分泌型酸性富含半胱氨酸样蛋白1型和整合素β1在致痫大鼠中的表达变化及相关性分析

Expression change and correlation analysis of SPARCL1 and Integrinβ1 in epileptic rats

目的研究癫痫大鼠海马和邻近颞叶皮层中分泌型酸性富含半胱氨酸样蛋白1型(secreted protein acidic and rich in cysteine1,SPARCL1)和整合素β1(Integrinβ1)的动态表达,以探讨二者对癫痫发展的影响。方法将雄性SD大鼠56只随机分为对照组(21只)和致痫组(35只)。致痫组予氯化锂(LiCl)匹罗卡品(PILO)进行造模,按癫痫发作后3 h、6 h、12 h、1 d、2 d、3 d、7 d时间点分为7个亚组,每亚组5只;对照组按对应时间分为7个亚组,每亚组3只。采用实时荧光定量PCR(qPCR)检测大鼠SPARCL1mRNA和Integrinβ1 mRNA表达水平,蛋白免疫印迹杂交实验(Western blot)检测大鼠SPARCL1和Integrinβ1的蛋白表达水平。比较致痫组和对照组大鼠海马组织和颞叶皮层组织中SPARCL1和Integrinβ1的mRNA和蛋白表达的变化,并分析大鼠癫痫发作后不同时间不同组织中SPARCL1和Integrinβ1的mRNA和蛋白表达水平的相关性。结果对照组7个时间亚组的海马组织和颞叶皮层样本中SPARCL1和Integrinβ1的mRNA和蛋白表达水平差异均无统计学意义(均P<0.05)。与对照组比较,致痫组3 h组、6 h组、12 h组和1 d组海马组织中SPARCL1 mRNA表达水平均降低(P<0.05),致痫组6 h组、12 h组、1 d组和2 d组海马组织中SPARCL1蛋白水平均降低(均P<0.05);致痫组海马组织3 h组和6 h组的Integrinβ1 mRNA表达水平明显下降(P<0.01),致痫组1 d组和2 d组Integrinβ1 mRNA表达水平显著增加(P<0.01);致痫组6 h、12 h组的Integrinβ1蛋白表达水平下降(P<0.05),3 d、7 d组Integrinβ1蛋白表达水平升高(均P<0.05)。与对照组比较,致痫组3 h组、6 h组和7 d组大鼠颞叶皮层中SPARCL1 mRNA表达水平显著升高(P<0.01),致痫组12 h组和1 d组则明显下降(P<0.01),致痫组12 h组、1 d组、2 d组、3 d组和7 d组SPARCL1蛋白表达水平显著降低(均P<0.05);致痫组3 h组、6 h组和7 d组大鼠颞叶皮层Integrinβ1的mRNA显著增加(P<0.01),除致痫组3 h组外,致痫组6 h组、12 h组、1 d组、2 d组、3 d组、7 d组大鼠颞叶皮层Integrinβ1蛋白表达均下调(均P<0.05)。总体上,与对照组相比,癫痫发作后1周内,致痫组海马组织中SPARCL1和Integrinβ1的mRNA和蛋白表达均呈先降低后升高趋势;而颞叶皮层SPARCL1和Integrinβ1的mRNA表达呈先升后降趋势,蛋白的表达则呈下降趋势。致痫组大鼠海马组织中SPARCL1和Integrinβ1两者的mRNA表达水平(r=0.684,P<0.01)、蛋白表达水平(r=0.724,P<0.01)分别成正相关,颞叶皮层组织中SPARCL1和Integrinβ1两者的mRNA表达水平(r=0.908,P<0.01)和蛋白表达水平(r=0.852,P<0.01)亦分别成正相关。结论与正常大鼠比较,癫痫大鼠癫痫发作后1周内海马组织中SPARCL1和Integrinβ1的mRNA和蛋白表达均呈先降低后升高趋势;颞叶皮层SPARCL1和Integrinβ1的mRNA表达呈先升后降趋势,蛋白的表达则呈下降趋势。大鼠癫痫的发生发展过程中,海马组织和颞叶皮层组织中SPARCL1和Integrinβ1的mRNA和蛋白表达水平存在相关性。

