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目的 探讨高密度脂蛋白(HDL)对氧糖剥离(OGD)小鼠心肌细胞的保护作用.方法 通过蛋白酶消化及差速贴壁法获得原代清道夫受体B1基因敲除小鼠(SR-B1-/-)的心肌细胞与普通C57小鼠(SR-B1+/+)的心肌细胞.① 将两种细胞均分为正常对照组(Con组)、OGD组、OGD+HDL组3组,培养4 h后用碘化丙啶(PI)染色测定细胞坏死情况.② 将SR-B1+/+心肌细胞分为Con组、OGD组、OGD+HDL组、磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)抑制剂LY294002组4组,用PI染色测定细胞坏死情况;用原位末端缺刻标记法(TUNEL)测定细胞凋亡情况;按照试剂盒步骤测定细胞上清液中肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)含量;用蛋白质免疫印迹试验(Western Blot)测定SR-B1、Akt蛋白表达.结果 ① 在SR-B1+/+心肌细胞中,给予HDL可抑制OGD致细胞坏死;而HDL对OGD的SR-B1-/-心肌细胞无保护作用.② 对SR-B1+/+细胞研究显示:与Con组比较,OGD组坏死细胞显著增多,细胞活性显著下降,上清液中LDH、CK-MB含量升高,磷酸化蛋白激酶B(p-Akt)、SR-B1表达显著下降.与OGD组比较,OGD+HDL组坏死细胞显著减少〔PI阳性细胞率:(26.71±5.94)％比(64.24±18.34)％〕,细胞活性显著升高〔(63.84±6.95)％比(26.71±5.13)％〕,上清液中LDH、CK-MB含量显著下降〔LDH(U/L):896.3±161.5比1568.3±243.5,CK-MB(U/L):304.3±72.9比583.6±81.6〕,p-Akt、SR-B1表达显著升高(p-Akt/t-Akt:0.84±0.13比0.18±0.06,SR-B1/β-actin:1.23±0.19比0.09±0.02),差异均有统计学意义(均P<0.05).与OGD+HDL组比较,LY294002组坏死细胞增多,细胞活性下降,上清液中LDH、CK-MB含量升高,p-Akt、SR-B1表达下降,且各指标与OGD组比较差异均无统计学意义.4组间细胞凋亡情况差异无统计学意义.结论 HDL对OGD小鼠心肌细胞具有保护作用,其机制可能与激活PI3K/Akt通路后上调SR-B1蛋白表达相关.

Objective To investigate the protective effect of high-density lipoprotein (HDL) on the mice cardiac myocytes induced by oxygen and glucose deprivation (OGD).Methods Cardiac cells of primary scavenger receptor-B1 knockout mice (SR-B1-/-) and normal C57 mice (SR-B1+/+) were obtained by protease digestion and differential adhesion method. ① The two kinds of cells were divided into normal control group (Con group), OGD group, OGD+HDL group. Propidium iodide (PI) staining were used to determine the necrosis of cardiac myocytes. ② SR-B1+/+cardiac cells were divided into Con group, OGD group, OGD+HDL group, and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) inhibitor LY294002 group. PI staining were used to determine the necrosis of cardiac myocytes. TUNEL staining was used to determine the cell apoptosis. The kit was used to determine the contents of MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) in the culture medium supernatant. The expressions of SR-B1 and Akt protein were determined by Western Blot.Results ① In SR-B1+/+ cardiomyocytes, HDL could inhibit cell necrosis induced by OGD. There was no protective effect of HDL on OGD in the SR-B1-/- cardiomyocytes.② The study of SR-B1+/+ cells was showed that compared with Con group, necrotic cells were significantly increased and cell activity were significantly decreased, the cell viability were significantly decreased, the contents of LDH and CK-MB in supernatant were significantly increased, the expressions of phosphorylated Akt (p-Akt) and SR-B1 were significantly decreased in OGD group. Compared with OGD group, the number of necrotic cells in the OGD+HDL group was significantly decreased [PI positive cells rate: (26.71±5.94)％ vs. (64.24±18.34)％], the cell activity was significantly increased [(63.84±6.95)％ vs. (26.71±5.13)％], the contents of LDH and CK-MB in supernatant were significantly decreased [LDH (U/L): 896.3±161.5 vs. 1568.3±243.5, CK-MB (U/L): 304.3±72.9 vs. 583.6±81.6], the expressions of p-Akt and SR-B1 were significantly increased (p-Akt/t-Akt: 0.84±0.13 vs. 0.18±0.06, SR-B1/β-actin: 1.23±0.19 vs. 0.09±0.02), with statistically significant differences (allP < 0.05). Compared with OGD+HDL group, necrotic cells in LY294002 group were increased, cell activity was decreased, LDH and CK-MB contents in supernatant were increased, p-Akt and SR-B1 expressions were decreased; there was no statistical difference between LY294002 group and OGD group. There was no significant difference in cell apoptosis among the 4 groups.Conclusions HDL has protective effect on the mice myocardial cells. The mechanism may be related with the up regulation of the expression of SR-B1 protein by the activation of PI3K/Akt pathway.