人参皂苷Rgl对慢性吗啡作用及纳洛酮催促戒断SK-N-SH细胞的影响及机制研究

Effects and mechanism of ginsenoside-Rg1 on SK-N-SH cell treated with chronic morphine and naloxone-precipitated withdrawal

目的探讨人参皂苷Rgl对吗啡慢性作用及纳洛酮催促戒断SK-N-SH细胞的影响及其机制。方法采用不同浓度人参皂苷Rgl1、2、4、8、16、32μmol·L-1对SK-N-SH细胞预处理24h后,用吗啡(100μmol·L-1培养基)孵育24h,采用MTT法研究人参皂苷Rgl对吗啡慢性作用SK-N-SH细胞增殖的影响;用相同浓度的吗啡作用于SK-N-SH细胞后,10μmol·L-1纳洛酮催促戒断前24h,加入1、2、4μmol·L-1的Rg1作用24h,采用荧光分光光度法、RT-PCR及Western blot技术分别检测人参皂苷Rgl对吗啡慢性作用纳洛酮催促戒断SK-N-SH细胞内[Ca2+]i、CaMKⅡβ mRNA及蛋白表达的影响。结果①与对照组相比,吗啡慢性作用48h可抑制SK-N-SH细胞的增殖;细胞内[Ca2+]i增高(P<0.01);细胞CaMKⅡβ mRNA及蛋白表达明显升高;②加入10μmol·L-1纳洛酮作用30min后,细胞内[Ca2+]i急剧降低(P<0.05);CaMKⅡβ mRNA及蛋白表达进一步升高(P<0.05);③2μmol·L-1人参皂苷Rg1对细胞进行预处理能缓解吗啡对细胞的增殖活性的抑制作用(P<0.01)。④慢性吗啡作用SK-N-SH细胞,在纳洛酮急性戒断前,加入不同浓度的Rg1可缓解细胞内钙离子浓度的降低;同时可降低CaMKⅡβ mRNA和蛋白表达的进一步升高。结论人参皂苷Rgl可缓解吗啡对SK-N-SH细胞的抑制作用,通过调控细胞内[Ca2+]i以及CaMKⅡβ mRNA和蛋白表达,从而缓解吗啡成瘾及戒断症状的产生。

Aim To explore the effects and mechanism of ginsenoside-Rg1 on SK-N-SH cells treated with chronic morphine and naloxone-precipitated withdrawal. Methods Cells were pretreated with ginsenoside-Rg1 1,2,4,8,16,32 μmol·L-1 for 24 h,then incubated for 24 h with morphine （ 100 μmol·L-1 ） . MTT colorimetr was used to study the effects of ginsenoside-Rg1 on the multiplication of the cells treated with chro-nic morphine. After stimulated by the same concentra-tion of morphine,cells were added with different concentrations of Rg1 1,2,4 μmol·L -1 for 24 h before stimulated with 10 μmol·L -1 NAL. Fuorospectrophotometry RT-PCR and Western blot techniques were used to detect the effects of ginsenoside-Rg1 on the [Ca2＋ ]i,CaMKⅡ β mRNA and protein expression of the SK-N-SH cells treated with chronic morphine and naloxone-precipitated withdrawal. Results ① Compared with control group,morphine significantly inhibited cell multiplication and resulted in calcium overload,and the expression of CaMKⅡ-β mRNA and protein noticeably increased （ P〈0. 01） . ② Compared with MOR group,30 minutes after naloxone-precipitated withdrawal,[Ca2 ＋ ]i noticeably decreased （ P〈0. 01） and protein and mRNA expressions of CaMKⅡ β further increased. ③ Hinsenoside-Rg1 （ 2 μmol· L -1） pretreatment could relieve the depressant effect of morphine on cell multiplication （ P〈0. 01） . ④ After chronic morphine exposure before naloxone-precipitated withdrawal,ginsenoside-Rg1 of different concentrations 1,2,4 μmol·L -1 could turn over the above mentioned phenomenon induced by NAL （ P〈0. 01） . Conclusion Ginsenoside-Rg1 can relieve the depressant effect of morphine on cell multiplication and regulate intracellular [Ca2 ＋ ]i,CaMK Ⅱβ mRNA and protein expres-sion,thus it alleviates morphine addiction and withdrawal symptoms by naloxone.