摘要
目的探讨μ阿片受体(MOR)羧基端^(375)STANT^(379)位点磷酸化对吗啡镇痛效果的影响。方法针对MOR羧基端^(375)STANT^(379)序列设计穿膜多肽,采用标准固相合成方法进行合成,采用高效液相色谱(HPLC)和高分辨质谱法(HRMS-ESI)分别测定合成多肽的纯度和相对分子质量,检测合成无误后用于后续实验。采用CCK-8法检测合成多肽对表达MOR的HEK-293细胞的毒性作用。采用热板法检测各组小鼠末次给药后不同时间点吗啡镇痛反应率,观察合成多肽对吗啡镇痛效果的影响。结果针对^(375)STANT^(379)关键磷酸化区域设计多肽TAT-Q368-T379,HPLC检测多肽纯度大于96%,HRMS-ESI检测多肽相对分子质量2894.4834(预期相对分子质量2894.5625),多肽合成正确,符合后期实验要求。CCK-8法检测结果显示各组细胞存活率差异无显著意义(P>0.05),多肽安全范围广。热板法检测结果显示多肽预处理组小鼠吗啡镇痛时间较吗啡组明显延长(Waldχ^(2)_(组别)=1519.252,Waldχ^(2)_(时间)=756.177,Waldχ^(2)_(组别*时间)=8657.036,P<0.05)。结论MOR羧基端^(375)STANT^(379)磷酸化区域与吗啡镇痛相关,针对这一区域氨基酸序列设计的多肽可作为工具竞争性抑制该位点磷酸化,有效改善吗啡镇痛效果。
Objective To investigate the effect of phosphorylation at the carboxy-terminal^(375)STANT^(379)site ofμ-opioid receptor(MOR)on the analgesic effect of morphine.Methods A penetrating polypeptide was designed according to the sequence of the carboxy-terminal^(375)STANT^(379)site of MOR and was synthesized by the standard solid-phase synthesis method.High-performance liquid chromatography(HPLC)and high-resolution mass spectrometry(HRMS-ESI)were used to determine the purity and relative molecular weight of the synthetic peptide,which was used for subsequent experiments after correct synthesis.CCK-8 assay was used to observe the cytotoxicity of the synthetic peptide on HEK-293 cells expres-sing MOR,and the hot plate method was used to measure the response rate of morphine analgesia at different time points after the last administration to observe the effect of the synthetic peptide on the analgesic effect of morphine.Results The peptide TAT-Q368-T379 was designed based on the key phosphorylation cassette of^(375)STANT^(379).HPLC showed that the purity of the peptide was>96%,and HRMS-ESI showed that the peptide had a relative molecular weight of 2894.4834(the expected relative molecular weight was 2894.5625),suggesting that the peptide was synthesized correctly and met the requirements for subsequent experiments.CCK-8 assay showed that there was no significant difference in cell viability between groups(P>0.05),suggesting that the peptide had a wide safety range.The hot plate method showed that compared with the mice in the morphine group,the peptide pretreatment group had a significantly prolonged analgesic time of morphine(Waldχ^(2)_(group)=1519.252,Waldχ^(2)_(time)=756.177,Waldχ^(2)_(group*time)=8657.036,P<0.05).Conclusion The carboxy-terminal^(375)STANT^(379)phosphorylation cassette of MOR is associa-ted with morphine analgesia,and the peptide designed based on the amino acid sequence of^(375)STANT^(379)phosphorylation cassette can effectively improve the analgesic effect of morphine by competitively inhibiting
作者
周筱慧
李笑妍
褚海辰
董铭心
ZHOU Xiaohui;LI Xiaoyan;CHU Haichen;DONG Mingxin(Department of Anesthe-siology,The Affiliated Hospital of Qingdao University,Qingdao 266003,China)
出处
《精准医学杂志》
2022年第3期262-266,共5页
Journal of Precision Medicine
基金
国家自然科学基金资助项目(81873729)。
关键词
受体
阿片样
μ
磷酰化
吗啡
镇痛
固态合成技术
Receptors,opioid,mu
Phosphorylation
Morphine
Analgesia
Solid-phase synthesis techniques
