ERS and participate in development of various diseases,such as lipid metabolism disorder,neurodegeneration,cancer,aging and ischemic heart diseases.In high-fat diet animals,changes in autophagy flux will lead to disorders of insulin signaling in peripheral tissues and ERS,resulting in obesity.Consistently,obese animals with autophagy dysfunction display oxidative stress,ERS,damaged NO bioavailability and attenuated endothelial protection against anti-atherosclerosis.Autophagy is regulated by a lot of molecular signaling pathways.Adenosine mono-phosphate-activated protein kinase(AMPK)is one of signaling pathways,which acts as both sensor of upstream energy and activator of downstream autophagy,and plays an important role in endocrine diseases especially diabetes.AMPK can regulate autophagy positively in TSC1/2-dependent or non-TSC1/2-dependent mechanisms,mainly through inhibiting mTOR complex and phosphorylating ULK1,then triggering autophagy downstream signaling molecules S6K1 and 4E-BP1 in energy balance process and metabolic stress.Our previous studies have shown that chronic intermittent hypobaric hypoxia(CIHH)has a protective effect on multiple tissues of body.For example,in heart,CIHH promotes cardiac function recovery from ischemia/reperfusion,reduces infarct size and antagonizes arrhythmia.In liver,CIHH improves insulin resistance,glycolipid metabolism disorders,andsteatosis,and protects liver agaist ERS-mediated damage.In blood vessels,CIHH has anti-hypertension effect through upregulating expression of eNOS,increasing nitric oxide(NO)production,and enhancing endothelium-dependent relaxation(EDR)in mesenteric arteries of renal hypertensive rats.Furthermore,CIHH has other beneficial effects,such as immune regulation,anti-inflammatory and anti-oxidation.Therefore,we proposed a hypothesis that CIHH may improve vascular and endothelial dysfunction through enhancing autophagy,reducing ERS,and improving NO production in metabolic syndrome rats.In this study,we aimed to investigate the effect of CIHH on vascular endothelium and vasomotor in metabolic syndrome rats by using morphological,physiologic and molecular biology methods in whole body,tissue,and cell levels.The study consists of three parts:(1)To confirm the protective effect of CIHH on vascular endothelium and vasomotor in metabolic syndrome rats by using morphological,biochemistry and microvessel recording techniques.(2)To investigate the role of anti-oxidative tress and anti-ERS in CIHH protective effect on vascular endothelium by using ex vivo vessel culture,microvessel recording and Western blot techniques.(3)To investigate the role of autophagy in CIHH protective effect on vascular endothelium by using ex vivo vessel culture,microvessel recording and Western blot techniques.Part I Protective effect of CIHH on vascular endothelial function inmetabolic syndrome ratsObjective: To confirm the protective effect of CIHH on vascular endothelium and vasomotor in metabolic syndrome rats.Methods: Sprague-Dawley rats were randomly divided into four groups:control group(CON),chronic intermittent hypobaric hypoxia treatment group(CIHH),metabolic syndrome model group(MS),MS plus CIHH group(MS+CIHH).CON and CIHH rats were fed with standard rat chow diet and tap water.MS rats and MS+CIHH rats were fed with high fat diet and 10%fructose water to induce MS.After 16 weeks,CIHH rats and MS+CIHH ratswere treated with hypobaric hypoxia simulating 5000 m altitude for 28 days,6hours per day in a hypobaric chamber,and placed in a normoxic environment in the rest of time.CON rats and MS rats were always placed in normoxic environment.During CIHH treatment,animal weight and systolic arterial pressure(SAP)were measured in the same time every week.After CIHH treatment,the body length,weight and fat were measured to get Lee's index and fat coefficient with formula.The size of adipocytes in perivascular adipose tissue was observed and measured under scanning electron microscopy.Blood samples were collected from vena cava for measurement of blood biochemicals(fasting glucose,insulin,C peptide,lipids),inflammatory