当归四逆汤成分组合预处理对MIRI大鼠MMVEC eNOS/NO信号通路的影响研究
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摘要
目的：建立SD大鼠心肌缺血再灌注损伤(myocardial ischemia reperfusion injury, MIRI)模型,观察当归四逆汤中四种单体成分甘草酸、阿魏酸、芍药苷、肉桂酸及其组合对MIRI大鼠心肌微血管内皮细胞(myocardial microvascular endothelial cell, MMVEC) eNOS/NO信号通路的影响,探讨当归四逆汤治疗MIRI的微血管机制,为防治MIRI提供新的思路。
方法：实验分两步进行：第一步,以正交设计法筛选当归四逆汤中甘草酸、阿魏酸、芍药苷、肉桂酸四种单体抗MIRI大鼠MMVEC损伤的最佳组合(best combination of glycyrrhizic acid, ferulic acid, paeoniflorin, cinnamic acid, GFPC)。药物预处理(pharmacological preconditioning, PPC)采用四种单体及其组合于造模前预防性灌服四天,每天一次,最后一次于造模前30min给药。正交设计采用四因素(四种单体)四水平(每个单体四个剂量水平),将112只SD大鼠按正交设计分为16组,每组7只,用结扎冠脉左前降支(Left anterior descending coronary artery, LAD)的方法造成SD大鼠MIRI模型,结扎30min,再灌注2h,观察心电图改变及心肌HE染色确定模型可靠性和稳定性；造模结束后,腹主动脉取血,药效学观察指标为血清中NO、ET、SOD、MDA含量,应用硝酸还原法检测血清NO含量；放射免疫法检测血清ET水平；硫代巴比妥酸(TBA)法检测MDA含量；氮蓝四唑(NBT)显色法检测SOD。
第二步,应用筛选出的最佳成分组合GFPC开展对MIRI大鼠MMVEC的eNOS/NO信号通路调节机制研究。将32只SD大鼠分为shame组(假手术组,开胸只穿线,不结扎),I/R组(MIRI模型),PPC组(MIRI+单体组合灌胃),PPC+L组(MIRI+单体组合灌胃+eNOS选择性阻断剂Nω-硝基L-精氨酸甲酯,再灌注前15min,30mg/kg股静脉给药),每组8只。造模结束后,取心尖部固定,切片,电镜观察MMVEC凋亡及形态学改变；TUNEL法检测MMVEC凋亡率,腹主动脉取血,应用硝酸还原法检测血清NO含量,血液流变仪检测血液流变学指标；取左心室,切取200mg提取MMVEC和鉴定,采用real-time RT-PCR检测大鼠MMVEC eNOS和iNOSmRNA的表达；切取左心室200mg提取MMVEC, western blot的方法检测eNOS和iNOS蛋白的表达。
所有数据均以均数±标准差表示,应用SPSS15.0对实验数据进行统计分析,正交设计用方差分析,组间数据比较用单因素方差分析,计数资料的分析用χ2检验,P<0.05有统计学意义。
结果：
1.MIRI模型的验证
心电图示,造模后SD大鼠心电图ST段显著抬高,恢复灌注后ST段显著下降,模型可靠性及可重复性高,心肌HE染色见shame组心肌纹理正常,横纹清晰,而I/R组可见明显的心肌细胞横纹不清,肌细胞空泡变性,肌丝溶解断裂,局部有炎细胞浸润,并可见间质水肿的存在。2.抗MIRI MMVEC损伤的最佳成分组合筛选
再灌注120min后,可有效升高对血清中NO含量的依次为阿魏酸>甘草酸>芍药苷>肉桂酸,即阿魏酸400mg/kg,甘草酸50 mg/kg,芍药苷0 mg/kg,肉桂酸0 mg/kg;可有效降低血清ET含量的依次为阿魏酸>肉桂酸>芍药苷>甘草酸,即阿魏酸300mg/kg,肉桂酸400 mg/kg,芍药苷0 mg/kg,甘草酸0 mg/kg,;可有效升高血清中SOD含量的依次为芍药苷>肉桂酸>阿魏酸>甘草酸,即芍药苷100 mg/kg,肉桂酸200 mg/kg,可魏酸Omg/kg,甘草酸0 mg/kg;可有效降低血清中MDA含量的依次为阿魏酸>芍药苷>肉桂酸>甘草酸,即阿魏酸200mg/kg,芍药苷50 mg/kg,肉桂酸0 mg/kg,甘草酸0 mg/kg o综合分析可以有效升高血清NO,降低血清ET,保护内皮功能；同时可以升高SOD减轻氧化应激损伤,降低脂质过氧化物MDA的产生,抗MIRI MMVEC损伤的最佳成分组合(Glycyrrhizic acid, Ferulic acid, Paeoniflorin, Cinnamic acid, GFPC)为甘草酸50mg/kg,阿魏酸400 mg/kg,芍药苷100 mg/kg,肉桂酸400 mg/kg o 3.GFPC对MIRI大鼠MMVEC的eNOS/NO信号通路的影响
