甲硫脑啡肽通过阿片受体促进脂多糖作用下奶牛子宫内膜基质细胞的增殖

Effect of Met-enkephalin on Lipopolysaccharide-stimulated Proliferation of Bovine Endometrial Stromal Cells

为明确甲硫脑啡肽(Met-enkephalin,MENK)调控原代奶牛子宫内膜基质细胞(bovine endometrial stromal cell,BESC)增殖的作用机制,采用脂多糖(lipopolysaccharide,LPS)、MENK和阿片受体拮抗剂共处理细胞进行了本试验。本研究使用的阿片受体拮抗剂包括非选择性阿片受体拮抗剂纳洛酮(naloxone,Nx)、δ阿片受体拮抗剂ICI 154129和μ阿片受体拮抗剂CTAP,分组情况:空白对照组,LPS处理组,LPS与MENK(10-10mol·L^(-1))共处理组(LM组),LPS、MENK和阿片受体拮抗剂(Nx、CTAP和ICI 154129,均为10-10mol·L^(-1))共处理组以及LPS与阿片受体拮抗剂共处理组。检测细胞活性、细胞周期、细胞增殖相关因子基因表达以及Wnt/β-catenin和PI3K/AKT相关通路变化。结果表明:阿片受体拮抗剂单独或与LPS共处理细胞24 h,对细胞活力无影响(P>0.05)。与LM组相比,LPS、MENK和Nx共处理组(LMN组)以及LPS、MENK和ICI 154129共处理组(LMI组)的G1期相对细胞数升高(P<0.05),LMN组以及LPS、MENK和CTAP共处理组(LMC组)S期相对细胞数降低(P<0.05)。与LM组相比,LMN组和LMI组细胞CCN2、TGFB1和TGFB3基因表达下调(P<0.01),LMC组细胞CCN2基因表达下调(P<0.01)。与LM组相比,LMN组cyclinD1和GSK-3β蛋白表达上调(P<0.05),LMI组细胞β-catenin和GSK-3β蛋白表达上调(P<0.01),LMC组细胞cyclinD1蛋白表达上调(P<0.05);与LM组相比,LMN组、LMI组和LMC组AKT磷酸化水平升高(P<0.05)。以上结果说明,μ阿片受体和δ阿片受体均参与MENK对BESC的促增殖作用。

To clarify the mechanism of Met-enkephalin(MENK)on the proliferation of bovine endometrial stromal cell(BESC),the BESC were co-treated with lipopolysaccharide(LPS),opioid receptor antagonists and MENK.The opioid receptor antagonists used in this study included the nonselective opioid receptor antagonist naloxone(Nx),theδopioid receptor antagonist ICI 154129 were theμopioid receptor antagonist CTAP,and the groups were as follows:the blank control group,the LPS treatment group,the LPS and MENK(10-10mol·L^(-1))co-treatment group(LM group),the LPS,MENK and opioid receptor antagonists(Nx,CTAP and ICI 154129 were all 10-10mol·L^(-1))co-treatment group and the LPS and opioid receptor antagonist co-treatment groups.Cell viability,cell cycle,the expression of cell proliferation-related genes and the expression of Wnt/β-catenin and PI3K/AK pathway proteins were detected.The results showed that the treatment of opioid receptor antagonist alone in treatment concentration or co-treatment of LPS for 24 h had no effect on cell viability(P>0.05).Compared with the LM group,the cell numbers of G1 phase increased(P<0.05)in the co-treatment groups of LPS,MENK and Nx(LMN)and LPS,MENK and ICI 154129(LMI),the cell numbers of S phase decreased(P<0.05)in LMN group and the co-treatment group of LPS,MENK and CTAP(LMC).Compared with LM group,the gene expressions of CCN2,TGFB1 and TGFB3 decreased(P<0.01)in the groups of LMN and LMI;the CCN2 expression decreased(P<0.01)in LMC group.Compared with LM group,there were increases in the protein expressions of cyclinD1 and GSK-3βin LMN group(P<0.05),theβ-catenin and GSK-3βin LMC group(P<0.01),and the cyclinD1 in LMC group(P<0.05).Compared with LM group,the phosphorylation level of AKT increased(P<0.05)in LMN,LMI,and LMC groups.In conclusion,bothδopioid receptor andμopioid receptor are involved in the proliferation of MENK on BESC.