PPARγ下调在AGEs诱导软骨细胞TNF-α及MMP-13表达中的作用与机制研究

发布时间：2018-06-12 22:52

  本文选题：晚期糖基化终末产物 + 软骨细胞　； 参考：《中南大学》2010年博士论文

【摘要】：第一部分AGEs对兔软骨细胞TNF-a和MMP-13表达的影响与意义 目的：以外源性晚期糖基化终末产物(advanced glycation end products, AGEs)为损伤因子,在体外培养的家兔软骨细胞模型上,观察AGEs对肿瘤坏死因子-a (tumor necrosisfactor-a,TNF-α)和基质金属蛋白酶-13(Matrix metalloproteinase 13, MMP-13)的表达和影响,探讨AGEs与骨关节炎(Osteoarthritis, OA)的关系及可能的信号通路及机制。 方法：在原代培养的家兔软骨细胞模型上,(1)不同浓度的AGEs与软骨细胞共孵育48h后检测软骨细胞TNF-a和MMP-13 mRNA的表达情况；(2)AGEs受体(Receptor for advanced glycation end products, RAGE)的抗体(anti-RAGE)及核因子，，B (nuclear factor-KB, NF-κB)的特异性阻断剂吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate, PDTC)与软骨细胞预孵12h后,再加入100μg/mlAGEs共同孵育48h,检测软骨细胞TNF-a及MMP-13mRNA的表达；(3)不同浓度的AGEs与软骨细胞共孵育48h后检测软骨细胞过氧化氢酶(Catalase,CAT)、超氧化物歧化酶(superoxide dismutase, SOD)活性及丙二醛(Malondialdehyde, MDA)、活性氧(reactive oxygen species, ROS)水平。采用RT-PCR方法检测TNF-α和MMP-13的mRNA表达量,试剂盒方法检测CAT、SOD活性及MDA水平,荧光探针法检测ROS水平。 结果：(1)不同浓度的AGEs (1,10,25,50,100μg/ml)与软骨细胞共孵育48h后,TNF-a及MMP-13mRNA的表达较正常对照组明显升高(P0.05或P0.01),且均以AGEs浓度为100μg/ml时作用最明显；(2)Anti-RAGE (5μg/ml)与PDTC(0.1mmol/L)能显著抑制由AGEs (100μg/ml)诱导的软骨细胞TNF-α及MMP-13表达增多(P0.01),而anti-RAGE (5μg/ml)和PDTC(0.1mmol/L)单独处理组与正常对照组相比差异均无统计学意义(P0.05)；(3)不同浓度的AGEs (1,10,25,50,100μg/ml)与软骨细胞共孵育48h后,浓度依赖性地使软骨细饱CAT、SOD活性降低, MDA、ROS含量增多,与正常对照组相比差异有统计学意义(P0.05) 结论：AGEs能显著刺激软骨细胞TNF-α和MMP-13表达增多,诱导软骨细胞损伤,其机制与激活RAGE,诱导活性氧(ROS)生成增多,激活NF-κB信号通路有关。 第二部分AGEs对兔软骨细胞PPARγ表达的影响与机制 目的：在体外培养的家兔软骨细胞模型上,观察AGEs对软骨细胞过氧化物酶体增生物激活受体γ(Peroxisome proliferator-activated receptor-γ, PPARγ)表达的影响,探讨AGEs与PPARγ的关系与机制。 方法：在原代培养的家兔软骨细胞模型上,(1)不同浓度的AGEs与软骨细胞共孵育48h后检测软骨细胞PPARγ的表达情况；(2)软骨细胞与AGEs共孵育不同时间后检测软骨细胞PPARγ的表达情况；(3) RAGE的抗体(anti-PAGE)与软骨细胞预孵1h后,再加入100μg/mlAGEs共同孵育48h,检测软骨细胞PPARγ的表达情况；(4)不同浓度的丝分裂原活化蛋白激酶(MAPK)信号路径的阻断剂(P38-MAPK阻断剂SB203580,JNK-MAPK阻断剂SP600125,ERK-MAPK阻断剂PD98059)与软骨细胞预孵30min,再加入100μg/mlAGEs共同孵育48h,检测软骨细胞PPARγ的表达情况。用RT-PCR方法检测PPARγ的mRNA水平,用Western blot方法检测PPARγ的蛋白含量。 