脊髓G蛋白表达在代谢型谷氨酸5受体参与吗啡耐受形成中的作用

目的:研究鞘内注射代谢型谷氨酸5受体(metabotropic glutamate receptor 5,mGluR5)反义寡聚脱氧核苷酸(oligodeoxynucleotide,ODN)时大鼠吗啡耐受发生情况及其对脊髓内G蛋白各亚型表达的影响。方法:将48只雄性SD大鼠随机分为6组:对照组(S组,n=14),反义链组(ANT组,n=6),错义链组(MIS组,n=6),吗啡耐受组(M组,n=6),反义链吗啡合用组(AM组,n=8)和错义链吗啡合用组(MM组,n=8)。采用鞘内给药方式注射寡聚核苷酸、吗啡和生理盐水。以5μl含30 nM的寡聚核苷酸(ANT组,MIS组,AM组和MM组)或0.9%生理盐水(S组和M组)每日给药2次,连续5天。于第6天取6只S组及ANT组和MIS组大鼠L4/5脊髓行real-time PCR,观察其mGluR5的mRNA表达。其余大鼠于第6天起连续3天每日给吗啡2次,每次15μg,S组以5μl0.9%生理盐水代替。给药后以热板试验测定其热痛阈,以缩爪阈值作为机械痛阈指标,并计算最大效应分数(maximum percent effect,MPE)进行比较。采用免疫印迹(western blot)方法检测并比较各组L4/5脊髓中G蛋白各亚型(Gαi,Gαo,Gαq和Gβ)的表达。结果:ANT组中mGluR5含量比S组下降48.2%(P<0.05),MIS组无此效果。M组和MM组大鼠MPE%在第8天与S组比较无统计学差异(P>0.05),AM组大鼠MPE%在第8天仍高于S组,有统计学意义(P<0.05)。AM组大鼠MPE%在第7~8天即显著高于M组(P<0.05)。第9天M组和MM组大鼠Gαi,Gαo,Gαq和Gβ的蛋白表达量显著高于S组(P<0.05)。而AM组大鼠G蛋白各亚型表达量均与S组无显著性差异(P>0.05),与M组相比显著下调(P<0.05)。结论:大鼠吗啡耐受发生时,大鼠腰段脊髓G蛋白各亚型表达均明显升高。鞘内注射mGluR5反义寡聚脱氧核苷酸能显著抑制大鼠吗啡耐受的发生并抑制G蛋白各亚型的表达升高。

THE ROLE OF G PROTEIN EXPRESSION IN THE PARTICIPATION OF METABOTROPIC GLUTAMATE RECEPTOR 5 IN MORPHINE TOLERANCE

Objective: To investigate the role of intrathecal injection of metabotropic glutamate receptor 5(mGluR5) antisense oligodeoxynucleotide(ODN) on the occurrence of morphine tolerance and expressions of spinal G proteins in rats.Methods: Forty-eight male Sprague-Dawley rats were randomly divided into six groups: control group(S group,n = 14),antisense ODN group(ANT group,n = 6),mismatch ODN group(MIS group,n = 6),morphine tolerance group(M group,n = 8),antisense ODN plus morphine group(AM group,n = 8) and mismatch ODN plus morphine group(MM group,n = 8).Rats in ANT group,MIS group,AM group and MM group received 5 μl of intrathecal 30 nM ODN twice a day for 5 days.Rats in S and M groups were received 5 μl of 0.9% saline twice a day for 5 days.On day 6,real-time PCR was used to investigate the mRNA of mGluR5 in S group,ANT group and MIS group.From day 6,15 μg morphine was added twice a day in M group,AM group and MM group.Rats in S group received 5 μl of 0.9% saline instead of any drugs.Hot-plate test and paw pressure test were used to assess nociception and maximum percent effect(MPE) was measured.The expressions of G proteins(Gαi,Gαo,Gαq and Gβ) were detected by western blot.Results: Compared with S group,mGluR5 was reduced by 48.2% in ANT group(P < 0.05).MPE% of M group and MM group decreased on day 8,with no significant difference with S group(P > 0.05).MPE% of AM group on day 8 was still higher than S group(P < 0.05).MPE% of AM group on day 7 and 8 was higher than M group(P < 0.05).The expressions of G proteins(Gαi,Gαo,Gαq and Gβ) in M group and MM group were up-regulated,as compared with S group(P < 0.05).The expressions of G proteins between AM and S groups were no significantly different(P > 0.05).The expression of G protein of AM group was decreased than M group.Conclusion: Intrathecal injection of mGluR5 antisense ODN can inhibit the occurrence of morphine tolerance,and reduce the up-regulated G protein(Gαi,Gαo,Gαq and Gβ) expression in the rat spinal cord which would happen when tolerance occurs.