Based On Estrogen Receptor GPR30 And Endoplasmic Reticulum Stress Response To Explore The Effect Of LWDHF On The Treatment Of Atherosclerosis In Menopausal Women

Objective:To increase the incidence of cardiovascular and cerebrovascular diseases caused by estrogen deficiency in postmenopausal women.Therefore,it is of great significance to evaluate the effect of drugs on the cardiovascular disease of the menopause with the help of the related pathological models.This topic combined with previous studies,using ApoE-/-mice to establish ovariectomized atherosclerosis model(atherosclerosis),from the point of view of homocysteine,blood lipid,endothelial function,inflammatory cytokines and other evaluation effect of Liuwei Dihuang Fang(LWDHF)on the model,and the use of homocysteine(Hcy)to establish the injury model of human umbilical vein endothelial cells(HUVECs)and to evaluate the protective effect of Liuweidihuangfang on endothelial cells.To explore the effect of six ingredient on the endoplasmic reticulum stress in the process of atherosclerosis,and to explore the possible mechanism of the anti atherosclerosis effect of six herbs from the angle of estrogen receptor.Method1 healthy female ApoE-/-and C57BL/6 mice and sham operation group selection of C57BL/6 mice under the bilateral ovarian resection a small amount of fat,ApoE-/-control group underwent bilateral ovariectomy.More than two feeding SPF mice pellet feed feeding.The rest of the ApoE-/-mice were ovariectomized and fed with high fat diet for 30 days,TC>7.8mmol/l model mice were randomly divided into four groups:model group,estradiol valerate group(1.3 x 10-4g/kg)and simvastatin group(4.3x10-3g/kg),Liuwei Dihuang group(9.75g/kg)intragastric administration,1 times a day,sham operation group,ApoE-/-control group and model group were administered with volume of physiological saline,a total of 90 days.Weigh mice every week,body weight and food intake,according to the weight adjustment to the drug dose,blood test every four weeks to detect blood TC.After 90 days,blood was taken to detect the expression of CRP,TG,HDL,LDL,TNF-,IL-1,IL-10,NO,eNOS,iNOS,Hcy,and TC in plasma.HE staining and transmission electron microscope were used to observe the morphological changes of aorta.The effect of LWDHF on estrogen receptor GPR30 and expression of Grp78 and CHOP was detected by Westen blot and Immunohistochemistry.2 Using the same 80mmol/L Hcy for cystine to establish HUVEC injury model,observe the protective effect of LWDHF containing serum on HUVEC.After successful model replication,MTT assay was used to detect cell survival,Hoechst staining was used to observe the cell apoptosis,and the apoptosis rate was detected by flow cytometry.The expression of estrogen receptor GPR30 was detected by blot Westen and immunofluorescence,and the effect of on the endoplasmic reticulum stress protein Grp78 was adhered to at the same time.G15(GPR30 inhibitor)on HUVEC blocking estrogen receptors westen after blotting and immunofluorescence assay endoplasmic reticulum stress and 25umol/L of ER stress protein Grp78 and CHOP expression.Results1 Effect of LWDHF on atherosclerosis model of ovariectomized ApoE-/-mice1.1 In ApoE-/-control group of mice plasma TC,TG,LDL-C and HDL-C levels were significantly increased,a further increase in the indicators in the model group,after 90 days of administration,the administration can decreased significantly the content of TC,TG and LDL-C and HDL-C content.There was no difference in the clinical application of simvastatin.1.2 Oil red staining:C57 Group aortic arch little plaque formation,ApoE-/-control group,a small amount of plaque formation,the model mice aortic arch plaques form dense,blocking blood vessels;each treatment group could reduce arterial varying degrees the formation of atherosclerotic plaques,LWDHF group effect is more obvious than the other two groups.HE staining:ApoE-/-mice in the control group only appears calm in membrane lipids and membrane thinning of vascular pathology of these two phenomena,mild vascular injury model group were all pathological blood vessels,the overall degree of vascular injury further deepened.Each treatment group could reduce the degree of vascular damage in varying degrees,which LWDHF and simvastatin has significantly decreased vascular calcification,vascular wall of atherosclerotic plaque area and the number of the fibrous cap,significantly slowing the process of atherosclerosis.1.3 In ApoE-/-control group in mice plasma NO and eNOS content decreased,increased levels of iNOS,further aggravate the mice in the model group the changes of