结果：(1)不同浓度的AGEs (1,10,25,50,100μg/ml)与软骨细胞共孵育48h后,软骨细胞PPARγ的mRNA水平及蛋白含量较正常对照组明显降低(P0.05),AGEs浓度越高PPARγ的mRNA水平及蛋白含量越低；(2)软骨细胞与100μg/ml AGE共孵育不同时间(0,3,6,12,24,48h)后,PPARγ的mRNA表达和蛋白含量均随时间的延长而降低,0h处理组与其他各组比较PPARγ的mRNA表达和蛋白含量差异均有统计学意义(P0.05)；(3)Anti-RAGE (5μg/ml) +AGEs处理组软骨细胞PPARγ的mRNA表达显著高于AGEs (100μg/ml)处理组(P0.05)；(4) P38-MAPK阻断剂SB203580+AGEs和JNK-MAPK阻断剂SP600125+AGEs处理组的软骨细胞PPARγ的mRNA水平及蛋白含量明显高于AGEs单独处理组(P0.05),阻断剂浓度越高PPARγ的mRNA水平及蛋白含量越高；ERK-MAPK阻断剂PD98059+AGEs处理组与AGEs单独处理组比较,软骨细胞PPARγ的mRNA水平及蛋白含量均无明显差异(P0.05) 结论：1.AGEs可诱导软骨细胞PPARγ表达下调,并具有浓度和时间依赖性；2.AGEs通过AGEs-RAGE-MAPK途径实现对软骨细胞PPARγ表达下调；3.MAPKs家族中P38-MAPK和JNK-MAPK信号通路参与了AGEs诱导软骨细胞PPARγ表达下调,ERK-MAPK信号通路与该效果无关。 第三部分PPARγ激动剂对AGEs诱导兔软骨细胞TNF-α和MMP-13表达的影响与机制 目的：在体外培养的家兔软骨细胞模型上,观察PPARγ激动剂吡格列酮(pioglitazone)对AGEs诱导兔软骨细胞TNF-a和MMP-13表达的影响,进一步探讨PPARγ表达下调在AGEs致骨关节炎的作用、机制与意义。 方法：在原代培养的家兔软骨细胞模型上,不同剂量的吡格列酮(1,10,50μM)与软骨细胞预孵2h后,再加入100μg/mlAGEs共同孵育48h, (1)RT-PCR方法检测软骨细胞TNF-α和MMP-13的mRNA水平,用Western blot方法检测TNF-α和MMP-13的蛋白含量；(2)试剂盒方法检测软骨细胞CAT、SOD活性及MDA水平；(3)荧光探针法检测软骨细胞ROS水平；(4)免疫荧光染色法检测软骨细胞的NF-κB-p65亚基转运情况。 结果：(1)不同剂量的吡格列酮(1,10,50μM)+AGEs处理组软骨细胞TNF-α和MMP-13mRNA水平及蛋白含量明显低于AGEs处理组(P0.05),吡格列酮剂量越大TNF-α和MMP-13mRNA水平及蛋白含量越低,50μM匹格列酮+AGEs处理组及50μM匹格列酮单独处理组与正常对照组相比差异无统计学意义(P0.05)；(2)1、10、50 u M口比格列酮与软骨细胞预孵育1h后,浓度依赖性的拮抗由AGEs所致软骨细胞CAT、SOD活性降低及MDA、ROS水平增高(P0.05),且均50μM毗格列酮作用最明显；(3)1、10、50μM吡格列酮与软骨细胞预孵育1h后,再加入100μg/mlAGEs共同孵育48h,软骨细胞NF-κBP65的核转位呈浓度依赖性明显抑制,100μg/mlAGEs单纯处理组则明显高于正常对照组(P0.05)。 结论：1.存在一条AGEs诱导软骨细胞TNF-a和MMP-13表达增多的信号通路,即：AGEs→RAGE→ROS↑→激活MAPK (P38-MAPK和JNK-MAPK)→下调PPARγ→F-κB活化→TNF-a和MMP-13↑; 2. PPARy下调在AGEs诱导软骨细胞TNF-a和MMP-13表达增多的信号通路中起到了重要作用；3.PPARy激动剂吡格列酮能显著抑制AGEs诱导软骨细胞TNF-a和MMP-13表达增多；4.吡格列酮通过抑制RAGE/ROS/NF-κB信号通路,抵抗AGEs所致软骨细胞损伤,从而起到防治骨关节炎病变的作用；5. AGEs致骨性关节炎病变可能存在以下细胞内信号传导途径：AGEs与RAGE结合→氧自由基↑→激活MAPK→下调PPARy→细胞内信号转导使NF-κB活化→一系列炎症和损伤因子表达→软骨损伤。
[Abstract]:Part one the effect and significance of AGEs on the expression of TNF-a and MMP-13 in rabbit chondrocytes
Objective: To observe the expression and influence of AGEs on the tumor necrosis factor -a (tumor necrosisfactor-a, TNF- a) and matrix metalloproteinase -13 (advanced glycation end products, AGEs) as the damage factor, and to observe the expression and effect of AGEs on the tumor necrosis factor -a (tumor necrosisfactor-a, TNF- a) and matrix metalloproteinase -13 (13). Objective to investigate the relationship between AGEs and Osteoarthritis (OA) and possible signaling pathways and mechanisms.
