更欣汤对围绝经期失眠模型大鼠下丘脑生物钟基因表达的影响

Effect of Geng Xin Decoction(更欣汤)on Hypothalamic Biological Clock Gene Expression in Perimenopausal Insomnia Model Rats

目的探讨更欣汤治疗围绝经期失眠的可能作用机制。方法60只SD雌性大鼠筛选出动情周期规律的大鼠52只,随机分为空白组13只和造模组39只。造模组大鼠采用雷公藤多苷片灌胃联合对氯苯丙氨酸(PCPA)混悬液腹腔注射建立围绝经期失眠大鼠模型。将26只造模成功的大鼠随机分为模型组和更欣汤组各13只。更欣汤组每日予更欣汤颗粒剂1.8 g/(kg·d)灌胃,空白组和模型组大鼠每日予10 ml/(kg·d)生理盐水灌胃,连续14天。观察大鼠一般行为学变化,采用戊巴比妥钠协同实验检测大鼠睡眠情况(睡眠潜伏期、睡眠持续期),采用Western blot法检测大鼠下丘脑生物钟基因CLOCK、芳基烃受体核转运体样蛋白(BMAL1)、Per1、Per2蛋白表达,RT-qPCR法检测大鼠下丘脑CLOCK、BMAL1、Per1、Per2 mRNA表达。结果戊巴比妥钠协同实验结果显示,模型组和更欣汤组大鼠睡眠持续期较空白组延长(P<0.05),各组大鼠睡眠潜伏期差异均无统计学意义(P>0.05)。与空白组比较,模型组大鼠精神状态差,前期白天及夜间均活跃、呈觉醒状态,摄食水减少,毛发竖起、色淡黄、色泽干枯、易脱落,大便呈黄绿色、质软或质稀不成形;下丘脑CLOCK、BMAL1蛋白表达水平升高,Per1、Per2蛋白表达水平降低(P<0.05或P<0.01)。与模型组比较,更欣汤组大鼠精神状态明显好转,白天活动较前减少,毛色稍黄、但有光泽,大便成形,下丘脑Per2蛋白表达水平升高(P<0.01),CLOCK、BMAL1及Per1蛋白表达水平均无统计学意义(P>0.05)。各组大鼠下丘脑CLOCK、BMAL1、Per1、Per2 mRNA相对表达量比较差异均无统计学意义(P>0.05)。结论更欣汤可改善围绝经期失眠模型大鼠睡眠状态,其作用机制可能与上调下丘脑生物钟基因Per2蛋白表达有关。

Objective To explore the possible mechanism of Geng Xin Decoction(更欣汤,GXD)in treating perimenopausal insomnia.Methods Fifty-two out of 60 SD female rats with regular estrous cycle were selected and randomly divided intoblank group(n=13)and modeling group(n=39).The rats in the modeling group were given tripterygium glycosides by gavage and intraperitoneal injection of p-chlorophenylalanine(PCPA)suspension to establish the perimenopausal insomnia rat model.Twenty-six successfully modeled rats were randomly divided into a model group and GXD group with 13 rats in each group.Rats in the GXD group were given 1.8 g/(kg·d)of Geng Xin Decoction granules by gavage every day,while those in the blank group and model group were given 10 ml/(kg·d)of normal saline by gavage every day,all for 14 days.The general behavioral changes of rats were observed.Pentobarbital sodium synergistic experiment was applied to detect the sleep status of the rats including sleep latency and sleep duration.Western blotting was used to detect the protein expression of biological clock gene CLOCK,BMAL1,Per1,and Per2 in the hypothalamus,and RT-qPCR method was used to detect the mRNA expression of CLOCK,BMAL1,Per1,and Per2.Results The results of pentobarbital sodium synergistic experiment showed that the sleep duration was prolonged in the model group and GXD group than in the blank group(P<0.05),while no significant difference was found among the three groups in the sleep latency(P>0.05).Compared to those in the blank group,the rats in the model group were in poor mental state and were active both in the daytime and at night in the early stage,showing an arousal state with decreased food and water intake,hair erection with light yellow,dry,and easy to fall off hair;the stool was yellow-green,soft or thin;the protein expression level of CLOCK and BMAL1 in hypothalamus increased,while the protein expression level of Per1 and Per2 decreased(P<0.05 or P<0.01).Compared to those in the model group,the mental state of the rats in GXD group was significantly improved,and the daytime activities of the rats decreased,with slightly yellow but shiny hair,and formed stool;the protein expression level of Per2 increased,while that of CLOCK,BMAL1 and Per1 protein was not statistically significant(P>0.05).There was no significant difference in the relative mRNA expression of CLOCK,BMAL1,Per1 and Per2 in the hypothalamus among the groups(P>0.05).Conclusion The mechanism of GXD in improving the sleep state of perimenopausal insomnia rats may be related to the up-regulation of protein expression of the biological clock gene Per2 in the hypothalamus.