血管紧张素Ⅱ-1型受体自身抗体生命早期暴露对子代大鼠脂代谢的影响

Effects of early-life exposure to angiotensinⅡ type 1 receptor autoantibody on lipid metabolism in offspring rats

目的探讨血管紧张素Ⅱ-1型受体自身抗体(angiotensin Ⅱ type 1 receptor autoantibody, AT_(1)-AA)生命早期(胎儿期和哺乳期)暴露对子代大鼠脂代谢的影响。方法将32只体重150~170 g、AT_(1)-AA阴性的健康雌性未孕无特定病原体级Sprague Dawley大鼠随机分为免疫组和对照组, 各16只。免疫组多次皮下注射人血管紧张素Ⅱ-1型受体细胞外第二环功能表位肽段与弗氏免疫佐剂的等体积混合物, 进行主动免疫建立AT_(1)-AA阳性雌性大鼠模型;对照组皮下注射与免疫组等量的生理盐水与弗氏免疫佐剂的等体积混合物。每次免疫前大鼠尾采集血液样本检测2组大鼠血清AT_(1)-AA水平, 免疫组血清AT_(1)-AA阳性且水平增长进入平台期即AT_(1)-AA阳性雌性大鼠模型建立成功。免疫8周后, 2组雌性大鼠分别与健康的AT_(1)-AA阴性雄性大鼠交配受孕。采集母代大鼠和子代大鼠孕18 d(G18)、生后21 d(P21)及子代大鼠断乳后正常喂养至12周龄(W12)的血液样本;整个实验期间不对子代大鼠进行主动免疫。采用酶联免疫吸附法检测大鼠血清AT_(1)-AA水平(吸光度A405), 免疫组与对照组大鼠A405值的比值≥2.1为AT_(1)-AA阳性;采用全自动生化分析仪测定母代大鼠和子代大鼠的血脂水平;并对子代大鼠及与其母代大鼠的血清AT_(1)-AA水平、总胆固醇(total cholesterol, TC)、高密度脂蛋白-胆固醇[间接反映高密度脂蛋白(high density lipoprotein, HDL)]、低密度脂蛋白-胆固醇和游离脂肪酸水平进行相关性分析。采用两独立样本t检验、线性回归分析、方差分析对数据进行统计学分析。结果 (1)免疫组母代大鼠在G18和P21的血清AT_(1)-AA水平分别为1.170±0.190和0.988±0.283, 均高于同期对照组母代大鼠(分别为0.114±0.016和0.084±0.006, t值分别为14.64和9.57, P值均<0.001)。(2)免疫组子代大鼠在G18、P21的血清AT_(1)-AA水平(子代大鼠G18:0.948±0.220、子代雄性大鼠P21:0.758±0.273、子代雌性大鼠P21:0.774±0.274), 均显著高于同期对照组子代大鼠(子代大鼠G18:0.105±0.010、子代雄性大鼠P21:0.080±0.002、子代雌性大鼠P21:0.084±0.005, t值分别为10.10、7.46、7.55, P值均<0.001), 且均与其同期母代大鼠血清AT_(1)-AA水平呈正相关(子代大鼠G18:R=0.78, P=0.038;子代雄性大鼠P21:R=0.82, P=0.006;子代雌性大鼠P21:R=0.82, P=0.007);而在W12的血清AT_(1)-AA水平与同期对照组子代大鼠比较, 差异均无统计学意义(P值均>0.05)。(3)免疫组母代大鼠在G18的血清TC水平[(2.36±0.32)mmol/L]显著高于同期对照组母代大鼠[(1.95±0.24)mmol/L, t=2.70, P=0.019], 在P21的血清TC、HDL水平[分别为(2.82±0.50)和(1.94±0.33)mmol/L]均显著高于同期对照组母代大鼠[分别为(2.18±0.26)和(1.57±0.23)mmol/L, t值分别为3.41和2.80, P值分别为0.004和0.013]。(4)与同期对照组子代大鼠比较, 免疫组子代大鼠在G18、W12脂代谢均无显著变化(P值均>0.05)。免疫组子代雄性和雌性大鼠在P21的血清TC、HDL水平[子代雄性大鼠TC:(2.38±0.52)mmol/L、HDL:(1.44±0.32)mmol/L, 子代雌性大鼠TC:(2.50±0.72)mmol/L、HDL:(1.41±0.33)mmol/L], 均显著高于同期对照组子代雄性和雌性大鼠[子代雄性大鼠TC:(1.83±0.30)mmol/L、HDL:(1.07±0.18)mmol/L, 子代雌性大鼠TC:(1.70±0.26)mmol/L、HDL:(1.00±0.14)mmol/L, t值分别为2.73、2.98、3.16和3.41, P值分别为0.015、0.009、0.006和0.004]。(5)免疫组子代雄性和雌性大鼠在P21的血清TC、HDL水平与其同期母代大鼠的血清TC、HDL水平之间均无相关性(R值均<0.5, P值均>0.05);免疫组子代雄性和雌性大鼠在P21的血清HDL水平与其自身血清TC水平均呈正相关(子代雄性大鼠R=0.98, 子代雌性大鼠R=0.97, P值均<0.001), 且与其自身血清AT_(1)-AA水平均呈正相关(子代雄性大鼠R=0.74, P=0.023;子代雌性大鼠R=0.91, P=0.001);免疫组子代雄性和雌性大鼠在P21的血清TC水平与其自身血清AT_(1)-AA水平均呈正相关(子代雄性大鼠R=0.72, P=0.030;子代雌性大鼠R=0.90, P=0.001)。结论 AT_(1)-AA的生命早期暴露可能导致子代大鼠TC和HDL的异常表达。