Methods: on the original cultured rabbit chondrocyte model, (1) the expression of TNF-a and MMP-13 mRNA in cartilage cells was detected by different concentrations of AGEs and cartilage cells for 48h; (2) the specificity of the AGEs receptor (Receptor for advanced glycation end products) and the nuclear factor. The sex blocker, pyrrolidine two thiocarbamate (pyrrolidine dithiocarbamate, PDTC), was incubated with cartilage cells for 12h, and then incubated with 100 mu g/mlAGEs to incubate 48h, and to detect the expression of TNF-a and MMP-13mRNA in cartilage cells. (3) different concentrations of AGEs and chondrocytes were incubated for 48h after 48h and detected the chondrocyte catalase (Catalase, CAT). Superoxide dismutase (SOD) activity and malondialdehyde (Malondialdehyde, MDA), active oxygen (reactive oxygen species, ROS) level. The RT-PCR method was used to detect the expression of TNF- alpha and MMP-13.
Results: (1) after different concentrations of AGEs (1,10,25,50100 g/ml) and cartilage cells were incubated for 48h, the expression of TNF-a and MMP-13mRNA was significantly higher than that in the normal control group (P0.05 or P0.01), and the effect was the most obvious when the concentration of AGEs was 100 mu g/ml. (2) Anti-RAGE (5 mu g/ml) could significantly inhibit the cartilage induced by 100 micron. The expression of TNF- alpha and MMP-13 increased (P0.01), but there was no significant difference between the anti-RAGE (5 u g/ml) and PDTC (0.1mmol/L) alone group as compared with the normal control group (P0.05). (3) the concentration of AGEs (1,10,25,50100 u g/ml) and cartilage cells were reincubated with cartilage cells. The amount increased significantly compared with the normal control group (P0.05).
Conclusion: AGEs can significantly stimulate the expression of TNF- alpha and MMP-13 in cartilage cells and induce cartilage cell damage. The mechanism is related to the activation of RAGE, the induced activity of reactive oxygen species (ROS) and the activation of NF- kappa B signaling pathway.
The second part is the effect and mechanism of AGEs on PPAR gamma expression in rabbit chondrocytes.
Objective: To observe the effect of AGEs on the expression of peroxisome activation receptor gamma (Peroxisome proliferator-activated receptor- gamma (PPAR gamma), PPAR gamma, and the relationship and mechanism between AGEs and PPAR gamma in the chondrocyte model of rabbit in vitro.
Methods: on the original cultured rabbit chondrocyte model, (1) the expression of PPAR gamma in cartilage cells was detected after incubating with different concentrations of AGEs and cartilage cells for 48h; (2) the expression of PPAR gamma in cartilage cells was detected after CO incubation with AGEs at different time; (3) RAGE antibody (anti-PAGE) was incubated with cartilage cells and then added to 1H, and then added to the cartilage cells. The expression of PPAR gamma in cartilage cells was detected by 100 micron g/mlAGEs, and (4) the blockers of different concentrations of mitogen activated protein kinase (MAPK) signaling pathway (P38-MAPK blocker SB203580, JNK-MAPK blocker SP600125, ERK-MAPK blocker PD98059) were incubated with soft bone cells, and then 100 micron g/mlAGEs was incubated together. The expression of PPAR gamma in chondrocytes was measured. The mRNA level of PPAR gamma was detected by RT-PCR method and the protein content of PPAR gamma was detected by Western blot.
