| Objective:To investigate the effect of Glycyrrhizic acid, Ferulic acid, Paeoniflorin, Cinnamic acid of dangguisini decotion on eNOS/NO pathway of MMVEC in MIRI rats after establishing an MIRI model in SD rats. And to investigate the pharmacodynamic effects and mechanisms of MMVEC protection of dangguisini decotion on MIRI rat. So as to Provide a new approach for prevention and treatment of MIRI.Methods:The experiment contains two chapters: 1st chapter, select the best combination of glycyrrhizic acid,ferulic acid, paeoniflorin, cinnamic acid (GFPC) from dangguisini decotion on the protecting effect against MIRI to MMVEC. Fed the rats with GFPC for PPC (pharmacological preconditioning), once a day, the last fed 30min before establishing the MIRI model. Orthogonal design with 4 factors (4 monomer) and 4 levels (4 doses).112 SD rats were divided into 16 groups,7 per group. establish the MIRI model by ligating LAD (Left anterior descending coronary artery), ischemia 30min,reperfusion 2h. The reliability and stability of the model was observed by ECG changes and myocardial HE staining. After the model, extracting blood from abdominal aortic, NO, ET, SOD, MDA of serum were tested. NO was tasted by methods of nitrate reduction, ET was measured by methods of radioimmunoassay, MDA was tested by methods of TBA, SOD was measured by means of NBT.2nd chapter, observe the effect on eNOS/NO pathway of MMVEC of MIRI rats of selected best combination of GFPC and the mechanism.32 SD rats were devided into shame group (non-ischemic conditions), I/R group (heart subject to ischemia-reperfusion), ppc group (heart subjected to ischemia-reperfusion treated with GFPC), ppc+L group (heart subjected to ischemia-reperfusion treated with GFPC and pre-treated with the eNOS inhibitor, Nco-Nitro-L-arginine methylester, L-NAME,15min before reperfusion,30mg/kg),8 per group. After the model, the Left ventricular apical was fixed and then sliced, morphological changes of MMVEC was observed by electron microscopy, AI of MMVEC was observed by the means of TUNEL extracting blood from abdominal aortic, NO was tasted by methods of nitrate reduction, hemorheologic changes was tested by hemorheological analyzer; taking the left ventricular, extract MMVEC from half of the left ventricular, eNOS mRNA, iNOS mRNA of MMVEC were measured by the methods of real-time RT-PCR, eNOS and iNOS protein of MMVEC were measured by the means of western blot.Statistical analysis:All results are reported as mean±S.D. Statistical analysis was performed using analysis of variance of orthogonal design,1-way and 2-way analysis of variance (ANOVA) for multiple-group comparisons, Chi-square test for data count. The probability of null hypothesis<0.05 (P<0.05) was considered statistically significant.Results:1.validation of MIRI model ECG:After ischemia, ST segment elevated significantly; when reperfusion, ST segment Significantly decreased; reliability and stability of the model were totally good. HE staining of myocardial:normal myocardial texture and clear stripes of shame group were seen; however, unclear stripes in myocytes, Vacuolar degeneration of muscle myofilament dissolution of fracture, locally infiltration of inflammatory cells and the presence of interstitial edema were seen in the MIRI group.2.select of GFPC with the effect anti MIRI MMVEC injuryAfter 2h reperfusion, ferulic acid> glycyrrhizic acid> paeoniflorin> cinnamic acid, that is the combination of ferulic acid 400mg/kg, glycyrrhizic acid 50 mg/kg, paeoniflorin 0 mg/kg, cinnamic acid 0 mg/kg could Significantly increased NO content of serum; ferulic acid> cinnamic acid> paeoniflorin> glycyrrhizic acid, that is the combination of ferulic acid 300mg/kg, cinnamic acid 400 mg/kg, paeoniflorin 0 mg/kg, glycyrrhizic acid 0 mg/kg could effectively reduce the ET levels of serum; paeoniflorin> cinnamic acid> ferulic acid> glycyrrhizic acid, that is the combination of paeoniflorin 100 mg/kg, cinnamic acid 200 mg/kg, ferulic acid Omg/kg, glycyrrhizic acid 0 mg/kg could effectively increas SOD levels of serum; ferulic