factor(interleukin-1?,interleukin-6 and tumor necrosis factor-?),adhesion molecules(intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1),NO and endothelin-1(ET-1).Microvessel recording technique was used to observe the effect of CIHH on contraction induced by phenylephrine(PE)and norepinephrine(NE),EDR induced by acetylcholine(ACh)and endothelium-independent relaxation induced by sodium nitroprusside(SNP)in mesenteric artery rings of rats.Results:1 Compared with CON rats,basiline SAP and body weight was increased in MS rats(P<0.05).CIHH treatment had no effect on basiline SAP and body weight in CON rats(P>0.05),but effectively antagonized the increase of baseline SAP and body weight in MS rats(P<0.05).2 Compared with CON rats,Lee's index,fat coefficient and the size of adipocytes were significantly increased in MS rats.CIHH treatment had no effect on Lee's index,fat coefficient and the size of adipocytes in CON rats(P>0.05),but effectively antagonized obesity symptom and adipocyte hyperplasia in MS rats(P<0.05-0.01).3 Compared with CON rats,there were metabolism disorders and insulin resistance in MS rats(P<0.05).CIHH treatment had no effect on the metabolism in CON rats(P>0.05),but effectively antagonize metabolism disorders and insulin resistance in MS rats(P<0.05-0.01).4 Compared with CON rats,there were an increase of inflammatory factor and adhesion molecules,and imbalance of NO and ET-1 in MS rats(P<0.05-0.01).CIHH treatment had no effect on inflammatory factor,adhesion molecules,and the balance of NO and ET-1 in CON rats(P>0.05),but effectively antagonized the changes in MS rats(P<0.05-0.01).5 Compared with CON rats,the contraction induced by PE and NE was increased in mesenteric artery rings of MS rats(P<0.01),but reduced in mesenteric artery rings of CIHH rats(P<0.05-0.01).The increased contraction by PE and NE in mesenteric artery rings of MS rats was antagonized by CIHH treatment(P<0.01).6 Compared with CON rats,the EDR was reduced in mesenteric artery rings of MS rats(P<0.01),but increased in mesenteric artery rings of CIHH rats(P<0.05-0.01).The reduced EDR in mesenteric artery rings of MS rats was antagonized by CIHH treatment(P<0.01).7 Compared with CON rats,the endothelium-independent relaxation by SNP was reduced in mesenteric artery rings of MS rats(P<0.05),but increased in mesenteric artery rings of CIHH rats(P<0.01).The reduced endothelium-independent relaxation by SNP in mesenteric artery rings of MS rats was antagonized by CIHH treatment(P<0.01).Summary:This part of the study indicated that there were significant disorder of glycolipid metabolism,insulin resistance,elevated SAP,endothelial and vasomotor dysfunction in MS rats induced by high fat and high fructose diet.CIHH treatment alleviated the disorder of glycolipid metabolism,improved abnormal vascular endothelial function and vasomotor,which might be carried out by improving insulin resistance,reducing inflammation,and increasing NO production.Part ? CIHH improves endothelium-dependent relaxation throughreducing endoplasmic reticulum stress in metabolic syndromeratsObjective: To investigate the role of oxidative stress and ERS inimprovement of CIHH on endothelium-dependent relaxation and underlying mechanism in mesenteric artery of metabolic syndrome rats.Methods: Sprague-Dawley rats were randomly divided into four groups:control group(CON),chronic intermittent hypobaric hypoxia treatment group(CIHH),metabolic syndrome model group(MS),MS plus CIHH group(MS+CIHH).CON and CIHH rats were fed with standard rat chow diet and tap water.MS and MS+CIHH rats were fed with high fat diet and 10%fructose water to induce MS.After 16 weeks,CIHH rats and MS+CIHH rats were treated with hypobaric hypoxia simulating 5000 m altitude for 28 days,6hours per day in a hypobaric chamber,and placed in a normoxic environment in the rest of time.CON and MS rats were always placed in normoxic environment.Microvessel recording technique was used to observe the effect of endothelial NO synthase(eNOS)inhibitor L-NAME,superoxide anion scavenger 4-Hydroxy-TEMPO and ERS inhibitor 4-phenylbutanoic