[1]陶振宇,张月雷,陈华,等.细胞自噬在酒精性股骨头坏死中的作用及相关机制研究[J].中医正骨,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.
点击复制

细胞自噬在酒精性股骨头坏死中的作用及相关机制研究()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期数:
2018年10期
页码:
4-11
栏目:
股骨头坏死
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; 张月雷; 陈华; 孙辽军
(温州医科大学附属第二医院,浙江 温州 325027)
Author(s):
TAO Zhenyu; ZHANG Yuelei; CHEN Hua; SUN Liaojun
The Second Affiliated Hospital of Wenzhou Medical University,Wenzhou 325027,Zhejiang,China
关键词:
股骨头坏死;  自噬;  西罗莫司;  小鼠;  动物实验
Keywords:
femur head necrosis;  autophagy;  sirolimus;  mice;  animal experimentation
摘要:
目的:探讨细胞自噬在酒精性股骨头坏死中的作用及相关机制。方法:将15只1月龄SPF级C57小鼠随机分为空白对照组、酒精组及雷帕霉素-酒精组,每组5只。空白对照组喂食饮用水6周,腹腔内注射5 mg·kg-1不含雷帕霉素的溶剂; 酒精组喂食含30%酒精的饮用水6周,腹腔内注射5 mg·kg-1不含雷帕霉素的溶剂; 雷帕霉素-酒精组喂食含30%酒精的饮用水6周,腹腔内注射5 mg·kg-1的雷帕霉素溶液。腹腔内注射每天1次,连续注射2周。酒精干预6周后,获取各组小鼠右侧股骨头,行微型CT平扫后将股骨头制成切片,苏木素-伊红染色后在显微镜下观察股骨头组织形态,并计算空骨陷窝百分比(总细胞凋亡率)。取MC3T3-E1细胞,传代培养至第3代,经含酒精浓度为0 mmol·L-1、25 mmol·L-1、50 mmol·L-1、100 mmol·L-1、200 mmol·L-1的α-MEM培养基孵育后,采用CCK8法检测其相对生长率。将培养好的第3代MC3T3-E1细胞随机分为4组,分别以普通α-MEM培养基(正常组)、含100 mmol·L-1酒精的α-MEM培养基(含酒精组)、含100 nmol·L-1雷帕霉素的α-MEM培养基(含雷帕霉素组)、含100 mmol·L-1酒精和100 nmol·L-1雷帕霉素的α-MEM培养基(含酒精-雷帕霉素组)培养,连续培养24 h; 采用Western blot法检测MC3T3-E1细胞中微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)-Ⅰ、LC3-Ⅱ、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、Beclin1蛋白表达量。结果:①股骨头CT扫描结果。空白对照组小鼠股骨头骨小梁大体形态良好,分布均匀,未见软骨下坏死带形成; 酒精组小鼠股骨头软骨下坏死带形成,骨小梁闭塞,髓腔内骨小梁变细、稀疏、断裂,骨皮质增厚; 雷帕霉素-酒精组小鼠股骨头CT影像学表现介于酒精组与空白对照组之间,可见髓腔内部分骨小梁出现断裂萎缩,软骨下骨部分坏死形成。②显微镜下股骨头组织形态观察结果。空白对照组小鼠股骨头骨小梁形态良好,未见空骨陷窝、脂肪细胞及脂质沉积,无组织坏死、纤维化; 酒精组小鼠股骨头骨小梁稀疏、紊乱,脂肪细胞增多,骨细胞内脂质沉积,可见大量空骨陷窝形成,局部组织坏死、纤维化,细胞数目减少; 雷帕霉素-酒精组小鼠股骨头部分骨小梁出现断裂、萎缩,可见少量空骨陷窝形成。③总细胞凋亡率。空白对照组总细胞凋亡率为(6.06±1.93)%,酒精组为(63.9±9.63)%,雷帕霉素-酒精组为(34.0±5.82)%; 3组总细胞凋亡率比较,差异有统计学意义(F=192.800,P=0.000); 进一步两两比较,酒精组和雷帕霉素-酒精组总细胞凋亡率均大于空白对照组(P=0.000,P=0.000),酒精组总细胞凋亡率大于雷帕霉素-酒精组(P=0.000)。④不同浓度酒精干预后MC3T3-E1细胞相对生长率。在含酒精浓度为0 mmol·L-1、25 mmol·L-1、50 mmol·L-1、100 mmol·L-1、200 mmol·L-1的α-MEM培养基中培养24 h后MC3T3-E1细胞相对生长率比较,差异有统计学意义(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)。未经酒精干预的MC3T3-E1细胞相对生长率高于浓度为25 mmol·L-1、50 mmol·L-1、100 mmol·L-1、200 mmol·L-1的酒精干预后的MC3T3-E1细胞相对生长率(P=0.011,P=0.000,P=0.000,P=0.000)。⑤MC3T3-E1细胞中LC3-Ⅱ/LC3-Ⅰ的比值及mTOR、Beclin-1的蛋白表达。4组小鼠MC3T3-E1细胞中LC3-Ⅱ/LC3-Ⅰ的比值比较,组间差异有统计学意义(1.16±0.10,0.71±0.06,1.50±0.06,1.23±0.05; F=86.600,P=0.000); 正常组MC3T3-E1细胞中LC3-Ⅱ/LC3-Ⅰ的比值高于含酒精组(P=0.000),低于含雷帕霉素组(P=0.000),与含酒精-雷帕霉素组比较差异无统计学意义(P=0.166); 含酒精组MC3T3-E1细胞中LC3-Ⅱ/LC3-Ⅰ的比值低于含雷帕霉素组及含酒精-雷帕霉素组(P=0.000,P=0.000); 含雷帕霉素组LC3-Ⅱ/LC3-Ⅰ的比值高于含酒精-雷帕霉素组(P=0.000)。4组小鼠MC3T3-E1细胞中mTOR蛋白表达量比较,组间差异有统计学意义(1.13±0.10,1.23±0.06,0.34±0.03,0.63±0.05; F=167.800,P=0.000); 正常组MC3T3-E1细胞中mTOR蛋白表达量低于含酒精组(P=0.033),高于含雷帕霉素组及含酒精-雷帕霉素组(P=0.000,P=0.000); 含酒精组MC3T3-E1细胞中mTOR蛋白表达量高于含雷帕霉素组及含酒精-雷帕霉素组(P=0.000,P=0.000),含雷帕霉素组mTOR蛋白表达量低于含酒精-雷帕霉素组(P=0.000)。4组小鼠MC3T3-E1细胞中Beclin-1蛋白表达量比较,组间差异有统计学意义(0.97±0.08,0.67±0.01,1.11±0.08,1.06±0.04; F=42.400,P=0.000); 正常组MC3T3-E1细胞中Beclin-1蛋白表达量高于含酒精组,而与含雷帕霉素组、含酒精-雷帕霉素组比较,组间差异均无统计学意义(P=0.077,P=0.127); 含酒精组MC3T3-E1细胞中Beclin-1蛋白表达量低于 含雷帕霉素组及含酒精-雷帕霉素组(P=0.000,P=0.000); 含雷帕霉素组MC3T3-E1细胞中Beclin-1蛋白表达量高于含酒精-雷帕霉素组(P=0.004)。结论:细胞自噬在酒精性股骨头坏死的病变过程中可能发挥着非常重要的保护作用。酒精刺激引起的细胞自噬水平的降低可能是引起股骨头坏死的原因之一,其作用机制可能与酒精刺激影响自噬关键调控分子mTOR、Beclin1的表达有关。
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.

