Protective Effects And Mechanisms Of Danzhi Jiangtang Capsule On Vascular Endothelial Cell Damages Induced By Hyperlipidemia

Objective:1.To study whether high fat diet?HFD?could induce vascular endothelium-dependent vasodilatation?EDV?,serum parameters,pathological and molecular biologicalchanges of thoracic aorta in male Sprauge-Dawley rats,and further to investigate the protective effects and mechanisms of Danzhi Jiangtang Capsule on vascular function.3.To observe whether palmitic acid could induce oxidative stress and endoplasmic reticulum stress in human umbilical vein endothelial cells and whether the serum containing Danzhi Jiangtang Capsule could reverse the toxic effect of palmitic acid.Methods:1.Animal experiment:After forty male SD rats of 8 weeks-old were acclimatized for 1 week,which were randomly assigned to 2 groups:standard chow diet?SCD?and high-fat diet?HFD?group.HFD group rats were given HFD for twelve weeks,then they were randomly assigned to 3 groups:HFD,HFD with low dose Danzhi Jiangtang Capsule?HFD+DJCL?and HFD with low dose Danzhi Jiangtang Capsule?HFD+DJCH?group.While the latter 3 groups were fed with HFD,HFD+DJCL and HFD+DJCH groups were given with Danzhi Jiangtang Capsule500mg/kg/d and 1000mg/kg/d respectively by gavage administration for 8 weeks.Then the serum levels of TNF-?,IL-1?,T-AOC,SOD,FFA,MDA and endothelial function indice?NO and ET-1?were detected.2.Tissue experiment:Thoracic aorta was separated and used for the tension test of vascular rings.The pathological changes of rat thoracic aorta were determined by HE staining;the changes of aortic elastic fibers and collagen fibers were detected by Masson staining.Oil red-O staining was used to detect rat aortic lipid deposition.Immunohistochemical assay of CD68 was used to detect macrophage infiltration.TUNEL method was used to detect apoptosis of endothelial cells in thoracic aorta.RT-PCR was used to detect the gene expressions of CPT1b,ACC,IRE1?,XBP1s,GRP78,CHOP,ATF6 and eNOS respectively.JNK,p-JNK,IRE1?,GRP78,CHOP,eNOS,and p-eNOS protein expressions in thoracic aorta were detected by Western blot.3.Cell experiment:The preparation of drug serum:thirty male SD rats were randomly divided into 3 groups,according to the principle of random distribution.The normal diet group were given water,the other two groups were given low dose of Danzhi Jiangtang Capsule?1g/kg/d?and high dose of Danzhi Jiangtang Capsule?2g/kg/d?respectively for 2 weeks.Then all rats was anesthetized and blood was taken from the carotid artery.Serum was separated under sterile condition,inactivated at 56?for 30minutes and stored at 80?.Human umbilical vein endothelial cells?HUVEC?were cultured in F12K medium containing 10%fetal bovine serum,0.1g/L heparin sodium and 20U/ml FGF-b,and assayed in 3 experiments:?1?Control group?5%normal rat serum+5%FBS?,PA group?5%normal rat serum+0.5mM/LPA+5%FBS?,PA+DJCL?5%FBS+5%DJCL serum+0.5mM/L PA?,PA+DJCH group?5%FBS+5%DJCH serum+0.5mM/L PA?,PA+4-PBA group?5%FBS+5%normal rat serum+0.5mM/L PA+10mM/L 4-PBA?,TM group?5%normal rat serum+5%FBS+1?g/L TM?.?2?Control group?5%FBS+5%normal rat serum?,H₂O₂ group?5%FBS+5%normal rat serum+300?M H₂O₂?,H₂O₂+DJCL group?5%FBS+5%DJCL serum+300?M H₂O₂?,H₂O₂+DJCH group?5%FBS+5%DJCH serum+300?M H₂O₂?,H₂O₂+GSH group?5%FBS+5%normal rat serum+300?M H₂O₂+400?M GSH?,?3?Control group?5%FBS+5%normal rat serum?,TM group?5%normal rat serum+1?g/L TM+5%FBS?,TM+DJCL?5%DJCL serum+1?g/L TM+5%FBS?,TM+DJCH?5%DJCH serum+1?g/L TM+5%FBS?,TM+PBA?5%normal rat serum+5%FBS+1?g/L TM+10 mM/L 4-PBA?.Expression of proteins related with oxidative stress and endoplasmic reticulum stress SIRT1,eNOS,P-eNOS,IRE1,GRP78 and CHOP detected by Western blot.Flow cytometry detect the levels of reactive oxygen species in experiment.Immunofluorescence detect the protein expression and localization of CHOP.Results:1.Animal experiment:Compared with the SCD group,?1?the body mass in HFD group was higher;?2?The serum levels of ET-1,MDA,FFA,IL-1?,TNF?in