The Mechanism Of LWDHF On Treating-AS Mediated By Menopause Based On The Correlation Between Estrogen Receptor And Kidney Essence Deficiency

ObjectiveThe cardiovascular disease incidence of postmenopausal women with low estrogen level is significantly higher than that of childbearing age women,indicating the important meaning of estrogen on maintaining and regulating the female normal cardiovascular function.The protective effects of estrogen are mainly through estrogen receptors(ER).ER of postmenopausal women are expressed abnormally,which is the important reason in menopausal atherosclerosis(AS).In this study,we evaluated the effect of Liuweidinghuangfang(LWDHF)in menopausal atherosclerosis model which was made by female menopausal rats.We.also explored the antimenopausal atherosclerosis mechanism of LWDHF from the change of blood lipids,the function of vascular endothelial cell and the expression of ER.MethodsThe healthy female rats were employed and they were randomly divided into two groups,one was sham operation group,the other was operation group.A few fats below bilateral ovaries in sham operation group rats were cut off and rats were fed with normal diet.Bilateral ovaries were removed in operation group rats and they were fed with high fat diet plus VD3 to set up AS model.LWDHF’s compatibility has three couple drugs:they are Rehmannia(SD)/Alismataceae(ZX),Dogwood(SZY)/paeonol(DP)and yams(SY)/poria(FL).After 30 days,operation group rats were divided into six groups according to lipid levels:model group,estradiol benzoate group(estradiol benzoate,1.8 × 10-6g/kg by intramuscular injection,once every three days),LWDHF group(6.75 g/kg by intragastric administration,once a day),SD/ZX group(2.97g/kg by intragastric administration,once daily),SZY/DP group(1.89g/kg by intragastric administration,one time per day),SY/FL group(1.89g/kg by intragastric administration,once a day).The same volume of 0.5%Sodium carboxymethyl cellulose(CMC-Na)were given by intragastric administration in sham operation group and model group for 11 weeks.The feed intake and body weight were weighted weekly,and the administered dose was adjusted based on body weight.11 weeks after administration,blood was draw to calculate the quantity of circulating endothelial cells(CECs).The contents of lipid(TC,TG,HDL,LDL),the level of CRP,TNF-a,IL-6,MDA,SOD in serum;and the level of Ang Ⅱ,6-k-PGF 1αΟ and TXB2 in plasma were detected by biochemical method and radioimmunoassay.Aortic morphological changes were observed by Oil red O dyeing,light and electron microscopy.The aortic expression of ERa,ERP,GPR30 were observed by immunohistochemical method and western blot.Results1.The effect of lipid levels with LWDHF and its compatibility on AS rats by ovariectomy(OVX):Compared with sham operation group,the levels of total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)in serum and atherosclerosis index(AI)in model group increased significantly,and HDL-C level is reduced(P<0.01).These data were improved significantly after given with LWDHF,which showed that LWDHF could prevent the development of menopausal AS through reducing the blood fat level of AS rats,and increasing the level of HDL-C.Three couple drugs all could reduce the levels of TC and LDL-C in serum,decrease AI index,increase the level of HDL-C,compared with model group(P<0.01);but the effect was weaker than LWDHF.The regulation of LWDHF on the level of blood fat was the result of the cooperation of three couple drugs.2.The effect of aortic morphology with LWDHF and its compatibility on AS rats by OVX:(1)The observation by Oil red O dyeing:The arterial intima in AS rats was rough,less flexible.The aortic wall had red-stained plaques after dyeing with punctate distribution,and the coloring of basement membrane was significantly heavier than that in sham operation group(P<0.01),LWDHF could significantly reduce these pathological changes.Three couple drugs could also improve aorta pathological changes,but the effect was weaker than LWDHF.(2)The light microscope observation by HE dyeing:The arterial intima in model group was thickened,the fiber was slightly elevated and the endothelial cells were swollen,degenerated,partial deleted.Medial membrane was thickened with the proliferation of smooth muscle cell(SMC),the increased volume of cells and the disorder of cells arrangement.LWDHF could obviously improve pathological changes of arterial structure.Three couple drugs could also change the structure of aortic pathology by HE stained,but it was not as effective as the LWDHF.