[1]胡子旋,姚楠,黄丹娥,等.补肾强筋胶囊含药血清对膝骨关节炎软骨细胞模型自噬影响的实验研究[J].中医正骨,2025,37(01):38-44.
　HU Zixuan,YAO Nan,HUANG Dan'e,et al.The effect of Bushen Qiangjin Jiaonang(补肾强筋胶囊)medicated serum on autophagy in knee osteoarthritis chondrocyte model:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(01):38-44.
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补肾强筋胶囊含药血清对膝骨关节炎软骨细胞模型自噬影响的实验研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年01期
页码:
38-44
栏目:
基础研究
出版日期:
2025-01-20

文章信息/Info

Title:
The effect of Bushen Qiangjin Jiaonang(补肾强筋胶囊)medicated serum on autophagy in knee osteoarthritis chondrocyte model:an experimental study
作者:
胡子旋1; 姚楠1; 黄丹娥1; 黄雪君2; 甘海宁2; 赵自明3; 陈玉兴3
1.广东省第二中医院,广东 广州 510095; 2.广东省中医药工程技术研究院,广东 广州 510095; 3.广东省中医药研究开发重点实验室,广东 广州 510095
Author(s):
HU Zixuan1; YAO Nan1; HUANG Dan'e1; HUANG Xuejun2; GAN Haining2; ZHAO Ziming3; CHEN Yuxing3
1.Guangdong Provincial Second Hospital of Traditional Chinese Medicine,Guangzhou 510095,Guangdong,China 2.Guangdong Provincial Engineering Technology Research Institute of Traditional Chinese Medicine,Guangzhou 510095,Guangdong,China 3.Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine,Guangzhou 510095,Guangdong,China
关键词:
骨关节炎; 膝;  补肾强筋胶囊;  白细胞介素-1β;  软骨细胞;  自吞噬;  腺苷一磷酸;  蛋白激酶类;  TOR丝氨酸-苏氨酸蛋白激酶
Keywords:
osteoarthritis; knee;  Bushen Qiangjin Jiaonang;  interleukin-1β;  chondrocytes;  autophagy;  adenosine monophosphate;  protein kinases;  TOR serine-threonine kinases
摘要:
目的:基于腺苷一磷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路探讨补肾强筋胶囊含药血清对膝骨关节炎(knee osteoarthritis,KOA)软骨细胞模型自噬的影响。方法:将体外培养的人软骨细胞分为4组。正常组给予10%空白血清,模型组给予10%空白血清和10 ng·mL-1白细胞介素-1β(interleukin-1β,IL-1β),补肾强筋胶囊组给予10%补肾强筋胶囊含药血清和10 ng·mL-1 IL-1β,AMPK抑制剂组给予10%补肾强筋胶囊含药血清、10 ng·mL-1 IL-1β及10 μmol·L-1 Dorsomorphin(AMPK抑制剂)进行干预。干预24 h后以CCK-8法检测细胞增殖率,以膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶双染法检测细胞凋亡率,以丹酰尸胺法检测细胞自噬率,以实时荧光定量PCR技术检测Beclin1、微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3B,LC3B)、UNC-51样自噬激活激酶1(UNC-51 like autophagy activating kinase 1,ULK1)的mRNA表达水平,以Western blot法检测Beclin1、LC3B-Ⅰ、LC3B-Ⅱ、ULK1、磷酸化AMPKα(phospho-AMPKα,p-AMPKα)、磷酸化mTOR(phospho-mTOR,p-mTOR)的蛋白表达水平。结果:①软骨细胞增殖率。模型组的细胞增殖率低于正常组(P=0.000),补肾强筋胶囊组的细胞增殖率高于模型组(P=0.019),AMPK抑制剂组的细胞增殖率低于补肾强筋胶囊组(P=0.002)。②软骨细胞凋亡率。模型组的细胞凋亡率高于正常组(P=0.000),补肾强筋胶囊组的细胞凋亡率低于模型组(P=0.000),AMPK抑制剂组的细胞凋亡率高于补肾强筋胶囊组(P=0.000)。③软骨细胞自噬率。模型组的细胞自噬率低于正常组(P=0.000),补肾强筋胶囊组的细胞自噬率高于模型组(P=0.000),AMPK抑制剂组的细胞自噬率低于补肾强筋胶囊组(P=0.000)。④软骨细胞自噬相关基因的mRNA表达水平。模型组Beclin1、LC3B、ULK1的mRNA表达水平均低于正常组(P=0.002,P=0.001,P=0.003),补肾强筋胶囊组Beclin1、LC3B、ULK1的mRNA表达水平均高于模型组(P=0.008,P=0.005,P=0.012),AMPK抑制剂组Beclin1、LC3B、ULK1的mRNA表达水平均低于补肾强筋胶囊组(P=0.000,P=0.038,P=0.004)。⑤软骨细胞自噬相关基因的蛋白表达水平。模型组Beclin1、ULK1、p-AMPKα的蛋白表达水平及LC3B-Ⅱ/LC3B-Ⅰ比值均低于正常组(P=0.028,P=0.019,P=0.007,P=0.044),p-mTOR的蛋白表达水平高于正常组(P=0.025); 补肾强筋胶囊组Beclin1、ULK1、p-AMPKα的蛋白表达水平及LC3B-Ⅱ/LC3B-Ⅰ比值均高于模型组(P=0.004,P=0.048,P=0.040,P=0.043),p-mTOR的蛋白表达水平低于模型组(P=0.031); AMPK抑制剂组Beclin1、ULK1、p-AMPKα的蛋白表达水平及LC3B-Ⅱ/LC3B-Ⅰ比值均低于补肾强筋胶囊组(P=0.032,P=0.005,P=0.035,P=0.047),p-mTOR的蛋白表达水平高于补肾强筋胶囊组(P=0.033)。结论:补肾强筋胶囊含药血清可能通过AMPK/mTOR通路上调KOA软骨细胞模型的自噬水平,从而延缓KOA病程。
Abstract:
Objective:To explore the effect of Bushen Qiangjin Jiaonang(补肾强筋胶囊,BSQJJN)medicated serum on autophagy in knee osteoarthritis(KOA)chondrocyte model based on the adenosine monophosphate activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)pathway.Methods:The human primary chondrocytes cultured in vitro were divided into normal group,model group,BSQJJN group,and AMPK inhibitor group.The chondrocytes in normal group were intervened with 10% blank serum,the ones in model group with 10% blank serum and 10 ng/mL interleukin-1β(IL-1β),the ones in BSQJJN group with 10% BSQJJN medicated serum and 10 ng/mL IL-1β,and the ones in AMPK inhibitor group with 10% BSQJJN medicated serum,10 ng/mL IL-1β and 10 μmol/L Dorsomorphin(an AMPK inhibitor).After 24-hour intervention,the chondrocyte proliferation rate,chondrocyte apoptosis rate,and chondrocyte autophagy rate were detected by CCK-8 assay,annexin V-fluorescein isothiocyanate/propidium iodide(FITC/PI)double staining,and dansyl cadaverine method,respectively.Furthermore,the mRNA expression levels of Beclin1,microtubule-associated protein 1 light chain 3B(LC3B),and UNC-51 like autophagy activating kinase 1(ULK1)were detected by real-time fluorescence quantitative PCR technology,and the protein expression levels of Beclin1,LC3B-I,LC3B-II,ULK1,phospho-AMPKα(p-AMPKα),and phospho-mTOR(p-mTOR)were detected by using Western blot.Results:①The chondrocyte proliferation rate.The chondrocyte proliferation rate was lower in model group compared to normal group(P=0.000),and was higher in BSQJJN group compared to model group(P=0.019),and was lower in AMPK inhibitor group compared to BSQJJN group(P=0.002).②The chondrocyte apoptosis rate.The chondrocyte apoptosis rate was higher in model group compared to normal group(P=0.000),and was lower in BSQJJN group compared to model group(P=0.000),and was higher in AMPK inhibitor group compared to BSQJJN group(P=0.000).③The chondrocyte autophagy rate.The chondrocyte autophagy rate was lower in model group compared to normal group(P=0.000),and was higher in BSQJJN group compared to model group(P=0.000),and was lower in AMPK inhibitor group compared to BSQJJN group(P=0.000).④The mRNA expression levels of chondrocyte autophagy-related genes.The mRNA expression levels of Beclin1,LC3B and ULK1 were lower in model group compared to normal group(P=0.002,P=0.001,P=0.003),and were higher in BSQJJN group compared to model group(P=0.008,P=0.005,P=0.012),and were lower in AMPK inhibitor group compared to BSQJJN group(P=0.000,P=0.038,P=0.004).⑤The protein expression levels of chondrocyte autophagy-related genes.The protein expression levels of Beclin1,ULK1,p-AMPKα and the ratio of LC3B-Ⅱto LC3B-Ⅰwere lower,while the protein expression level of p-mTOR was higher in model group compared to normal group(P=0.028,P=0.019,P=0.007,P=0.044,P=0.025).The protein expression levels of Beclin1,ULK1,p-AMPKα and the ratio of LC3B-Ⅱto LC3B-Ⅰwere higher,while the protein expression level of p-mTOR was lower in BSQJJN group compared to model group(P=0.004,P=0.048,P=0.040,P=0.043,P=0.031).The protein expression levels of Beclin1,ULK1,p-AMPKα and the ratio of LC3B-Ⅱto LC3B-Ⅰwere lower,while the protein expression level of p-mTOR was higher in AMPK inhibitor group compared to BSQJJN group(P=0.032,P=0.005,P=0.035,P=0.047,P=0.033).Conclusion:BSQJJN medicated serum may up-regulate the autophagy level of KOA chondrocyte model through the AMPK/mTOR pathway,thereby delaying the progression of KOA.

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备注/Memo

备注/Memo:
基金项目:广东省中医药局科研项目(20222011); 广东省医学科研基金项目(B2022208); 广东省第二中医院科研创新基金项目(SEZYY2023A02)
通讯作者:姚楠 E-mail:nanyao2000@126.com
更新日期/Last Update: 1900-01-01