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[1]陶振宇,張月雷,陳華,等.細胞自噬在酒精性股骨頭壞死中的作用及相關機制研究[J].中醫(yī)正骨,2018,30(10):4-11.
　TAO Zhenyu,ZHANG Yuelei,CHEN Hua,et al.Roles of cell autophagy in alcohol-induced osteonecrosis of the femoral head and its related mechanisms of action:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(10):4-11.
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細胞自噬在酒精性股骨頭壞死中的作用及相關機制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期數(shù):
2018年10期
頁碼:
4-11
欄目:
股骨頭壞死
出版日期:
2018-10-20

文章信息/Info

Title:
Roles of cell autophagy in alcohol-induced osteonecrosis of the femoral head and its related mechanisms of action:an experimental study
作者:
陶振宇; 張月雷; 陳華; 孫遼軍
(溫州醫(yī)科大學附屬第二醫(yī)院,浙江 溫州 325027)
Author(s):
TAO Zhenyu; ZHANG Yuelei; CHEN Hua; SUN Liaojun
The Second Affiliated Hospital of Wenzhou Medical University,Wenzhou 325027,Zhejiang,China
關鍵詞:
股骨頭壞死;  自噬;  西羅莫司;  小鼠;  動物實驗
Keywords:
femur head necrosis;  autophagy;  sirolimus;  mice;  animal experimentation
摘要:
目的:探討細胞自噬在酒精性股骨頭壞死中的作用及相關機制。方法:將15只1月齡SPF級C57小鼠隨機分為空白對照組、酒精組及雷帕霉素-酒精組,每組5只。空白對照組喂食飲用水6周,腹腔內(nèi)注射5 mg·kg-1不含雷帕霉素的溶劑; 酒精組喂食含30%酒精的飲用水6周,腹腔內(nèi)注射5 mg·kg-1不含雷帕霉素的溶劑; 雷帕霉素-酒精組喂食含30%酒精的飲用水6周,腹腔內(nèi)注射5 mg·kg-1的雷帕霉素溶液。腹腔內(nèi)注射每天1次,連續(xù)注射2周。酒精干預6周后,獲取各組小鼠右側(cè)股骨頭,行微型CT平掃后將股骨頭制成切片,蘇木素-伊紅染色后在顯微鏡下觀察股骨頭組織形態(tài),并計算空骨陷窩百分比(總細胞凋亡率)。取MC3T3-E1細胞,傳代培養(yǎng)至第3代,經(jīng)含酒精濃度為0 mmol·L-1、25 mmol·L-1、50 mmol·L-1、100 mmol·L-1、200 mmol·L-1的α-MEM培養(yǎng)基孵育后,采用CCK8法檢測其相對生長率。將培養(yǎng)好的第3代MC3T3-E1細胞隨機分為4組,分別以普通α-MEM培養(yǎng)基(正常組)、含100 mmol·L-1酒精的α-MEM培養(yǎng)基(含酒精組)、含100 nmol·L-1雷帕霉素的α-MEM培養(yǎng)基(含雷帕霉素組)、含100 mmol·L-1酒精和100 nmol·L-1雷帕霉素的α-MEM培養(yǎng)基(含酒精-雷帕霉素組)培養(yǎng),連續(xù)培養(yǎng)24 h; 采用Western blot法檢測MC3T3-E1細胞中微管相關蛋白1輕鏈3(microtubule-associated protein 1 light chain 3,LC3)-Ⅰ、LC3-Ⅱ、哺乳動物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、Beclin1蛋白表達量。結(jié)果:①股骨頭CT掃描結(jié)果。空白對照組小鼠股骨頭骨小梁大體形態(tài)良好,分布均勻,未見軟骨下壞死帶形成; 酒精組小鼠股骨頭軟骨下壞死帶形成,骨小梁閉塞,髓腔內(nèi)骨小梁變細、稀疏、斷裂,骨皮質(zhì)增厚; 雷帕霉素-酒精組小鼠股骨頭CT影像學表現(xiàn)介于酒精組與空白對照組之間,可見髓腔內(nèi)部分骨小梁出現(xiàn)斷裂萎縮,軟骨下骨部分壞死形成。②顯微鏡下股骨頭組織形態(tài)觀察結(jié)果。空白對照組小鼠股骨頭骨小梁形態(tài)良好,未見空骨陷窩、脂肪細胞及脂質(zhì)沉積,無組織壞死、纖維化; 酒精組小鼠股骨頭骨小梁稀疏、紊亂,脂肪細胞增多,骨細胞內(nèi)脂質(zhì)沉積,可見大量空骨陷窩形成,局部組織壞死、纖維化,細胞數(shù)目減少; 雷帕霉素-酒精組小鼠股骨頭部分骨小梁出現(xiàn)斷裂、萎縮,可見少量空骨陷窩形成。③總細胞凋亡率。空白對照組總細胞凋亡率為(6.06±1.93)%,酒精組為(63.9±9.63)%,雷帕霉素-酒精組為(34.0±5.82)%; 3組總細胞凋亡率比較,差異有統(tǒng)計學意義(F=192.800,P=0.000); 進一步兩兩比較,酒精組和雷帕霉素-酒精組總細胞凋亡率均大于空白對照組(P=0.000,P=0.000),酒精組總細胞凋亡率大于雷帕霉素-酒精組(P=0.000)。④不同濃度酒精干預后MC3T3-E1細胞相對生長率。在含酒精濃度為0 mmol·L-1、25 mmol·L-1、50 mmol·L-1、100 mmol·L-1、200 mmol·L-1的α-MEM培養(yǎng)基中培養(yǎng)24 h后MC3T3-E1細胞相對生長率比較,差異有統(tǒng)計學意義(1.00±0.00,0.88±0.04,0.67±0.09,0.31±0.04,0.28±0.02; F=106.900,P=0.001)。未經(jīng)酒精干預的MC3T3-E1細胞相對生長率高于濃度為25 mmol·L-1、50 mmol·L-1、100 mmol·L-1、200 mmol·L-1的酒精干預后的MC3T3-E1細胞相對生長率(P=0.011,P=0.000,P=0.000,P=0.000)。⑤MC3T3-E1細胞中LC3-Ⅱ/LC3-Ⅰ的比值及mTOR、Beclin-1的蛋白表達。