Homer1a竞争性解聚mGluR5-Homer复合体介导吗啡的抗伤害性效应－－吗啡耐受的新机制

As an effective analgesic, morphine was widely used in clinical practice. But the tolerance damages its efficacy and the mechanism remains unknown. Our preliminary data suggest that morphine may induce the expression of Homer1a, an immediately early gene, through μ opioid receptor. The induced Homer1a could potentiate acute morphine efficacy and knockout of Homer1a may lead to decreased antinociception by acute morphine. As a short chain Homer protein, which just includes an EVH1 domain, Homer1a could compete with Homer1 to bind with the TPPSPF at the C-terminal of mGluR5 and lead to the dissociation of mGluR5-Homer crosslinking, and affect the mGluR5-mediated modulation of ion channels. These may have effect on the intracellular calcium level. Overexpression of Homer1a has been proved to have protective effect on inflammatory pain. We also got the similar results in our preliminary tests: overexpression of Homer1a by MECS could potentiate acute morphine efficacy. So we hypothesize that acute morphine-induced Homer1a through μ opioid receptor may potentiate morphine-mediated modulation of transmembrane N-type calcium and M-type potassium currents, and then strengthen acute morphine-induced antinociception; Repeated morphine treatments lead to desensitization of μ opioid receptor, which may result in decreased expression of morphine-induce Homer1a. Decreased expression of Homer1a may hurt morphine-induced antinociception, and then tolerance appears. The current project is helpful in building new mechanism of morphine antinociceptive tolerance and also it has a potential value in peptide synthesis to treat morphine analgesic tolerance in future.

吗啡作为疼痛治疗的常用药物，在临床应用广泛，但耐受阻碍了其有效应用，机制不明。我们预实验提示，吗啡可经μ受体诱导Immediately Early Gene-Homer1a的表达，且Homer1a基因敲除可损伤吗啡的镇痛效力。Homer1a作为短链Homer蛋白，可竞争性结合在mGluR5胞内段解聚mGluR5-Homer复合体，影响mGluR5对离子通道的调节及胞内游离钙水平。过表达Homer1a被证明可有效对抗炎性痛的进展，而我们在前期预实验研究中也得到类似的结果，过表达Homer1a可有效增强吗啡的镇痛效力。为此，我们提出如下假说：吗啡经μ受体诱导的Homer1a表达可有效协同吗啡对胞内外钙离子及钾离子电流的调节，增强吗啡的急性镇痛效应，反复吗啡处理所致的μ受体脱敏失活将降低吗啡诱导的Homer1a的表达，导致吗啡的镇痛效力丧失，引发耐受。申报课题的完成将有助于形成吗啡耐受的新理论。

本研究发现首次小鼠鞘内给予单剂量吗啡能够诱导脊髓内显著的即早基因homer1a的表达，同时伴ERK1/2磷酸化的明显增加；ERK1/2磷酸化增加能够明显增加homer蛋白在mGluR5胞内C端的结合力，形成mGluR5-homer复合体，由于首次吗啡诱导homer1a的显著表达，继而降低mGluR5-homer1b/c复合体的形成，阻断mGluR5信号对胞内内质网钙离子释放的调节，经电休克诱导即早基因homer1a的表达或修饰mGluR5胞内C端homer结合位点（TPPSPF），降低mGluR5-homer的连接（mGluR5 KO, mGluR5 F/R, mGluR5 TS-AA, Homer1 KO)，均可明显的协同增强吗啡的抗伤害性效应；homer1a敲除的小鼠中，吗啡急性抗伤害性效应明显受损，不能够达到野生型小鼠的cut-off值；通过使用homer1a KO小鼠与mGluR5 KO、mGluR5 F/R或mGluR5 TS-AA小鼠杂交形成双敲或双修饰小鼠，均可明显逆转homer1a KO小鼠吗啡镇痛效力损伤表型；长期给予吗啡后，可致吗啡诱导的homer1a表达降低，但仍显著的增强小鼠脊髓背角ERK1/2磷酸化，增加mGluR5-homer复合体形成。通过修饰mGluR5胞内homer结合段，降低mGluR5与homer间的连接，能够明显延缓吗啡耐受的形成。此外，我们还通过免疫荧光组织化学与原位杂交技术，证实了mGluR5、pERK1/2及homer1a在同一神经元中的共表达。使用设计的homer1a的短肽，经鞘内注射，可有效的逆转已经形成的吗啡耐受行为学表型。本研究有助于从新的角度理解阿片类药物的急性镇痛及慢性耐受机制，对于开发新的镇痛及阿片类协同镇痛药物具有重要的科学意义。