木瓜蛋白酶对MPA诱导的单核细胞分泌和黏附功能的抑制作用及分子机制初步研究

目的: 观察木瓜蛋白酶对单核细胞-血小板聚集物（MPA）诱导的单核细胞分泌和黏附功能的抑制作用，并初步探讨其分子机制。方法: 分离人外周血单核细胞和富血小板血浆（PRP），检测20 U/L木瓜蛋白酶不同作用方式下凝血酶和花生四烯酸（AA）诱导的MPA形成及肿瘤坏死因子-α（TNF-α）水平。将实验分为0（对照）、20、80 U/L木瓜蛋白酶组，分别以ELISA、流式细胞术和显微镜检查测定木瓜蛋白酶与单核细胞共孵育后MPA诱导的单核细胞趋化蛋白-1（MCP-1）和组织因子（TF）释放、CD11b和CC趋化因子受体2（CCR2）表达水平以及单核细胞与人脐静脉内皮细胞黏附率，再以Western blot检测单核细胞Akt磷酸化（p-Akt）和环氧化酶-2（COX-2）表达水平。结果: 20 U/L木瓜蛋白酶与单核细胞和血小板共孵育时显著降低凝血酶和AA诱导的MPA形成和TNF-α 水平（P<0.01）。20 U/L木瓜蛋白酶组MCP-1和TF水平，CD11b和CCR2表达率，单核细胞与内皮细胞黏附率均显著低于对照组（P<0.01），80 U/L组对五者的抑制率显著高于20 U/L组（P<0.01）。木瓜蛋白酶显著抑制p-AKT和COX-2表达，80 U/L组p-Akt和COX-2光密度比值显著低于20 U/L组（P<0.01）。结论:木瓜蛋白酶可通过抑制Akt磷酸化和COX-2表达途径而抑制MPA诱导单核细胞活化后的释放和黏附功能，从而可能有助于预防动脉粥样硬化。

Effects and preliminary molecular mechanisms of papain on the inhibition of monocyte secretory and adherent ability induced by monocyte-platelet aggregates

AIM: To observe the inhibitory effects of papain on monocyte secretory and adherent ability induced by monocyte-platelet aggregates (MPA), and to explore the preliminary molecular mechanisms. METHODS: Monocyte and platelet rich plasma were separated form human peripheral blood. Platelet were activated by thrombin and arachidonic acid, then mixed with monocyte in 20 U/L of papain,respectively, and levels of MPA and TNF-α were determined.The study was further divide into three groups: 0 (control), 20, 80 U/L of papain group activated by 10 U/L thrombin, MCP-1, tissue factor (TF), CD11b and CCR2 expression, and adherent monocyte percent were measured by ELISA, flow cytometry, and microscopic examination, respectively.Western blot analysis was used to detect the phosphorylated Akt and COX-2 expression.RESULTS:In incubated with monocyte and platelet, 20 U/L of papain significantly decreased MPA formation and TNF-α levels induced by thrombin and arachidonic acid (P<0.01) . In 20 U/L of papain group, MCP-1, TF, CD11b and CCR2 expression levels, and adherent monocyte percent were significantly decreased compared with control group (P<0.01).The inhibitory percent of the abovementioned five markers were increased in 80 U/L group than that in 20 U/L group (P<0.01). Papain significantly inhibited Akt phosphorylation and COX-2 expression, there were lower photodensity ratios in 80 U/L group than that in 20 U/L group (P<0.01). CONCLUSION: Papain may inhibit monocyte secretory and adherent ability induced by monocyte-platelet aggregates through the inhibition of Akt phosphorylation and COX-2 expression, and can be used to prevent atherosclerosis.