邻苯二甲酸二(2-乙基)己酯染毒对雄性糖尿病大鼠肝脏损伤及糖脂代谢的影响

Effects of di-2-ethylhexyl phthalate exposure on liver and glycolipid metabolism in male diabetic rats

【目的】通过邻苯二甲酸二(2-乙基)己酯(DEHP)染毒,观察氧化应激对雄性糖尿病大鼠肝脏损伤和营养物质代谢过程中的影响,并进一步探究DEHP的作用机制。【方法】32只5周龄无特定病原动物(SPF)级雄性Wistar大鼠,体重150~170 g,适应性喂养1周后建立2型糖尿病大鼠模型。造模方式为给予大鼠高糖高脂饲料喂养,喂养4周后腹腔注射链脲佐菌素(STZ)25 mg/kg,间隔2 d后再次腹腔注射;测得2次随机血糖>16.7 mmol/L为建模成功。将20只成模大鼠按体重随机分为4组:玉米油对照组和100、300、900 mg/kg DEHP染毒组,染毒方式为经口灌胃5 mL/kg,1次/d,连续染毒30 d。染毒期间给予糖尿病大鼠普通饲料喂养。分别于染毒第0、14、28天剪尾采血检测随机血糖,染毒第29天进行口服葡萄糖耐量试验,染毒第30天禁食16 h后次日测空腹血糖(FPG)。应用1.2%戊巴比妥钠麻醉后腹主动脉取血,处死大鼠后取肝脏称质量,计算肝脏系数并制作肝脏病理切片;采用全自动生化仪测定血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、三酰甘油(TG)、白蛋白(ALB);测定血清胰岛素以及氧化应激相关因子,包括还原型谷胱甘肽(GSH)、过氧化氢(H2O2)、丙二醛(MDA)、超氧化物歧化酶(SOD)等。【结果】不同剂量DEHP染毒对2型糖尿病大鼠体重和随机血糖值的影响差异均无统计学意义(均P>0.05)。葡萄糖耐量曲线显示,各剂量DEHP染毒组各时间点血糖值均高于玉米油对照组,且在葡萄糖溶液灌胃后15 min时,大鼠血糖值达到或超过上限值后出现“假平台期”,葡萄糖溶液灌胃后120 min时各组血糖水平均高于灌胃前。300、900 mg/kg DEHP染毒组糖尿病大鼠肝脏系数高于玉米油对照组(均P<0.01),且肝脏系数与DEHP染毒剂量呈正相关(r=0.80,P<0.0001)。DEHP染毒的糖尿病大鼠光学显微镜下可见肝细胞肿胀,细胞质淡染,细胞内空泡。900 mg/kg DEHP染毒组糖尿病大鼠血清ALP水平高于玉米油对照组(P<0.01),血清ALP水平与DEHP染毒水平呈正相关(r=0.75,P<0.01)。300、900 mg/kg DEHP染毒组糖尿病大鼠血清MDA水平均高于玉米油对照组(均P<0.01),血清MDA水平与DEHP染毒剂量呈正相关(r=0.84,P<0.0001)。900 mg/kg DEHP染毒组大鼠血清SOD水平高于玉米油对照组(P<0.01)。【结论】DEHP染毒可引起糖尿病大鼠肝脏损伤、糖脂代谢异常、氧化应激水平和抗氧化水平升高,但对胰岛素抵抗的影响不显著。

[Objective]Phthalates(PAEs)are common environmental endocrine disruptors.In this study,the effects of oxidative stress on liver and nutrient metabolism were determined in male diabetic rats exposed to di-2-ethylhexyl phthalate(DEHP),and the mechanism of DEHP toxicity was explored.[Methods]Thirty-two SPF male Wistar rats aged five weeks,weighing 150-170 g,were fed adaptively for one week to establish the model of type 2 diabetes.The model was established by intraperitoneal injection of streptozotocin(STZ)(25 mg/kg)after feeding with high sugar and high fat diet for four weeks.Second STZ injection was given two days later.The model was considered to be established successfully when the random blood glucose level was found to be higher than 16.7 mmol/L in two separate tests.Twenty diabetic rats were then randomly divided into four groups,including control group(corn oil),100,300 and 900 mg/kg DEHP groups.The rats were treated with DEHP by gavage(5 mL/kg)once a day for 30 days.They were fed with normal diet during the treatment period.Caudal venous blood was collected on the 1st,14th,and 28th days to measure the random blood glucose level.The changes of glucose tolerance were determined by oral glucose tolerance test on the 29th day.Fasting blood glucose(FPG)was measured on the next day of the last exposure.After the rats were anesthetized with pentobarbital and killed,the liver was weighed,the liver coefficient was calculated and the liver pathological section was made.Blood was taken from the abdominal aorta.The levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),triacylglycerol(TG)and albumin(ALB)in serum were measured by spectrophotometry,and the levels of insulin,glutathione(GSH),H2O2,malondialdehyde(MDA)and superoxide dismutase(SOD)in fasting serum were measured by radioimmunoassay.[Results]There was no significant difference in body weight and random blood glucose in the type 2 diabetic rats exposed to different concentrations of DEHP(all P>0.05).At each time point of the glucose tolerance curve,the blood glucose value of the exposure groups was higher than that of the control group.A"false plateau period"appeared after the blood glucose value reached or exceeded the upper limit at 15 minutes,and the blood glucose level in each group was higher than that of the control group at 120 minutes.The liver organ coefficient of 300 and 900 mg/kg DEHP groups was higher than that of the control group(both P<0.01),and the liver organ coefficient was positively correlated with the exposure concentration of DEHP(r=0.80,P<0.0001).Under the microscope,the liver cells in diabetic rats were swollen,the cytoplasm was light stained,and there were vacuoles in the cells.The serum ALP level in diabetic rats of 900 mg/kg DEHP group was significantly higher than that in the control group(P<0.01).The serum ALP level was positively correlated with the concentration of DEHP(r=0.75,P<0.01).The serum MDA level in diabetic rats of 300 mg/kg and 900 mg/kg DEHP groups was significantly higher than that of the control group(both P<0.01),and the serum MDA level was positively correlated with the concentration of DEHP(r=0.84,P<0.0001).The serum SOD level of 900 mg/kg DEHP group was significantly higher than that of control group(P<0.01).[Conclusion]DEHP exposure could lead to liver damage,abnormal glycolipid metabolism,and increase the level of oxidative stress and antioxidant level in male diabetic rats,but did not show a significant effect on insulin resistance.