| Objective:Part 1:To establish the primary culture of rats cardiac microvascular endothelial cells?CMVECs?and cell identification.To study the effects of NCBEE and NCBAE on Ang ? injured CMVECs' vitality,apoptosis rate,apoptosis related proteins Bcl-2,caspase-3 expression and NO,LDH,GSH-Px,MDA and SOD in vitro.Part 2:To establish 2K1C renovascular hypertensive rats models.To study the effects of NCBEE and NCBAE on hypertensive cardiac injury by the analysis of SBP,echocardiography,expressions of apoptosis related proteins in ventricular tissue,and serum NO,LDH,GSH-Px,MDA,SOD in vivo.Part 3:To study the effects of NCBEE and NCBAE on the change of serum metabolites of 2K1C renovascular hypertensive rats using metabonomics analysis.Methods:Part 1:?1?CMVECs were isolated and cultured from Wistar rats' left ventricular myocardial tissue by mincing and enzymatic digestion.The purified cells were identified as endothelial cells by fluoroimmunoassay of cellular markers?factor?and vWF related antigen?.?2?MTT method was used to detect the effect of Ang ? on CMVECs vitality.Flow cytometry was used to study the effects of Ang ? in different intervention time on apoptosis rate of CMVECs.Western blot was used to detect the effects of Ang ? in different intervention time on Bcl-2,caspase-3 expression of CMVECs.?3?The experiment was divided into con,Ang ?,Ang ?+Ator,Ang ?+NCBEE?H?,Ang ?+NCBEE?M?,Ang ?+NCBEE?L?,Ang ?+NCBAE?H?,Ang ?+NCBAE?M?and Ang ?+NCBAE?L?group.MTT method was used to detect the effects of NCBEE and NCBAE on cell vitality of Ang ? injured CMVECs.TUNEL method was used to study the effects of NCBEE and NCBAE on apoptosis of injured CMVECs.Western blot was used to detect the effects of NCBEE and NCBAE on expression of Bcl-2 and caspase-3 in injured CMVECs.The cell SOD and supernatant NO,LDH,GSH-Px and MDA were detected as well.Part 2:108 Wistar rats were randomly divided into sham group?n=12?and 2K1C model group?n=96?.The 2K1C hypertensive rat models were set up by clipping the left renal artery?SBP?150mmHg considered as the model has been made successfully?.Sham-operated rats underwent the same surgical procedure except for renal arterial clipping.The experiment was divided into sham group,2K1C group,2K1C+Ator group,2K1C+NCBEE?H?group,2K1C+NCBEE?M?group,2K1C+NCBEE?L?group,2K1C+NCBAE?H?group,2K1C+NCBAE?M?group and 2K1C+NCBAE?L?group.Rats were orally administrated with NCBEE and NCBAE for 6 weeks.Tail-Cuff Blood Pressure System was used to measure the systolic blood pressure before and once a week after left renal artery clipping and treatments.Twelve weeks after surgery,rat SBP and echocardiographic parameters were measured.Cardiac histopathology was assessed by staining paraffin sections of the left ventricular tissues with hematoxylin and eosin.The expression of Bcl-2 and caspease-3 of left ventricular tissue were assessed by western blot.Serum NO,LDH,GSH-Px,MDA and SOD levels were detected as well.Part 3:The establishment of 2K1C renovascular hypertensive rat models same as Part 2.After prepared the serum sample of each group,1H-NMR spectrum of serum samples were determined by NMR spectrometer.The effects of NCBEE and NCBAE on the change of serum metabolites of 2K1C hypertensive rats were analyzed.Results:Part 1:?1?CMVECs of adult rat were cultured successfully