(1)与shame组比较,I/R组大鼠血清中NO含量明显降低(P<0.05)；与I/R组比较,PPC组血清中NO含量明显升高(P<0.05)；与PPC组比较,PPC+L组血清中NO含量明显降低(P<0.05)；
(2)与shame组比较,I/R组MMVEC eNOS的mRNA表达及eNOS蛋白磷酸化下降明显(P<0.05)；与I/R组比较,PPC组MMVEC eNOS mRNA表达及eNOS蛋白磷酸化明显增加(P<0.05)；与PPC组比较,PPC+L组MMVEC eNOS的mRNA表达未见明显改变(P>0.05),但eNOS蛋白磷酸化明显减少(P<0.05)；
(3)与shame组比较,I/R组MMVEC iNOS mRNA和iNOS蛋白的表达明显升高(P<0.05),与I/R比较,PPC组MMVEC iNOS mRNA和iNOS蛋白表达明显降低(P<0.05)；PPC组与PPC+L组MMVEC iNOS mRNA和iNOS蛋白表达无明显差异(P>0.05)。
4. GFPC对MIRI大鼠MMVEC RA的影响
与shame组比较,I/R组心律失常发生率显著升高(P<0.05)；与I/R组比较,PPC组的发生率显著下降(P<0.05),PPC组与PPC+L组心律失常发生率无明显差异(P>0.05)。
5. GFPC对MIRI大鼠血液流变学的影响
与shame组比较,I/R组红细胞压积显著升高(P<0.05),与I/R组比较,PPC组红细胞压积显著降低(P<0.05)；PPC组和PPC+L组红细胞压积无明显差异(P>0.05)。与shame组比较,I/R组纤维蛋白原水平明显升高(P<0.05),与I/R组比较,PPC组及PPC+L组纤维蛋白原水平明显下降(P<0.05),PPC组与PPC+L组纤维蛋白原水平未见明显差异(P>0.05)。
6. GFPC对MIRI大鼠MMVEC形态学的影响
电镜观察shame组MMVEC线粒体膜规整,嵴粒存在,内皱紧密,无空泡,核膜规整,染色质均匀,无浓集现象,核仁存在；I/R组线粒体肿胀,膜不规整,内皱疏松有空泡,嵴粒消失,核膜不规整,染色质浓集,边集,核仁消失,甚至出现凋亡小体；PPC组与I/R组MMVEC损伤较轻；与PPC组比较,PPC+L组MMVEC损伤加重。
7. GFPC对MIRI大鼠MMVEC凋亡的影响
与shame组比较,I/R组MMVEC凋亡率明显升高(P<0.05)；与I/R组比较,PPC组MMVEC凋亡率明显下降(P<0.05)；与PPC组比较,PPC+L组MMVEC凋亡率有所升高(P<0.05)。
结论：
1.在当归四逆汤四种有效成份中,可提高NO含量、降低ET含量,对抗脂质过氧化,抗MIRI保护MMVEC的GFPC为甘草酸50mg/kg,可魏酸400mg/kg,芍药苷100mg/kg,肉桂酸400 mg/kg。
2.用GFPC药物预处理MIRI大鼠,可升高血清中NO,降低再灌注心律失常的发生率,改善血液流变学,降低MMVEC凋亡率,对抗MIRI引起的MMVEC凋亡。其机制是通过eNOS蛋白磷酸化,激活eNOS/NO信号通路,并上调eNOS mRNA的表达,下调iNOS mRNA和iNOS蛋白的表达,调节MIRI模型中MMVEC功能。
3. GFPC药物预处理对MMVEC的保护机制是：通过增强保护性eNOS基因表达和eNOS蛋白磷酸化,抑制损伤性iNos基因表达和iNOS蛋白合成来实现的。
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 injury
After 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 rat
Compared 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 rat
Electron 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 injury
2. 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.
