骨骼肌钝挫伤后线粒体自噬相关基因及自噬体变化研究
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摘要
目的:探讨骨骼肌钝挫伤后线粒体自噬相关基因与自噬体变化的分子机制。方法:Wistar雄性大鼠64只,随机分为对照组、钝挫伤组(按照取材时间分为12 h组、2 d组、5 d组、7 d组、10 d组、15 d组、30 d组)。采用重物下落装置导致右小腿内侧面(其皮下为腓肠肌中段)一次打击伤,建立急性骨骼肌钝挫伤模型,通过Western-Blotting和qRT-PCR技术分别检测钝挫伤后腓肠肌缺氧及自噬相关因子,包括低氧诱导因子-1α(HIF-1α)、腺苷酸活化蛋白激酶α2亚基(AMPKα2)、Bcl-2腺病毒E1B19ku相关蛋白3(BNIP3)和Bcl-2家族的类NIP3蛋白(NIX)的蛋白和mRNA表达变化情况;透射电镜观察受损部位不同恢复时间的超微结构及自噬体形成情况。结果:(1)钝挫伤后,HIF-1α、AMPKα2蛋白表达在12 h、2 d达到峰值,至伤后10 d,HIF-1α表达均明显高于对照组(P<0.05);AMPKα2表达在伤后5 d仍显著高于对照组(P<0.05),至10 d回落至正常水平;BNIP3持续高表达至5 d后开始回落,但至10 d仍高于对照组;NIX表达峰值出现在伤后12 h、2 d,持续高表达至7 d。(2)钝挫伤后,相关基因表达水平,Hif-1α与Ampkα2mRNA在2 d显著高于对照组(P<0.01),至5 d表达下降但仍高于对照组(P<0.05),随后回落至正常水平;Bnip3、Nix的mRNA变化情况与其蛋白表现基本一致。损伤后12 h在单个网孔视野中即可见到多个自噬体,至2～7 d自噬体数量逐渐增多,10 d后自噬体数量比钝挫伤后12 h～7 d组相比呈现减少趋势,至15 d后自噬体数量逐渐减少。结论:骨骼肌钝挫伤发生后,损伤早期代谢调控因子AMPKα2、缺氧敏感因子HIF-1α表达变化情况表明损伤后受损骨骼肌内部出现能量危机并形成缺氧环境;线粒体自噬、细胞凋亡相关因子BNIP3、NIX表达变化表明损伤后线粒体自噬被启动,缺氧诱导了骨骼肌钝挫伤早期的线粒体自噬;缺氧诱导的线粒体自噬可及时清除受损线粒体,维持线粒体质量、为新生线粒体提供原料,从而减小损伤程度,以利于受损骨骼肌的快速恢复,这可能是损伤发生后机体的一种代偿性机制。
Objective To investigate the molecular mechanism of mitochondrial autophagy-related genes and autophagosome changes after the blunt trauma of skeletal muscles. Methods Sixty-four male Wistar rats were randomly divided into a control and a blunt trauma group,each of 32. The acute skeletal muscle contusion was induced by a single blow on the middle segment of the gastrocnemius muscle,using the heavy objects dropping device to rats of the blunt trauma group,while no trauma was induced in the control group. The expression of hypoxia and autophagy-related factors in the skeletal muscle of the blunt trauma,including the hypoxia inducible factor-1α(HIF-1α),α2 subunit of AMP-activated protein kinase(AMPKα2),BCL2 interacting protein 3(BNIP3) and NIP3-like protein X(NIX) were measured using the Western blotting and qRT-PCR 12 h,2 d,5 d,7 d,10 d,15 d and 30 d after the modeling for both groups. The ultrastructure and autophagosomes at different time points were observed using a transmission electron microscopy(TEM). Results The expression of HIF-1α and AMPKα2 reached the peak at 12 h and 2 d after the trauma,and the expression of HIF-1αwas significantly higher than that of the control group 10 days after the modeling(P<0.05). The expression of AMPKα2 was significantly higher than the control group 5 days after the injury(P<0.05),but reached the normal level 5 days later. The expression of BNIP3 remained at a high level at the beginning,and began to decline till 5 days after the modeling,but was still higher than the control group till 10 days after the trauma(P<0.05). The NIX expression peaked at 12 h and 2 d after injury,keeping high-level expression till 7 days after injury. The expression of Hif-1α and Ampkα2 mRNA was significantly higher than that of the control group 2 days after the modeling,and began to decrease 3 days later,but still significantly higher than the control group. The mRNA expression of Bnip3 and Nix was basically the same as their proteins. A number of autophagosomes were observed at 12 h after injury,and the number of autophagosomes increased gradually from 2 to 7 days after the modeling,followed by a decrease from 10 days after the modeling. Moreover,15 days after the modeling,the number of autophagosomes decreased gradually. Conclusion The changes of the early-stage metabolic regulator AMPKα2 and hypoxia-sensitive factor HIF-1α after the blunt trauma of skeletal muscles indicate that an energy crisis occurs in the skeletal muscle after injury,and the hypoxic environment was formed. The mitochondrial autophagy and the expression of BNIP3 and NIX show that the mitochondrial autophagy is activated by the hypoxia. Hypoxia-induced mitochondrial autophagy can remove the damaged mitochondria,maintain mitochondrial quality and provide raw materials for the new generation of mitochondrias and facilitate the rapid recovery of damaged skeletal muscles,which may be a compensatory mechanism of the body response to injury.