acid> paeoniflorin> cinnamic acid> glycyrrhizic acid, that is the combination of ferulic acid 200mg/kg, paeoniflorin 50 mg/kg, cinnamic acid 0 mg/kg, glycyrrhizic acid 0 mg/kg could effectively reduce the MDA levels of serum.Comprehensive analysis, the combination of glycyrrhizic acid 50mg/kg, ferulic acid 400mg/kg, paeoniflorin 100mg/kg, cinnamic acid 400mg/kg, is the GFPC have the best anti effect on MIRI MMVEC injury, which could increase NO and SOD reduce ET and MDA of serum.3. Effect of GFPC on the eNOS/NO pathway of MMVEC in MIRI rat(1) Compared with shame group, I/R group rat NO content of serum reduced significantly (P<0.05), compared with I/R group, ppc group's NO of serum increase significantly (P<0.05), compared with PPC group, PPC+L group's NO content of serum decreased significantly (P<0.05);(2) Compared with shame group, eNOS mRNA and protein of I/R group significantly increased(P<0.05); compared with I/R group, eNOS mRNA and eNOS protein of PPC group significantly increased (P<0.05); compared with PPC group, eNOS mRNA of PPC+L group changes isn't significant(P>0.05), but eNOS protein significantly reduced(P<0.05);(3) Compared with shame group, iNOS mRNA and protein of I/R group significantly increased(P<0.05), compared with I/R group, iNOS mRNA and protein of PPC group decreased significantly (P<0.05); compared with PPC group, iNOS mRNA and protein of PPC+L group changes no significantly (P>0.05).4. Effect of GFPC on RA of MIRI ratCompared with the shame, incidence of arrhythmia of I/R group was signific-antly higher (P<0.05); compared with I/R group, PPC group were significantly decreased (P<0.05).5. Effect of GFPC on hemorheologic changes of MIRI rat compared with shame group, hematocrit of I/R group was significantly higher (P<0.05), compared with I/R group, hematocrit of PPC group were significantly lower (P<0.05); Hematocrit of PPC group and PPC+L group had no significant difference (P> 0.05). Compared with shame group, fibrinogen levels of I/R group was significantly higher(P<0.05); compared with I/R group, fibrinogen levels of PPC and PPC+L group was significantly decreased(P<0.05); fibrinogen levels of PPC and PPC+L group had no Significant difference(P>0.05).6. Effect of GFPC on morphological changes of MMVEC in MIRI ratElectron microscope:in shame group, mitochondrial membrane integrity of MM-VEC, crest particle exists, within the folds close, no bubble, regular nuclear memb-rane, chromatin uniform, no concentration phenomenon, nucleolus exists; mitoch-ondrial of I/R group swelling, membrane Irregular, loose vacuoles within the wrin-kle, ridge particles disappeared, and irregular nuclear membrane, chromatin conden-sation and margination, nucleolar disappearance, or even apoptotic bodies; compared with I/R group,PPC group significantly improved in symptoms; PPC+L group worse than PPC group.7. Effect of GFPC on the apoptosis of MMVEC in MIRI rat Compared with shame group, MMVEC apoptosis rate of I/R group was significa-ntly higher (P<0.05); compared with I/R group, MMVEC apoptosis rate of PPC group decreased (P<0.05); compared with PPC group, MMVEC apoptosis rate of PPC+L group was increased (P<0.05).Conclusions:1. GFPC as glycyrrhizic acid 50mg/kg, ferulic acid 400 mg/kg, paeoniflorin 100 mg/kg, cinnamic acid 400 mg/kg could increase the content of NO, reduce the content of ET, resist lipid peroxidation best, GFPC has the best effect on anti-MIRI MMVEC injury2. pharmacological preconditioning MIRI rats with GFPC, which could increase NO content of serum, increased the eNOS gene expression and protein phosphorylation, decrease the expression of iNOS mRNA and protein, reduce the incidence of reperfusion arrhythmias and improve the hemorheologic, decrease apoptosis rate of MMVEC, the functional mechanism is through regulating the the eNOS/NO signal pathway and of MIRI MMVEC. 3.The mechanism of protective effect of PPC with GFPC is through enhancing the expression of protective eNOS mRNA and eNOS phosphorylation, and suppressing the expression of iNOS mRNA and iNOS.
|
PDF Full Text Request