4組小鼠MC3T3-E1細胞中LC3-Ⅱ/LC3-Ⅰ的比值比較,組間差異有統(tǒng)計學意義(1.16±0.10,0.71±0.06,1.50±0.06,1.23±0.05; F=86.600,P=0.000); 正常組MC3T3-E1細胞中LC3-Ⅱ/LC3-Ⅰ的比值高于含酒精組(P=0.000),低于含雷帕霉素組(P=0.000),與含酒精-雷帕霉素組比較差異無統(tǒng)計學意義(P=0.166); 含酒精組MC3T3-E1細胞中LC3-Ⅱ/LC3-Ⅰ的比值低于含雷帕霉素組及含酒精-雷帕霉素組(P=0.000,P=0.000); 含雷帕霉素組LC3-Ⅱ/LC3-Ⅰ的比值高于含酒精-雷帕霉素組(P=0.000)。4組小鼠MC3T3-E1細胞中mTOR蛋白表達量比較,組間差異有統(tǒng)計學意義(1.13±0.10,1.23±0.06,0.34±0.03,0.63±0.05; F=167.800,P=0.000); 正常組MC3T3-E1細胞中mTOR蛋白表達量低于含酒精組(P=0.033),高于含雷帕霉素組及含酒精-雷帕霉素組(P=0.000,P=0.000); 含酒精組MC3T3-E1細胞中mTOR蛋白表達量高于含雷帕霉素組及含酒精-雷帕霉素組(P=0.000,P=0.000),含雷帕霉素組mTOR蛋白表達量低于含酒精-雷帕霉素組(P=0.000)。4組小鼠MC3T3-E1細胞中Beclin-1蛋白表達量比較,組間差異有統(tǒng)計學意義(0.97±0.08,0.67±0.01,1.11±0.08,1.06±0.04; F=42.400,P=0.000); 正常組MC3T3-E1細胞中Beclin-1蛋白表達量高于含酒精組,而與含雷帕霉素組、含酒精-雷帕霉素組比較,組間差異均無統(tǒng)計學意義(P=0.077,P=0.127); 含酒精組MC3T3-E1細胞中Beclin-1蛋白表達量低于 含雷帕霉素組及含酒精-雷帕霉素組(P=0.000,P=0.000); 含雷帕霉素組MC3T3-E1細胞中Beclin-1蛋白表達量高于含酒精-雷帕霉素組(P=0.004)。結(jié)論:細胞自噬在酒精性股骨頭壞死的病變過程中可能發(fā)揮著非常重要的保護作用。酒精刺激引起的細胞自噬水平的降低可能是引起股骨頭壞死的原因之一,其作用機制可能與酒精刺激影響自噬關鍵調(diào)控分子mTOR、Beclin1的表達有關。
Abstract:
Objective:To explore the roles of cell autophagy in alcohol-induced osteonecrosis of the femoral head(ONFH)and its related mechanisms of action.Methods:Fifteen 1-month-old SPF-grade C57 mice were randomly divided into blank control group,alcohol group and rapamycin(RAPA)-alcohol group,5 mice in each group.The mice in blank control group,alcohol group and RAPA-alcohol group were fed with drinking water,drinking water added with 30% alcohol and drinking water added with 30% alcohol for 6 weeks respectively,and were intraperitoneal injected with solvent(5 mg/kg)without RAPA,solvent(5 mg/kg)without RAPA and RAPA solution(5 mg/kg)respectively.The intraperitoneal injection was performed once a day for consecutive 2 weeks.After six-week alcohol intervention,the right femoral heads were obtained from mice of each group and were sectioned after micro-CT plain scanning.The tissue morphology of femoral heads were observed under microscope after hematoxylin-eosin(HE)staining,and the percentage of empty bone lacuna(total cell apoptosis rate)was calculated.MC3T3-E1 cells were fetched out from femoral head tissues for subculturing.The relative growth rate(RGR)of the third-generation MC3T3-E1 cells was measured by using CCK8 method after the cells were cultured in α-MEM culture medium supplemented with alcohol with concentrations of 0,25,50,100 and 200 mmol/L respectively.The third-generation MC3T3-E1 cells were randomly divided into 4 groups and were cultured for continuous 24 hours in normal α-MEM culture medium(normal group),α-MEM culture medium supplemented with alcohol with concentration of 100 mmol/L(alcoholic group),α-MEM culture medium supplemented with RAPA with concentration of 100 nmol/L(RAPA group),and α-MEM culture medium supplemented with alcohol and RAPA with concentration of 100 mmol/L and 100 nmol/L(alcoholic-RAPA group)respectively.The protein expressions of microtubule-associated protein 1 light chain 3(LC3)-Ⅰ,LC3-Ⅱ,mammalian target of rapamycin(mTOR)and Beclin1 in MC3T3-E1 cells were detected by using Western blot assays.Results:The results of CT scanning