bymincing and enzymatic digestion,and the cells showed their endothelial cell-specific,cobblestone-like morphology.?2?The morphology of CMVECs became lathy or shrinkage deformed,and cell vitality decreased after intervention of Ang ? with concentration from 0.01?mol·L-1 to 10?mol·L-1 compared with control group?P<0.05;P<0.01;P<0.01;P<0.01 respectively?.Ang ? start lost its ability to induce tube formation at intervention time>20h in concentration of1?mol·L-1.The apoptosis rates of CMVECs were increased time dependently?4h,8h to24h?after intervention of Ang ??P<0.05;P<0.01;P<0.01;P<0.01;P<0.01;P<0.01respectively?.Western blot analysis showed that Ang ? intervention?8h,12h to 20h?decreased Bcl-2 expression and increased cleaved caspase-3 level time-dependently in CMVECs?all P<0.01?.?3?The morphology of CMVECs became lathy or shrinkage deformed,and cell vitality decreased in Ang ? group compared with control group?P<0.01?.The morphology of injured CMVECs was improved and cell vitalities were significantly increased after intervention of NCBEE?NCBAE compared with Ang ? group?all P<0.01?.The apoptosis rate of Ang ? group was increased compared with control group?25.55±3.23%vs 2.26±0.89%,P<0.01?.The apoptosis rates of injured CMVECs were decreased after intervention of NCBEE?H??P<0.01?,NCBEE?M??P<0.01?,NCBEE?L??P<0.01?,NCBAE?H??P<0.01?and NCBAE?M??P<0.01?.In western blot analysis,Ang ? intervention showed the decrease of Bcl-2 expression?0.22±0.03 vs0.71±0.09,P<0.01?and increase of cleaved caspase-3 level?0.55±0.06 vs 0.20±0.02,P<0.01?compared with control group.NCBEE?H?,?M?and NCBAE?H?,?M?intervention showed significant increase of Bcl-2 expression?all P<0.01?and decrease of cleaved caspase-3 level?all P<0.01?dose-dependently in CMVECs compared with Ang ? group.Moreover,in Ang ? group,cell SOD?113.89±9.34 U/mgprot vs 165.05±23.05 U/mgprot,P<0.01?,supernatant NO?30.67±2.42?mol/L vs 59.03±8.99?mol/L,P<0.01?,GSH-Px?21.49±2.56 U/ml vs 55.42±5.95 U/ml,P<0.01?were decreased,LDH?864.61±45.94U/L vs 456.01±46.89 U/L,P<0.01?and MDA?2.41±0.29nmol/ml vs 1.42±0.24nmol/ml,P<0.01?were increased compared with control group.But after intervention of NCBEE and NCBAE,cell SOD?P<0.05?,supernatant NO?P<0.01?,GSH-Px?P<0.01?were increased,LDH?P<0.05?and MDA?P<0.01?were decreased compared with Ang ? group.The drug intervention can effectively improve the indicators.Part 2:Six weeks after surgery,SBP in 2K1C-induced hypertensive model group was higher than sham group?P<0.01?.Six-week treatment of NCBEE?H?,?M?and NCBAE?H?,?M?caused reduction in the development of SBP in 2K1C rats?P<0.01,P<0.01,P<0.01,P<0.05respectively?.The results of echocardiography showed the increases in LVPWd?1.72±0.28mm vs 1.55±0.24mm,P<0.05?,LVPWs?2.78±0.16mm vs 2.52±0.31mm,P<0.01?,IVSd?1.89±0.28mm vs 1.44±0.23mm,P<0.01?,IVSs?2.92±0.29mm vs2.51±0.31mm,P<0.01?,and decreases in LVDd?5.83±0.39mm vs 6.62±0.52mm,P<0.01?,LVEF?59.71±7.65%vs 70.20±5.05%,P<0.01?and LVFS?33.89±6.81%vs39.17±3.73%,P<0.05?in 2K1C group compared to sham group.However,six-week treatment of NCBEE?H?and NCBAE?H?reduced LVPWd?all P<0.05?,LVPWs?P<0.05;P<0.01?,IVSd?all P<0.01?and IVSs?all P<0.05?,and enhanced LVEF?all P<0.01?of 2K1C hypertensive rats.Besides,LVW,LVW/BW ratio and cardiomyocyte CSA in the 2K1C group were significantly increased