acid(PBA)on EDR in mesenteric artery rings of rats.Western blot analysis was used to examine the phosphorylation of eNOS and the expression of ERS-related proteins.Ex vivo culture,microvessel recording and Western blot techniques were used to observe the effect of ERS inducer Tu and inhibitor PBA on EDR and protein expression.Results:1 Compared with CON rats,the blocking of L-NAME on EDR was decreased in mesenteric artery rings of MS rats(P<0.01),but increased in mesenteric artery rings of CIHH rats(P<0.01).The decreased blocking of L-NAME on EDR in mesenteric artery rings of MS rats was antagonized by CIHH treatment(P<0.01).2 Compared with CON rats,the enhancement of 4-Hydroxy-TEMPO on EDR was significantly increased in mesenteric artery rings of MS rats(P<0.01),but decreased in mesenteric artery rings of CIHH rats(P<0.01).The increased enhancement of 4-Hydroxy-TEMPO on EDR in mesenteric artery rings of MS rats was antagonized by CIHH treatment(P<0.01).3 Compared with CON rats,the enhancement of PBA on EDR wassignificantly increased in mesenteric artery rings of MS rats(P<0.01),but not changed significantly in mesenteric artery rings of CIHH rats(P>0.05).The increased enhancement of PBA on EDR in artery rings of MS rats was antagonized by CIHH treatment(P<0.01).4 Compared with CON rats,phosphorylation of eNOS(Ser1177)was decreased in mesenteric arteries of MS rats(P<0.01),but not changed significantly in mesenteric arteries of CIHH rats(P>0.05).The reduced phosphorylation of eNOS(Ser1177)in mesenteric arteries of MS rats was antagonized by CIHH treatment(P <0.01).5 Compared with CON rats,the expression of GRP78 and CHOP was upregulated in mesenteric arteries of MS rats(P<0.01),but not changed significantly in mesenteric arteries of CIHH rats(P>0.05).The increased expression of GRP78 and CHOP in mesenteric arteries of MS rats was antagonized by CIHH treatment(P<0.05-0.01).6 The EDR was significantly inhibited by ERS inducer Tu(P <0.01),while increased by ERS inhibitor PBA in mesenteric artery rings of MS+CIHH rats(P <0.01).7 ERS inducer Tu increased protein expression of GRP78 and CHOP(P<0.05-0.01),down-regulated the phosphorylation of eNOS(Ser1177)(P<0.01);while ERS inhibitor PBA decreased protein expression of GRP78 and CHOP(P<0.05-0.01),up-regulated the phosphorylation of eNOS(Ser1177)in mesenteric arteries of MS+CIHH rats.Summary:This part of the study indicated that there were excessive oxidative stress and ERS in MS rats induced by high fat plus high fructose diet.Also the vasodilatation dysfunction in MS rats might be resulted from reduction of phosphorylation of eNOS and NO production.CIHH treatment may improve endothelial dysfunction and enhance vasodilatation function through decreasing oxidative stress and ERS,increasing phosphorylation of eNOS and NO production in MS rats.Part ? CIHH improves endothelium-dependent relaxations throughenhancing autophagy in metabolic syndrome ratsObjective: To investigate the role of autophagy in CIHH improvement on endothelium-dependent relaxations and underlying mechanisms in metabolic syndrome rats.Methods: Sprague-Dawley rats were randomly divided into four groups:control group(CON),chronic intermittent hypobaric hypoxia treatment group(CIHH),metabolic syndrome model group(MS),MS plus CIHH group(MS+CIHH).CON rats and CIHH rats were fed with standard rat chow diet and tap water.MS rats and MS+CIHH rats were fed with high fat diet and10% fructose water to induce MS.After 16 weeks,CIHH and MS+CIHH rats were treated with hypobaric hypoxia simulating 5000 m altitude for 28 days,6hours per day in a hypobaric chamber,and placed in an normoxic environment in the rest of time.CON and MS rats were always placed in normoxic environment.Western blot analysis was used to examine protein expression of autophagy related proteins,AMPK phosphorylation and mTOR phosphorylation.Ex vivo culture,microvessel recording and Western blot technique were used to observe effects of ERS inducer Tu and inhibitor PBA,autophagy inducer RAPA and inhibitor 3MA,AMPK? inhibitor Com C and mTOR inhibitor RAPA,on EDR and protein expression.Ex vivo vessel culture,transmission electron microscopy and Western blot analysis were used to observe effect of autophagy degradation blocker CQ on autophagy flux.Results:1 ERS inducer Tu increased the protein expression of Beclin-1 and LC3-?