on femoral heads showed that(1)the trabecular bones had good gross morphology and were uniformly distributed,and no subchondral necrotic zone was found in femoral heads in blank control group;(2)the subchondral necrotic zone,occluded trabecular bones,incrassated bone cortex and thin,sparse and fractured intramedullary trabecular bones were found in femoral heads in alcoholic group;(3)the CT imaging manifestations of femoral heads of mice in RAPA-alcohol group were between alcoholic group's and blank control group's,and some fractured and atrophic trabecular bones were found in medullary cavity and partial osteonecrosis of subcartilaginous bone formed.The results of morphological observation on femoral heads under the microscope showed that(1)the trabecular bone had good morphology and no empty bone lacuna,adipocyte,lipid deposition,tissue necrosis and fibrosis were found in femoral heads in blank control group;(2)the trabecular bones were sparse and disordered,and the adipocytes increased in number,and lipidoses was found in boen cells,meanwhile,a large number of empty bone lacuna formed,and necrosis and fibrosis were found in partial tissues,and cells decreased in number in femoral heads in alcoholic group;(3)some fractured and atrophic trabecular bones and a small number of empty bone lacuna were found in femoral heads in RAPA-alcohol group.The total cell apoptosis rates of blank control group,alcohol group and RAPA-alcohol group were 6.06+/-1.93,63.9+/-9.63 and 34.0+/-5.82% respectively.There was statistical difference in the total cell apoptosis rates between the 3 groups(F=192.800,P=0.000).Further pairwise comparison showed that the total cell apoptosis rates were higher in alcohol group and RAPA-alcohol group compared to blank control group,and were higher in alcohol group compared to RAPA-alcohol group(P=0.000,P=0.000; P=0.000).After 24-hour cultivation in α-MEM culture medium supplemented with alcohol with concentrations of 0,25,50,100 and 200 mmol/L,there was statistical difference in RGR of MC3T3-E1 cells(1.00+/-0.00,0.88+/-0.04,0.67+/-0.09,0.31+/-0.04,0.28+/-0.02; F=106.900,P=0.001).The RGR of MC3T3-E1 cells without alcohol intervention was higher than that of MC3T3-E1 cells intervened by alcohol with concentrations of 25,50,100 and 200 mmol/L(P=0.011,P=0.000,P=0.000,P=0.000).There was statistical difference in the ratio of LC3-Ⅱ to LC3-Ⅰin MC3T3-E1 cells of mice between the 4 groups(1.16+/-0.10,0.71+/-0.06,1.50+/-0.06,1.23+/-0.05; F=86.600,P=0.000).The ratio of LC3-Ⅱ to LC3-Ⅰin MC3T3-E1 cells was lower in alcoholic group and was higher in RAPA group compared to normal group(P=0.000; P=0.000),and there was no statistical difference between normal group and alcoholic-RAPA group(P=0.166).The ratio of LC3-Ⅱ to LC3-Ⅰin MC3T3-E1 cells was lower in alcoholic group compared to RAPA group and alcoholic-RAPA group,and was higher in RAPA group compared to alcoholic-RAPA group(P=0.000,P=0.000; P=0.000).There was statistical difference in the protein