方法：实验分两步进行：第一步,以正交设计法筛选当归四逆汤中甘草酸、阿魏酸、芍药苷、肉桂酸四种单体抗MIRI大鼠MMVEC损伤的最佳组合(best combination of glycyrrhizic acid, ferulic acid, paeoniflorin, cinnamic acid, GFPC)。药物预处理(pharmacological preconditioning, PPC)采用四种单体及其组合于造模前预防性灌服四天,每天一次,最后一次于造模前30min给药。正交设计采用四因素(四种单体)四水平(每个单体四个剂量水平),将112只SD大鼠按正交设计分为16组,每组7只,用结扎冠脉左前降支(Left anterior descending coronary artery, LAD)的方法造成SD大鼠MIRI模型,结扎30min,再灌注2h,观察心电图改变及心肌HE染色确定模型可靠性和稳定性；造模结束后,腹主动脉取血,药效学观察指标为血清中NO、ET、SOD、MDA含量,应用硝酸还原法检测血清NO含量；放射免疫法检测血清ET水平；硫代巴比妥酸(TBA)法检测MDA含量；氮蓝四唑(NBT)显色法检测SOD。
第二步,应用筛选出的最佳成分组合GFPC开展对MIRI大鼠MMVEC的eNOS/NO信号通路调节机制研究。将32只SD大鼠分为shame组(假手术组,开胸只穿线,不结扎),I/R组(MIRI模型),PPC组(MIRI+单体组合灌胃),PPC+L组(MIRI+单体组合灌胃+eNOS选择性阻断剂Nω-硝基L-精氨酸甲酯,再灌注前15min,30mg/kg股静脉给药),每组8只。造模结束后,取心尖部固定,切片,电镜观察MMVEC凋亡及形态学改变；TUNEL法检测MMVEC凋亡率,腹主动脉取血,应用硝酸还原法检测血清NO含量,血液流变仪检测血液流变学指标；取左心室,切取200mg提取MMVEC和鉴定,采用real-time RT-PCR检测大鼠MMVEC eNOS和iNOSmRNA的表达；切取左心室200mg提取MMVEC, western blot的方法检测eNOS和iNOS蛋白的表达。
所有数据均以均数±标准差表示,应用SPSS15.0对实验数据进行统计分析,正交设计用方差分析,组间数据比较用单因素方差分析,计数资料的分析用χ2检验,P<0.05有统计学意义。
结果：
1.MIRI模型的验证
心电图示,造模后SD大鼠心电图ST段显著抬高,恢复灌注后ST段显著下降,模型可靠性及可重复性高,心肌HE染色见shame组心肌纹理正常,横纹清晰,而I/R组可见明显的心肌细胞横纹不清,肌细胞空泡变性,肌丝溶解断裂,局部有炎细胞浸润,并可见间质水肿的存在。2.抗MIRI MMVEC损伤的最佳成分组合筛选
再灌注120min后,可有效升高对血清中NO含量的依次为阿魏酸>甘草酸>芍药苷>肉桂酸,即阿魏酸400mg/kg,甘草酸50 mg/kg,芍药苷0 mg/kg,肉桂酸0 mg/kg;可有效降低血清ET含量的依次为阿魏酸>肉桂酸>芍药苷>甘草酸,即阿魏酸300mg/kg,肉桂酸400 mg/kg,芍药苷0 mg/kg,甘草酸0 mg/kg,;可有效升高血清中SOD含量的依次为芍药苷>肉桂酸>阿魏酸>甘草酸,即芍药苷100 mg/kg,肉桂酸200 mg/kg,可魏酸Omg/kg,甘草酸0 mg/kg;可有效降低血清中MDA含量的依次为阿魏酸>芍药苷>肉桂酸>甘草酸,即阿魏酸200mg/kg,芍药苷50 mg/kg,肉桂酸0 mg/kg,甘草酸0 mg/kg o综合分析可以有效升高血清NO,降低血清ET,保护内皮功能；同时可以升高SOD减轻氧化应激损伤,降低脂质过氧化物MDA的产生,抗MIRI MMVEC损伤的最佳成分组合(Glycyrrhizic acid, Ferulic acid, Paeoniflorin, Cinnamic acid, GFPC)为甘草酸50mg/kg,阿魏酸400 mg/kg,芍药苷100 mg/kg,肉桂酸400 mg/kg o 3.GFPC对MIRI大鼠MMVEC的eNOS/NO信号通路的影响