/?(P<0.01);while ERS inhibitor PBA decreased the protein expression of Beclin-1 and LC3-?/? in mesenteric arteries of MS+CIHH rats(P<0.05-0.01).2 Autophagy inducer RAPA increased EDR(P<0.01),while autophagy inhibitor 3MA inhibited EDR in mesenteric artery rings of MS+CIHH rats(P<0.01).3 Autophagy inducer RAPA increased the protein expression of Beclin-1and LC3-?/?(P<0.01),decreased the protein expression of GRP78 andCHOP(P<0.01),up-regulated the phosphorylation of eNOS(Ser1177)(P<0.05);while autophagy inhibitor 3MA decreased the protein expression of Beclin-1 and LC3-?/?(P<0.05),increased the protein expression of GRP78 and CHOP(P<0.05-0.01),and down-regulated the phosphorylation of eNOS(Ser1177)in mesenteric arteries of MS+CIHH rats(P<0.01).4 Compared with CON rats,the protein expression of Beclin-1,LC3-?/? and p62 was upregulated in mesenteric artery of MS rats(P<0.05-0.01).Compared with MS rats,those proteins expression was downregulated in MS+CIHH rats(P <0.05).5 CQ,autophagy degradation blockers,increased the accumulation of LC3-?/? and p62 in all rats.Compared with CON rats,the blocking effect of autophagy degradation was the highest in CIHH rats(P<0.05-0.01)and the lowest in MS rats(P<0.05-0.01).6 Compared with CON rats,the phosphorylation of AMPK(Thr172)was decreased,while the phosphorylation of mTOR(Ser2448)was increased in mesenteric arteries of MS rats(P<0.01).Compared with MS rats,the phosphorylation of AMPK(Thr172)was up-regulated and the phosphorylation of mTOR(Ser2448)was down-regulated in mesenteric arteries of MS+CIHH rats(P<0.01).7 EDR was significantly inhibited by AMPK? inhibitor Com C(P<0.01),while increased by mTOR inhibitor RAPA in mesenteric arteries of MS+CIHH rats(P<0.01).8 AMPK? inhibitor Com C down-regulated the phosphorylation of AMPK(Thr172)(P<0.01),up-regulated the phosphorylation of mTOR(Ser2448)(P<0.01),decreased the protein expression of Beclin-1 and LC3-?/?(P<0.05-0.01),increased the protein expression of GRP78 and CHOP(P<0.01),down-regulated the phosphorylation of eNOS(Ser1177)(P<0.01);while mTOR inhibitor RAPA up-regulated the phosphorylation of AMPK(Thr172)(P<0.01),down-regulated the phosphorylation of mTOR(Ser2448)(P<0.01),increased the protein expression of Beclin-1 and LC3-?/?(P<0.01),decreased the protein expression of GRP78 and CHOP(P<0.05-0.01),up-regulated the phosphorylation of eNOS(Ser1177)in mesenteric arteries of MS+CIHH rats(P<0.01).Summary:This part of the study indicated that the activity of AMPK-mTOR signaling pathway was decreased,the autophagy degradation pathway was damaged,the expression of ERS-mediated autophagy-related proteins was upregulated,the expression of phosphorylated eNOS was downregulated,the endothelial function was impaired,and the EDR was reduced in MS rats induced by high fat plus high fructose diet.CIHH treatment protected vascular endothelium and enhanced EDR through activating AMPK-mTOR signaling pathway,improving autophagy flux,reducing ERS,promoting expression of phosphorylated eNOS,and increasing NO production in MS rats.Conclusion:There were glycolipid metabolism disorders,insulin resistance,inflammation,oxidative stress in MS rats induced by high fat plus high fructose diet.And there were ERS,autophagy dysfunction,vascular endothelial dysfunction and vasomotor disorder in mesenteric arteries of MS rats.CIHH treatment has a protective effect on vascular endothelium and improves EDR in mesenteric artery rings of metabolic syndrome rats,which may be carried out through reducing oxidative stress and ERS,improving the autophagy dysfunction,up-regulating the phosphorylation of eNOS and imcreasing NO production.In addition,the protect effects of CIHH may be related to the improvement of glycolipid metabolism disorders,the reduction of insulin resistance and inflammation. |
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