Results: (1) after different concentrations of AGEs (1,10,25,50100 g/ml) and chondrocytes were incubated with chondrocytes for 48h, the mRNA level and protein content of PPAR gamma in chondrocytes were significantly lower than that in the normal control group (P0.05), the higher the AGEs concentration was, the lower the mRNA level and the protein content of PPAR gamma, and (2) the time of incubation of the soft bone cells and 100 mu g/ml AGE was different. The expression of mRNA and protein content of PPAR gamma decreased with the prolongation of time. The mRNA expression and protein content of PPAR gamma in 0h treatment group were statistically significant (P0.05). (3) mRNA table of PPAR gamma in Anti-RAGE (5 u g/ml) +AGEs treatment group was significantly higher than that of AGEs (100 mu) treatment group; (4) The mRNA level and protein content of PPAR gamma in the chondrocyte of the blocker SB203580+AGEs and the JNK-MAPK blocker SP600125+AGEs treatment group was significantly higher than that of the AGEs alone group (P0.05). The higher the concentration of the blocker was, the higher the mRNA level and the protein content of PPAR gamma; the ERK-MAPK blocker was compared with the AGEs alone treatment group. The level of mRNA and protein content of gamma had no significant difference (P0.05).
Conclusion: 1.AGEs can induce down regulation of PPAR gamma expression in chondrocytes, and it has concentration and time dependence; 2.AGEs can reduce the expression of PPAR gamma in chondrocytes by AGEs-RAGE-MAPK pathway; P38-MAPK and JNK-MAPK signaling pathway in 3.MAPKs family is involved in AGEs induced down regulation of PPAR gamma in cartilage cells, ERK-MAPK signaling pathway and the effect of this effect are not. Close.
The third part is the effect and mechanism of PPAR gamma agonist on AGEs induced TNF- alpha and MMP-13 expression in rabbit chondrocytes.
Objective: To observe the effect of PPAR gamma agonist pioglitazone (pioglitazone) on the expression of TNF-a and MMP-13 in rabbit chondrocytes induced by AGEs in vitro, and further explore the effect, mechanism and significance of the down regulation of PPAR gamma expression in AGEs induced osteoarthritis.
Methods: on the original cultured rabbit chondrocyte model, after incubating 2H with different doses of pioglitazone (1,10,50 mu M) and cartilage cells, 48h was incubated with 100 mu g/mlAGEs, and (1) RT-PCR method was used to detect mRNA level of TNF- alpha and MMP-13 in cartilage cells. Western blot method was used to detect the content of TNF- alpha and protein. (2) reagent box The CAT, SOD activity and MDA level of cartilage cells were detected by the method. (3) the fluorescence probe method was used to detect the ROS level of cartilage cells, and (4) the transport of NF- kappa B-p65 subunits of cartilage cells was detected by immunofluorescence staining.
Results: (1) the level and protein content of TNF- alpha and MMP-13mRNA in the chondrocytes of different doses of pioglitazone (1,10,50 mu M) +AGEs treatment group were significantly lower than that in AGEs treatment group (P0.05). The higher the dose of pioglitazone, the lower the level of TNF- A and MMP-13mRNA and the protein content, the 50 micron pioglitazone + AGEs treatment group and 50 mu M pioglitazone alone treatment group and normal pairs. There was no significant difference in the contrast group (P0.05); (2) the concentration dependent antagonism of AGEs induced cartilage cells CAT, SOD activity decreased and ROS level increased (P0.05), and the action of 50 mu M vishaglitazone was the most obvious. (3) after incubating with chondrocytes, 1,10,50 u M mouth was preincubated with chondrocytes. (3) The nuclear transposition of NF- kappa BP65 in chondrocytes was significantly inhibited by the addition of 100 mu g/mlAGEs and co incubated for 48h. The 100 u g/mlAGEs treatment group was significantly higher than that of the normal control group (P0.05).
Conclusion: 1. there is a signal pathway of increased expression of TNF-a and MMP-13 in cartilage cells induced by AGEs, that is, AGEs, RAGE, ROS, MAPK (P38-MAPK and JNK-MAPK), down PPAR gamma, F- kappa B activation, etc. 3.PPARy agonist pioglitazone significantly inhibits the increased expression of TNF-a and MMP-13 in cartilage cells induced by AGEs; 4. pioglitazone can resist the injury of cartilage cells induced by AGEs by inhibiting the RAGE/ROS/NF- kappa B signaling pathway, and thus plays a role in the prevention and treatment of osteoarthritis, and 5. AGEs induced osteoarthritis may exist in the following cell The signal transduction pathway: the combination of AGEs and RAGE, oxygen free radical, activates MAPK, down PPARy and intracellular signal transduction makes NF- kappa B activation, a series of inflammation and damage factor expression, and cartilage damage.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【引证文献】

相关期刊论文 前1条

1 张林;陆军;李永刚;;骨关节炎中介导软骨细胞代谢失衡的相关信号通路研究进展[J];东南大学学报(医学版);2013年04期

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