护心方含药血清对THP-1源性泡沫细胞自噬相关蛋白的影响

Effects of Huxin Fang (护心方) Drug-Containing Serum on Autophagy-Related Protein of THP-1 Derived Foam Cells

目的探讨护心方抗动脉粥样硬化的可能作用机制。方法用160 nmol/L佛波醇酯类多克隆刺激剂孵育THP-1细胞24 h后诱导分化成巨噬细胞,加入50 mg/L氧化低密度脂蛋白(ox-LDL)培养48 h,使其转化为泡沫细胞。以40%护心方含药血清作用12 h。采用蛋白质印迹检测自噬相关蛋白微管相关蛋白轻链3Ⅱ(LC3-Ⅱ)、微管相关蛋白轻链3I(LC3-Ⅰ)、Beclin-1及p62表达,激光共聚焦显微镜观察自噬蛋白指标LC3B在细胞内分布及含量。结果与空白组比较,模型组LC3-Ⅱ/LC3-Ⅰ和Beclin-1表达显著增加,p62表达显著降低,LC3B表达平均荧光强度显著升高(P<0.05);与模型组比较,护心方组LC3-Ⅱ/LC3-Ⅰ和Beclin-1表达显著降低,p62表达显著升高,LC3B表达平均荧光强度降低(P<0.05)。结论护心方能够逆转ox-LDL诱导的细胞自噬相关蛋白的表达,从而抵抗动脉硬化过程中过度自噬。这可能是其抗动脉粥样硬化的机制之一。

Objective To explore the potential anti-atherosclerosis mechanism of Huxin Fang （护心方,TY, HF）. Methods THP-1 cells were incubated with 160nmol/L phorbol esters polyclonal stimulants for 48 hours to differenti- ate into macrophages, and then 50mg/L oxidized low-density lipoprotein （ox-LDL） were added for another 48 hours to make them transfer into foam cells. After that, serum containing 40% HF were added for 12 hours. Autophagy- associated proteins microtube-associated protein light chain 311 （LC3-U ）, microtubule-associated protein light chain 3I （LC3- I ）, Beclin and p62 expressions were detected by Western blotting, while the distribution and content of autophagic protein index LC3B were observed by laser eonfocal microscopy. Results Compared with the blank group, the expression of LC3- II/LC3- I and Beelin-1 in the model group significantly increased, the expression of p62 decreased and the average fluorescence intensity of LC3 B significantly increased （P 〈 0. 05 ）. Compared with the model group, the expression of LC3-II/LC3-I and Beclin-1 in the HF group significantly decreased, the expression of p62 significantly increased and the average fluorescence intensity of LC3B decreased （P 〈 0.05）. Conclusion HF could reverse the expression of the autophagy-related proteins induced by ox-LDL, thereby resisting excessive autoph- agy during arteriosclerosis. This might be one of the possible anti-atherosclerosis mechanism of HF.