Objective To investigate the molecular mechanism of mitochondrial autophagy-related genes and autophagosome changes after the blunt trauma of skeletal muscles. Methods Sixty-four male Wistar rats were randomly divided into a control and a blunt trauma group,each of 32. The acute skeletal muscle contusion was induced by a single blow on the middle segment of the gastrocnemius muscle,using the heavy objects dropping device to rats of the blunt trauma group,while no trauma was induced in the control group. The expression of hypoxia and autophagy-related factors in the skeletal muscle of the blunt trauma,including the hypoxia inducible factor-1α(HIF-1α),α2 subunit of AMP-activated protein kinase(AMPKα2),BCL2 interacting protein 3(BNIP3) and NIP3-like protein X(NIX) were measured using the Western blotting and qRT-PCR 12 h,2 d,5 d,7 d,10 d,15 d and 30 d after the modeling for both groups. The ultrastructure and autophagosomes at different time points were observed using a transmission electron microscopy(TEM). Results The expression of HIF-1α and AMPKα2 reached the peak at 12 h and 2 d after the trauma,and the expression of HIF-1αwas significantly higher than that of the control group 10 days after the modeling(P<0.05). The expression of AMPKα2 was significantly higher than the control group 5 days after the injury(P<0.05),but reached the normal level 5 days later. The expression of BNIP3 remained at a high level at the beginning,and began to decline till 5 days after the modeling,but was still higher than the control group till 10 days after the trauma(P<0.05). The NIX expression peaked at 12 h and 2 d after injury,keeping high-level expression till 7 days after injury. The expression of Hif-1α and Ampkα2 mRNA was significantly higher than that of the control group 2 days after the modeling,and began to decrease 3 days later,but still significantly higher than the control group. The mRNA expression of Bnip3 and Nix was basically the same as their proteins. A number of autophagosomes were observed at 12 h after injury,and the number of autophagosomes increased gradually from 2 to 7 days after the modeling,followed by a decrease from 10 days after the modeling. Moreover,15 days after the modeling,the number of autophagosomes decreased gradually. Conclusion The changes of the early-stage metabolic regulator AMPKα2 and hypoxia-sensitive factor HIF-1α after the blunt trauma of skeletal muscles indicate that an energy crisis occurs in the skeletal muscle after injury,and the hypoxic environment was formed. The mitochondrial autophagy and the expression of BNIP3 and NIX show that the mitochondrial autophagy is activated by the hypoxia. Hypoxia-induced mitochondrial autophagy can remove the damaged mitochondria,maintain mitochondrial quality and provide raw materials for the new generation of mitochondrias and facilitate the rapid recovery of damaged skeletal muscles,which may be a compensatory mechanism of the body response to injury.
引文
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[16] Sun Y,Hong J,Chen M,et al. Ablation of Lgr4 enhances energy adaptation in skeletal muscle via activation of Ampk/Sirt1/Pgc1 alpha pathway[J]. Biochem Biophys Res Commun,2015,464(2):396-400.