each index,different treatment group can increase in plasma NO and eNOS content,reduce the content of iNOS,effect of LWDHF on improving eNOS expression is superior to that of female 2 alcohol group and simvastatin group.1.4 Control ApoE-/-mice CRP,TNF alpha and IL-1 beta increased,feeding high fat model group all the indices of the mice increased further,exacerbated inflammatory response,the treatment group can different degrees of decrease in plasma CRP,TNF alpha and IL-1 beta content and elevated IL-10 content,there are obvious anti-inflammatory effect and effect of Liuwei Dihuang Decoction on the inflammatory index to is better than the other two treatment groups,showing the better anti-inflammatory effect.1.5 In ApoE-/-control mice plasma Hcy levels were significantly increased,in model group mice plasma Hcy levels further increased,the drug group in LWDHF were not significantly reduced plasma Hcy levels.1.6 Western blot results showed that ApoE-/-control mice aortic endoplasmic reticulum stress protein GRP78 and chop expression increased,a further increase in the model group mice aorta protein expression and produces surface endoplasmic reticulum stress;estrogen group and simvastatin group decreased the expression of GRP78 and chop,LWDHF group further increases in the expression of GRP78,while reducing the expression of chop.1.7 Western blot results showed that ApoE-/-control group mice aorta estrogen receptor GPR30 expression decreased,after fed with high fat diet,model group mice GPR30 expression decreased,the treatment group can increased to different extent,GPR30 expression,except estradiol valerate group,other each give medicine group of GPR30 expression did not exceed the normal level.Effect of LWDHF serum on endothelial cell damage induced by Hcy2.1 MTT results showed in 80mmol/L Hcy of HUVECs injury rate reached 50%,then gave 20%of the drug containing serum,the protection of drugs on HUVECs rate is approximately 99.97%.2.2 Hoechst staining:after Hcy,the color of the fluorescent dye was brighter than that of the control group,which showed typical apoptotic morphological changes,and the number of cells was significantly decreased.Adding LWDHF containing serum can significantly change the morphology of apoptosis induced by Hcy,the number of cells increased significantly.2.3 Apoptosis was determined by annexin V/PI staining:The apoptosis rate increased significantly after 24h Hcy,after adding 20%LWDHF serum,apoptosis decreased significantly compared with model group.2.4 Effect of LWDHF containing serum on endoplasmic reticulum stress protein:Western blot and immunofluorescence results show that after treated with 80mmol/LHcy,elevated expression of endoplasmic reticulum stress protein GRP78 and chop in HUVECs,to give 20 LWDHF containing serum,further increased the expression of GRP78,CHOP expression significantly decreased.2.5 Effect of LWDHF containing serum on estrogen receptor GPR30:according to the results of Western blot and immunofluorescence,adminsted Hcy after 24h,the expression of GPR30 decreased significantly,LWDHF can up-regulated expression of GPR30,but no more than normal level.2.6 Effect of estrogen receptor GPR30 on endoplasmic reticulum stress protein GRP78 and CHOP:Western blot results showed that give to give 80mmol/LHcy 24h,up-regulated expression of endoplasmic reticulum stress protein GRP78 and chop,Liuwei Dihuang Pill medicated serum can be further elevated GRP78 expression,and reduce the chop expression and alleviate the endoplasmic reticulum stress reaction;in the use of 25umol/L G15 blocking receptor GPR30 also give Hcy at 24h after,two protein expression with the use of a separate Hcy consistent,but given Liuwei Dihuang decoction containing serum,can not change the protein expression,no approach to alleviate ER stress response;immunofluorescence staining and Western blot results are consistent.conclusion1 LWDHF has a good therapeutic effect on ovariectomized atherosclerosis in ApoE-/-mice model,through the influence of homocysteine,blood lipids,inflammatory factor,endothelial function reached the role of anti atherosclerosis.2 LWDHF can significantly inhibit the apoptosis of endothelial cells,and alleviate the occurrence of atherosclerosis.3 LWDHF can improve the expression of endoplasmic reticulum stress protein Grp78 and CHOP in order to alleviate the effect of endoplasmic reticulum stress reaction to protect the aorta blood vessel.4 The impact of LWDHF on endoplasmic reticulum stress reaction to protect endothelial cells,alleviate atherosclerosis effect may be achieved through estrogen receptor GPR30.