HFD group were significantly increased,and serum levels of NO,SOD and T-AOC were decreased obviously.Compared with HFD rats,the body mass in DJCL and DJCH groups were significantly reduced,and the serum levels of ET-1,MDA,FFA,IL-1?and TNF?were significantly decreased,meanwhile the serum levels of NO,SOD and T-AOC were increased significantly.Moreover,the serum levels of ET-1,NO,SOD,T-AOC and IL-1?in HFD+DJCH group were more obvious than that in HFD+DJCL group,thus exhibited dose-dependent effect.2.Tissue experiment:Compared with the SCD group,the endothelium-dependent relaxation of thoracic aorta in the HFD group was significantly reduced.Secondly,HE and Masson staining showed that the thoracic aorta wall thickened,the elastic fibers decreased,and the collagen fibers increased significantly in the HFD group.Oil red-O staining showed that the blood lipid deposition in thoracic aorta was increased in HFD group.The immunohistochemical results showed that the expression of CD68 protein in the HFD group was significantly increased.TUNEL assay showed that apoptosis of vascular endothelial cells existed in the HFD group.RT-PCR results showed that gene expressions of ACC,IRE1?,GRP78,CHOP and XBP1s were increased significantly,while CPT1b gene expression in HFD group was significantly decreased;ATF6 and eNOS gene expressions had no significant difference between the 2 groups.WB results showed that protein expressions of GRP78,CHOP,IRE1?,BAX and p-JNK in HFD group were increased significantly,while protein expressions of BCL-2 and p-eNOS were decreased significantly.DJCL and DJCH treatment improved the endothelium-dependent relaxation of thoracic aorta,alleviated their pathological changes,reduced significantly the expressions of ER stress-related molecules and p-JNK,increased the protein expressions of BAX and p-eNOS.Moreover,compared with HFD+DJCL group,improvement of the expressions of IRE1?,GRP78 mRNA and BAX,BCL-2,IRE1?,CHOP protein were significantly higher than that in HFD+DJCH group.3.Cell experiment:?1?Compared with CON group,the protein expressions of P-eNOS and SIRT1 were significantly decreased,while protein expressions of IRE1?,GRP78 and CHOP were increased significantly in HUVEC in PA and TM groups.Compared with PA group,protein expressions of P-eNOS,and SIRT1 were significantly higher,while protein expressions of IRE1?GRP78 and CHOP were significantly decreased in PA+PBA,PA+DJCL and PA+DJCH groups.?2?Compared with CON group,the protein expressions of P-eNOS and SIRT1 in H₂O₂ group were significantly lower;compared with H₂O₂ group,the protein expressions of P-eNOS and SIRT1 were significantly higher in H₂O₂+GSH,H₂O₂+DJCL and H₂O₂+DJCH groups respectively.?3?Compared with CON group,the protein expressions of IRE1?,GRP78 and CHOP in TM group were increased significantly;compared with that in TM group,the protein expressions of and IRE1?,GRP78 and CHOP were decreased significantly in TM+PBA,TM+DJCL,TM+DJCH groups respectively.?4?Compared with CON group,PA treatement could increase the ROS level in HUVEC cells,and DJCL and DJCH treatment could reduce the ROS levels.?5?CHOP was expressed and distributed in cytoplasm and cell nucleus.The protein expression of CHOP in PA group was increased obviously,meanwhile the protein expressions of CHOP protein in PA+DJCL and PA+DJCH groups were decreased significantly by immunofluorescence.Conclusion:1.Twenty weeks of high fat diet could induce metabolic syndrome in male SD rats with decreased endothelium-dependent relaxation in thoracic aorta.Danzhi Jiangtang Capsule could obviously alleviate the above symptoms,the mechanisms may be related with reduced lipid deposition,oxidative stress and endoplasmic reticulum stress.2.Palmitic acid could cause oxidative stress and endoplasmic reticulum stress in HUVEC.The effects of Danzhi Jiangtang Capsule on endothelial cells was probably through inhibiting endothelial cell apoptosis,reducing oxidative stress and endoplasmic reticulum stress.