expression of mTOR in MC3T3-E1 cells between the 4 groups(1.13+/-0.10,1.23+/-0.06,0.34+/-0.03,0.63+/-0.05; F=167.800,P=0.000).The protein expression of mTOR in MC3T3-E1 cells was higher in alcoholic group and was lower in RAPA group and alcoholic-RAPA group compared to normal group(P=0.033; P=0.000,P=0.000).The protein expression of mTOR in MC3T3-E1 cells was higher in alcoholic group compared to RAPA group and alcoholic-RAPA group,and was lower in RAPA group compared to alcoholic-RAPA group(P=0.000,P=0.000; P=0.000).There was statistical difference in the protein expression of Beclin-1 in MC3T3-E1 cells between the 4 groups(0.97+/-0.08,0.67+/-0.01,1.11+/-0.08,1.06+/-0.04; F=42.400,P=0.000).The protein expression of Beclin-1 in MC3T3-E1 cells was higher in normal group compared to alcoholic group,and there was no statistical difference between normal group and RAPA group and between normal group and alcoholic-RAPA group(P=0.077,P=0.127).The protein expression of Beclin-1 in MC3T3-E1 cells was lower in alcoholic group compared to RAPA group and alcoholic-RAPA group,and was higher in RAPA group compared to alcoholic-RAPA group(P=0.000,P=0.000; P=0.004).Conclusion:Cell autophagy may play a very important protective role in the pathological process of alcohol-induced ONFH.The decreased autophagy level caused by alcohol stimulation may be one of the reasons for causing ONFH,and its mechanism of action may be related to the influence of alcohol stimulation on the expression of key regulatory molecules(mTOR and Beclin1)in autophagy.

參考文獻/References:


[1] 陳志偉,王振濤,李泰賢,等.酒精相關性股骨頭壞死的發(fā)病特征分析[J].中醫(yī)正骨,2017,29(12):23-25.
[2] HERRERA-SOTO JA,PRICE CT.Core decompression for juvenile osteonecrosis[J].Orthop Clin North Am,2011,42(3):429-436.
[3] 惠銀銀,劉又斌,王晶,等.非創(chuàng)傷性股骨頭壞死病因的研究進展[J].中醫(yī)正骨,2018,30(2):33-36.
[4] MAUREL DB,BOISSEAU N,BENHAMOU CL,et al.Alcohol and bone:review of dose effects and mechanisms[J].Osteoporos Int,2012,23(1):1-16.
[5] KANG JS,PARK S,SONG JH,et al.Prevalence of osteonecrosis of the femoral head:a nationwide epidemiologic analysis in Korea[J].J Arthroplasty,2009,24(8):1178-1183. 中醫(yī)正骨2018年10月第30卷第10期 J Trad Chin Orthop Trauma,2018,Vol.30,No.10(總731) (總732)中醫(yī)正骨2018年10月第30卷第10期 J Trad Chin Orthop Trauma,2018,Vol.30,No.10
[6] YANG Z,KLIONSKY DJ.Eaten alive:a history of macroautophagy[J].Nat Cell Biol,2010,12(9):814-822.
[7] CARAMES B,HASEGAWA A,TANIGUCHI N,et al.Autophagy activation by rapamycin reduces severity of experimental osteoarthritis[J].Ann Rheum Dis,2012,71(4):575-581.
[8] LOTZ MK,CARAMÉS B.Autophagy and cartilage homeostasis mechanisms in joint health,aging and OA[J].Nat Rev Rheumatol,2011,7(10):579-587.
[9] SASAKI H,TAKAYAMA K,MATSUSHITA T,et al.Autophagy modulates osteoarthritis-related gene expression in human chondrocytes[J].Arthritis Rheum,2012,64(6):1920-1928.
[10] YANG RT,ZHANG C,LIU Y,et al.Autophagy prior to chondrocyte cell death during the degeneration of Meckel's cartilage[J].Anat Rec(Hoboken),2012,295(5):734-741.
