脑信号蛋白3A及其受体神经菌毛素-1在慢性肝性脑病大鼠模型中的表达及意义

Expression and significance of semaphorin 3A and its receptor NRP-1 in a rat model of chronic hepatic encephalopathy

目的探究肝性脑病(HE)大鼠大脑前额叶皮层、海马CA1、CA3、DG区中脑信号蛋白3A(Sema3A)及受体神经菌毛素(NRP-1)的表达变化及可能的意义。方法SD大鼠40只随机分为4组:对照组、HE 1 d、15 d、30 d组,每组各10只。胆管结扎并辅以乙酸铵灌胃的方法制备慢性HE(C型)大鼠模型,进行行为学、神经病学改变的检测;ELISA检测大鼠血清中血氨及其他生化指标;HE染色观察大鼠肝脏和脑组织形态学变化;Real-time PCR检测各脑区Sema3A、NRP-1 mRNA的表达;免疫组化、Western Blot分别检测大脑皮层、海马CA1、CA3、DG区中Sema3A及NRP-1蛋白的表达变化。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果4组间神经反射等级、血氨、HE等级均有统计学差异(F值分别为76.39、417.07、71.56,P值均<0.001),与对照组比较,HE 1 d、15 d、30 d组神经反射得分、血氨、HE等级均明显升高(P值均<0.05)。4组间TBA、Alb、TBil、AST、ALT、ALP比较差异均有统计学意义(F值分别为1022.61、171.56、685.18、421.01、675.20、2405.04,P值均<0.001),与对照组相比,HE 1 d、15 d、30 d组Alb水平明显降低(P<0.05),其余指标明显升高(P值均<0.05)。病理学结果显示,与对照组相比,造模后大鼠肝脏出现不同程度的变性坏死,炎细胞浸润,大脑前额叶皮层及海马CA1、CA3、DG区体等部位观察到神经细胞不同程度的肿胀、破裂、排列紊乱等病理征象。4组间Sema3A、NRP-1 mRNA比较差异均有统计学意义(Sema3A mRNA:F值分别为273.83、158.84、103.59、128.90,P值均<0.001;mRNA:F值分别为88.78、173.02、156.44、289.09,P值均<0.001);与对照组比较,HE 1 d、15 d、30 d组大脑前额叶皮层、海马CA1、CA3、DG区中Sema3A、NRP-1 mRNA相对表达量均较高(P值均<0.05)。建模后Sema3A、NRP-1蛋白表达水平上调,免疫组化显示,HE组大鼠前额叶皮层和海马CA1、CA3、DG区Sema3A及NRP-1呈阳性染色。Western Blot显示,4组间大脑前额叶皮层及海马CA1、CA3、DG区Sema3A、NRP-1蛋白表达差异均有统计学意义(Sema3A:F值分别为835.14、774.16、282.60、856.29,P值均<0.001;NRP-1:F值分别为876.59、472.48、402.36、207.47,P值均<0.001);与对照组相比较,HE 1 d、15 d、30 d组大脑前额叶皮层及海马CA1、CA3、DG区内Sema3A、NRP-1蛋白表达均明显升高(P值均<0.05)。结论Sema3/NRP-1在慢性HE大鼠脑组织内表达上调,参与HE神经损伤的病理生理过程。

Objective To investigate the changes in the expression of semaphorin 3A(Sema3A)and receptor neuropilins-1(NRP-1)in the prefrontal cortex and the hippocampal CA1,CA3,and DG regions in rats with hepatic encephalopathy(HE)and their possible significance.Methods A total of 40 Sprague-Dawley rats were randomly divided into control group and 1-,15-,and 30-day HE groups,with 10 rats in each group.Bile duct ligation and ammonium acetate by gavage were used to establish a rat model of chronic HE(type C).The behavioral and neurological changes were observed;ELISA was used to measure blood ammonia and related biochemical parameters;HE staining was used to observe morphological changes of the liver and brain tissue;real-time PCR was used to measure the mRNA expression of Sema3A and NRP-1 in each brain region;immunohistochemistry and Western blot were used to measure the protein expression of Sema3A and NRP-1 in the cerebral cortex and the hippocampal CA1,CA3,and DG regions.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results There were significant differences between the four groups in nerve reflex level,blood ammonia,and HE grade(F=76.39,417.07,and 71.56,all P<0.001),and compared with the control group,the 1-,15-,and 30-day HE groups had significant increases in nerve reflex score,blood ammonia,and HE grade(all P<0.05).There were significant differences between the four groups in total bile acid,albumin,total bilirubin,aspartate aminotransferase,alanine aminotransferase,and alkaline phosphatase(F=1022.61,171.56,685.18,421.01,675.98,and 2405.04,all P<0.001),and compared with the control group,the 1-,15-,and 30-day HE groups had a significant reduction in albumin(P<0.05)and significant increases in the other indices(all P<0.05).The pathological results showed that compared with the control group,the HE groups had varying degrees of degeneration,necrosis,and inflammatory cell infiltration in the liver,with the pathological signs of swelling,tortuous,and ruptured nerve axons in the prefrontal cortex and the hippocampal CA1,CA3,and DG regions.There were significant differences between the four groups in the mRNA expression of Sema3A and NRP-1(Sema3A:F=273.83,158.84,103.59,and 128.90,all P<0.001;NRP-1:F=88.78,173.02,156.44,and 289.09,all P<0.001),and compared with the control group,the 1-,15-,and 30-day HE groups had significantly higher relative mRNA expression of Sema3A and NRP-1 in the prefrontal cortex and the hippocampal CA1,CA3,and DG regions(all P<0.05).There were increases in the protein expression of Sema3A and NRP-1 after modeling,and immunohistochemistry showed that the HE groups had positive staining of Sema3A and NRP-1 in the prefrontal cortex and the hippocampal CA1,CA3,and DG regions.Western blot showed that there were significant differences between the four groups in the protein expression of Sema3A and NRP-1 in the prefrontal cortex and the hippocampal CA1,CA3,and DG regions(Sema3A:F=835.14,774.16,282.60,and 856.29,all P<0.001;NRP-1:F=876.59,472.48,402.36,and 207.47,all P<0.001),and compared with the control group,the 1-,15-,and 30-day HE groups had significant increases in the protein expression of Sema3A and NRP-1 in the prefrontal cortex and the hippocampal CA1,CA3,and DG regions(all P<0.05).Conclusion Sema3A/NRP-1 is upregulated in the brain tissue of chronic HE rats and is involved in the pathophysiological process of nerve injury in HE.