(1)与shame组比较,I/R组大鼠血清中NO含量明显降低(P<0.05)；与I/R组比较,PPC组血清中NO含量明显升高(P<0.05)；与PPC组比较,PPC+L组血清中NO含量明显降低(P<0.05)；
(2)与shame组比较,I/R组MMVEC eNOS的mRNA表达及eNOS蛋白磷酸化下降明显(P<0.05)；与I/R组比较,PPC组MMVEC eNOS mRNA表达及eNOS蛋白磷酸化明显增加(P<0.05)；与PPC组比较,PPC+L组MMVEC eNOS的mRNA表达未见明显改变(P>0.05),但eNOS蛋白磷酸化明显减少(P<0.05)；
(3)与shame组比较,I/R组MMVEC iNOS mRNA和iNOS蛋白的表达明显升高(P<0.05),与I/R比较,PPC组MMVEC iNOS mRNA和iNOS蛋白表达明显降低(P<0.05)；PPC组与PPC+L组MMVEC iNOS mRNA和iNOS蛋白表达无明显差异(P>0.05)。
4. GFPC对MIRI大鼠MMVEC RA的影响
与shame组比较,I/R组心律失常发生率显著升高(P<0.05)；与I/R组比较,PPC组的发生率显著下降(P<0.05),PPC组与PPC+L组心律失常发生率无明显差异(P>0.05)。
5. GFPC对MIRI大鼠血液流变学的影响
与shame组比较,I/R组红细胞压积显著升高(P<0.05),与I/R组比较,PPC组红细胞压积显著降低(P<0.05)；PPC组和PPC+L组红细胞压积无明显差异(P>0.05)。与shame组比较,I/R组纤维蛋白原水平明显升高(P<0.05),与I/R组比较,PPC组及PPC+L组纤维蛋白原水平明显下降(P<0.05),PPC组与PPC+L组纤维蛋白原水平未见明显差异(P>0.05)。
6. GFPC对MIRI大鼠MMVEC形态学的影响
电镜观察shame组MMVEC线粒体膜规整,嵴粒存在,内皱紧密,无空泡,核膜规整,染色质均匀,无浓集现象,核仁存在；I/R组线粒体肿胀,膜不规整,内皱疏松有空泡,嵴粒消失,核膜不规整,染色质浓集,边集,核仁消失,甚至出现凋亡小体；PPC组与I/R组MMVEC损伤较轻；与PPC组比较,PPC+L组MMVEC损伤加重。
7. GFPC对MIRI大鼠MMVEC凋亡的影响
与shame组比较,I/R组MMVEC凋亡率明显升高(P<0.05)；与I/R组比较,PPC组MMVEC凋亡率明显下降(P<0.05)；与PPC组比较,PPC+L组MMVEC凋亡率有所升高(P<0.05)。
结论：
1.在当归四逆汤四种有效成份中,可提高NO含量、降低ET含量,对抗脂质过氧化,抗MIRI保护MMVEC的GFPC为甘草酸50mg/kg,可魏酸400mg/kg,芍药苷100mg/kg,肉桂酸400 mg/kg。
2.用GFPC药物预处理MIRI大鼠,可升高血清中NO,降低再灌注心律失常的发生率,改善血液流变学,降低MMVEC凋亡率,对抗MIRI引起的MMVEC凋亡。其机制是通过eNOS蛋白磷酸化,激活eNOS/NO信号通路,并上调eNOS mRNA的表达,下调iNOS mRNA和iNOS蛋白的表达,调节MIRI模型中MMVEC功能。
3. GFPC药物预处理对MMVEC的保护机制是：通过增强保护性eNOS基因表达和eNOS蛋白磷酸化,抑制损伤性iNos基因表达和iNOS蛋白合成来实现的。
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 injury
After 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 rat
Compared 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 rat
Electron 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 injury
2. 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.
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