Endoplasmic Reticulum Stress Involved In High-fat Diet And Palmitic Acid-induced Vascular Damage And Fenofibrate Intervention

Objective:1. To observe whether high fat diet(HFD) can induce vascular endothelium-dependent vasodilatation (EDV) and the pathological changes in female Sprauge Dawley rats, and further to investigate the beneficial effects of fenofibrate(FF) on vascular function.2. To investigate whether palmitic acid(PA) can induce endoplasmic reticulum stress (ERS) and vascular endothelium-dependent vasodilatation in mouse aortic endothelial cells (MAEC) and normal female rats, and further to investigate whether the protective effects of fenofibric acid (FA) on endothelium-dependent vasodilatation related with decreased endoplasmic reticulum stress.Methods:1. In-vivo studies:Female Sprauge Dawley rats were divided into standard chow diets (SCD), high-fat diets (HFD) and FF treatment group (HFD+FF) randomly. The rats of latter two groups were given HFD feeding for 5 months to make them obese, then HFD+FF rats were treated with FF (30 mg/kg, once daily) via gavage for another 2 months. Body weight was measured weekly. The levels of serum lipid indices including TG, TC, HDL-C and LDL-C were detected. Free fatty acid (FFA) and palmitic acid (PA) levels were measured by gas chromatography. The total nitrite and nitrate concentration levels were measured according to the commercial kit instructions. The pathological changes of rat thoracic aorta were analyzed with HE and Weigert staining.Oil-red O staining was used to determine neutral lipid. The mRNA expressions of GRP78 and CHOP were assessed by RT-PCR.The protein expressions of GRP78, CHOP, ERK, p-ERK, JNK, p-JNK,eNOS and p-eNOS were evaluated by Western blot. Isometric tension was used to observe vasodilatation induced by cumulative concentration response of ACh.2. In-vitro studies:Then influences of palmitic acid and fenofibrate on the vascular rings of thoracic aorta from normal female SD rats was analyzed.MAEC were assigned into negative control(CON)group,4-PBA group(PBA),palmitic acid group(PA),4-PBA+PA group(PBA+PA), FA+PA group(FA+PA) and positive normal control TM group(TM). Cell proliferation in PA and FA group were determined by methyl thiazolyl tetrazolium(MTT) arrays. NO secretion of supernatant fluid was detected by the total nitrite and nitrate concentration levels.The gene expressions of GRP78, CHOP, IRE1α and XBP1-s were assessed by RT-PCR,and Western blot was used to detect the protein expressions of GRP78, CHOP, ERK,p-ERK,JNK, p-JNK,eNOS and p-eNOS.Results:1. In vivo studies:Compared with that in SCD group,①five months of HFD feeding caused obvious obesity, as indicated by increased body mass.②when compared with age-matched SCD-fed female rats, significantly higher levels of plasma TG, TC, LDL-C lower level of HDL-C were found in the HFD-fed rats than that in the SCD-fed rats.③The serum levels of FFA and PA were obviously elevated in HFD group. ④The serum levels of NO was elevated significantly.⑤Further examination of microscopic structure of thoracic aorta showed pathological characteristics including irregular thickening of thoracic aortic wall, disorders and fractures of elastic fibers in the HFD-fed female rats.⑥Oil red-O staining confirmed that more lipid deposited in the vascular walls of HFD group. ⑦The mRNA expressions of genes GRP78 and CHOP in thoracic aorta of the HFD-fed rats were significantly elevated. ⑧The protein expressions of GRP78, CHOP, p-ERK and p-JNK were significantly increased, while the protein expression of p-eNOS were decreased obviously.⑨EDV to ACh were much weaker in aortic rings of the HFD-fed rats compared with SCD-fed rats.　⑩enofibrate could decrease body weight and serum lipid indices and improve pathological changes via gavage for another 2 months.The mRNA expressions of GRP78 and CHOP were decreased significantly in thoracic aortas in HFD+FF group. The protein expressions of GRP78, CHOP, p-ERK, p-JNK were decreased significantly but the protein expression of p-eNOS was increased obviously in HFD+FF group. FF treatment significantly ameliorated the impaired relaxation to ACh.2. In vitro studies:　Compared with the CON group, ①When incubated with different concentrations of PA for 90 min,we found that when concentration of PA was increased to 0.2 mmol/L, the relaxation of aorta was reduced obviously. After pretreatment with the endoplasmic reticulum stress inhibitors 4-PBA or FA, relaxation to ACh was improved significantly compared with that in PA group. when incubated with TM for 90 min, the response to Ach was significantly reduced compared to that in vehicle (Krebs buffer).②PA inhibited the vitality on MAEC and the most appropriate concentration and time was 0.5 mmol/L for 24 h. ③NO secretion of supernatant fluid was decreased induced by PA. FA pretreatment obviously increased secretion of NO decreased by PA treatment in supernatant. ④The mRNA expressions of GRP78, CHOP, IREla and XBP1-s were increased obviously. ⑤The protein expressions of GRP78, CHOP, p-ERK, p-JNK were increased but p-eNOS was decreased in PA group.⑥After pretreatment with the endoplasmic reticulum stress inhibitors 4-PBA or FA,the genes and protein expressions were reversed obviously. ⑦The gene and protein expressions of CHOP and GRP78 in positive normal control of ER stress TM group were significantly increased.Conclusion:1. HFD diet for 5 months successfully induced the model of hyperlipemia and reduced thoracic aorta endothelium-dependent vasodilatation reaction,fenofibrate could significantly relieve the symptoms, this effect maybe related with decreased ER stress and increased phosphorylation of eNOS.2. PA could inhibit the cellular proliferation, induce ER stress in MAEC. The beneficial effect of FA probably involve promoting cell proliferation, inhibition of endoplasmic reticulum stress, improving endothelial-dependent vasodilation response.