乳酸和氢盐水联合后适应减轻心肌细胞凋亡

Pharmacological post-conditioning with lactic acid and saturated hydrogen saline could attenuate myocardial apoptosis

目的 探讨乳酸和饱和氢盐水能否模拟后适应通过丝裂原活化蛋白激酶途径（MAPK）减轻心肌细胞凋亡.方法 在济南军区总医院108只SD大鼠随机（随机数字法）分为假手术组、缺血-再灌注组（R/Ⅰ组,移除球囊后立即在缺血部位分三点注射生理盐水共计60μL,而后持续再灌注）、后适应组（M-Post组,后适应处理方案为20/20 s× 4,移除球囊后立即在缺血部位分三点注射生理盐水共计60 μL,而后持续再灌注）、乳酸组（Lac组,再灌注即刻微量注射器在缺血心肌部位分三点注射乳酸60 μL,而后持续再灌注）、饱和氢盐水组（Hyd组,再灌注即刻微量注射器在缺血心肌部位分三点注射氢盐水60μL,而后持续再灌注）、乳酸＋饱和氢盐水组（Lac＋ Hyd组,再灌注即刻微量注射器在缺血心肌部位分三点注射乳酸和氢盐水各60μL,而后持续再灌注）,每组18只.心肌缺血45 min制作急性心肌梗死模型,测定每只大鼠再灌注3min后右心房血浆pH值.再灌注3min后每组取6只处死,取心肌组织采用硫代巴比妥酸法和分光光度计法测定心肌组织MDA含量和SOD活性.另再灌注30 min后每组取6只处死,取心肌组织用Western blot方法分别测定磷酸化MAPK（p38/JNK和ERK）、TNF-α、Caspase-8的表达.另再灌注24h后各组剩余6只大鼠测定血流动力学,处死后取心脏进行TUNEL凋亡检测.统计学多组间比较应用单因素方差分析,如组间差异显著则两两比较应用q检验.结果 Lac＋ Hyd组再灌注后3rain右心房血浆pH值显著低于R/I组（7.32±0.06）vs.（7.43 ±0.03）,P＜0.05; MDA含量显著低于R/I组（1.14±0.16） nmol/mgpro vs..（1.56±0.21）nmol/mgpro,P＜0.05;SOD含量显著高于R/I组（57.92±15.12） U/mgpro vs.（35.48 ±12.46） U/mgpro,P＜0.05.Lac＋ Hyd组再灌注30 min后心肌组织P-p38 （0.46±0.06）vs.（2.18±0.32）,P＜0.05;和P-JNK含量（0.59±0.03）vs.（1.62±0.29）,P＜0.05,显著低于R/I组,TNF-α含量（0.34±0.08）vs..（1.78±0.31）,P＜0.05;和Caspase-8含量（0.31±0.07）vs.（1.52±0.28）,P＜0.05,均显著低于R/I组;心肌细胞凋亡指数显著低于R/I组（9.50±1.51）％vs.（15.21±1.91）％,P＜0.05.以上指标均与M-Post组相比均差异无统计学意义（P＞0.05）.再灌注30 min后缺血心肌组织P-ERK含量Lac＋ Hyd组与R/I组相似（0.55 ±0.13）vs.（0.57±0.05）,P＞0.05;Hyd组显著低于R/I组（0.30 ±0.09）vs（0.57±0.05）,P＜0.05;Lac、Hyd和Lac＋Hyd组P-ERK表达均显著低于M-Post组（1.21±0.13）,（0.30±0.09）,（0.55±0.13）vs.（1.96±0.39）,P＜0.05.结论 乳酸和饱和氢盐水联合使用可较好模拟后适应上游触发因子,抑制p38/JNK和ERK的磷酸化,减轻心肌细胞凋亡.

