胰岛素强化治疗拮抗烫伤大鼠心肌细胞凋亡的机制研究

Intensive insulin treatment protected the cardiac myocytes against apoptosis in severely scalded rats

目的探讨大鼠重度烫伤后胰岛素强化治疗拮抗心肌细胞凋亡的作用及机制。方法将18只SD大鼠按随机数字表法分为假伤组、烫伤组和胰岛素强化治疗组（强化治疗组），每组6只。制作30％总体表面积（TBSA）Ⅲ度烫伤模型及胰岛素强化治疗模型。伤后6h取大鼠左室心肌组织，采用原位末端缺刻标记法（TUNEL）检测心肌细胞凋亡，用免疫组化和蛋白质免疫印迹法（Western blotting）分别观察3种凋亡相关基因天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）、Bax和Bcl-2的蛋白表达。结果与假伤组比较，烫伤后心肌凋亡细胞明显增多（（13．1±3．4）％比（0．6±0．4）％，P〈0．01]caspase-3、Bax的蛋白表达明显增高（免疫组化caspase-3：13．72±4．13比1．36±0．95，Bax：29．64±5．42比2．24±1．04；Westernblottingcaspase-3：5．72±2．13比1，Bax：4．64±1．42比1），而Bcl-2表达水平显著降低（免疫组化：3．39±1．52比8．01±2．56；Westernblotting：0．69±0．42比1，均P〈O．01）。经胰岛素强化治疗后，心肌细胞的凋亡率较烫伤组明显降低[（6．7±1．8）％比（13．1±3．4）％，P〈0．01]，3种凋亡相关基因的蛋白表达水平正好与烫伤组的变化相反（免疫组化caspase-3：8．88±3．36比13．72±4．13，Bax：14．43±3．69比29．64±5．42，Bcl-2：8．61±3．72比3．39±1．52；Westernblottingcaspase-3：2．18±0．86比5．72±2．13，Bax：2．87±1．35比4．64±1．42，Bcl-2：3．57±1．70比0．69±0．42，P〈0．05或P〈0．01）。结论胰岛素强化治疗可能通过调节caspase-3、Bax和Bcl-23种细胞凋亡相关基因的表达，对烫伤后心肌细胞发挥抗凋亡作用。

Objective To investigate the effect of intensive insulin treatment, in the protection of myocardiocytes against apoptosis in severely scalded rats and its underlying mechanism. Methods Eighteen Sprague-Dawley （SD） rats were randomly divided into three groups （6/each） to receive： sham surgery, burn damage （on the back of the animals, degree ⅢI , to 30% of total body surface area）, and burn damage＋ intensive insulin treatment. Tissue samples were collected from the left ventricle 6 hours after infliction of the burn damage for the examination of myocardial cell apoptosis [by terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） staining] and the expression of apoptosis-related molecules caspase-3, Bax, and Bcl-2 （by immuno-histochemistry and Western blotting）. Results In comparison with the animals in sham treated group, the myocardioeyte apoptosis rate in animals in burn damage only group increased significantly[（13.1 ± 3.4） %vs. （0. 6 ± 0. 4） %, P〈0. 01]. The expression of caspase-3 and Bax both significantly increased while the level of Bel-2 expression significantly decreased （immuno-histochemistry caspase-3：13.72±4.13 vs. 1.36±0. 95, Bax： 29.64±5.42 vs. 2.24±1.04, Bel-2.. 3.39±1.52 vs. 8. 01± 2.56% Western blotting caspase-3： 5. 72±2.13 vs. 1, Bax：4. 64±1.42 vs. 1, Bcl-2： 0. 69±0. 42 vs. 1, all P〈0.01）. The animals received intensive insulin treatment showed significantly less myocardiocyte apoptosis [（6.7 ± 1.8） % vs. （13.1 ± 3.4）%, P 〈 0. 013, significantly lower expression in caspase-3, Bax, and significantly higher level of Bcl-2 expression as compared to the animals in burn damage only group （immuno-histoehemistry caspase-3： 8.88 ± 3. 36 vs. 13.72 ± 4. 13, Bax： 14. 43 ± 3.69 vs. 29.64 ± 5.42, Bcl-2： 8. 61±3.72 vs. 3.3±1.52; Western blotting easpase-3：2.18±0. 86 vs. 5.72±2.13, Bax： 2.87± 1.35 vs. 4.64±1.42, Bel-2： 3. 57±1. 70 vs. 0.69±0.42, P〈0.05 or P〈0.01）. Conclusion Intensive insulin therapy may protect myocardiocytes against apoptosis in severely burned animals through the regulation of the expression of apoptosis-related molecules caspase-3, Bax and Bel-2.