[17] Huafeng Z, Marta BM, Shimoda ea. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[J]. J Biol Chem, 2008, 283(16):10892-10903.
[18] Zhu Y, Massen S, Terenzio M, et al. Modulation of serines 17 and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis[J]. J Biol Chem,2013,(288):1099-1113.
[19] Tam ZY, Gruber J, Halliwell B, et al. Mathematical modeling of the role of mitochondrial fusion and fission in mitochondrial DNA maintenance[J]. PloS One,2013,8(10):157-169.
[20] Zhang BB,Zhou G,Li C. AMPK:an emerging drug target for diabetes and the metabolic syndrome[J]. Cell Metab,2009,(9):407-416.
[21] Minibayeva F,Dmitrieva S,Ponomareva A,et al. Oxidative stress-induced autophagy in plants:The role of mitochondria[J]. Plant Physiol Biochem,2012,59:11-9.
[22]侯振海,余斌,汪志忠.急性低氧及低氧复合运动对骨骼肌及代谢酶活性的影响[J].中国临床康复,2004,5:902-903.
[23]刘树坤,于晓华,吴耀义,等. SD大鼠骨骼肌钝挫伤早期的组织病理变化[J].中国组织工程研究,2012,(24):4371-4375.
[24] Parone PA, Da Cruz S, Tondera D, et al. Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA[J]. PloS one,2008,3(9):e3257.
[25] Nakamura Y, Kitamura N, Shinogi D, et al. BNIP3and NIX mediate Mieap-induced accumulation of lysosomal proteins within mitochondria[J]. PloS one, 2012,(7):475-479.
[26] Sandoval H,Thiagarajan P,Dasgupta SK,et al. Essential role for Nix in autophagic maturation of erytroid cells[J]. Nature,2008,454(7201):232-235.
[27] Fisher BD, Baracos VE, Shnitka TKea. Ultrastructural events following acute muscle trauma[J]. Med Sci Sports Exerc,1990,22(2):185-193.
[28] Fan Z,Wu X,Zhang L,et al.HDAC4 mediates IFN-γinduced disruption of energy expenditure-related gene expression by repressing SIRT1 transcription in skeletal muscle cells[J]. Biochimica et Biophysica Acta, 2016,1859(2):294-305.
[29] Stiles L,Shirihai OS. Mitochondrial dynamics and morphology in beta-cells[J]. Best Pract Res Clin Endocrinol Metab,2012,26(6):725-738.
[30] Skurvydas A,Brazaitis M,Venckunas T,et al. The effect of sports specialization on musculus quadriceps function after exercise-induced muscle damage[J]. Appl Physiol Nutr Metab,2011,36(6):873-880.
[31] TE S,McBride HM. Staying cool in difficult times:mitochondrial dynamics, quality control and the stress response[J]. Biochim Biophys Acta,2013,1833(2):417-424.
[2] Ciechanover A. The ubiquitin-proteasome pathway:on protein death and cell life[J]. EMBO J,1998,17(24):7151-7160.
[3] Gomes LC,Scorrano L. High levels of Fis1,a pro-fission mitochondrial protein, trigger autophagy[J]. Biochim Biophys Acta,2008,1777(7-8):860-866.
[4] Lemasters JJ. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress,mitochondrial dysfunction, and aging[J]. Rejuvenation Res,2005,8(1):3-5.
[5]董贵俊,葛新发,李可峰,等.磁场定位磁纳米化bFGF治疗大鼠腓肠肌钝挫伤恢复效果研究[J].武汉体育学院院报,2011,45(2):43-46.
[6] Leeder J,Spence J,Taylor E,et al. The effect of electrical stimulation on recovery from exercise-induced muscle damage[J]. J Appl Physiol. 2008,105(4):1146-1155.
[7] Chen R,Lai UH,Zhu L,et al. Reactive oxygen species formation in the brain at different oxygen levels:The role of hypoxia inducible factors[J]. Front Cell Dev Biol. 2018,6:132.
[8] Stothers CL,Luan L,Fensterheim BA,et al. Hypoxiainducible factor-1αregulation of myeloid cells[J]. J Mol Med(Berl). 2018,96(12):1293-1306.
[9] Silwal P,Kim JK,Yuk JM,et al. AMP-activated protein kinase and host defense against infection[J]. Int J Mol Sci. 2018,19(11):1-26.
