| Purpose:1.To discuss the mechanism of Uncaria rhynchophylla compound recipe(URCR) on keeping blood pressure down,through studying URCR influencing on blood pressure and plasma endothelin-1 (ET-1),angiotentionⅡ(AngⅡ) and nitrous oxide(NO),and expressions of artery tissue eNOS.2.To discuss the mechanism of URCR preventing hypertensive blood-vessel from remodeling,by Studying on URCR influencing the change of major arteries parietes morphology and ultrastructure, and the protein expressions of C1CF,Basic fibroblast growth factor(bFGF) and Osteopontin(OPN) in artery tissues,and the expressions of bFGF and OPNmRNA in RH rats.3.To reveal in cell,molecule and gene sides the physiological influence of URCR working on releasing symptoms of hypertension,improving and preventing hypertensive blood-vessel reconstruction,and adjusting cardiovascular system functions,and to provide theory basis for best clinical cure methods,throtigh studying the cause of hypertension,combined relative Chinese medicine theories,as well as applying for URCR influencing renovascular hypertensive(RH) rats.Materials and Methods:1.Experimental medicines:(1) Uncaria rhynchophlla(miq.) Jacks,Rhizoma Gastrodia,Astragalus membranaceus(Fisch.) Bunge,Salvia miltiorrhiza Bge.,Semen Cassiae obtusifoliae,Paeony Root,Tadix Achyranthis Bidentatae,Rubus kawakamii Hayata.All above-mentioned raw medicines are supplied by LiaoNing University of Traditional Chinese Medicine Hospital I.Raw-material medicine mixed according to recipe portion after a second dry,dipped in distilled water for 1 hour at air temperature; and decocted in slow fire for three times,each time cooking one hour long;mixed all filtrate and concentrated to liquid containing raw medicine 2g per ml,reserved at -4℃in refrigerator.(2) Cow-bezoar Anti-hypertension Bolus(CbAhB):1.6g/bolus;Product No.:8015000;Beijing TongRenTang Technology Development Company Ltd.Factory supplied.Made into liquid with distilled water containing 0.2g/ml,supply for rats gavages.(3) Captopril(CAP):25mg/tablet,product No.:080315,Shanghai XuDongHaiPu Medicine Co.Ltd supplied.Made into liquid with distilled water containing 2.5mg/ml,supply for rats gavages.2.Experimental Animals:The animals used in this experiment are Gold blatt classic hypertensive rat models and had been improved and made into 2K1C renovascular hypertensive rat models.3.Groups:Experimental animals had been divided into 6 groups,each group 10 rats as:Sham-operation group(SOG),Model group(MG),URCR High-dosage medicine group(HDG),URCR Low-dosage medicine group(LDG),Chinese medicine group(CMG),and Western medicine group(WMG).4.Methods:URCR HDG was supplied with 20g raw medicine/kg/d;LDG was supplied with 6.6g raw medicine/kg/d;WMG was supplied with 7.5mg/kg/d;CMG was supplied with 0.96g/kg/d;SOG and MG both were supplied with 1ml/100g distilled water gavage.Administered once at 10:00am per day,and continued 12 weeks.Fasted 24 hours after the last time administered but non-fasting water.The rats were executed after anesthesia with 10%chloral hydrate by given intraperitoneal injection at 0.4ml/100g dosage.5.Study Contents:(1) The foundation and evaluation of RH models(2) The influence of URCR made to the blood pressure of RH rats(3) Thee influence of URCR made to the levels of ET-1,AngⅡand NO of RH rats(4) The observation of Aortic wall pathematology,the detection of morphology indexes and the ultrastructure observation of vascular smooth muscle cell(VSMC)(5) Using euzymelinked immunosorbent assay(Elisa) to detect the contents of C1CF,eNOS,bFGF and OPN in artery tissues of RH rats(6) Using RT-PCR to detect bFGF and OPNmRNA expressions in aortic wallResults:1.The foundation and evaluation of RH modelsCompared with SOG,the operation groups,that is,after the left renal artery partly stenosis the systolic blood pressures(SBP) of 2K1C RH rat models increased within 3 days,and sharply increased after 7 days,and hypertension had been preliminary formed after 14 days.And at 28 days the aortic SBP increased from before operation(101.8±4.18)mmHg to(152.32±4.53)mmHg, which had the remarkable difference(P＜0.05).2.The influence of URCR made to the