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[1]沈默金,鄭珍萍,吳偉欣,等.龜鹿二仙膠含藥血清對膝骨關(guān)節(jié)炎小鼠軟骨細(xì)胞模型自噬和凋亡的影響及作用機(jī)制研究[J].中醫(yī)正骨,2025,37(01):30-37.
　SHEN Mojin,ZHENG Zhenping,WU Weixin,et al.Exploring the effects and mechanism of Guilu Erxian Jiao(龜鹿二仙膠)medicated serum on autophagy and apoptosis of knee osteoarthritis mice chondrocyte model:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(01):30-37.
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龜鹿二仙膠含藥血清對膝骨關(guān)節(jié)炎小鼠軟骨細(xì)胞模型自噬和凋亡的影響及作用機(jī)制研究()
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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:
30-37
欄目:
基礎(chǔ)研究
出版日期:
2025-01-20

文章信息/Info

Title:
Exploring the effects and mechanism of Guilu Erxian Jiao(龜鹿二仙膠)medicated serum on autophagy and apoptosis of knee osteoarthritis mice chondrocyte model:an experimental study
作者:
沈默金1; 鄭珍萍1; 吳偉欣1; 楊美鑫1; 顧富城1; 耿秋東1; 王和鳴1; 李楠2
1.福建中醫(yī)藥大學(xué)中醫(yī)學(xué)院,福建 福州 350122; 2.中醫(yī)骨傷及運動康復(fù)教育部重點實驗室,福建 福州 350122
Author(s):
SHEN Mojin1; ZHENG Zhenping1; WU Weixin1; YANG Meixin1; GU Fucheng1; GENG Qiudong1; WANG Heming1; LI Nan2
1.School of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 2.Key Laboratory of Orthopedics&Traumatology and Rehabilitation of Traditional Chinese Medicine of Ministry of Education,Fuzhou 350122,Fujian,China
關(guān)鍵詞:
骨關(guān)節(jié)炎; 膝;  龜鹿二仙膠;  軟骨細(xì)胞;  自吞噬;  細(xì)胞凋亡;  信號傳導(dǎo);  TOR絲氨酸-蘇氨酸蛋白激酶;  小鼠
Keywords:
osteoarthritis; knee;  Gui Lu Er Xian Jiao;  chondrocytes;  autophagy;  apoptosis;  signal transduction;  TOR serine-threonine kinases;  mice
摘要:
目的:探討龜鹿二仙膠含藥血清對白細(xì)胞介素-1β誘導(dǎo)的膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)小鼠軟骨細(xì)胞模型自噬和凋亡的影響及作用機(jī)制。方法:將10只SD大鼠隨機(jī)分為龜鹿二仙膠含藥血清制備組和空白血清制備組,分別給予龜鹿二仙膠藥液和生理鹽水灌胃,連續(xù)灌胃1周后,采集腹主動脈血,制備龜鹿二仙膠含藥血清和空白血清。取培養(yǎng)至第2代的C57BL/6小鼠膝關(guān)節(jié)軟骨細(xì)胞,分為空白血清組、模型組、龜鹿二仙膠含藥血清組。模型組、龜鹿二仙膠含藥血清組加入終濃度為10 ng·mL-1的白細(xì)胞介素-1β,誘導(dǎo)培養(yǎng)24 h后,空白血清組、模型組加入10%空白血清,龜鹿二仙膠含藥血清組加入10%龜鹿二仙膠含藥血清,繼續(xù)培養(yǎng)24 h。采用流式細(xì)胞分析儀檢測各組軟骨細(xì)胞凋亡率,采用電子顯微鏡觀察各組軟骨細(xì)胞自噬小體,分別采用實時定量PCR和Western Blot法檢測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)、UNC-51樣自噬激活激酶1(UNC-51 like autophagy activating kinase 1,ULK1)、自噬相關(guān)蛋白5(autophagy related protein 5,ATG5)、微管相關(guān)蛋白1輕鏈3B(microtubule associated protein 1 light chain 3B,LC3B)、B淋巴細(xì)胞瘤-2相關(guān)X蛋白(Bcl-2 associated X protein,Bax)、基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)-13、趨化因子配體2(chemokine ligand 2,CCL2)的mRNA和蛋白表達(dá)水平。結(jié)果:①軟骨細(xì)胞凋亡率檢測結(jié)果。模型組軟骨細(xì)胞凋亡率高于空白血清組(P=0.000),龜鹿二仙膠含藥血清組軟骨細(xì)胞凋亡率低于模型組(P=0.000)。②軟骨細(xì)胞自噬小體觀察結(jié)果。空白血清組可見雙層膜封閉的球狀自噬小體,模型組自噬小體數(shù)量較空白血清組減少,龜鹿二仙膠含藥血清組自噬小體數(shù)量較模型組增加。③軟骨細(xì)胞自噬和凋亡相關(guān)基因mRNA和蛋白表達(dá)水平檢測結(jié)果。模型組軟骨細(xì)胞AMPK、ULK-1、ATG5、LC3B、Bcl-2的mRNA和蛋白表達(dá)水平均低于空白血清組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000; P=0.002,P=0.000,P=0.000,P=0.000,P=0.000),mTOR、Bax、MMP-13、CCL2的mRNA和蛋白表達(dá)水平均高于空白血清組(P=0.002,P=0.000,P=0.000,P=0.000; P=0.004,P=0.000,P=0.000,P=0.000); 龜鹿二仙膠含藥血清組軟骨細(xì)胞AMPK、ULK1、ATG5、LC3B、Bcl-2的mRNA和蛋白表達(dá)水平均高于模型組(P=0.000,P=0.003,P=0.001,P=0.001,P=0.001; P=0.020,P=0.002,P=0.000,P=0.008,P=0.004),mTOR、Bax、MMP-13、CCL2的mRNA和蛋白表達(dá)水平均低于模型組(P=0.008,P=0.001,P=0.000,P=0.000; P=0.040,P=0.000,P=0.001,P=0.000)。結(jié)論:龜鹿二仙膠含藥血清能夠增強(qiáng)KOA軟骨細(xì)胞自噬,抑制其凋亡與細(xì)胞外基質(zhì)降解,且龜鹿二仙膠含藥血清可能通過激活A(yù)MPK/mTOR/ULK1信號通路發(fā)揮作用。
Abstract:
