Abstract：
BACKGROUND: Uncaria rhynchophylla (Miq.) Miq. ex Havil. is a plant species that is routinely devoted in traditional Chinese medicine to treat central nervous system disorders. Rhynchophylline (Rhy), a predominant alkaloid isolated from Uncaria rhynchophylla (Miq.) Miq. ex Havil., has been demonstrated to reverse methamphetamine-induced (METH-induced) conditioned place preference (CPP) effects in mice, rats and zebrafish. The precise mechanism is still poorly understood, thus further research is necessary.
OBJECTIVE: This study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence.
METHODS: A rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence.
RESULTS: Rhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro.
CONCLUSIONS: This finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.
OBJECTIVE: This study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence.
METHODS: A rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence.
RESULTS: Rhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro.
CONCLUSIONS: This finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.
摘要：
背景：钩藤(Miq.）Miq。前Havil.是一种植物，通常用于中药治疗中枢神经系统疾病。钩藤碱(Rhy),从钩藤中分离出的一种主要生物碱（Miq。）Miq。前Havil.,已被证明可以逆转小鼠中甲基苯丙胺诱导的（METH诱导的）条件位置偏爱（CPP）效应，老鼠和斑马鱼.精确的机制仍然知之甚少，因此需要进一步的研究。
目的：本研究旨在研究miRNAs在Rhy对METH依赖的抑制作用中的作用。
方法：建立大鼠CPP模型和PC12细胞成瘾模型。微阵列测定用于筛选和鉴定候选miRNA。行为评估,实时PCR，双荧光素酶报告分析，西方印迹,进行了立体定向注射antagomir/agomir和细胞转染实验,以阐明候选miRNA的作用和Rhy对METH依赖的干预机制.
结果：Rhy成功逆转了METH诱导的CPP效应，并上调了METH依赖性大鼠海马和PC12细胞中miR-181a-5p的表达。此外，antagomir181a抑制miR-181a-5p可逆转METH诱导的CPP效应。同时,与低剂量METH（0.5mg/kg）组合的agomir181a过表达miR-181a-5p引起明显的CPP效应，通过抑制miR-181a-5p被Rhy阻断。最后,结果表明，miR-181a-5p通过在体内和体外靶向γ-氨基丁酸A受体α1（GABRA1）发挥其调节作用。
结论：这一发现揭示了Rhy通过调节miR-181a-5p/GABRA1轴抑制METH依赖，这可能是治疗药物依赖的一个有希望的目标。
目的：本研究旨在研究miRNAs在Rhy对METH依赖的抑制作用中的作用。
方法：建立大鼠CPP模型和PC12细胞成瘾模型。微阵列测定用于筛选和鉴定候选miRNA。行为评估,实时PCR，双荧光素酶报告分析，西方印迹,进行了立体定向注射antagomir/agomir和细胞转染实验,以阐明候选miRNA的作用和Rhy对METH依赖的干预机制.
结果：Rhy成功逆转了METH诱导的CPP效应，并上调了METH依赖性大鼠海马和PC12细胞中miR-181a-5p的表达。此外，antagomir181a抑制miR-181a-5p可逆转METH诱导的CPP效应。同时,与低剂量METH（0.5mg/kg）组合的agomir181a过表达miR-181a-5p引起明显的CPP效应，通过抑制miR-181a-5p被Rhy阻断。最后,结果表明，miR-181a-5p通过在体内和体外靶向γ-氨基丁酸A受体α1（GABRA1）发挥其调节作用。
结论：这一发现揭示了Rhy通过调节miR-181a-5p/GABRA1轴抑制METH依赖，这可能是治疗药物依赖的一个有希望的目标。