Objective To study the dynamic expression of secreted protein acidic and rich in cysteine-like protein type 1(SPARCL1)and integrinβ1 in the hippocampus and adjacent temporal cortex of epileptic rats for investigating their effects on the development of epilepsy.Methods Fifty-six male SD rats were randomly divided into a control group(21 rats)and an epileptogenic group(35 rats).The epileptogenic group was modeled with lithium chloride(LiCl)and pilocarpine(PILO)and the epileptogenic group was divided into 7 subgroups according to the observation time(3 h,6 h,12 h,1 d,2 d,3 d and 7d)after seizure,with 5 animals in each subgroup.The control group was divided into 7 subgroups according to the corresponding time with 3 rats in each subgroup.Real-time fluorescence quantitative PCR(qPCR)was used to detect the mRNA expression levels of both SPARCL1 and integrinβ1 transcripts and protein immunoblot hybridization assay(western blot)was used to detect the protein expression levels of SPARCL1 and integrinβ1 translations in the hippocampus and adjacent temporal cortex of the rats.The correlation between the mRNA and protein expression of SPARCL1 and integrinβ1 in different tissue at different time points after seizures in the rats was analyzed.Results The differences in mRNA and protein expression levels of SPARCL1 and integrinβ1 in hippocampal tissue and temporal cortex samples from the seven temporal subgroups of the control group were not statistically significant(all P<0.05).Compared with control group,SPARCL1 mRNA expression levels were reduced in hippocampal tissue in the 3 h,6 h,12 h and 1 d epileptogenic groups(P<0.05),and SPARCL1 protein levels were reduced in hippocampal tissue in the 6 h,12 h,1 d and 2 d epileptogenic groups(all P<0.05).Compared with the control group,SPARCL1 mRNA expression levels were significantly higher in the temporal cortex of the rats in the 3 h,6 h and 7 d epileptogenic groups(P<0.01),significantly lower in the 12 h and 1 d epileptogenic groups(P<0.01).SPARCL1 protein expression levels were significantly lower in the 12 h,1 d,2 d,3 d and 7 d epileptogenic groups(all P<0.05)compared with control group.Compared with the control group,the expression levels of integrinβ1 mRNA in hippocampal tissue of the 3 h and 6 h epileptogenic groups were significantly decreased(P<0.01)and the 1 d and 2 d groups epileptogenic groups significantly increased(P<0.01).The expression levels of SPARCL1 protein decreased in the 6 h and 12 h epileptogenic groups(P<0.05)and increased in the 3 d and 7 d epileptogenic groups(both P<0.05).Compared with the control group,the mRNA of integrinβ1 in the temporal cortex of rats in the 3 h,6 h and 7 d epileptogenic groups groups were significantly increased(P<0.01).Except for the 3 h group,the protein expression of integrinβ1 in the temporal cortex of the rats in the 6 h,12 h,1 d,2 d,3 d and 7 d groups were down-regulated(P<0.05 for all).The expression levels of SPARCL1 mRNA and integrinβ1 mRNA were positively correlated in hippocampal tissues of rats in the epileptogenic group(r=0.684,P<0.01),and the expression levels of SPARCL1 and integrinβ1 protein were also positively correlated(r=0.724,P<0.01).SPARCL1 mRNA and integrinβ1 mRNA were positively correlated in temporal cortex tissue(r=0.908,P<0.01),and expression levels of both were positively correlated(r=0.852,P<0.01).Conclusions Compared with normal rats,the mRNA and protein expressions of SPARCL1 and integrinβ1 decreased at first and then increased within 1 week after epileptic seizure in the hippocampus of epileptic rats.At the same time,the mRNA expressions of SPARCL1 and integrinβ1 in the temporal cortex of epileptic rats increased first and then decreased,but the protein expression showed a downward trend.During the development of epilepsy in rats,the mRNA and protein expression levels of SPARCL1 and integrinβ1 in the hippocampus and temporal cortex were correlated.