谷氨酰胺对脓毒症大鼠心肌细胞Bcl-2/Bax表达的影响

目的 研究脓毒症大鼠心肌细胞凋亡与Bcl-2/Bax基因及蛋白表达的关系,探讨谷氨酰胺(Gin)对脓毒症心肌细胞凋亡的保护作用.方法采用内毒素(LPS 4 ms/ks)腹腔注射法制作脓毒症大鼠模型,实验地点在中山大学北校区动物实验中心.健康Sprague-Dawley(sD)大鼠72只随机分为对照组、LPS组及LPS+Gln(0.3g/kg)组,再分为0 h,6 h,12 h,24 h亚组(腹腔注射后每组成功存活至观察时间点的大鼠累积达到6只,n=6),检测各时点心肌细胞凋亡率和Bcl-2及Bax蛋白的含量,同时检测心肌Bcl-2及Bax mRNA表达.对结果进行方差分析和相关性统计分析.结果 LPS组大鼠心肌细胞凋亡率6 h、12 h及24 h均明显高于埘照组(F=186.786,P<0.01);心肌细胞Bax蛋白表达术后6 h降低(F=9.027,P<0.01),之后高于对照组;而Bcl-2蛋白表达6 h,12 h及24 h均低于对照组(F=301.142,P<0.01);Bax/Bcl-2蛋白表达比明显高于对照组(F=527.373,P<0.01);BaxmRNA和Bcl-2 6 h,12 h及24 h较对照组均明显上调(F=126.157,80.745,P<0.01).LPS+Gln组与LPS组比较,6 h,12 h及24 h心肌细胞凋亡率明显低于LPS组(F=75.187,P<0.01);Bax蛋白表达下降(F=20.981,P<0.01),而Bcl-2蛋白却升高(F 164.969,P<0.000);Bax/Bcl-2蛋白表达比降低(F=141.426,P<0.01);Bax mRNA和Bcl-2 6 h,12 h及24 h组较LPS组均明显下调(F=103.463,89.373,P<0.01).结论 Bcl-2抗凋亡基因及Bax促凋亡基因及其蛋白的表达参与脓毒症心肌细胞凋亡;应用Gln能影响凋亡相关基因及蛋白表达,减轻脓毒症心肌细胞的凋亡,改善脓毒症预后.

Effects of glutamine on expressions of the myocardial Bcl-2/Bax in rats with sepsis

Objective To investigate the relationship between apoptosis of myocardial cell in the rats with sepsis and the expressions of Bcl-2 and Bax protein,and the protective effect of Glutamine (Gln) on apoptosis of myocardial cell. Method The sepsis models were produced by injecting lipopolysaccharide (LPS) (4 mg/kg) via peritoneal. A total of 72 SD rats were divided into three groups, including the control group, LPS group and LPS + Gln(0.3 g/kg) group. Every group was further divided into four subgroups according to the period from injec-tion to death, 0 h,6 h, 12 h and 24 h (n = 6). Apoptosis of myocardial cells was detected using TUNEL staining, Bcl-2 and Bax protein expressions were measured with immunohistologieal staining and Bcl-2 and Bax mRNA ex-pressions were determined with RT-PCR. Results Apoptosis index of myocardial cells in LPS group was signifi-cantlyhigher than that in the control group (F = 186.786, P < 0.01) at all timepoint. Compared with the control group,the amount of Bax protein in the LPS group was lower at 6 h after administration (F = 9.027, P < 0.01), and higher at 12 h and 24 h after administration; however, the level of Bcl-2 protein expression was significantly lower at all timepoint (F = 301. 142, P < 0.01); and the ratio of Bax/Bcl-2 in the LPS group was obviously higher at all timepoint (F = 527.373, P < 0.01). The expression level of Bax and Bcl-2 mRNA in LPS group gradually elevated and was higher than that in the control group from 6 h (F = 126.157, 80.745, P < 0.01). Compared with the LPS group, at all timepoint, the apoptosis index of apoptotic myocardial cells in the LPS + Gln group was significantly lower (F = 75.187, P < 0.01) ; amount of Bax protein was lower but amout of Bcl-2 pro-tein was higher (F = 20.981, 164.969, P < 0.01) ; the ratio of Bax/Bcl-2 protein expression was lower (F = 141.426, P < 0.01); and the Bax and Bcl-2 mRNA expression levels were lower (F = 103.463,89.373, P < 0.01). Conclusions Bcl-2, anti-apoptosis gene, and Bax, pro-apoptosis gene, play an important role in apopto-sis of myocardial cell in the rats with sepsis. Gin reduces the apoptosis of myocardial cell and improves prognosis of the rat with sepsis via regulation of apoptosi-related gene and protein expressions.