Objective:To explore the effects and underlying mechanism of Guilu Erxian Jiao(龜鹿二仙膠,GLEXJ)medicated serum on autophagy and apoptosis of interleukin(IL)-1β-induced knee osteoarthritis(KOA)mice chondrocyte model.Methods:Ten Sprague-Dawley(SD)rats were randomized into GLEXJ medicated serum preparation group and blank serum preparation group,with 5 ones in each group.The rats in the 2 groups were intervened by intragastric administration with GLEXJ solution and normal saline,respectively,twice a day for consecutive 7 days.Two hours after the end of the last intervention,the blood was drawn from the abdominal aorta of rats in each group for making GLEXJ medicated serum and blank serum.The chondrocytes extracted from the knee cartilage tissues of C57BL/6 mice were cultured,and the second-generation ones were collected and divided into blank serum group,model group and GLEXJ medicated serum group.The second-generation chondrocytes in model group and GLEXJ medicated serum group were intervened by IL-1β at a final concentration of 10 ng/mL.After 24-hour culture,the chondrocytes in blank serum group and model group were further intervened by 10% blank serum,whereas,the ones in GLEXJ medicated serum group by 10% GLEXJ medicated serum.After 24-hour culture,the apoptosis rate of chondrocytes in each group was detected by flow cytometry,and the autophagosomes in chondrocytes in each group were observed by electron microscopy.Furthermore,the mRNA and protein expression levels of adenosine monophosphate activated protein kinase(AMPK),mammalian target of rapamycin(mTOR),UNC-51 like autophagy activating kinase 1(ULK1),autophagy related protein 5(ATG5),microtubule associated protein 1 light chain 3B(LC3B),B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),matrix metalloproteinase(MMP)-13,and chemokine ligand 2(CCL2)were detected by using real-time quantitative PCR(RT-qPCR)and Western blotting,respectively.Results:①The apoptosis rate of chondrocytes.The apoptosis rate of chondrocytes was higher in model group compared to blank serum group and GLEXJ medicated serum group(P=0.000; P=0.000).②The autophagosomes in chondrocytes.The spherical autophagosomes enclosed by double membrane were observed in chondrocytes of blank serum group,besides,the number of autophagosomes decreased in model group compared to blank serum group,while that increased in GLEXJ medicated serum group compared to model group.③The mRNA and protein expression levels of autophagy and apoptosis-related genes in chondrocytes.The mRNA and protein expression levels of AMPK,ULK1,ATG5,LC3B and Bcl-2 in chondrocytes were lower,while,that of mTOR,Bax,MMP-13 and CCL2 were higher in model group compared to blank serum group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000; P=0.002,P=0.000,P=0.000,P=0.000,P=0.000; P=0.002,P=0.000,P=0.000,P=0.000; P=0.004,P=0.000,P=0.000,P=0.000).The mRNA and protein expression levels of AMPK,ULK1,ATG5,LC3B and Bcl-2 in chondrocytes were higher,while,that of mTOR,Bax,MMP-13 and CCL2 were lower in GLEXJ medicated serum group compared to model group(P=0.000,P=0.003,P=0.001,P=0.001,P=0.001; P=0.020,P=0.002,P=0.000,P=0.008,P=0.004; P=0.008,P=0.001,P=0.000,P=0.000; P=0.040,P=0.000,P=0.001,P=0.000).Conclusion:GLEXJ medicated serum can enhance the autophagy of KOA chondrocytes,thereby inhibiting their apoptosis and extracellular matrix degradation.It may exert the effects by activating the AMPK/mTOR/ULK1 signaling pathway.

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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金項目(81973880)
通訊作者:李楠 E-mail:[email protected]
更新日期/Last Update: 1900-01-01