Effect Of Dracocephalum Heterophyllum Benth Flavone On Hypertensive Rats And Mechanisms Research

Objective:Dracocephalum heterophyllum Benth in traditional Uyger medicine is wildly used for the treatment of hypertension and so on.In this study,the anti-hypertension effect were confirmed through Dracocephalum heterophyllum Benth extract(DHBE)were extracted and the rat model of renal hypertension were established.Explore the possible active ingrediens.Dracocephalum heterophyllum Benth flavone(DHBF)were extracted and the effect on blood pressure and myocardial hypertrophy were observed,and the possible mechanism of DHBF was futher explored,which provide a theoretical data and basic information for the development and effective use of Uighur local herbs.Methods:①The effect DHBE on hypertension rats:DHBE was extracted for 3 times,each extraction time cover 3 hour,the solution with 70%ethanol and solid-liquid was mixed as 1:30(g:ml).The Renal hypertension rat model was established by the left renal artery stenosis method(2 kidney 1 clip,2K 1C).5 groups were divided as Sham,Model,DHBE low dose group(DHBE-L,300mg·kg-1·d-1)and DHBE high dose group(DHBE-H,600mg·kg-1·d-1),and captopril group(20mg·kg-1·d-1).The drugs were given by intragastric administration for 5 weeks.Systolic blood pressure(SBP)was measured by tail cuff approach every week.After 5 weeks later,hemodynamic parameters was measured and end organ damage(EOD)were valued.②Identify and inspections on the chemical composition of Dracocephalum heterophyllum Benth;ethyl acetate and n-butanol extract were extracted,of which vasorelaxation effect were observed and flavonoid content were measured.③AB-8 macroporous resin purification method was used to extract DHBF;the flavone content was measured by spectrophotometry method,with rutin as the standard.DHBF was extracted for 3 times,each extraction time cover 2 hour,the solution with 70%ethanol and solid-liquid was mixed as 1:50(g:ml).The Renal hypertension rat model was established by the left renal artery stenosis method.5 groups were divided as Sham,Model,DHBF low dose group(DHBF-L,300mg·kg-1·d-1)and DHBF high dose group(DHBF-H,600mg·kg-1·d-1),and captopril group(20mg·kg-1·d-1).The drugs were given by intragastric administration for 6 weeks.SBP was measured by tail cuff approach every week.After 6 weeks later,the echocardiography,hemodynamics and histological examination of the heart were performed and EOD were valued.④The possible mechanism of DHBF were explored from the following several aspects:Effect of DHBF on rennin-angiotensin-adosterone-system(RAAS):The content of Angiotensin II(Ang II)in heart and kidney,Aldosterone(ALD)in plasma were all measured by radioimmunoassay.The expressions of Angiotensin II type 1 receptor(AT1a)and Angiotensin II type 2 receptor(AT2)mRNA were measured by Real-time RT-PCR,The protein expressions of AT1 and AT2 were measured by Western blot;Effect of DHBF on inflammatory cytokines:The content of Interleukin-1 beta(IL-1β),tumor necrosis factor(TNF-α)and Interleukin-10(IL-10)were all measured by radioimmunoassay;Effect of DHBF on Endothelial function:The content of Nitric oxide(NO)was measured by nitrate reductase method,Endothelin-1(ET-1),Thromboxane B2(TXB2),6-keto prostaglandin F1α(6-keto-PGF1α)were all measured by radioimmunoassay and TXB2/6-keto-PGF1α was calculated;Effect of DHBF on Calcineurin phosphatase(CaN)pathway:The expressions of Calpain-I and CaN mRNA were measured by Real-time RT-PCR;Effect of DHBF on Myocardial remodeling:The expressions of Matrix metallopeptidase 9(MMP-9)and Tissue inhibitor of metalloproteinases 1(TIMP-1)mRNA were measured by Real-time RT-PCR.Results:①The effect of DHBE on hypertension rats:Compared with Model group,each group in DHBE all revealed the effectiveness of reduced SBP,Diastolic blood pressure(DBP),Left ventricular systolic pressure(LVSP)and Left ventricular end-diastolic pressure(LVEDP),extend the time for Diastolic duration(DD)and minimized the left ventricular weight/body weight(LVW/BW).