Objective To study the hypothesis about the pharmacological post-conditioning with lactic acid and saturated hydrogen saline after ischemic injury of myocardium instead of post-conditioning with mechanical dilatation of severely occluded coronary vessels to attenuate apoptosis of cardiocyte by mitogenactivated protein kinases （MAPK） pathway.Methods A total of 108 rats were randomly （random number） divided into 6 groups （n =18 in each group）：sham operated group （received 60 μL normal saline without ischemia）,reperfusion/injury group （R/I,received 60 μL normal saline solution and routine ischemicreperfusion [IR] procedure）,post-conditioning group （M-Post,received 60 μL normal saline and postconditioning treatment,4 cycles of 20/20 s of reperfusiorr/re-occlusion）,lactic acid group （Lac,received 60 μL lactic acid and routine IR procedure）,saturated hydrogen saline group （Hyd,received 60 μL hydrogen rich saline and routine IR procedure）,and lactic acid ＋ saturated hydrogen saline group （Lac ＋ Hyd,received a combination of 60 μL of lactic acid and 60 μL of hydrogen rich saline along with routine IR procedure）.Acute myocardial infarction model was made by ischemia for 45 min,and pH value of blood from right atrium was detected in rats of all groups.After 3 min reperfusion,6 rats of each group were sacrificed and myocardial tissue was taken out to measure the level of MDA and SOD.After 30 min reperfusion,other 6 rats of each group were sacrificed and myocardial tissue was taken out to measure the level of phosphorylated MAPK （p38/JNK and ERK）,TNF-α,Caspase-8 by Western-blot method.After 24 h reperfusion,there were only 6 rats in each group,and hemodynamics were measured in each rat,and then rats were sacrificed and hearts were taken out to detect cell apoptosis by TUNEL method.A one-way analysis of variance （ANOVA） was used,and q tests were employed to determine if any significant differences in individual variable existed between groups.Results The pH of blood from right atrium after 3 min of reperfusion in Lac ＋ Hyd group was significantly lower than that in R/I group （7.32 ± 0.06 vs.7.43 ± 0.03,P 〈 0.05）,the content of MDA was lower （1.14 ± 0.16 vs.1.56 ± 0.21,P 〈 0.05） and the content of SOD was higher in Lac ＋ Hyd group than those in R/I group （57.92 ± 15.12 vs.35.48 ± 12.46,P 〈 0.05）.Apoptotic index of Lac ＋ Hyd group was much lower than that of R/I group （9.50 ± 1.51） % vs.（15.21 ± 1.91）%,P〈0.05.After 30 min of reperfusion,the level of P-p38 in ischemic myocardia in Lac ＋ Hyd group was significantly lower than that in R/I group （0.46 ±0.06 vs.2.18 ±0.32,P 〈0.05）,the levels of P-JNK （0.59±0.03 vs.1.62 ±0.29,P 〈0.05）,TNFα （0.34 ±0.08 vs.1.78 ±0.31,P 〈0.05） and Caspase-8 （0.31 ±0.07 vs.1.52 ±0.28,P 〈0.05） were all lower than those in R/I group.However,there were no significant differences in levels of all above variables between Lac ＋ Hyd group and M-Post group （P 〉 0.05）.After 30 rmin of reperfusion,there was no significant difference in the level of P-ERK between Lac ＋ Hyd group and R/I group （0.55 ± 0.13 vs.0.57 ± 0.05,P 〉 0.05）,and the level of PERK in Hyd group was significantly lower than that in R/I group （0.30 ± 0.09 vs.0.57 ± 0.05,P 〈0.05）,And the level of P-ERK in Lac、Hyd and Lac ＋ Hyd groups was significantly lower than that in MPost group （1.21 ±0.13,0.30 ±0.09,0.55 ±0.13 vs.1.96 ±0.39,P 〈0.05）.Conclusion Pharmacological post-conditioning with lactic acid and saturated hydrogen saline could be used instead of mechanical post-conditioning to inhibit the phosphorylation of p38/JNK and ERK,attenuating myocardial cell apoptosis.