【作者】 李小强；

【导师】 梅其炳； 招明高；

【作者基本信息】 第四军医大学 ， 药理学， 2007， 博士

【摘要】 目的:①建立MN9202手性固定相HPLC拆分方法,制备单一对映体,比较MN9202对映体对心肌细胞收缩、舒张功能及钙瞬变的影响,及其对离子通道的立体选择性;②探讨MN9202对心肌的保护作用及其机制。为正确评价MN9202消旋体及其对映体作用的差异性,为二氢吡啶类钙通道拮抗剂的临床应用提供科学依据。方法:①采用Daicel OJ-H手性固定相色谱柱,通过对流动相中正己烷与乙醇的比例、添加三乙胺的量对MN9202对映体的保留时间及分离度的影响进行考察,优化色谱条件,拆分MN9202消旋体。②采用IonOptix单细胞动缘探测系统同步检测MN9202消旋体、S-(-)-MN9202和R-(+)-MN9202作用后心肌细胞收缩、舒张和钙瞬变的变化。③采用全细胞膜片钳方法,比较MN9202消旋体、S-(-)-MN9202和R-(+)-MN9202对心肌细胞L型钙通道电流密度的影响。进而探讨各药物对L型钙通道稳态激活动力学和稳态失活动力学特性的影响。④通过股静脉注射LPS建立感染性休克致动物心肌损伤模型,研究MN9202对心肌的保护作用;RM-6200型四道生理记录仪实时监测血压等参数;用ELISA法测定血浆TNF-α水平;取心肌组织样本,进行HE染色,比较心肌形态学改变。⑤原代培养新生大鼠心肌细胞,用LPS和不同浓度的MN9202消旋体、对映体及氨氯地平作用,采用ELISA法测定培养上清液中TNF-α含量,RT-PCR及免疫荧光法分别测定心肌细胞中TNF-αmRNA和蛋白的表达。⑥RT-PCR和western blot法分别测定LPS刺激不同时间及DHPs作用后心肌细胞iNOS、COX-2 mRNA和蛋白的表达。Western blot法测定磷酸化Akt和总Akt的表达。结果:①本研究建立的MN9202 HPLC手性拆分条件为:Daicel OJ-H手性分析柱,正己烷-乙醇-三乙胺(93︰7︰0.5,V︰V︰V)为流动相,流速为0.5 ml/min,检测波长为237 nm。用此法MN9202对映异构体可达到基线分离,并可少量制备S-(-)-MN9202和R-(+)-MN9202。②不同浓度的MN9202消旋体和S-(-)-MN9202可剂量依赖性抑制大鼠单个心肌细胞的收缩:在1×10-5 mol/l浓度时,百分峰值收缩幅度(PTA %baseline)与对照组相比分别降低了73.8 %和92.7 %;而R-(+)-MN9202则呈剂量依赖性增强心肌细胞的收缩,1×10-5 mol/l时, PTA %baseline升高了18.2 %。MN9202消旋体和S-(-)-MN9202剂量依赖性抑制单个心肌细胞钙瞬变幅度(△FFI)。与对照组相比,1×10-5 mol/l MN9202消旋体和S-(-)-MN9202,细胞钙瞬变分别由0.45±0.04下降到0.07±0.02,及由0.45±0.04下降到0.05±0.01(P<0.01)。而R-(+)-MN9202则增加了钙瞬变幅度:在最大浓度1×10-5 mol/l时,细胞钙瞬变由0.45±0.01升高到0.58±0.05(P<0.01)。③MN9202消旋体剂量依赖性的降低了L型钙通道电流密度。检测电压为0 mV,MN9202浓度为0.03、0.1、0.3、1和3μmol/l时,CaL电流峰值的密度较对照组分别减少了6.7 %、15.0 %(P<0.01)、27.5 %(P<0.01)、50.7 %(P<0.01)和69.5 %(P<0.01);但药物并未改变L型钙通道激活的阈电位(-40 mV)和峰值电流电位(0 mV)。S-(-)-MN9202也抑制了L型钙通道电流密度,检测电压为0 mV时,1μmol/l S-(-)-MN9202使CaL电流峰值的密度从对照组的-9.8±1.0减少到-3.2±0.9(减少了67 %)(P<0.01)。而1μmol/l R-(+)-MN9202则引起了L型钙通道电流密度增加:检测电压为0 mV时,使CaL电流峰值的密度从对照组的-9.3±0.7增加到-10.3±0.6(P<0.01)。MN9202消旋体,S-(-)-MN9202和R-(+)-MN9202均未明显改变L型钙通道的激活特性。但各药物均对通道稳态失活有一定的影响:MN9202消旋体和S-(-)-MN9202均使稳态失活曲线左移,而R-(+)-MN9202使稳态失活曲线轻微右移。④LPS在30 min内引起大鼠平均动脉压的快速下降,从119±4降至85±2 mmHg(P<0.05);60 min后,大鼠平均动脉压仍持续下降,在240 min时为81±3 mmHg。10及30μg/kg MN9202消旋体给药后,延缓了LPS对大鼠平均动脉压的下降。但MN9202的应用并未改变LPS对心率的影响。给予LPS使血浆中TNF-α的水平发生了变化,先升高,至1小时达到峰值,而后逐渐降低。MN9202给药后可使LPS引起的大鼠血浆TNF-α升高的峰值显著降低(P<0.05)。⑤正常心肌细胞几乎不表达TNF-α,但细胞培养液中TNF-α的含量可随LPS浓度的增加而提高;TNF-α的释放与mRNA表达也随着LPS刺激时间的延长而显著升高,在刺激后12小时达高峰。0.1 ~ 10μmol/l的氨氯地平、MN9202消旋体、R-(+)-MN9202对映体可浓度依赖性的抑制LPS刺激的TNF-α的释放和mRNA的表达,S-(-)-MN9202对映体对TNF-α的释放及mRNA的表达无明显影响。1.0μmol/l的氨氯地平、MN9202消旋体、R-(+)-MN9202、S-(-)-MN9202对TNF-α释放的抑制率分别为52.41、31.62、49.81和2.25 %。⑥MN9202消旋体可浓度依赖性地抑制LPS刺激所致iNOS和COX-2转录和翻译水平的表达;1.0μmol/l的MN9202消旋体对COX-2的抑制作用与氨氯地平相当,对iNOS的抑制作用强于氨氯地平;R-(+)-MN9202明显抑制iNOS和COX-2的表达(P<0.05),而S-(-)-MN9202对iNOS和COX-2的表达均无影响。⑦LPS刺激的同时,用MN9202消旋体、R-(+)-MN9202对映体或氨氯地平处理心肌细胞,均可明显增加pAkt的表达(P<0.05);但S-(-)-MN9202对Akt的活化无明显影响。用PI3K抑制剂wortmannin或LY294002预处理2 h后,LPS和DHPs(MN9202消旋体、R-(+)-MN9202对映体和氨氯地平)共处理心肌细胞,各DHPs处理组Akt的活化均受到抑制;3种DHPs类化合物对TNF-α释放的抑制作用均可被wortmannin或LY294002不同程度的降低;Cox-2和iNOS蛋白的表达与无PI3K抑制剂预处理组相比无显著性差异。结论:①本研究建立的手性固定相拆分条件为MN9202单一对映异构体的分离、制备及含量测定等提供了简单、快捷的方法。②两种MN9202对映体对心肌细胞收缩与舒张特性、钙瞬变和L型钙通道均具有立体选择性。S-(-)-MN9202表现出了拮抗特性,而R-(+)-MN9202则具有一定的激动活性。③MN9202对LPS致大鼠心肌损伤有保护作用,此作用与其抑制LPS引起的TNF-α、iNOS和COX-2表达升高密切相关,其中R-(+)-MN9202是抑制炎症相关基因表达的优映体。④MN9202通过激活PI3K,引起下游Akt的活化,参与了TNF-α的调节过程,但对Cox-2和iNOS的调节作用可能依赖于其它信号转导通路。更多还原

