| Objective: To study the isolation of 17-Methoxyl-7-Hydroxy-Benzene-Furanchalcone(MHBFC)from the petroleum ether extract of YLS,and its effect on the progression of cardiac hypertrophy induced by pressure overload in eNOS knock–out mice,in order to provide theoretical and experimental basis for its clinical application.Methods:(1)Separation of MHBFC from YLS Dried roots of YLS were soaked with 70% Et OH for extraction,then leached by petroleum ether and ethyl acetate respectively,and the petroleum ether(PE)fraction and ethyl acetate(Et OAc)fraction were obtained.The petroleum ether fraction was subjected to column chromatography over silica gel(200-300 mesh),and TLC identification of the same fraction,after multiple silica gel column chromatography and recrystallization,3 monomers were identified by IR,1HNMR,13 CNMR and X-ray single crystal diffraction(the compound 1 is MHBFC).(2)The establishment of the cardiac hypertrophy model 60 wild type C57BL/6j mice and eNOS knock-out mice each,were randomly divided into the operation group and the sham group according to the weight.The mice were anaesthetized with 4% chloral hydrate(0.01 ml.g-1,intraperitoneal injection),and the abdominal aorta was dissected above the kidneys.A 27-gauge needle was placed alongside the artery,and the needle and artery were tightly tied together.At this moment,we could observe the kidney becoming obviously pale.The needle was then quickly removed.A standardized reduction in the size of the aortic lumen was successfully created.A similar operation was performed on the sham animals but no ligature was made.Finally,the muscle and skin of the mice were sutured by line 4-0,and the wound was sterilized again,then put the mice back in the cage to awaken.(3)Post operation grouping,drug delivery and index detection After 4 days of surgery,the wild-type mice and eNOS knock-out mice were randomly divided into 4 groups respectively(n=10): sham group;model group;MHBFC 24 mg/kg;MHBFC 12 mg/kg.All animal were administered orally via a gastric tube for treatment for one times a day for 6 weeks,and the sham and model group received 0.5% CMCCNa.The arterial systolic blood pressure(SBP)and heart rate(HR)were measured at the week 1,3,5 after administration by the tail-cuff method using an animal blood pressure analyzer.At the end of the study,collection of related specimens,the level of IL-6 inflammatory factor was measured by ELISA,the pathological changes of the heart and blood vessels were observed by HE staining,the collagen deposition in the heart was observed by Masson's staining,the myocardial ultrastructure was observed by the transmission electron microscopy,the protein level of ET-1 in myocardial tissue was observe by immunohistochemistry,atrial natriuretic peptide(ANP),?-myosin heavy chains(?-MHC),brain natriuretic peptide(BNP),Serum amyloid A(SAA)m RNA were measured by the method of RT-PCR.Results:(1)3 compounds were isolated from petroleum ether of YLS:Compound 1: MHBFC,orange square crystal,the molecular formula is C18H14O4,and the relative molecular mass is 294.30.Compound 2: Karanjin,white needle crystal,the molecular formula is C18H12O4,and the relative molecular mass is 292.30.Compound 3: 2?,2?-Dimethylpyrano-[5?,6?:8,7] flavone,white needle crystal,the molecular formula is C20H16O3,and the relative molecular mass is 304.34.The 3 compounds were isolated from petroleum ether of YLS for the first time.(2)Effects of MHBFC on SBP and HR: After treatment with MHBFC,compared with the model group,the SBP and HR of wild-type mice decreased significantly(P<0.05),but there was no significant difference between SBP and HR in eNOS knockout mice.(3)Effects of MHBFC on the viscera index: After drug treatment,the cardiac weight index,left ventricular mass index,right ventricular mass index,lung weight index and kidney weight index of MHBFC 24 mg/kg group in wild-type mice were significantly decreased,however,the above index of eNOS knockout mice had no significant difference.(4)Effects of MHBFC on the inflammatory factor IL-6: Compared with the model group,the plasma IL-6 level in wild-type mice of MHBFC 24 mg/kg group was significantly decreased(P<0.01),however,there was no significant change in the above index of eNOS knockout mice.(5)Effects of MHBFC on myocardial cells and myocardial collagen deposition: Compared with the model group,the myocardial cell area,collagen volume fraction(CVF)and Perivascular collagenvolume area(PVCA)of the wild-type mice treated with MHBFC were significantly decreased(P < 0.01,P < 0.01,P < 0.01),however,there was no significant change in the myocardial cell area,CVF and PVCA of eNOS knockout mice.(6)Effects of MHBFC on vascular remodeling: After treatment with MHBFC,the degree of vascular hypertrophy of wild-type mice in MHBFC 12 mg/kg and MHBFC 24 mg/kg group were significantly decreased(P < 0.05 and P < 0.05),the vascular cross-sectional area decreased significantly(P < 0.05 and P < 0.01),the vascular thickness decreased significantly(P < 0.01 and P < 0.01),the tube radius,inner radius and outer radius were significantly decreased(P < 0.05);the vascular remodeling index of eNOS knockout mice were decreased slightly,but compared with the model group had no significant difference.(7)Effects of MHBFC on BNP??-MHC?ANP?SAA m RNA and ET-1 protein: Compared with the model group,the levels of the four genes and ET-1 protein in wild-type mice of MHBFC treatment group were significantly decreased,however,the levels of the four genes and ET-1 protein in eNOS knockout mice did not change significantly.(8)Effects of MHBFC on the myocardial ultrastructure: In the wild type mice,the mitochondria volume increased significantly,the Z line was fuzzy,the myocardial fibers dissolved and broken,and even became punctate.The mitochondria arranged in disorder.They were not parallel to the myocardial fibers,and occasionally necrotic mitochondria.After treatment with MHBFC,the symptoms were obviously improved,the mitochondria were parallel to the arrangement of the myocardial fibers,and the Z line was clear.However,the volume and number of mitochondrial of eNOS knockout mice increased significantly,the Z line blurred,the myocardial fibers were broken and dissolved,and the mitochondria had large area necrosis and different shapes.After treatment with MHBFC,the myocardial ultrastructure of the mice was not significantly improved.Conclusions:(1)MHBFC exists in the petroleum ether of YLS,the separation method is simple and the yield is high.(2)The effect of MHBFC 12 and 24 mg/kg on cardiac hypertrophy in wild-type mice was obviously reversed,however,there was not obvious effect of reversing cardiac hypertrophy in eNOS knockout mice.(3)The mechanism of MHBFC reversing cardiac hypertrophy is related to the eNOS gene,and its reversal effect on cardiac hypertrophy in mice depends on the eNOS gene. |
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