光生物调节作用对类固醇肌病预防效应的细胞模型研究
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摘要
研究背景：长期使用或大量使用糖皮质激素(glucocorticoid，GC)可以导致类固醇肌病的发生，但还没有有效的无毒副作用的绿色治疗方法。延迟性肌肉酸痛(Delayed onset muscle soreness，DOMS)的康复分为三个阶段，内源性GC促进第二阶段损伤蛋白质的分解，但抑制第三阶段蛋白质的合成，目前还没有促进DOMS功能康复的有效方法。GC可以从对骨骼肌能量代谢、氨基酸平衡、蛋白质代谢和成肌等方面的干扰而破坏骨骼肌细胞的内稳态，这可能是GC引起类固醇肌病的机制。激活丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)信号途径或G蛋白耦联受体信号途径可能抑制GC的信号转导。光生物调节作用是单色光或激光对生物系统的非损伤调节作用，可以激活细胞MAPKs信号途径和G蛋白耦联受体信号途径，有可能发展成为治疗类固醇肌病和促进DOMS功能康复的一种绿色疗法。
研究目的：建立一个简单的类固醇肌病的细胞模型。探究光生物调节作用对地塞米松(dexamethasone，DEX)引起肌管萎缩和抑制成肌细胞增殖的预防作用，以及涉及其中的信号转导途径。
研究方法：用噻哗蓝【3-(4，5)-dimethylthiahiazo(-z-yl)-3，5-di-phenytetrazoliumromide，MTT】比色法研究移殖浓度为1000细胞／孔-4000细胞／孔的C2C12成肌细胞增殖特性。用二辛可宁酸(bicinchoninic acid，BCA)法研究C2C12在分化为肌管的过程中蛋白总量的变化特性。在此基础上，建立一个简单的类固醇肌病的细胞模型。用MTT比色法研究了不同浓度的DEX对成肌细胞增殖的抑制效应。利用BCA法和WesternBlot，研究了孵育DEX以不同浓度或不同时间对C2C12肌管蛋白代谢的影响。由于实验需要，设计并建立了一套程控化的红光范围(640±17nm)的发光二极管系统【red light(640±17nm) from light emitting diode array,RLED】，并对该系统的稳定性和均一性作了基本的评估。在以上工作基础上，研究了RLED对DEX引起肌管萎缩和抑制成肌细胞增殖的预防作用。最后，利用MAPK／细胞外信号调节激酶激酶(MAPK／extracellular signal-regulated kinase kinase，MEK)抑制剂PD98059和磷脂酰肌醇-3激酶(phosphatidyl-inositol 3 kinase activity，PI3K)抑制剂Wortmannin，初步研究光生物调节作用可能涉及的信号转导途径。
研究结果：1．各浓度的细胞均出现了典型的生长曲线。以初始浓度为2000／孔、3000／孔和4000／孔的细胞在培养2-3天后进入对数增殖期，而1000／孔组的细胞在移殖后4-5天才开始对数增长。以4000／孔的细胞到培养第5天后开始进入平台期。选择3000细胞／孔移殖浓度，培养4天的C2C12细胞为对象研究DEX对细胞增殖的影响；使用BCA法在分化后每天检测肌细胞的蛋白总量，结果显示分化后2-4天蛋白总量不断增加，分化4天后维持在一个相对稳定的水平。选择分化8天的C2C12肌管为对象研究DEX对肌管蛋白代谢的影响。2．孵育3天25nmol／L—10μmol／L的DEX可以显著的抑制C2C12成肌细胞增殖；孵育24小时25nmol／L—10μmol／L的DEX导致C2C12肌管蛋白总量或α-actin蛋白量显著下降。孵育DEX(1μmol／L)6小时-24小时可以引起C2C12肌管总蛋白量下降。而孵育时间为4小时的时候，未能发现肌管总蛋白量显著性下降；3．在照射强度为3.5mW／cm~2条件下RLED照射300s或900s均可以部分预防DEX(1μmol／L)引起肌管的总蛋白和收缩蛋白α-actin的蛋白量下降(P＜0.05)。而在照射强度为0.5-1.5mW／cm~2，照射时间为900s的条件下，不能观察到RLED对DEX抑制成肌细胞增殖的显著的预防效果(P＞0.05)；4．DI组(DEX+PI3K抑制剂Wortmannin)细胞蛋白总量和DEX组相比没有显著性差异，提示PI3K信号受到抑制对DEX的效应没有显著性影响。和DL组(DEX+3.5mW／cm~2×300s光照)相比，DIL组(DEX+抑制剂+光照)的蛋白总量显著性下降(P＜0.05)，提示RLED对DEX致C2C12肌管萎缩的保护效应可能涉及PI3K相关的信号转导途径。收缩蛋白α-actin的蛋白印迹显示出相似的结果。然而，使用抑制剂的方法不能证明光生物调节作用是否涉及MEK途径。
最终结论：本研究建立了类固醇肌病的细胞模型。在此基础上，首次发现RLED对GC引起肌管萎缩的预防效应，该效应可能涉及PI3K相关的信号转导途径。本研究为光生物调节作用用于防治类固醇肌病和DOMS功能的康复的应用研究提供了基础性的支持。
Background: Long term or high dose glucocorticoid treatment could result insteroid myopathy on which there is no green effective therapeytic approaches with noside effects. There might be three phases of delayed onset muscle soreness (DOMS)recovery, endogenous glucocorticoids might premote proteolysis of damaged proteinsin the second phase, but inhibit protein synthesis in the third phase. There is akso feweffective therapeytic approaches on DOMS functional recovery. After being activatedby glucocorticoids, glucocorticoid receptors could mediate several gene expressionpositively or negatively, which followed by many biological responses such asaccelerating protein breakdown and slow down protein systhesis. The results ofstudies relative to steroid myopathy showed that glucocorticoids could interfere withenergy metabolism, amino acid balance, protein metabolism of skeletal muscle cellsand myogenesis and destroy the cellular homeostasis, which might be the mechanismof myopathy induced by glucocorticoids. The mitogen-activated protein kinase(MAPK) signals or G proteins might inhibit the signal transduction of glucocorticoidsand its receptors. Photobiomodulation is a non-damage modulation of monochromaticlight or laser irradiation on biosystems, and could activate the signal transductioninvolved in MAPKs or G proteins, indicating the potential application ofphotobiomodulation to glucocorticoid induced steroid myopathy.
