摘要
背景:前期研究发现,酸枣仁提取物通过提高脑组织5-羟色胺1A受体(serotonin 1A receptor,5-HT1AR)表达,使之与5-羟色胺结合,延长小鼠慢波睡眠,促进生长激素的分泌,从而使骨生长。5-HT1AR作为一种蛋白质,其表达丰度受到miRNA的调控。作者推测酸枣仁提取物可能通过miRNA调控5-HT1AR的表达从而发挥药物作用。目的:观察酸枣仁提取物通过干预小鼠脑组织中miR-7b-3p/5-HT1AR通路对骨骼生长的影响。方法:①将昆明种小鼠分为正常对照组、用药组(灌胃酸枣仁提取物0.320 mg/g),阳性对照组(灌胃酸枣仁皂甙0.013 mg/g)和酸枣仁提取物+5-HT1AR抑制剂组(灌胃酸枣仁提取物最后3 d同时每天侧脑室注射5-HT1AR选择性抑制剂P-MPPF 8μg),25 d后观察酸枣仁提取物对小鼠骨生长、血清生长激素水平及脑组织5-HT1AR表达的影响;②基因芯片法筛选由酸枣仁提取物引起的骨生长小鼠与普通小鼠脑组织差异表达的miRNAs,并通过PCR验证和双荧光素酶报告基因实验证实筛选的miR-7b-3p与5-HT1AR的调控关系;③体外培养小鼠脑皮质细胞并鉴定,利用Western blot法观察酸枣仁提取物对脑皮质细胞中5-HT1AR表达的影响;④将昆明种小鼠分为正常对照组、用药组、miR-7b-3p inhibitor组、用药+miR-7b-3p mimics组、阳性对照组,观察各组小鼠脑组织5-HT1AR表达及5-羟色胺与5-HT1AR结合活性;⑤将SD大鼠分为正常对照组、用药组、miR-7b-3p inhibitor组、用药+miR-7b-3p mimics组、阳性对照组,观察各组大鼠慢波睡眠的变化。结果与结论:①酸枣仁提取物可以促进小鼠体长、胫骨增长,促进生长激素分泌,提高脑组织5-HT1AR含量;②基因芯片筛选出差异表达的miRNAs个数为16个,其中上调有13个,下调有3个;生物信息学预测下调miR-7b-3p可以调控5-HT1AR表达,且双荧光素酶报告基因实验证实二者有直接调控关系;③酸枣仁提取物和沉默脑皮质细胞中miR-7b-3p表达可以引起5
BACKGROUND:Previous studies found that Semen Ziziphi Spinosae extract prolongs slow-wave sleep and promotes growth hormone secretion in mice by increasing the expression of brain tissue serotonin 1A receptor(5-HT1AR),which binds to serotonin,thus leading to bone growth.Serotonin 1A receptor acts as a protein,and its expression abundance is regulated by miRNAs.The authors hypothesized that Semen Ziziphi Spinosae extract may regulate the expression of 5-HT1AR through miRNAs and thus exert drug effects.OBJECTIVE:To observe the effect of Semen Ziziphi Spinosae extract on bone growth by regulating the miR-7b-3p/5-HT1AR pathway in mouse brain tissue.METHODS:(1)Kunming mice were divided into a normal control group,a drug administration group(gavage administration of Semen Ziziphi Spinosae extract 0.320 mg/g),a positive control group(gavage administration of jujuboside 0.013 mg/g),a Semen Ziziphi Spinosae extract+5-HT1AR selective inhibitor group(8μg of P-MPPF,a 5-HTIAR selective inhibitor,was injected into the lateral ventricle every day for the last 3 days during the gavage administration of Semen Ziziphi Spinosae extract).After 25 days,the effects of Semen Ziziphi Spinosae extract on bone growth,serum growth hormone level and brain 5-HT1AR expression were observed.(2)Then the chip method was used to observe differentially expressed miRNAs in the brain tissues of growing mice and ordinary mice.PCR validation and dual luciferase reporter gene assay confirmed the regulatory relationship between the screened Mir-7B-3p and 5-HT1AR.(3)Mouse cerebral cortical cells were cultured and identified in vitro,and the effect of Semen Ziziphi Spinosae extract on 5-HT1AR expression in cerebral cortical cells was observed using western blot.(4)Kunming mice were divided into a normal control group,a medication group,a miR-7b-3p inhibitor group,a medication+5-HT1AR inhibitor group,and a positive control group.The expression of 5-HT1AR in brain tissues of mice and the binding activity of 5-HT and 5-HT1AR were observed.(5)Sprague-Dawley r
作者
罗石任
吴晓龙
谢艳
Luo Shiren;Wu Xiaolong;Xie Yan(Luoyang Orthopedic-Traumatological Hospital of Henan Province(Henan Provincial Orthopedic Hospital),Zhengzhou 450000,Henan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第12期2450-2457,共8页
Chinese Journal of Tissue Engineering Research
基金
河南省中医药科学研究专项(2022ZY2051),项目负责人:罗石任。
