溶血磷脂酸对创伤失血性休克大鼠肝细胞凋亡的影响

Effect of lysophosphatidic acid on hepatocyte apoptosis in rats with traumatic hemorrhagic shock

目的 观察溶血磷脂酸(LPA)对创伤失血性休克(THS)模型大鼠肝细胞凋亡的影响.方法 选择69只健康雄性SD大鼠,按随机数字表法分为正常对照组(n=9)、THS模型组(n=30)、LPA预处理组(n=30).采用将大鼠双侧股骨中段折断,右侧股动脉插管放血,将血压维持在35-40mmHg(1mmHg≈0.133kPa)的方法复制大鼠THS模型.LPA预处理组制模前20min和制模后10min静脉注射LPA(1mg/kg);正常对照组和THS模型组给予等量生理盐水.于制模后1、6、12h处死大鼠取血,检测各组大鼠丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBil)水平;采用原位末端缺刻标记试验(TUNEL)检测肝脏组织细胞凋亡情况;采用蛋白质免疫印迹试验(WesternBlot)检测各组肝组织天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)的蛋白表达水平.结果 光镜下可见正常对照组肝细胞结构正常,排列整齐;THS模型组肝细胞肿胀、坏死;LPA预处理组肝细胞形态学变化较THS模型组明显减轻.THS模型组肝细胞凋亡较正常对照组明显增加,凋亡指数(AI)较正常对照组升高,caspase-3蛋白表达较正常对照组明显增加,ALT、AST、TBil水平较正常对照组明显升高,术后12h达到峰值〔AI:(93.20±3.32)%比(3.44±1.32)%,caspase-3/3-磷酸甘油醛脱氢酶(GAPDH):0.46±0.02比0.13±0.01,ALT(U/L):75.81±7.10比20.83±2.72,AST(U/L):69.14±8.41比22.28±3.12,TBil(μmol/L):139.71±15.90比82.25±8.38,均P<0.05〕;LPA预处理组细胞凋亡较THS模型组明显减少,AI较THS模型组明显降低,caspase-3蛋白表达水平较THS模型组明显减少,ALT、AST、TBil水平较THS模型组明显降低,制模后1h即出现统计学差异〔AI:(25.29±4.75)%比(45.69±10.70)%,caspase-3/GAPDH:0.17±0.01比0.26±0.02,ALT(U/L):28.36±9.60比49.21±9.49,AST(U/L):33.71±10.80比54.30±18.41,TBil(μmol/L):95.59±10.60比107.34±13.51,均P<0.05〕.结论 LPA可抑制THS大鼠肝细胞凋亡并改善肝功能.LPA有望成为治疗THS导致肝功能损害的潜在策略.

Objective To observe the eleet of lysophosphatidie acid(ILPA)on hepatoryte apoptosis induced by traumatic hemorrhagic shock(THS)in rat models.Methods The 69 male healthy Sprague-Dawley(SD)rats were selected and randomly divided into three groups:normal control(n=9).THS model(n=30)and LPA pretreatment(n=30)groups.The rat THS models were duplicated by breaking fractures al the middle parts of bilateral femur bones and an intravenus catheter was used 10 withdraw blood from right femoral artery to let the blood pressure maintain at 35-40 mmHg(1 mmHg≈0133 kPa).LPA(1 mg/kg)was intravenously injected 20 minutes before and 10 minutes after modeling in the LPA pretreatment group;the normal control and THS model groups were given equal amount of normal saline.The rats were killed and hlood samples were collected after 1.6 and 12 hours of model establishment.The alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total hilirubin(TBil)levels in each group were detected;the hepatocyte apoptosis was detected by terminal deoxynucleotidyI transferase-mediated dUTP-biotin nick end labeling assay(TUNEL),and the protein expression level of liver tissue aspartate specifie cysteine protease 3(caspase-3)was detected by Western Blot.Results Under light microscope,the structure of hepatocytes in normal control group was normal and arranged orderly;hepatocytes in THS model group were swollen and necrotic;the morphological changes of hepatocytes in LPA pretreatment group were significantly less than those in THS model group.In THS model group,the hepatoeytes apoptosis was obviously inereased,AI was elevated and the expression of caspase-3 protein was significantly higher than those in the normal control group;the levels of ALT,AST and TBil in THS model group were also significantly higher than those in the normal control group,reaching the peak at 12 hours after operation[Al:(93.20±3.32)%vs.(3.44±1.32)%,caspase-3/glyceraldehyde 3-phosphate dehydrogenase(GAPDH):0.46±0.02 vs.0.13±0.01,ALT(U/L):75.81±7.10 vs.20.83±2.72,AST(U/L):69.14±8.41 vs.22.28±3.12,TBil(μmol/L):139.71±15.90 vs.82.25±8.38.all P<0.05]:compared with those in the THS model group,the hepatoeyte apoptnais,AI and the expression of caspase-3 protein were significantly lower in LPA pretrealment group,and the levels of ALT,AST and TBil were also signifceanly lower in LPA pretreatment group,the difference was statistically signifcant 1 hourafter modeling[Al:(25.29±4.75)%vs.(45.69±10.70)%,caspase-3/GAPDH:0.17±0.01 vs.0.26±0.02,ALT(U/L):28.36±9.60 vs.49.21±9.49,AST(U/L):33.71±10.80 vs.54.30±18.41.TBil(μmol/L):95.59±10.60 vs.107.34±13.51,all P<0.05].Conclusions LPA can inhibit the hepatocyte apoptosis induced by THS and improve the liver function.LPA might be a potential trealment stralegy for liver function damage caused by THS in future.