Object: To set up a cellular model of steroid myopathy. To study the preventiveeffects of photobiomodulation on myotube atrophy or the dexamethasone inducedinhibition of myoblast proliferation, and the involved potential signal transductionpathways.
Method: First, we evaluated the proliferation of C2C12 myoblasts by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay, andthe myotube total protein content by bicinchoninic acid (BCA) assay. To set up acellular model of steroid myopathy, the inhibition effects of dexamethasone on themyoblast proliferation and myotube protein metabolism was also studied by MTTassay, BCA assay and Western Blot, respectively. Second, we build up a set ofcomputer program controlled light emitting diode array of red light (640±17nm)(RLED) for photobiomodulation and the stability and homogenization of the systemwere evaluated by a power meter. Third, we observated the preventive effects ofRLED on the glucocorticoid induced myotube atrophy by BCA assay andWesternBlot. By MTT assay, we also studied the preventive effects of RLED on theglucocorticoid induced inhibition of myoblast proliferation. Four, usingMAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD98059and the phosphatidyl-inositol 3 kinase activity (PI3K) inhibitor Wortmannin, westudied the possible signal transduction pathways involved in the photobiomodulationwhich reduced the glucocorticoid induced myotube atrophy.
Results: 1. The cells transplanted in 2000/well, 3000/well and 4000/well steppedin the exponential phase 2-3 days after transplant while the cells of 1000/well groupdid not get into the exponential phase until day 4. The cells of 4000/well groupstepped in the stable phase at day 5. We chose the cell transplanted in 3000/well and 4days for growth to investigate the inhibition effect ofdexamethasone on the myoblastproliferation. On the other hand, the total protein content of differentiated myotubeincreased from day 2 to day 4 after differentiation, and stepped into a stable level afterday 4. We chose the 8 days differentiated myotube for the further investigation of thenegative effect of dexamethasone on protein metabolism. 2. After 3 days incubationwith 25nmol/L—10μmol/L of dexamethasone, the proliferation of C2C 12 myotubewas decrease with increasing glucocorticoid concentration. 24 hours incubation with25nmol/L—10μmol/L of dexamethasone resulted in the decrease of C2C12 myotubetotal protein content orα-actin content. 6-24 hours incubation ofdexamethasone(1μmol/L) induced significant decrease in myotube total proteincontent. While myotube incubated with dexamethasone(1μmol/L) for 4 hours, no significant decrease in total protein was observed. 3. RLED Irradiation at 3.5mW/cm~2for 300s and 900s after dexamethasone(1μmol/L) addition could partial prevent thedecrease of myotube total protein content andα-actin content induced byglucocorticoid(P＜0.05). On the other hand, no significant benefic preventive effect onthe dexamethasone(100nmol/L) inducing inhibition of myoblast proliferation(P＞0.05).4. There was no significant difference between the total protein content of myotube inDI(DEX+Wortmannin) group and DEX group. The total protein content of myotubein DL(DEX+3.5mW/cm~2×300s irradiation) group was significantly higher that inDIL(DEX+Wortmannin+irradiation) group, suggesting PI3K might involve in thephotobiomodulation, which supported by the similar result from WestemBlot ofα-actin.