【Abstract】 Aim In this study, we attempt to set up a resolution approach to get single enantiomer of MN9202 on a chiral stationary phase column; then, to determine the stereoselectivity effects of the optical enantiomers of MN9202 on cell shortening and relengthening, calcium transient and L-type calcium channels in rat ventricular myocytes; in addition, to pursue the effects and mechanisms of the optical enantiomers of MN9202 on myocardium injury induced by LPS. From above, we want to get a scientific evaluation for MN9202, which will be helpful for enlarging our understanding of clinic applications of dihydropyridine-type drugs.Methods①The enantiomers of MN9202 were separated on Daicel OJ-H column by optimizing conditions of chromatogram, such as the proportion of hexane or ethanol in the mobile phase.②Mechanical contraction properties and calcium transient were recorded in myocytes and assessed by video-edge detection system in absence or presence of racemic MN9202, S-(-)-MN9202 and R-(+)-MN9202.③To investigate the stereoselectivity of the optical enantiomers of MN9202 to ion channels, the actions (including ICa,L, steady-state activation and steady-state inactivation curves) of the optical enantiomers of MN9202 and racemic MN9202 were studied on L-type calciun channels in single isolateded adult rat ventricular myocytes, using the whole-cell configuration of the patch clamp technique.④Sepsis model was set up by LPS (5 mg/kg, i.v.) in anesthetized rats. The right carotid artery was connected to a pressure transducer (RM-6200) for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). After recorded baseline hemodynamic parameters, animals received vehicle or LPS were monitored for 240 min. 0.5 ml blood was taken to measure the plasma levels of TNF-αby ELISA. Finally, myocardial samples were taken to stain with hematoxylin and eosin.⑤The primary cultured neonatal rat cardiomyocytes were treated with LPS, alone or in the presence of various concentrations of racemic MN9202, MN9202 enantiomers or amlodipine. The TNF-αrelease in the culture medium was measured by ELISA. TNF-αmRNA and proteins in myocytes were measured by RT-PCR and immunofluorescent staining analysis, respectively.⑥The iNOS and COX-2 mRNA or protein levels in LPS-treated cardiomyocytes at different times or cotreated with DHPs were assessed by RT-PCR and western blot assays, respectively. Regulation of phospho-Akt (pAkt) and Akt was assessed by western blot assays.Results①The chromatographic separations were performed using a OJ-H column and using a mobile phase, hexane: ethanol: triethylamine (93︰7︰0.5, V ︰V︰V). The flow-rate of the mobile phase was 0.5 ml/min, and the samples were monitored with UV detection at 237 nm. Under the optimized conditions, the enantiomers of MN9202 were well separated from the baseline and several milligrams two enantiomers, S-(-)-MN9202 and R-(+)-MN9202 were obtained.