CAJ | 학술논문

目的观察受体诱导一氧化碳(CO)对移植心冷缺血再灌注(I/R)损伤中细胞凋亡的影响,并探讨其机制.方法以BALB/C小鼠建立同系移植心冷I/R损伤模型.受体麻醉前3 h以二氯甲烷(MC)500 mg/kg灌胃诱导CO(MC组,n=12),或橄榄油灌胃(IR组,n=12);在MC组的基础上受体在移植心恢复血供前1 h腹腔注射PI3K抑制剂LY294002(40 mg/kg,LY组,n=10)或二甲亚砜(DMSO组,n=10);检测移植后3、24 h移植心细胞凋亡指数(AI)、磷酸化Akt(p-Akt)蛋白、bcl-2与bax蛋白表达比值;设正常对照组(N组,n=5).结果受体以MC灌胃后血液中碳氧血红蛋白(COHb)浓度与心肌组织CO含量均在3 h达到峰值,分别为(9.82±0.84)%和(2.25±0.08)pmol/mg;与IR组比较,MC组明显降低移植心AI[3 h:(8.65±2.01)%比(19.28±4.94)%,P＜0.01;24 h:(5.82±2.36)%比(10.54±3.66)%,P＜0.05]、激活Akt蛋白(3 h:P＜0.01;24 h:P＜0.05)、上调bcl-2/bax比值(3 h:1.97±0.16比0.46±0.07,P＜0.01;24 h:1.89±0.10比0.51±0.04,P＜0.01);与MC组比较,LY组明显增加AI[3 h:(17.95±4.92)%,P＜0.01;24 h:(9.75±3.14)%;P＜0.01]、抑制Akt蛋白激活(P＜0.01)、下调bcl-2/bax比值(3 h:0.47±0.06,P＜0.01;24 h:0.52±0.03,P＜0.01);DMSO组与MC组的各个指标差异无统计学意义(P＞0.05).结论受体诱导C0能明显抑制冷I/R诱导的移植心细胞凋亡,其机制可能与通过PI3K/Akt信号途径上调bcl-2/bax比值有关.
목적 관찰수체유도일양화탄(CO)대이식심랭결혈재관주(I/R)손상중세포조망적영향,병탐토기궤제.방법 이BALB/C소서건립동계이식심랭I/R손상모형.수체마취전3 h이이록갑완(MC)500 mg/kg관위유도CO(MC조,n=12),혹감람유관위(IR조,n=12);재MC조적기출상수체재이식심회복혈공전1 h복강주사PI3K억제제LY294002(40 mg/kg,LY조,n=10)혹이갑아풍(DMSO조,n=10);검측이식후3、24 h이식심세포조망지수(AI)、린산화Akt(p-Akt)단백、bcl-2여bax단백표체비치;설정상대조조(N조,n=5).결과 수체이MC관위후혈액중탄양혈홍단백(COHb)농도여심기조직CO함량균재3 h체도봉치,분별위(9.82±0.84)%화(2.25±0.08)pmol/mg;여IR조비교,MC조명현강저이식심AI[3 h:(8.65±2.01)%비(19.28±4.94)%,P＜0.01;24 h:(5.82±2.36)%비(10.54±3.66)%,P＜0.05]、격활Akt단백(3 h:P＜0.01;24 h:P＜0.05)、상조bcl-2/bax비치(3 h:1.97±0.16비0.46±0.07,P＜0.01;24 h:1.89±0.10비0.51±0.04,P＜0.01);여MC조비교,LY조명현증가AI[3 h:(17.95±4.92)%,P＜0.01;24 h:(9.75±3.14)%;P＜0.01]、억제Akt단백격활(P＜0.01)、하조bcl-2/bax비치(3 h:0.47±0.06,P＜0.01;24 h:0.52±0.03,P＜0.01);DMSO조여MC조적각개지표차이무통계학의의(P＞0.05).결론 수체유도C0능명현억제랭I/R유도적이식심세포조망,기궤제가능여통과PI3K/Akt신호도경상조bcl-2/bax비치유관.
Objective To investigate whether induction of carbon monoxide (CO) in recipients could inhibit cold ischemia/reperfusion (I/R)-induced apoptosis of cardiac grafts and the possible anti-apoptotic mechanisms. Methods Inbred BALB/C mice were used as donors and recipients to establish transplant-induced cold I/R injury model. Recipients were treated with either methylene chloride (500 mg/kg, per os, group MC,n = 12) or olive oil ( group IR,n = 12) 3 h prior to anesthesia, or treated with MC plus either LY294002 of PI3 K inhibitor (40 mg/kg, i. p, group LY, n = 10) or DMSO ( group DMSO, n =10) 1 h prior to reperfusion of cardiac grafts. Recipients were killed at 3rd and 24th h after transplantation for cardiac graft samples. The apoptosis index (AI) and the protein of phosphorylated Akt, bcl-2 and bax were measured, respectively. Age-matched normal mice served as controls (group N,n =5). Results Following MC application serum COHb[(9. 82 ± 0. 84) %]and tissue CO[(2. 25 ± 0. 08 ) pmol/mg]peaked within 3 h in recipients. As compared with group IR, induction of CO in recipients decreased significantly the level of AI[3 h: (8.65 ±2.01)% vs. (19.28 ±4.94)%,P＜0.01; 24 h: (5.82 ±2.36)% vs.( 10. 54 ± 3.66) %, P ＜ 0. 05], increased the Akt phosphorylation ( 3 h: P ＜ 0. 01; 24 h: P ＜ 0. 05 ) and up-regulated the ratios of bcl-2/bax (3 h: 1.97 ±0. 16 vs. 0. 46 ±0. 07,P ＜0. 01; 24 h: 1.89 ±0. 10 vs.0. 51 ±0. 04, p ＜ 0. 01 ) in cardiac grafts. However, as compared with group MC, recipients pretreated with LY294002 could reverse the anti-apoptotic effects of MC by increasing the level of AI[3 h: ( 17.95 ±4.92)% ,P＜0. 01; 24 h: (9.75 ±3. 14)% ,P＜0. 01], inhibiting the Akt phosphorylation (P＜0. 01)and down-regulating the ratio of bcl-2/bax (3 h: 0. 47 ±0. 06,P ＜0. 01; 24 h: 0. 52 ±0. 03 ,P ＜0. 01 ).And the DMSO had no impact on the anti-apoptotic effects of MC ( P ＞ 0. 05 ). Conclusion Induction of carbon monoxide in recipients inhibits cold I/R-induced apoptosis of cardiac grafts by regulating bcl-2 and bax proteins via the activation of PI3K/Akt signal pathway.