[11] 陳鎮(zhèn)秋,何偉,魏秋實.股骨頭壞死中醫(yī)證型與血液學指標的關系研究[J].中醫(yī)正骨,2013,25(3):35-40.
[12] ZHANG Q,YANG YJ,WANG H,et al.Autophagy activation:a novel mechanism of atorvastatin to protect mesenchymal stem cells from hypoxia and serum deprivation via AMP-Activated protein kinase/mammalian target of rapamycin pathway[J].Stem Cells Dev,2012,21(8):1321-1332.
[13] XIA X,KAR R,GLUHAK-HEINRICH J,et al.Glucocorticoid-induced autophagy in osteocytes[J].J Bone Miner Res,2010,25(11):2479-2488.
[14] PATTINGRE S,ESPERT L,BIARD-PIECHACZYK M,et al.Regulation of macroautophagy by mTOR and Beclin 1 complexes[J].Biochimie,2008,90(2):313-323.
[15] ESKELINEN EL.New insights into the mechanisms of macroautophagy in mammalian cells[J].Int Rev Cell Mol Biol,2008,266:207-247.
[16] SCOTT RC,SCHULDINER O,NEUFELD TP.Role and regulation of starvation-induced autophagy in the drosophila fat body[J].Dev Cell,2004,7(2):167-178.
[17] ROSENFELDT MT,RYAN KM.The role of autophagy in tumour development and cancer therapy[J].Expert Rev Mol Med,2009,11:36.
[18] SCARLATTI F,MAFFEI R,BEAU I,et al.Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells[J].Cell Death Differ,2008,15(8):1318-1329.
[19] GAYTÁN M,MORALES C,SÁNCHEZ-CRIADO JE,et al.Immunolocalization of beclin 1,a bcl-2-binding,autophagy-related protein,in the human ovary:possible relation to Life span of corpus luteum[J].Cell Tissue Res,2008,331(2):509-517.
[20] KOROLCHUK VI,SAIKI S,LICHTENBERG M,et al.Lysosomal positioning coordinates cellular nutrient responses[J].Nat Cell Biol,2011,13(4):453-460.
[21] POÜS C,CODOGNO P.Lysosome positioning coordinates mTORC1 activity and autophagy[J].Nat Cell Biol,2011,13(4):342-344.

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[5]張兵,馬凰富,劉波,等.非創(chuàng)傷性股骨頭壞死的舌象定量研究[J].中醫(yī)正骨,2015,27(04):8.
　ZHANG Bing,MA Huangfu,LIU Bo,et al.Quantitative study on tongue manifestation of patients with nontraumatic osteonecrosis of femoral head[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(10):8.
[6]李剛,程春生.股骨頭壞死血瘀證癥狀與平樂郭氏正骨方藥藥物組成 最大頻繁關聯(lián)模式挖掘[J].中醫(yī)正骨,2015,27(04):21.
　LI Gang,CHENG Chunsheng.Maximal frequent association pattern mining for studying the relationship between BLOOD STASIS syndrome in patients with osteonecrosis of femoral head and drug components of Pingle Guo's orthopedics prescription[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(10):21.
[7]李文龍,梅沉成,杜貴強,等.微創(chuàng)減壓植骨多孔鉭棒植入治療 ARCOⅡ期非創(chuàng)傷性缺血性股骨頭壞死[J].中醫(yī)正骨,2016,28(02):43.
[8]張磊,金紅婷,童培建.骨健口服液早期干預非創(chuàng)傷性股骨頭壞死的臨床研究[J].中醫(yī)正骨,2016,28(03):14.
　ZHANG Lei,JIN Hongting,TONG Peijian.Clinical study on Gujian Koufuye(骨健口服液)for early intervention of nontraumatic osteonecrosis of femoral head[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(10):14.
[9]鮑榮華,王國平,夏曉斌,等.直接前入路微創(chuàng)全髖關節(jié)置換術治療晚期股骨頭壞死[J].中醫(yī)正骨,2016,28(03):61.
[10]張超,姚晨,沈計榮.微創(chuàng)髓芯減壓打壓支撐植骨術結(jié)合補腎活血湯口服治療ARCOⅡ、Ⅲ期股骨頭壞死的近期療效觀察[J].中醫(yī)正骨,2016,28(03):20.
　ZHANG Chao,YAO Chen,SHEN Jirong.[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(10):20.

備注/Memo

備注/Memo:
基金項目:浙江省醫(yī)藥衛(wèi)生科技計劃項目(2014KYB160)
通訊作者:孫遼軍 E-mail:[email protected]
更新日期/Last Update: 2019-02-25