②The exposure of racemic MN9202 and S-(-)-MN9202 (1×10-8 mol/l ~ 1×10-5 mol/l) caused concentration-dependent inhibition of PTA and ?FFI in single rat ventricular myocytes. The extent of maximal percentage inhibition of PTA %baseline was significantly reduced by 73.8 % and 92.7 %, respectively. ?FFI was reduced from 0.45±0.04 to 0.07±0.02 and from 0.45±0.04 to 0.05±0.01 in presence of 1×10-5 mol/l racemic MN9202 and S-(-)-MN9202, respectively. However, R-(+)-MN9202 induced concentration-dependent increases of PTA and ?FFI in single rat ventricular myocytes. The extent of maximal percentage inhibition of PTA %baseline was significantly increased by 18.2 %, and ?FFI was increased from 0.45±0.01 to 0.58±0.05 in presence of 1×10-5 mol/l R-(+)-MN9202 (P<0.01, compared with control group).③In response to depolarizations positive to 0 mV, ICa,L density was reduced by 6.7 %,15.0 % (P<0.01), 27.5 % (P<0.01), 50.7 % (P<0.01) and 69.5 % (P<0.01), respectively in 0.03, 0.1, 0.3, 1 and 3μmol/l racemic MN9202 groups. S-(-)-MN9202 (10-6 mol/l) reduced the ICa,L density from -9.8±1.0 to -3.2±0.9 (P<0.01, 67% decrease) in response to depolarizations positive to 0 mV, exhibiting more effective antagonist activity than racemic MN9202. However, R-(+)-MN9202 (10-6 mol/l) increased the ICa,L density from -9.3±0.7 to -10.3±0.6 (P<0.01) in response to depolarizations positive to 0 mV. In ventricular myocytes, racemic MN9202, S-(-)-MN9202 and R-(+)-MN9202 didn’t affect the steady-state activation curve of ICa, L. However, a shift of the midpoint of the steady-state inactivation curve was produced in the hyperpolarizing direction from ?28.8±0.2 mV to ?35.4±0.7 mV in the presence of 1μmol/l racemic MN9202 and from?28.8±0.2 mV to ?37.7±0.6 mV in the presence of 1μmol/l S-(-)-MN9202. But, in contrast, R-(+)-MN9202 at the same concentration did cause only a small shift of the midpoint of the steady-state inactivation curve (to the right) in the depolarizing direction from ?28.8±0.2 mV to ?27.1±0.6.④Administration of LPS caused a rapid fall in MAP from 119±4 to 85±2 mmHg (P<0.05)within 30 min. After 60 min, there was a continuous further fall in MAP. 10 and 30μg/kg MN9202 prevented the delayed fall in MAP observed in LPS groups. Administration of LPS resulted in an increase of heart rate, and in the MN9202 group, there was no significant difference of heart rate compared with LPS group. The injection of LPS resulted in bell-shape changes in the plasma levels of TNF-αwhich reached a peak at 60 min after LPS injection and subsequently decreased slowly (P<0.05). Treatment of 10μg/kg and 30μg/kg MN9202 decreased the TNF-αlevel in plasma. In LPS group, increase and oedema of interstitial cell, vacuolar degeneration of cardiomyocytes, inflammatory cell infiltrate have been significantly found, compared with control group. Administration of 10μg/kg and 30μg/kg MN9202 mitigated these above symptoms.