Conelution: Based on the cellular model, we found the preventive effects ofphotobiomodulation on steroid myopathy for the first time. The photobiomodulationmay involded in PI3K signal transduction. This study as a basic research supports theapplication ofphotobiomodulation to steroid myopathy and DOMS recovery.
研究目的：建立一个简单的类固醇肌病的细胞模型。探究光生物调节作用对地塞米松(dexamethasone，DEX)引起肌管萎缩和抑制成肌细胞增殖的预防作用，以及涉及其中的信号转导途径。
研究方法：用噻哗蓝【3-(4，5)-dimethylthiahiazo(-z-yl)-3，5-di-phenytetrazoliumromide，MTT】比色法研究移殖浓度为1000细胞／孔-4000细胞／孔的C2C12成肌细胞增殖特性。用二辛可宁酸(bicinchoninic acid，BCA)法研究C2C12在分化为肌管的过程中蛋白总量的变化特性。在此基础上，建立一个简单的类固醇肌病的细胞模型。用MTT比色法研究了不同浓度的DEX对成肌细胞增殖的抑制效应。利用BCA法和WesternBlot，研究了孵育DEX以不同浓度或不同时间对C2C12肌管蛋白代谢的影响。由于实验需要，设计并建立了一套程控化的红光范围(640±17nm)的发光二极管系统【red light(640±17nm) from light emitting diode array,RLED】，并对该系统的稳定性和均一性作了基本的评估。在以上工作基础上，研究了RLED对DEX引起肌管萎缩和抑制成肌细胞增殖的预防作用。最后，利用MAPK／细胞外信号调节激酶激酶(MAPK／extracellular signal-regulated kinase kinase，MEK)抑制剂PD98059和磷脂酰肌醇-3激酶(phosphatidyl-inositol 3 kinase activity，PI3K)抑制剂Wortmannin，初步研究光生物调节作用可能涉及的信号转导途径。
研究结果：1．各浓度的细胞均出现了典型的生长曲线。以初始浓度为2000／孔、3000／孔和4000／孔的细胞在培养2-3天后进入对数增殖期，而1000／孔组的细胞在移殖后4-5天才开始对数增长。以4000／孔的细胞到培养第5天后开始进入平台期。选择3000细胞／孔移殖浓度，培养4天的C2C12细胞为对象研究DEX对细胞增殖的影响；使用BCA法在分化后每天检测肌细胞的蛋白总量，结果显示分化后2-4天蛋白总量不断增加，分化4天后维持在一个相对稳定的水平。选择分化8天的C2C12肌管为对象研究DEX对肌管蛋白代谢的影响。2．孵育3天25nmol／L—10μmol／L的DEX可以显著的抑制C2C12成肌细胞增殖；孵育24小时25nmol／L—10μmol／L的DEX导致C2C12肌管蛋白总量或α-actin蛋白量显著下降。孵育DEX(1μmol／L)6小时-24小时可以引起C2C12肌管总蛋白量下降。而孵育时间为4小时的时候，未能发现肌管总蛋白量显著性下降；3．在照射强度为3.5mW／cm~2条件下RLED照射300s或900s均可以部分预防DEX(1μmol／L)引起肌管的总蛋白和收缩蛋白α-actin的蛋白量下降(P＜0.05)。而在照射强度为0.5-1.5mW／cm~2，照射时间为900s的条件下，不能观察到RLED对DEX抑制成肌细胞增殖的显著的预防效果(P＞0.05)；4．DI组(DEX+PI3K抑制剂Wortmannin)细胞蛋白总量和DEX组相比没有显著性差异，提示PI3K信号受到抑制对DEX的效应没有显著性影响。和DL组(DEX+3.5mW／cm~2×300s光照)相比，DIL组(DEX+抑制剂+光照)的蛋白总量显著性下降(P＜0.05)，提示RLED对DEX致C2C12肌管萎缩的保护效应可能涉及PI3K相关的信号转导途径。收缩蛋白α-actin的蛋白印迹显示出相似的结果。然而，使用抑制剂的方法不能证明光生物调节作用是否涉及MEK途径。
最终结论：本研究建立了类固醇肌病的细胞模型。在此基础上，首次发现RLED对GC引起肌管萎缩的预防效应，该效应可能涉及PI3K相关的信号转导途径。本研究为光生物调节作用用于防治类固醇肌病和DOMS功能的康复的应用研究提供了基础性的支持。
Background: Long term or high dose glucocorticoid treatment could result insteroid myopathy on which there is no green effective therapeytic approaches with noside effects. There might be three phases of delayed onset muscle soreness (DOMS)recovery, endogenous glucocorticoids might premote proteolysis of damaged proteinsin the second phase, but inhibit protein synthesis in the third phase. There is akso feweffective therapeytic approaches on DOMS functional recovery. After being activatedby glucocorticoids, glucocorticoid receptors could mediate several gene expressionpositively or negatively, which followed by many biological responses such asaccelerating protein breakdown and slow down protein systhesis. The results ofstudies relative to steroid myopathy showed that glucocorticoids could interfere withenergy metabolism, amino acid balance, protein metabolism of skeletal muscle cellsand myogenesis and destroy the cellular homeostasis, which might be the mechanismof myopathy induced by glucocorticoids. The mitogen-activated protein kinase(MAPK) signals or G proteins might inhibit the signal transduction of glucocorticoidsand its receptors. Photobiomodulation is a non-damage modulation of monochromaticlight or laser irradiation on biosystems, and could activate the signal transductioninvolved in MAPKs or G proteins, indicating the potential application ofphotobiomodulation to glucocorticoid induced steroid myopathy.
