Chaihu Extract modulated miR-33-5p expression in brain tissue and affected skeletal growth in mice
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 dif-ferentially expressed miRNAs and TPH2.Murine brain cells were cultured in vitro to observe the impact of CE on TPH2 con-tent.Fifty Kunming mice were randomized into five groups of normal control,treatment,miR-33-5p mimics,miR-33-5p inhibi-tor and positive control(n=10 each).The effects of drug on 5-HT/TPH2 expression and GH secretion through miR-33-5p inter-vention was observed.RESULTS After 20-day gavage,low/medium/high-dose groups showed greater body length as com-pared 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.
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