龟鹿二仙胶激活PINK1/Parkin介导线粒体自噬诱导成骨细胞分化  

Guilu Erxian Gel activates PINK1/Parkin-mediated mitochondrial autophagy to induce osteoblast differentiation

作　　者：修禹 宋超 张榕升 邓志博 戴晗豪 苏议斌 杨林海 罗骏 徐杰 XIU Yu;SONG Chao;ZHANG Rongsheng;DENG Zhibo;DAI Hanhao;SU Yibin;YANG Linhai;LUO Jun;XU Jie(College of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350100,China;Second Department of Orthopedics,Fujian Provincial Hospital,Fuzhou 350001,China;Provincial Clinical Medical College,Fujian Medical University,Fuzhou 350001,China)

机构地区：[1]福建中医药大学中医学院,福建福州350100 [2]福建省立医院骨二科,福建福州350001 [3]福建医科大学省立临床医学院,福建福州350001

出　　处：《中国骨质疏松杂志》2025年第2期169-174,共6页Chinese Journal of Osteoporosis

基　　金：福建省科技创新联合基金项目重大项目(2021Y9023);福建省财政厅重大科技项目[闽财指(2022)783号;闽财指(2021)964号];福建省卫生健康科技计划重大项目(2021ZD01003)。

摘　　要：目的探究龟鹿二仙胶对MC3T3-E1细胞成骨分化及机制的影响。方法制备含龟鹿二仙胶含药血清,运用CCK-8法确定最适宜干预浓度和时间。将细胞分为胎牛血清组(FBS组)、经典诱导组(JDYD组)、空白血清组(KB组)、龟鹿二仙胶组(GL组)。使用WB检测Runx2等成骨分化相关蛋白、COMPLEXⅠ~Ⅴ等线粒体功能相关蛋白,PINK1、Parkin等线粒体自噬相关蛋白表达情况;通过IF染色检测COL1A1、OCN等成骨分化相关蛋白表达;利用ALP和ARS染色检测成骨分化和矿化程度;通过激光共聚焦显微镜检测PINK1、Parkin与Mitotracker共定位情况。结果最适浓度和干预时间分别为10%和72 h(P<0.0001);GL组COL1A1、Runx2、OCN、COMPLEXⅡ~Ⅴ、PINK1、Parkin、LC3BⅠ和LC3BⅡ蛋白表达显著高于FBS组和KB组(P<0.05),与JDYD组持平;GL组COL1A1、Runx2、OCN的荧光强度高于FBS组和KB组,与JDYD组相当;GL组ALP和ARS染色阳性区域比例显著高于FBS组和KB组,与JDYD组持平;GL组PINK1、Parkin与Mitotracker的共定位程度显著高于FBS组和KB组,与JDYD组相近。结论龟鹿二仙胶通过激活PINK1/Parkin通路介导线粒体自噬,进而起到促进成骨细胞分化的作用。Objective To investigate the effect of Guilu Erxian Gel on osteogenic differentiation of MC3T3-E1 cells and mechanism.Methods Guilu Erxian Gel-containing serum was prepared,and the CCK-8 method was used to determine the optimal intervention concentration and time.Cells were divided into FBS group,JDYD group,KB group,GL group.WB was used to detect the expression of COL1A1,Runx2,OCN,COMPLEX I-V,PINK1,Parkin,p62,LC3BⅠ/Ⅱ;IF staining was used to detect the expression of COL1A1,Runx2 and OCN;ALP and ARS staining were used to detect osteogenic differentiation and mineralization;Laser confocal microscopy was used to detect the colocalization of PINK1,Parkin and Mitotracker.Results The optimal concentration and intervention time were 10%and 72 h(P<0.0001);The expression of COL1A1,Runx2,OCN,COMPLEX II-V,PINK1,Parkin,LC3BⅠ,and LC3BⅡproteins in GL group was significantly higher than that in FBS group and KB group(P<0.05),and comparable to JDYD group;The fluorescence intensity of COL1A1,Runx2,OCN in GL group was higher than that in FBS group and KB group,and comparable to JDYD group;The proportion of positive areas in ALP and ARS staining in GL group was significantly higher than that in FBS group and KB group,and comparable to JDYD group;The colocalization degree of PINK1,Parkin,and Mitotracker in GL group was significantly higher than that in FBS group and KB group,and similar to JDYD group.Conclusion Guilu Erxian Gel may promote osteoblast differentiation by activating the PINK1/Parkin pathway to mediate mitochondrial autophagy.

关 键 词：成骨细胞 龟鹿二仙胶 线粒体自噬 PINK1 PARKIN 

分 类 号：R285.5[医药卫生—中药学]