Object: To set up a cellular model of steroid myopathy. To study the preventiveeffects of photobiomodulation on myotube atrophy or the dexamethasone inducedinhibition of myoblast proliferation, and the involved potential signal transductionpathways.
Method: First, we evaluated the proliferation of C2C12 myoblasts by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay, andthe myotube total protein content by bicinchoninic acid (BCA) assay. To set up acellular model of steroid myopathy, the inhibition effects of dexamethasone on themyoblast proliferation and myotube protein metabolism was also studied by MTTassay, BCA assay and Western Blot, respectively. Second, we build up a set ofcomputer program controlled light emitting diode array of red light (640±17nm)(RLED) for photobiomodulation and the stability and homogenization of the systemwere evaluated by a power meter. Third, we observated the preventive effects ofRLED on the glucocorticoid induced myotube atrophy by BCA assay andWesternBlot. By MTT assay, we also studied the preventive effects of RLED on theglucocorticoid induced inhibition of myoblast proliferation. Four, usingMAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD98059and the phosphatidyl-inositol 3 kinase activity (PI3K) inhibitor Wortmannin, westudied the possible signal transduction pathways involved in the photobiomodulationwhich reduced the glucocorticoid induced myotube atrophy.
Results: 1. The cells transplanted in 2000/well, 3000/well and 4000/well steppedin the exponential phase 2-3 days after transplant while the cells of 1000/well groupdid not get into the exponential phase until day 4. The cells of 4000/well groupstepped in the stable phase at day 5. We chose the cell transplanted in 3000/well and 4days for growth to investigate the inhibition effect ofdexamethasone on the myoblastproliferation. On the other hand, the total protein content of differentiated myotubeincreased from day 2 to day 4 after differentiation, and stepped into a stable level afterday 4. We chose the 8 days differentiated myotube for the further investigation of thenegative effect of dexamethasone on protein metabolism. 2. After 3 days incubationwith 25nmol/L—10μmol/L of dexamethasone, the proliferation of C2C 12 myotubewas decrease with increasing glucocorticoid concentration. 24 hours incubation with25nmol/L—10μmol/L of dexamethasone resulted in the decrease of C2C12 myotubetotal protein content orα-actin content. 6-24 hours incubation ofdexamethasone(1μmol/L) induced significant decrease in myotube total proteincontent. While myotube incubated with dexamethasone(1μmol/L) for 4 hours, no significant decrease in total protein was observed. 3. RLED Irradiation at 3.5mW/cm~2for 300s and 900s after dexamethasone(1μmol/L) addition could partial prevent thedecrease of myotube total protein content andα-actin content induced byglucocorticoid(P＜0.05). On the other hand, no significant benefic preventive effect onthe dexamethasone(100nmol/L) inducing inhibition of myoblast proliferation(P＞0.05).4. There was no significant difference between the total protein content of myotube inDI(DEX+Wortmannin) group and DEX group. The total protein content of myotubein DL(DEX+3.5mW/cm~2×300s irradiation) group was significantly higher that inDIL(DEX+Wortmannin+irradiation) group, suggesting PI3K might involve in thephotobiomodulation, which supported by the similar result from WestemBlot ofα-actin.
Conelution: Based on the cellular model, we found the preventive effects ofphotobiomodulation on steroid myopathy for the first time. The photobiomodulationmay involded in PI3K signal transduction. This study as a basic research supports theapplication ofphotobiomodulation to steroid myopathy and DOMS recovery.
引文
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