摘要
目的:筛选由酸枣仁提取物调控的靶向5-羟色胺1A受体(5-HT1AR)影响骨骼增长的微小核糖核酸(miRNA),并阐明酸枣仁提取物通过该miRNA调控5-HT1AR影响骨骼增长的作用机制。方法:将大小鼠分别分为正常对照组、用药组、阳性对照组和五羟色胺1A受体选择性抑制剂组,并用不同溶液灌胃。一段时间后观察药物对小鼠体长、血清生长激素和脑组织五羟色胺1A受体表达以及对大鼠慢波睡眠的影响。筛选骨骼增长的小鼠与普通小鼠脑组织差异表达miRNAs,优选以五羟色胺1A受体为靶基因的miRNA并进行qRT-PCR验证。通过双荧光素酶报告基因实验验证miR-200c-3p与5-HT1AR之间的调控关系,进而培养小鼠脑皮层细胞并鉴定。通过给予高剂量和低剂量药物和过表达及沉默miR-200c-3p,ELISA法观察5-HT1AR的表达,并通过qRT-PCR法观察给药后细胞内miR-200c-3p的表达。结果:用药组小鼠血清生长激素、体长及脑组织五羟色胺1A受体表达均高于其他组;慢波睡眠时长用药组与阳性对照组长于正常对照组和抑制剂组;药物对异相睡眠时期无影响;两个实验组有13个miRNAs表达上调,3个miRNAs表达下调;经qRT-PCR验证调控五羟色胺1A受体的为下调的miR-200c-3p,且双荧光素酶报告基因实验证明miR-200c-3p可以调控5-HT1AR表达。体外培养小鼠脑皮层细胞,过表达miR-200c-3p引起5-HT1AR表达下降,而沉默miR-200c-3p后则5-HT1AR表达升高,且药物对体外培养的脑皮层细胞中miR-200c-3p的表达有调控作用。结论:小鼠体内可能存在酸枣仁提取物,通过调控miR-200c-3p的表达上调五羟色胺1A受体的表达,延长慢波睡眠,进而促进生长激素的分泌,最终促进体长增长。
Objective:To screen for micro ribonucleic acids(miRNAs)targeting the serotonin 1A receptor(5-HT1AR)that affect bone growth regulated by jujube kernel extract,and elucidating the mechanism by which jujube kernel extract regulates the effect of 5-HT1AR on bone growth through this miRNA.Methods:Rats and mice were divided into normal control group,drug treatment group,positive control group and selective inhibitor group of serotonin 1A receptor,and gavage with corresponding solution in each group.After a period of time,the effects of the drug on the body length of mice,the expression of serum growth hormone,serotonin-1A receptor in brain tissue and the slow-wave sleep of rats were observed.The differential expression of miRNAs in the brain tissues of mice with bone growth and ordinary mice was screened,and the miRNA with serotonin-1A receptor as the target gene was selected and verified by qRT-PCR.The regulatory relationship between miR-200c-3p and 5-HT1AR was verified by dual-luciferase reporter assay,and then the mouse cerebral cortex cells were cultured and identified.The expression of 5-HT1AR was observed by ELISA by giving high-dose and low-dose drugs,overexpressing and silencing miR-200c-3p.The expression of miR-200c-3p was observed by qRT-PCR.Results:The expression of serum growth hormone,body length and serotonin-1A receptor in brain tissue of the treated group were higher than those of the other groups.The duration of slow-wave sleep in medication group and positive control group was longer than that in normal control group and inhibitor group.Drugs had no effect on the phase of sleep.The expression of 13 miRNAs was up-regulated and 3 miRNAs was down-regulated in the two groups.The regulation of serotonin 1A receptor was verified by qRT-PCR as down-regulated miR-7b-3p.The dual-luciferase reporter assay showed that miR-200c-3p can regulate the expression of 5-HT1AR.The overexpression of miR-200c-3p in cultured mouse cortical cells caused a decrease in 5-HT1AR expression,while silencing miR-200c-3p led to an increase in 5-HT1AR expression.Moreover,drugs can regulate the expression of miR-200c-3p in cultured cortical cells in vitro.Conclusion:It is possible that the jujube kernel extract may upregulate the expression of 5-HT1A receptor by regulating miR-200c-3p in mice,prolong slow-wave sleep and promote growth hormone secretion,ultimately promoting body growth.
作者
梁国辉
王桂芝
谢艳
毛雯正
罗石任
张丽
吴晓龙
LIANG Guohui;WANG Guizhi;XIE Yan;MAO Wenzheng;LUO Shiren;ZHANG Li;WU Xiaolong(Luoyang Orthopedic-Traumatological Hospital of Henan Province(Henan Provincial Orthopedic Hospital),Luoyang 471002,Henan China)
出处
《中国中医骨伤科杂志》
2023年第12期7-14,共8页
Chinese Journal of Traditional Medical Traumatology & Orthopedics
基金
河南省中医药科学研究专项(2018ZY2114,2022ZY2051)。