[10] Adams DO,Johnson WJ,Marino PA. Mechanisms of target recognition and destruction in macrophage-mediated tumor cytotoxicity[J]. Fed Proc,1982,41(6):2212-2221.
[11] Mackaness GB. The monocyte in cellular immunity[J].Semin Hematol,1970,7(2):172-184.
[12]漆正堂,郭维,张媛,等.不同运动方式对大鼠骨骼肌线粒体融合分裂基因及Mfn2、Drp1蛋白表达的影响[J].中国运动医学杂志,2011,30(2):143-148.
[13]于天水,官大威,程子惠,等.大鼠骨骼肌钝挫伤分级模型的建立[J].法医学杂志,2008:168-171.
[14] Ashrafi G,Schwarz TL. The pathways of mitophagy for quality control and clearance of mitochondria[J]. Cell Death Differ,2013,20(1):31-42.
[15] Bhatia-Ki??ováI, Camougrand N. Mitophagy:a process that adapts to the cell physiology[J]. Int J Biochem Cell Biol,2013,45(1):30-33.
[16] Sun Y,Hong J,Chen M,et al. Ablation of Lgr4 enhances energy adaptation in skeletal muscle via activation of Ampk/Sirt1/Pgc1 alpha pathway[J]. Biochem Biophys Res Commun,2015,464(2):396-400.
[17] Huafeng Z, Marta BM, Shimoda ea. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[J]. J Biol Chem, 2008, 283(16):10892-10903.
[18] Zhu Y, Massen S, Terenzio M, et al. Modulation of serines 17 and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis[J]. J Biol Chem,2013,(288):1099-1113.
[19] Tam ZY, Gruber J, Halliwell B, et al. Mathematical modeling of the role of mitochondrial fusion and fission in mitochondrial DNA maintenance[J]. PloS One,2013,8(10):157-169.
[20] Zhang BB,Zhou G,Li C. AMPK:an emerging drug target for diabetes and the metabolic syndrome[J]. Cell Metab,2009,(9):407-416.
[21] Minibayeva F,Dmitrieva S,Ponomareva A,et al. Oxidative stress-induced autophagy in plants:The role of mitochondria[J]. Plant Physiol Biochem,2012,59:11-9.
[22]侯振海,余斌,汪志忠.急性低氧及低氧复合运动对骨骼肌及代谢酶活性的影响[J].中国临床康复,2004,5:902-903.
[23]刘树坤,于晓华,吴耀义,等. SD大鼠骨骼肌钝挫伤早期的组织病理变化[J].中国组织工程研究,2012,(24):4371-4375.
[24] Parone PA, Da Cruz S, Tondera D, et al. Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA[J]. PloS one,2008,3(9):e3257.
[25] Nakamura Y, Kitamura N, Shinogi D, et al. BNIP3and NIX mediate Mieap-induced accumulation of lysosomal proteins within mitochondria[J]. PloS one, 2012,(7):475-479.
[26] Sandoval H,Thiagarajan P,Dasgupta SK,et al. Essential role for Nix in autophagic maturation of erytroid cells[J]. Nature,2008,454(7201):232-235.
[27] Fisher BD, Baracos VE, Shnitka TKea. Ultrastructural events following acute muscle trauma[J]. Med Sci Sports Exerc,1990,22(2):185-193.
[28] Fan Z,Wu X,Zhang L,et al.HDAC4 mediates IFN-γinduced disruption of energy expenditure-related gene expression by repressing SIRT1 transcription in skeletal muscle cells[J]. Biochimica et Biophysica Acta, 2016,1859(2):294-305.
[29] Stiles L,Shirihai OS. Mitochondrial dynamics and morphology in beta-cells[J]. Best Pract Res Clin Endocrinol Metab,2012,26(6):725-738.
[30] Skurvydas A,Brazaitis M,Venckunas T,et al. The effect of sports specialization on musculus quadriceps function after exercise-induced muscle damage[J]. Appl Physiol Nutr Metab,2011,36(6):873-880.
[31] TE S,McBride HM. Staying cool in difficult times:mitochondrial dynamics, quality control and the stress response[J]. Biochim Biophys Acta,2013,1833(2):417-424.
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