参考文献/References:


[1] 陈志伟,王振涛,李泰贤,等.酒精相关性股骨头坏死的发病特征分析[J].中医正骨,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] 惠银银,刘又斌,王晶,等.非创伤性股骨头坏死病因的研究进展[J].中医正骨,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. 中医正骨2018年10月第30卷第10期 J Trad Chin Orthop Trauma,2018,Vol.30,No.10(总731) (总732)中医正骨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] 陈镇秋,何伟,魏秋实.股骨头坏死中医证型与血液学指标的关系研究[J].中医正骨,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.

相似文献/References:

[1]陈雷雷,张颖.何伟教授采用中医药疗法治疗股骨头坏死的经验[J].中医正骨,2015,27(10):74.
[2]徐西林,赵永兰,张晓峰,等.活骨注射液髋关节腔灌注对兔股骨头坏死模型 血管内皮生长因子表达的动态影响[J].中医正骨,2015,27(08):1.
　XU Xilin,ZHAO Yonglan,ZHANG Xiaofeng,et al.Dynamic effect of intra-articular hip injection of Huogu injection on the expression of vascular endothelial growth factor in rabbit models with femur head necrosis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(10):1.
[3]周勇,任菲菲,丰凡翔,等.血管内皮生长因子和骨形态发生蛋白2 在非创伤性股骨头坏死不同区域的表达[J].中医正骨,2015,27(08):7.
　ZHOU Yong,REN Feifei,FENG Fanxiang,et al.Expressions of vascular endothelial growth factor and bone morphogenetic protein 2 in different zones of femoral head with non-traumatic osteonecrosis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(10):7.
[4]鲍荣华,王国平,夏晓斌,等.钽棒植入治疗非创伤性股骨头坏死的疗效观察[J].中医正骨,2015,27(02):28.
[5]张兵,马凰富,刘波,等.非创伤性股骨头坏死的舌象定量研究[J].中医正骨,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]李刚,程春生.股骨头坏死血瘀证症状与平乐郭氏正骨方药药物组成 最大频繁关联模式挖掘[J].中医正骨,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]李文龙,梅沉成,杜贵强,等.微创减压植骨多孔钽棒植入治疗 ARCOⅡ期非创伤性缺血性股骨头坏死[J].中医正骨,2016,28(02):43.
[8]张磊,金红婷,童培建.骨健口服液早期干预非创伤性股骨头坏死的临床研究[J].中医正骨,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]鲍荣华,王国平,夏晓斌,等.直接前入路微创全髋关节置换术治疗晚期股骨头坏死[J].中医正骨,2016,28(03):61.
[10]张超,姚晨,沈计荣.微创髓芯减压打压支撑植骨术结合补肾活血汤口服治疗ARCOⅡ、Ⅲ期股骨头坏死的近期疗效观察[J].中医正骨,2016,28(03):20.
　ZHANG Chao,YAO Chen,SHEN Jirong.[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(10):20.

备注/Memo

备注/Memo:
基金项目:浙江省医药卫生科技计划项目(2014KYB160)
通讯作者:孙辽军 E-mail:sunlj797110@163.com
更新日期/Last Update: 2019-02-25