blood pressure of RH rats,the content of AngⅡ,ET-1,NO in blood,and the activity of eNOS in the artery tissues(1) URCR influence to the blood pressure of RH ratsAfter continuous intervention and treatment with medicine,from the beginning of the 3rd week, the blood pressure of WMG began to decreased firstly,which had remarkable difference(P＜0.05) compared with MG and treatment before,then gradually showed decrease trend(P＜0.01).The blood pressure of other treatment groups began to continuously decrease from the 6th week,and the blood pressure of URCR HMG had notable difference(P＜0.01) compared with treatment before and MG.After the 9th week treatment,the blood pressure of LDG and CMG began to remarkable decrease(P＜0.01).(2) The change of AngⅡcontent in plasmaCompared with rats in SOG,the concentration of AngⅡin rats plasma of MG increased notably, which had the remarkable difference(P＜0.01).Except WMG,the concentration of AngⅡin rat plasma of other administering medicine groups all increased higher(P＜0.01).Except URCR LDG, all the concentration of AngⅡin rats plasma among administering medicine groups were lower than MG,which had statistical difference(P＜0.01).The concentration of AngⅡin rats plasma of WMG were higher than other administering medicine groups(P＜0.05),and remarkable higher than LDG(P＜0.01).(3) The change of ET-1 content in plasmaCompared with rats in SOG,the concentration of ET-1 in rats plasma of MG and WMG increased notably(P＜0.01).Except WMG,the concentration of ET-1 in rat plasma of each treatment group all decreased down compared with MG.Among them,HDG decreased the most remarkable(P＜0.01),then in turn was LDG,and CMG(P＜0.01).Compared with WMG,HDG also decreased remarkable(P＜0.05).(4) The comparison of NO content in blood seraCompared with rats in SOG,the NO content in blood sera in MG rats was clearly lower than SOG,the difference had the statistical meaning(P＜0.01).Compared with MG,The NO content in blood sera of other treatment groups all were higher,and among them HDG increased highest (P＜0.01),then in turn was LDG and CMG(P＜0.05).Compared with MG,WMG increased but no remarkable difference(P>0.05).Compared with WMG,HDG had statistical difference(P＜0.05).(5) The comparison of eNOS activity in artery tissueCompared with rats in SOG,the eNOS activity in artery tissue of MG were obviously lower than SOG,the difference had the statistical meaning(P＜0.01).The eNOS activity in artery tissue of other treatment groups all increased remarkably(P＜0.05) compared MG.Compared with MG, WMG increased but had no remarkable difference(P>0.05).Compared with WMG,HDG had statistical difference(P＜0.05). 3.The influence of URCR on aortic wall morphology,ultrastructure and C1CF of RH rats(1) Comparison of aortic morphology observation under light microscopeThe HE staining section revealed:Compared with SOG,the thickness of aortic media in MG obviously increased(P＜0.01).Compared with MG,the thickness of aortic media in each administering groups decreased(P＜0.05).The thickness of aortic media in HDG decreased lowest compared with WMG(P＜0.01).However,the difference among each group were not remarkable(P＞0.05).(2) The comparison of Masson stain observation under light microscopeMasson strain showed that the vascular smooth muscle cells were strained in red and the collagen were strained in green.The collagenous tissues of rat aortic wall in SOG were homogeneous,and the intima-media elastic fibers were continuous,regular,and clear.The collagenous tissues in rat vessel wall of MG visibly increased,the intima-media smooth cells hyperplasia remarkable,and the elastic fibers were shown stiff,chaos and even ruptured.Compared with MG,the collagenous tissues in rat vessel wall of administering medicine groups reduced obviously,but the intima-media elastic fibers were comparatively regular.(3) The percent comparison of the volume fraction of collagen(VFC)Compared with SOG,the area percent of VFC in MG obviously increased(P＜0.01).Compared with MG,the area percent of VFC in each administering medicine group evidently reduced (P＜0.05),and HDG and LDG reduced the most(P＜0.01).