②Flavonoids,volatile oil,amino acids,peptides,proteins,sugars,tannins,organic acids,phenols,coumarin,lactone,terpenoids were found in Dracocephalum heterophyllum Benth,which also might contained saponins,and the anthraquinone,alkaloids,grease were not checked out.Ethyl acetate extract and n-butanol extract could relax vascular contraction which caused by KCl,IC50 were 0.055g·L-1,0.165 g·L-1 and the flavonoid content were 45.73%and 17.82%respectively.③The flavonoid content of DHBF were 57.97%.The effect of DHBF on hypertension rats:Compared with the Model group,the result of hemodynamic on DHBF revealed that SBP、LVSP、LVEDP、+dp/dtmax were all reduced in the group of DHBF-H(P<0.01,P<0.05),DD was prolonged(P<0.05);The result of the echocardiogram revealed that interventricular septum diameter(IVSd)、left ventricle posterior wall thickness at end-diastole(LVPWd)and LVW/BW were decreased,left venricular fractional shorting(FS)and ejection fraction(EF)were rised(P<0.01,P<0.05);The histological examination pointed that LVW/BW and arotic weight/legth(AW/length)were minimized(P<0.05,P<0.01);The pathological examination indicated that cross-sectional area of cardiac myocyte(CSA)were minimized(P<0.01),inflammatory cells infiltration were reduced,interstitial fibrosis and intramyocardial vessel thicken were inhibit patently.④Effect of DHBF on rennin-angiotensin-adosterone-system(RAAS):The content of AngⅡ in kidney were reduced but not reduced in heart,the expression of AT1a mRNA were reduced(P<0.05),the protein expression of AT1 was declined(P<0.05).The levels of ALD in plasma were declined(P<0.01);Effect of DHBF on inflammatory cytokines:The content of IL-1β、TNF-α were all decraseed(P<0.01)and IL-10 were not changed obviously;Effect of DHBF on Endothelial function:The content of NO were increased while ET-1 were decreased,The ratio of TXB2/6-keto-PGF1α were minimized(P<0.01,P<0.05);Effect of DHBF on Calcineurin phosphatase pathway:The expressions of Calpain-I and CaN mRNA were all reduced(P<0.01,P<0.05);Effect of DHBF on Myocardial remodeling:The expressions of MMP-9 and TIMP-1 mRNA were all declined(P<0.01).Conclusions:Flavonoids,volatile oil,amino acids,and other ingredients were found in Dracocephalum heterophyllum Benth;it could relax vascular contraction which caused by KCl;DHBF could decline the blood pressure,might be ①Reduced Ang Ⅱ in kidney and ALD in plasma,then inhibit the vasoconstrictor effect and water sodium retention,anti diuretic effect,anti-vascular smooth muscle cell proliferation,thereby regulating blood pressure,inhibit vascular remodeling.②Decreased ET-1,inhanced NO,adjusted the balance of TXA2/PGI2,reduced TNF-α、IL-1β,protected the endothelial function,inhibit vascular remodeling.DHBF could inhibit myocardial hypertrophy,might be①Reduced The mRNA and protein expression of AT1 in heart.②Decreased the level of ALD.③Declined TNF-α、IL-1β.Lowered this kind of stimulus,on the one hand,could inhibit the expression of Caplain-I and CaN,inhibit cardiomyocyte hypertrophy;on the other hand,could adjusted the balance of the MMP-9/TIMP-1,inhibit myocardial matrix remodeling.The effect of inhibit CaN channel might be related to inhanced NO,then inhibit L-Ca2+channel.