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[1]胡子旋,姚楠,黃丹娥,等.補腎強筋膠囊含藥血清對膝骨關(guān)節(jié)炎軟骨細胞模型自噬影響的實驗研究[J].中醫(yī)正骨,2025,37(01):38-44.
　HU Zixuan,YAO Nan,HUANG Dan'e,et al.The effect of Bushen Qiangjin Jiaonang(補腎強筋膠囊)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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補腎強筋膠囊含藥血清對膝骨關(guān)節(jié)炎軟骨細胞模型自噬影響的實驗研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期數(shù):
2025年01期
頁碼:
38-44
欄目:
基礎(chǔ)研究
出版日期:
2025-01-20

文章信息/Info

Title:
The effect of Bushen Qiangjin Jiaonang(補腎強筋膠囊)medicated serum on autophagy in knee osteoarthritis chondrocyte model:an experimental study
作者:
胡子旋1; 姚楠1; 黃丹娥1; 黃雪君2; 甘海寧2; 趙自明3; 陳玉興3
1.廣東省第二中醫(yī)院,廣東 廣州 510095; 2.廣東省中醫(yī)藥工程技術(shù)研究院,廣東 廣州 510095; 3.廣東省中醫(yī)藥研究開發(fā)重點實驗室,廣東 廣州 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
關(guān)鍵詞:
骨關(guān)節(jié)炎; 膝;  補腎強筋膠囊;  白細胞介素-1β;  軟骨細胞;  自吞噬;  腺苷一磷酸;  蛋白激酶類;  TOR絲氨酸-蘇氨酸蛋白激酶
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)/哺乳動物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路探討補腎強筋膠囊含藥血清對膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)軟骨細胞模型自噬的影響。方法:將體外培養(yǎng)的人軟骨細胞分為4組。正常組給予10%空白血清,模型組給予10%空白血清和10 ng·mL-1白細胞介素-1β(interleukin-1β,IL-1β),補腎強筋膠囊組給予10%補腎強筋膠囊含藥血清和10 ng·mL-1 IL-1β,AMPK抑制劑組給予10%補腎強筋膠囊含藥血清、10 ng·mL-1 IL-1β及10 μmol·L-1 Dorsomorphin(AMPK抑制劑)進行干預。干預24 h后以CCK-8法檢測細胞增殖率,以膜聯(lián)蛋白Ⅴ-異硫氰酸熒光素/碘化丙啶雙染法檢測細胞凋亡率,以丹酰尸胺法檢測細胞自噬率,以實時熒光定量PCR技術(shù)檢測Beclin1、微管相關(guān)蛋白1輕鏈3B(microtubule-associated protein 1 light chain 3B,LC3B)、UNC-51樣自噬激活激酶1(UNC-51 like autophagy activating kinase 1,ULK1)的mRNA表達水平,以Western blot法檢測Beclin1、LC3B-Ⅰ、LC3B-Ⅱ、ULK1、磷酸化AMPKα(phospho-AMPKα,p-AMPKα)、磷酸化mTOR(phospho-mTOR,p-mTOR)的蛋白表達水平。結(jié)果:①軟骨細胞增殖率。模型組的細胞增殖率低于正常組(P=0.000),補腎強筋膠囊組的細胞增殖率高于模型組(P=0.019),AMPK抑制劑組的細胞增殖率低于補腎強筋膠囊組(P=0.002)。②軟骨細胞凋亡率。模型組的細胞凋亡率高于正常組(P=0.000),補腎強筋膠囊組的細胞凋亡率低于模型組(P=0.000),AMPK抑制劑組的細胞凋亡率高于補腎強筋膠囊組(P=0.000)。③軟骨細胞自噬率。模型組的細胞自噬率低于正常組(P=0.000),補腎強筋膠囊組的細胞自噬率高于模型組(P=0.000),AMPK抑制劑組的細胞自噬率低于補腎強筋膠囊組(P=0.000)。④軟骨細胞自噬相關(guān)基因的mRNA表達水平。模型組Beclin1、LC3B、ULK1的mRNA表達水平均低于正常組(P=0.002,P=0.001,P=0.003),補腎強筋膠囊組Beclin1、LC3B、ULK1的mRNA表達水平均高于模型組(P=0.008,P=0.005,P=0.012),AMPK抑制劑組Beclin1、LC3B、ULK1的mRNA表達水平均低于補腎強筋膠囊組(P=0.000,P=0.038,P=0.004)。⑤軟骨細胞自噬相關(guān)基因的蛋白表達水平。模型組Beclin1、ULK1、p-AMPKα的蛋白表達水平及LC3B-Ⅱ/LC3B-Ⅰ比值均低于正常組(P=0.028,P=0.019,P=0.007,P=0.044),p-mTOR的蛋白表達水平高于正常組(P=0.025); 補腎強筋膠囊組Beclin1、ULK1、p-AMPKα的蛋白表達水平及LC3B-Ⅱ/LC3B-Ⅰ比值均高于模型組(P=0.004,P=0.048,P=0.040,P=0.043),p-mTOR的蛋白表達水平低于模型組(P=0.031); AMPK抑制劑組Beclin1、ULK1、p-AMPKα的蛋白表達水平及LC3B-Ⅱ/LC3B-Ⅰ比值均低于補腎強筋膠囊組(P=0.032,P=0.005,P=0.035,P=0.047),p-mTOR的蛋白表達水平高于補腎強筋膠囊組(P=0.033)。結(jié)論:補腎強筋膠囊含藥血清可能通過AMPK/mTOR通路上調(diào)KOA軟骨細胞模型的自噬水平,從而延緩KOA病程。
Abstract:
Objective:To explore the effect of Bushen Qiangjin Jiaonang(補腎強筋膠囊,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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備注/Memo

備注/Memo:
基金項目:廣東省中醫(yī)藥局科研項目(20222011); 廣東省醫(yī)學科研基金項目(B2022208); 廣東省第二中醫(yī)院科研創(chuàng)新基金項目(SEZYY2023A02)
通訊作者:姚楠 E-mail:[email protected]
更新日期/Last Update: 1900-01-01