Objective:To investigate the effects of early-life(intrauterine and breastfeeding period)exposure to angiotensinⅡtype 1 receptor autoantibody(AT 1-AA)on lipid metabolism in offspring rats.Methods:Thirty-two AT 1-AA negative healthy nonpregnant specific pathogen free female Sprague Dawley rats weighing 150-170 g were randomly divided into two groups.Those in the immune group(n=16)were subcutaneously injected with the mixture of an equal volume of Freund's adjuvant and the second extracellular loop of human-derived angiotensinⅡreceptor type 1(AT_(1)R-ECⅡ)repeatedly to establish the AT 1-AA-positive rat model by active immunization and those in the control group(n=16)with normal saline solution.Before each immunization,blood samples were collected from the tail of rats to detect serum AT 1-AA levels of those rats in both groups,and the AT 1-AA-positive rat model was successfully established when the serum AT 1-AA was positive and its level reached a plateau.After eight weeks of immunization,the female rats in the two groups were mated with healthy AT 1-AA-negative male rats to conceive.Serum samples were collected from the maternal and offspring rats at the gestation of 18 days(G18),postnatal 21 days(P21),and from the normally fed offspring rats from the time of weaning to 12 weeks old(W12).Active immunization was not performed on the offspring throughout the experiment.The serum AT 1-AA levels of maternal and offspring rats were determined by enzyme-linked immunosorbent assay,and serum AT_(1)-AA was positive when the ratio of AT_(1)-AA level of the immune group over the control group≥2.1.The blood lipid levels of maternal and offspring rats were measured by an automatic biochemical analyzer.Serum AT 1-AA levels,total cholesterol(TC),high-density lipoprotein-cholesterol[instead of high-density lipoprotein(HDL)],low-density lipoprotein-cholesterol,and free fatty acid levels of the offspring and maternal rats were determined for correlation analysis.Two independent sample t-test,linear regression analysis,and analysis of variance were adopted for statistical analysis.Results:(1)The serum levels of AT 1-AA in maternal rats at G18 and P21 in the immune group were significantly higher than those in the control group(G18:1.170±0.190 vs 0.114±0.016,t=14.64;P21:0.988±0.283 vs 0.084±0.006,t=9.57;both P<0.001).(2)The serum levels of AT 1-AA in the offspring at G18 and P21 in the immune group were significantly higher than those in the control group(offspring at G18:0.948±0.220 vs 0.105±0.010,t=10.10;male offspring at P21:0.758±0.273 vs 0.080±0.002,t=7.46;female offspring at P21:0.774±0.274 vs 0.084±0.005,t=7.55;all P<0.001),which showed a positive correlation with those in maternal rats at the same period(offspring at G18:R=0.78;male offspring at P21:R=0.82;female offspring at P21:R=0.82;all P<0.05).However,there was no significant difference in the serum AT 1-AA level in offspring at W12 between the immune and control group(P>0.05).(3)The serum levels of TC at G18 and P21,and HDL at P21 in maternal rats in the immune group were all higher than those in the control group[TC at G18:(2.36±0.32)vs(1.95±0.24)mmol/L,t=2.70;P21:(2.82±0.50)vs(2.18±0.26)mmol/L,t=3.41;HDL at P21:(1.94±0.33)vs(1.57±0.23)mmol/L,t=2.80;all P<0.05].(4)Compared with the offspring in the control group,there was no significant change in lipid metabolism at G18 and W12 in the offspring in the immune group(both P>0.05).The serum levels of TC and HDL in male and female offspring at P21 in the immune group were higher than their counterparts in the control[TC in male offspring:(2.38±0.52)vs(1.83±0.30)mmol/L,t=2.73;HDL in male offspring:(1.44±0.32)vs(1.07±0.18)mmol/L,t=2.98;TC in female offspring:(2.50±0.72)vs(1.70±0.26)mmol/L,t=3.16;HDL in female offspring:(1.41±0.33)vs(1.00±0.14)mmol/L,t=3.41;all P<0.05].(5)The serum levels of TC and HDL in male and female offspring at P21 in the immune group showed no correlation with those in maternal rats at P21(all R<0.5,all P>0.05).The serum levels of HDL in male and female offspring at P21 in the immune group had a positive correlation with their own serum TC levels(male offspring:R=0.98;female offspring:R=0.97;both P<0.001)and also with their own serum AT 1-AA levels(male offspring:R=0.74,P=0.023;female offspring:R=0.91,P=0.001).The serum levels of TC in male and female offspring at P21 in the immune group had a positive correlation with their serum AT 1-AA levels(male offspring:R=0.72,P=0.030;female offspring:R=0.90,P=0.001).Conclusion:The early-life exposure to AT 1-AA may cause abnormal expression of TC and HDL in offspring rats.