(4) The observation on ultrastructure change of smooth muscle cells in aortic media by Transmission electron microscope(TEM)In SOG,the smooth muscle cells in aortic media were shown fusiform shape,and the cells were entire and had regular nucleus.Also there are full of myofilament in cells.The nucleus gathered large numbers of mitochondria and a small quantity of endoplasmic reticulum.The shape of mitochondria is normal,and ridge arranged sternly as Z-shape.The smooth muscle cells in aortic media of MG obviously hyperplasia;cell nucleus had non-regular shapes;the mitochondria were swollen,round,and groundplasm dense lower,also ridge changed and became shorten and number reduced even disappeared to swollen.Nucleus chromatin shrift near to inside membrane,further nucleus chromatin condensed around nucleus or non-regularly piled up in nucleus,then solid-state polycondensated.The smooth muscle cells in aortic media of other treatment groups were near to those of SOG,and compared with MG,the swollen and deformed mitochondria obviously reduced, and the shape were regular.Nucleus chromatin equally distributed,and the numbers of endoplasmic reticulum also reduced.(5) The comparison of C1CF content in artery tissuesCompared with SOG,the C1CF content in RHR vascular wall of MG significantly increased (P＜0.01).Compared with MG,the C1CF content in artery tissues in each treatment groups obviously decreased,among them,HDG remarkable decreased(P＜0.01),then in turn LDG,CMG and WMG(P＜0.05).And there were no significantly differences among treatment groups.4.The influence of URCR made to the content of bFGF,OPN and mRNA expression in artery tissues of RH rats(1) The comparison of the content of bFGF in artery tissuesCompared with SOG,the content of bFGF in RHR vascular wall of MG significantly increased (P＜0.01);Compared with MG,the content of bFGF in artery tissues in each administrating group all decreased obviously(P＜0.05),HDG significantly decreased(P＜0.01) similar to SOG,but still had statistical difference(P＜0.05).Compared with HDG,CMG also had remarkably difference (P＜0.05).(2)The comparison of the content of OPN protein in artery tissuesCompared with SOG,the content of OPN in RHR vascular wall of MG significantly increased (P＜0.01).Compared with MG,the content of OPN in each treatment group remarkably decreased (P＜0.05),the content OPN protein of HDG decreased most remarkably(P＜0.01);Compared with HDG and LDG,CMG and WMG all had significantly differences(P＜0.05).(3) The comparison of bFGF mRNA expression in artery tissuesThe bFGF mRNA expression in artery tissues of MG were significantly higher than SOG (P＜0.01).Compared with MG,the bFGF mRNA expressions in artery tissues in HDG and LDG decreased significantly(P＜0.01).Compared MG,CMG and WMG had remarkable difference (P＜0.01).Compared HDG,the mRNA protein expression of CMG and WMG had remarkable difference(P＜0.05).(4) The comparison of OPN mRNA protein expression in artery tissuesOPN mRNA expression in artery tissues of MG were significantly higher than SOG(P＜0.01). Compared with MG,OPN mRNA expressions in artery tissues of HDG and LDG decreased significantly(P＜0.01).Compared with MG,CMG and WMG had remarkable difference(P＜0.01). Compared with HDG,mRNA protein expression of CMG and WMG had remarkable difference (P＜0.05).Conclusions: 1.URCR can improve hypertension treatment in RH rats,and the mechanism of anti-hypertensive probably improved pathological mechanisms in RH rats through decreasing AngⅡand ET-1 level in plasma,increasing NO level and ENOS expression in blood vessels,which also illustrated the reasonability of URCR.2.URCR could improve the phenotype and transfer change of VSMCs in aortic media of RH rats. The mechanism was associated with inhibitive effect of URCR on the promotion of bFGF and OPN to the transformation of VSMCs phenotype.3.URCR could improve the morphological index of aortic wall,and decrease VFC in the aortic media.A reversing effect of URCR on vascular remodeling in RHR was observed.The mechanisms were associated with suppressive effect of URCR on expression of bFGF and OPN mRNA protein in aortic wall.
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