compared with sham group?all P<0.01?.However,these pathological changes of LVW,LVW/BW ratio and cardiomyocyte CSA were reduced significantly in NCBEE?H??all P<0.01?and NCBAE?H?group?all P<0.01?compared with 2K1C hypertensive rats.Western blot analysis showed that 2K1C hypertension led to down-regulation of Bcl-2 and up-regulation of cleaved caspase-3 in left ventricular tissue of rats compared with sham group?0.21±0.03 vs 0.65±0.07,P<0.01;0.58±0.04 vs 0.19±0.02,P<0.01?.And both increased cleaved caspase-3 and decreased Bcl-2 expressions observed in 2K1C group were significantly attenuated in2K1C+NCBEE?H?,?M?and 2K1C+NCBAE?H?,?M?groups?all P<0.01?.Moreover,2K1C group showed a significant decreases in serum NO?36.56±5.32?mol/L vs83.43±5.08?mol/L,P<0.01?,SOD?24.38±4.15 U/ml vs 43.92±3.26 U/ml,P<0.01?,GSH-Px?57.92±4.18 U/ml vs 139.23±15.74 U/ml,P<0.01?,and increases in serum LDH?944.09±44.10 U/L vs 582.45±19.97 U/L,P<0.01?,MDA?13.56±1.39nmol/ml vs5.51±0.75nmol/ml,P<0.01?level as compared to sham group.Treatment with NCBEE,NCBAE for 6 weeks resulted in significant increases in serum NO,SOD and GSH-Px?all P<0.01?,decreases in serum LDH and MDA?all P<0.01?.These results suggesting that regulation of SBP,improvement of cardiac hypertrophy,modulation of apoptotic process,and the regulation of serum indicators by NCBEE,NCBAE might contribute to the beneficial effect on 2K1C hypertensive hearts.Part 3:Serum metabolomics identified 13differences compounds including leucine,isoleucine,valine,tyrosine,phenylalanine,?-glucose,?-glucose,glycoprotein,unsaturated lipid,creatine,?-hydroxy butyrate,pyruvate and citric acid in 2K1C group.Specifically,leucine,isoleucine,valine,tyrosine,phenylalanine,?-glucose,?-glucose,glycoprotein,unsaturated lipid were decreased?all P<0.05?,and creatine,?-hydroxy butyrate,pyruvate,citric acid were increased in serum of2K1C hypertensive rats?all P<0.05?.However,Intervention of NCBEE,NCBAE increased serum leucine,isoleucine,tyrosine?all P<0.05?.NCBAE also increased serum valine and phenylalanine?all P<0.05?.These results indicating that regulation of amino acid by NCBEE,NCBAE might useful on improvement of body pathology in 2K1C hypertensive rats.NCBEE,NCBAE increased serum glycoprotein,?-glucose,?-glucose?all P<0.05?,and decreased serum?-hydroxy butyrate?P<0.05?.NCBEE decreased serum pyruvate,citric acid and creatine as well?all P<0.05?.Moreover,NCBEE also increased serum unsaturated lipid?all P<0.05?.Conclusions:Part 1:NCBEE and NCBAE might protect Ang ? injured CMVECs via the improvement of cell viability,inhibition of apoptosis,regulations of the expression of Bcl-2 and caspase-3,and cell NO,LDH,GSH-Px,MDA,SOD.Part 2:2K1C renovascular hypertensive rat models were established successfully by clipping the left renal artery.NCBEE,NCBAE present cardioprotective properties in 2K1C hypertensive rat models by regulating SBP,ameliorating cardiac hypertrophy,improving serum NO,LDH,GSH-Px,MDA,SOD,and modulating the expression of Bcl-2 and caspase-3.Part 3:Differences metabolites between 2K1C hypertension group and sham group may become the disease markers of renovascular hypertension.NCBEE,NCBAE might improve the body pathology by regulating amino acid metabolism,glycometabolism,and fat metabolism. |
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