柴胡提取物抑制小鼠脑组织miR-33-5p表达对小鼠骨骼生长的影响

Chaihu Extract modulated miR-33-5p expression in brain tissue and affected skeletal growth in mice

目的:研究柴胡提取物抑制小鼠脑组织miR-33-5p表达,上调TPH2促进5-HT表达最终促进骨骼增长的作用及机制。方法:(1)60只昆明小鼠,随机分为正常对照组、低、中、高剂量组、阳性对照组、阴性对照组,每组10只,观察柴胡提取物对小鼠体长、胫骨长度、脑组织5-HT、TPH2及血清GH表达的影响。(2)利用3个数据库预测调控TPH2的共同miRNAs,并用qRT-PCR验证这些miRNA在正常小鼠和给药小鼠体内差异表达,进而采用双荧光素酶报告基因实验验证差异表达的miRNA与TPH2的调控关系。(3)体外培养小鼠脑细胞,观察药物对细胞中TPH2含量的影响。(4)昆明种小鼠50只,随机分为正常对照组、用药组、miR-33-5p mimics组、miR-33-5p inhibitor组、阳性对照组,每组10只。观察药物通过干预miR-33-5p对小鼠脑组织TPH2、5-HT表达及GH分泌的影响。结果:(1)灌胃20 d后,低、中、高剂量组体长均较正常对照组和阴性对照组提高(均为P<0.01)。胫骨长度中剂量组和高剂量组长于正常对照组(均为P<0.01)。脑组织5-HT含量低、中、高剂量组与正常对照组比较均有所升高(P<0.05,P<0.01,P<0.01),也高于阴性对照组(均为P<0.01),中剂量组较低剂量组表达升高(P<0.01)。血清GH表达水平中、高剂量组较正常对照组升高(均为P<0.01)。脑组织TPH2含量,中、高剂量组及阳性对照组均较正常对照组提高(均为P<0.01)。中剂量组高于低剂量组(P<0.01)。(2)在3个生物信息学网站预测的目标miRNAs中,只有miR-33-5p呈现差异表达(0.967±0.157,1.400±0.144;P=0.024)。双荧光素酶报告基因实验证明miR-33-5p与TPH2存在调控关系。(3)细胞实验证明药物和miR-33-5p对小鼠脑组织TPH2的表达有影响。(4)动物实验发现,用药组、miR-33-5p inhibitor组和阳性对照组较正常对照组脑组织TPH2表达均升高,而miR-33-5p mimics组则降低。同时影响5-HT和血清GH的表达。结论:柴胡提取物可以通过沉默脑组织miR-33-5p的表达来提高TPH2的表达,从而提高脑组织5-HT丰度,最终促进GH分泌及小鼠骨骼增长。

OBJECTIVE To explore the role and mechanism of Chaihu Extract(CE)in inhibiting miR-33-5p expression in murine brain tissue,up-regulating tryptophan hydroxylase 2(TPH2),promoting 5-hydroxytryptamine(5-HT)expression and ultimately enhancing skeletal growth.METHODS Sixty Kunming mice were randomized into six groups of normal control,low-dose,medium-dose,high-dose,positive control and negative control(n=10 each).The effects of CE on body length,tibia length,brain tissue 5-HT/TPH2 and serum GH expression were observed.Three databases were utilized for predicting common miRNAs regulating TPH2.Quantitative real-time polymerase chain reaction(qRT-PCR)validated differential expression of these miRNAs in normal and treated mice.Dual-luciferase reporter gene assays confirmed the regulatory relationship between differentially expressed miRNAs and TPH2.Murine brain cells were cultured in vitro to observe the impact of CE on TPH2 content.Fifty Kunming mice were randomized into five groups of normal control,treatment,miR-33-5p mimics,miR-33-5p inhibitor and positive control(n=10 each).The effects of drug on 5-HT/TPH2 expression and GH secretion through miR-33-5p intervention was observed.RESULTS After 20-day gavage,low/medium/high-dose groups showed greater body length as compared to normal control and negative control groups(P<0.01).Tibial length was longer in medium/high-dose group than that in normal control group(P<0.01).Brain tissue 5-HT content rose in low/medium/high-dose group as compared to normal control group(P<0.05,P<0.01,P<0.01)and it was higher than that in negative control group(P<0.01).Medium-dose group showed a higher expression of 5-HT than that in low-dose group(P<0.01).Serum GH expression was higher in medium/high-dose group than that in normal control group(P<0.01).Brain tissue TPH2 content increased in medium/high-dose and positive control groups as compared to normal control group(P<0.01).Medium-dose group was higher than low-dose group(P<0.01).Among target miRNAs predicted by three bioinformatic websites,only miR-33-5p showed differential expression(0.967±0.157,1.400±0.144;P=0.024).Dual-luciferase reporter gene assays confirmed the regulatory relationship between miR-33-5p and TPH2.Cell experiments indicated that CE and miR-33-5p affected TPH2 expression in murine brain tissue.As compared to normal control group,brain tissue TPH2 expression increased in treatment,miR-33-5p inhibitor and positive control groups and yet declined in miR-33-5p mimics group.The expressions of 5-HT and serum GH were also affected.CONCLUSION Enhancing TPH2 expression in murine brain tissue through silencing the expression of miR-33-5p,CE may boost the abundance of brain tissue 5-HT,promote GH secretion and stimulate skeletal growth.