(3)The observation by electron microscope:CECs in model group was severely damaged.Vascular endothelial cells were degenerated and swollen,and lipid drops could be seen in cytoplasm.SMCs were disordered and mixed thrombosis could be seen.Endothelial cells were slightly damaged in LWDHF group,and lipid in cytoplasm was reduced;SMCs were in order,and no thrombosis was attached.Three couple drugs also could improve aortic disease,but it was not as good as the LWDHF.3.The effect of endothelial functions with LWDHF and its compatibility on AS rats by OVX:(1)The number of peripheral blood CECs in model group was increased significantly;the level of NO and NOS was decreased,compared with sham operation group(P<0.01).LWDHF could reverse this appearance to improved the injury of endothelial cells on blood vessel caused by AS.In three couple drugs,reduced number of peripheral blood CECs with SY/FL is weaker than LWDHF;SZY/DP has no effect on increasing the level of NO.Therefore,effects of reduced the number of peripheral blood CECs with SY/FL was the weakest.The increased concentration of NO was mainly by SD/XF and SY/FL;and increased NOS levels were completed by the collaboration of three couple drugs.(2)The level of 6-k-PGF1α in model group was decreased significantly,and TXB2 level was’increased,compared with sham operation group(P<0.01).LWDHF could significantly increase 6-k-PGF1α level and decrease TXB2 level.It could prevent the development of menopausal AS by improving their balance,protecting endothelial cells,and inhibiting the proliferation of SMCs.SD/ZX had no effect on the balance between 6-k-PGF1α and TXB2.SY/FL could increase 6-k-PGF1αlevel,but SZY/DP and SY/FL could reduce TXB2 level.(3)The level of SOD in AS rats was decreased and the MDA level was significantly escalated,compared with sham operation group(P<0.01).LWDHF could regulate the balance of SOD and MDA to protect the oxidation system in endothelial cell.Three couple drugs could significantly improve the balance of SOD and MDA,but the effect was not as good as LWDHF.The regulated balance between SOD and MDA with LWDHF is the cooperation of three couple drugs,and the effect of SY/FL was the least.(4)The level of angiotensin Ⅱ(AngⅡ)in AS rats was higher than that in sham operation group(P<0.01).LWDHF could significantly reduce the level of AngⅡ,maintain normal vascular function by regulating renin-angiotensin-aldosterone system(RAAS)system.The effect of SD/ZX and SY/FL was weaker than LWDHF,so the key role of Ang II reduction was from SZY/DP.4.The effect of inflammatory factors with LWDHF and its compatibility on OVX AS rats:The contents of CRP,TNF-a and IL-6 in AS rats were higher than that in sham operation group(P<0.01).LWDHF could significantly reduce the levels of CRP,TNF-a and IL-6,indicating that LWDHF could delay the development of the AS by inhibiting inflammatory factors production and reducing inflammatory response.Three couple drugs all could decrease inflammatory factors level,SY/FL had no effect on reducing TNF-a and IL-6 levels.The anti-inflammatory effect was come from SD/ZX and SZY/DP.5.The effect of gonadal axis hormone levels with LWDHF and its compatibility on OVX AS rats:The level of estradiol(E2)in serum and uterine index in AS rats were significantly lower than that in sham operation group,and the levels of LH and FSH were significantly increased through the negative feedback regulation of gonadal axis.LWDHF had significant functions on E2 and uterine index,and had no effect on LH and FSH.LWDHF could partially improve the E2 secretion in female rats after OVX,which may had the same effect of estrogen.Three couple drugs had no affect on E2,LH and FSH,but they had a trend on increasing the uterine index.There was a significant difference between SD/ZX group and model group,which showed LWDHF could inhibit the development of AS may not be completely through the adjustment of gonadal axis.6.The expression of ERα,ERβ,G-protein coupled estrogen receptor(GPR30)with LWDHF and its compatibility on OVX AS rats:The expression of aortic ER in AS rats was significantly lower than that in sham operation group,indicating the expression of ER in menopause AS rats significantly reduced.LWDHF could significantly increase ER expression and reduce the pathological changes of artery,which described that LWDHF could delay the development of the AS through the increased ER expression.Three couple drugs all could increase significantly ER expression;SD/ZX were stronger than SZY/DP and SY/FL,indicating that LWDHF’s regulation of ER expression was the synergy of three couple drugs.SD/ZX had the strongest effect on ER expression.Conclusions1.LWDHF has a good therapeutic effect on OVX AS rats.2.LWDHF can improve lipid metabolism in serum,regulate the balance of body s antioxidant capacity,inhibit inflammatory response,improve platelet aggregation,and reduce the level of AngⅡ by regulating ER expression.3.The effect of anti-AS with LWDHF on OVX AS rats was the synergy of three couple drugs.SD/ZX has the strongest effect on adjusting lipid levels,adjusting the expression of ER α,ER β and GER30.SZY/DP has the highest effect on inhibiting inflammatory cytokine production,reducing AngⅡ level,adjusting the oxidation systems balance.SY/FL has better effect in adjusting 6-k-PGFl α/TXB2 balance than other two couple drugs.4.Kidney essence deficiency is related to the reduced ER expression,which is the pathological base of vascular remodeling on the QVX AS rats.Regulation of ER expression is an important way of AS vascular remodeling with LWDHF interference,and SD/ZX plays a key role.