⑤Neonatal rat cardiomyocytes did not release detectable amounts of TNF-αin the absence of LPS. However, in the presence of LPS, TNF-αrelease in the culture medium increased in a concentration-depended manner and mRNA levels also increased rapidly, reaching a peak at 12 h. Amlodipine, racemic MN9202 and R-(+)-MN9202, in concentrations ranging from 0. 1 to 10μmol/l, could concentration-dependently inhibit the release and mRNA expression of TNF-αin LPS-stimulated cardiomyocytes, but S-(-)-MN9202 had no obvious effect on the TNF-αrelease and mRNA expression. The inhibition rates of TNF-αrelease from LPS-stimulated neonatal rat cardiomyocytes in 1.0μmol/l amlodipine, racemic MN9202, R-(+)-MN9202 and S-(-)-MN9202 groups were 52.41, 31.62, 49.81 and 2.25 %, respectively.⑥Racemic MN9202 significantly attenuated the iNOS and TNF-αmRNA and protein levels in cardiomyocytes stimulated with LPS in a concentration-dependent manner. While the inhibition rate of 1.0μmol/l MN9202 on COX-2 expression was almost as same as the one of amlodipine, MN9202 showed more effective regulation on the iNOS levels in same concentration. Furthermore, R-MN9202 significantly depressed the levels of iNOS and COX-2, but S-MN9202 could not significantly attenuate these inflammation-relevant genes expression.⑦Compared with LPS-stimulated cells, when cardiomyocytes were cotreated with LPS and racemic MN9202, R-(+)-MN9202 or amlodipine, the phosphorylation levels of Akt were markedly increased (P<0.05), but S-(-)-MN9202 had no effect on the pAkt levels. Pre-treatment with selective inhibitors of the PI3K/Akt pathway, wortmannin or LY294002, in LPS and DHPs (racemic MN9202, R-(+)-MN9202 and amlodipine)-treated cells, Akt phosphorylation was partially inhibited. To elucidate the contribution of PI3K/Akt pathway to the regulation of TNF-α, Cox-2 and iNOS protein levels, cardiomyocytes were pretreated with wortmannin or LY294002 for 2 h and then cotreated with LPS and DHPs. The inhibition of PI3K with wortmannin or LY294002 partly attenuated the depression effects of DHPs on TNF-αprotein expression, but there was no significiant difference on the Cox-2 and iNOS expression between the groups which pre-treated with the PI3K/Akt inhibitors or not.Conclusion①The proposed chromatographic method is easy and accurate. It is reliable in the separation, preparation and quantification of MN9202.②S-(-)-MN9202 and R-(+)-MN9202 produce differential stereoselectivity effects in mechanical contraction properties (shortening and relengthening of individual myocytes), myocyte Ca2+ transients and L-type calcium channels. S-(-)-MN9202 displayed an antagonist activation, whereas R-(+)-MN9202 exerted an agonist effect.③MN9202 protected the impaired myocardium induced by LPS through inhibiting the LPS-induced expression of TNF-α, iNOS and COX-2. R-(+)-MN9202 has more markedly inhibitory effects on these LPS-induced inflammation-relevant genes expression.④The anti-TNF-αeffects of MN9202 were partly mediated by activation of Akt, downstream of the PI3K signal cascade, but anti-iNOS and COX-2 effects might be mediated by other signal pathway.更多还原