参芪复方调控GK大鼠生物钟相关基因Id2、Usp2的实验研究

Experimental Study on Regulation of Circadian Clock-related Genes Id2 and Usp2 of GK Rats by Shenqi Compound

目的采用基因芯片技术探索参芪复方减少2型糖尿病血糖波动的内在机制。方法30只自发性2型糖尿病(GK)大鼠适应性喂养4周后,随机分为模型组、参芪复方组(参芪组)和西格列汀组(西药组),每组10只,另设10只Wistar大鼠为正常组。参芪复方组给予参芪复方浸膏灌胃,西药组给予西格列汀混悬液灌胃,空白组、模型组给予生理盐水灌服,连续给药8周。药物干预期间观察大鼠一般状态,并根据体重变化及时调整灌胃量,每周1次测1日内8:00、10:00、14:00、16:00、18:005个时间点的血糖情况,计算每日血糖平均水平(MBG)、每日平均血糖的标准差(SDBG)、最大血糖波动幅度(LAGE)。实验第8周处死大鼠,采集血液标本,采用放免法和ELISA法测定血清胰岛素(INS)、胰高血糖素(GC)水平。应用基因芯片技术检测DNA结合抑制剂2(Id2)及泛素特异性加工酶2(Usp2)水平,同时采用实时荧光PCR检测Id2、Usp2 mRNA表达水平。结果造模期间,各组GK大鼠随着周龄增长,出现精神萎靡,反应迟缓,动作缓慢的现象,参芪组能改善GK大鼠多食、多饮现象。与正常组比较,模型组INS水平、Usp2 mRNA表达水平降低,GC、MBG、SDBG、LABG均明显升高(P<0.05,P<0.01);与模型组比较,西药组INS水平升高,GC水平降低(P<0.01);参芪组、西药组Usp2 mRNA表达升高,MBG、SDBG、LABG及Id2 mRNA表达均明显降低(P<0.05,P<0.01)。同时参芪组GC、SDBG、LABG水平高于西药组(P<0.01)。结论参芪复方可能通过调节生物钟相关基因Id2、Usp2减少血糖波动。

Objective To explore the internal mechanism of Shenqi Compound to reduce blood glucose fluctuation of type 2 diabetes by gene chip technology.Methods Thirty spontaneous type 2 diabetes mellitus(GK)rats were randomly divided into model group,Shenqi Compound group(Shenqi group)and sitagliptin group with adaptive feeding for 4 weeks,10 rats in each group.And another 10 Wistar rats were recruited as normal group.The Shenqi group was given the Shenqi compound extract,the sitagliptin group was given the sitagliptin suspension,and the blank group and the model group were given normal saline.All drugs were administered by gavage,and the course was 8 weeks.During the drug intervention,the general state of the rats was observed,and the amount of gavage was adjusted in time according to the weight change.Once a week,blood glucose was measured at 8:00,10:00,14:00,16:00,and 18:00 in a day.At the same time,the average daily blood glucose level(MBG),the standard deviation of the average daily blood glucose(SDBG),and the maximum blood glucose fluctuation range(LAGE)were calculated.In the 8 th week of the experiment,the rats were sacrificed and blood samples were collected.The levels of serum insulin(INS)and glucagon(GC)were measured by radioimmunoassay and ELISA.The gene chip technology was used to detect DNA binding inhibitor 2(Id2)and ubiquitin-specific processing enzyme 2(Usp2),and real-time fluorescent PCR was used to detect the expression levels of Id2 and Usp2 mRNA.Results During the modeling period,it could be seen that as the age of each group of GK rats increases,the rats showed signs of lethargy,slow response,and slow movement.However,the Shenqi group could improve the phenomenon of polyphagia and polydipsia of GK rats.Compared with the normal group,the expression levels of INS and Usp2 mRNA in the model group decreased,and the GC,MBG,SDBG,and LABG all increased significantly(P<0.05,P<0.01).Compared with the model group,the level of INS in the sitagliptin group increased,while the level of GC decreased(P<0.01).The expression of Usp2 mRNA in the Shenqi group and the sitagliptin group increased,and the expression of MBG,SDBG,LABG and Id2 mRNA were significantly decreased(P<0.05,P<0.01).At the same time,the levels of GC,SDBG and LABG in the Shenqi group were higher than those in the sitagliptin group(P<0.01).Conclusion Shenqi Compound may reduce blood glucose fluctuations by regulating the circadian clock-related genes Id2 and Usp2.