| Part I The effect and meaning of AGEs on expression of tumor necrosis factor-a and matrix metalloproteinase-13 in rabbit chondrocytesObjectives In the present study, we used the preparation AGEs as a damage factor to investigate the effects of AGEs on expression of TNF-a and MMP-13 on the cultured rabbit chondrocytes and explored the relationship between AGEs and OA as well as involved singaling pathway and mechanism.Methods The primary cultured rabbit chondrocytes were used in the present study. (1) The chondrocytes were incubated with different concentrations of AGEs for 48h, and detected the expression level of RNA for TNF-a and MMP-13. (2) The chondrocytes were incubated with PDTC (pyrrolidine dithiocarbamate) which is a kind of anti-RAGE (Receptor for advanced glycation end products) and the inhibitor of NF-κB for 12h, then exposed to AGEs at a concentration of 100μg/ml for 48h, and detected the expression level of RNA for TNF-αand MMP-13. (3) The chondrocytes were incubated with different concentrations of AGEs for 48h, and detected the level of CAT(Catalase), activity of SOD(superoxide dismutase), MDA (Malondialdehyde), ROS(reactive oxygen species). The expression levels of mRNA for TNF-a and MMP-13 were detected by RT-PCR, the level of CAT, activity of SOD and MDA were determined by Kits, the level of ROS were tested by pyrene fluorescence probe method.Results (1) The chondrocytes were treated with AGEs (1,10, 25,50,100μg/ml) for 48h, compared with the normal control group, the expression levels of TNF-a and MMP-13 increased significantly (P<0.05 or P<0.01) and the maximum stimulation of chondrocytes were found to be at 100μg/ml AGEs; (2) Both anti-RAGE (5μg/ml) and PDTC (O.1mmol/L) can significantly inhibit the AGEs (100μg/ml)-induced expression of TNF-a and MMP-13 (P＜0.01), however, here are no significant different betweem the groups of treated with anti-RAGE (5μg/ml) or PDTC(0.1mmol/L) alone with the normal control (P> 0.05); (3) The chondrocytes were treated with AGEs (1,10,25, 50,100μg/ml) for 48h, AGEs dose-dependently decreased the activity of CAT and SOD, increased the content of MDA and SOD. Compared with the normal control group, difference have statistical sense.CONCLUSION AGEs can stimulate the TNF-a and MMP-13 high-expression in the chondrocytes, thus damage the normal function of cells; AGEs can induce the expression of TNF-a and MMP-13 via a mechanism involving the activation of RAGE, the production of ROS and the activation of the signaling pathway of NF-κB. Part II The effect and related mechanism of AGEs on expression of PPARy in rabbit chondrocytesObjectives In the present study, we investigated the effects of AGEs on expression of PPARy (Peroxisome proliferator-activated receptor-y) on the cultured rabbit chondrocytes, and explored the relationship between AGEs and PPARy as well as involved mechanism.Methods The primary cultured rabbit chondrocytes were used in the present study. (1) The chondrocytes were incubated with different concentrations of AGEs for 48h, and detected the expression level of PPARy. (2)The chondrocytes were incubated with AGEs for different time periods, and detected the expression level of PPARy. (3) The chondrocytes were incubated with anti-RAGE for 1h, then exposed to AGEs at a concentration of 100μg/ml for 48h, and detected the expression level of PPARy. (4) The chondrocytes were pretreated for 30 min with different concentrations of selective inhibitors of MAPK signaling pathways (inhibitors of P38-MAPK:SB203580, inhibitors of JNK-MAPK:SP600125, inhibitors of ERK-MAPK:PD98059), then exposed to AGEs at a concentration of 100μg/ml for 48h, and detected the expression level of PPARy. The mRNA level of PPARy was detected by RT-PCR, the protein levels of PPARγwas determined using Western blot. Results (1) The chondrocytes were treated with AGEs (1,10, 25,50,100μg/ml) for 48h, compared with the normal control group the mRNA level and proteins level of PPARy decreased significantly (P <0.05). The higher the concentration of AGEs, the lower the PPARγmRNA expression level and protein level. (2) After the chondrocytes were incubated with AGEs at a concentration of 100μg/ml for different time periods (0,3,6,12,24,48h), the PPARy mRNA expression level and protein level decreased time-dependently. In the treated group of Oh, compared with other groups, difference of the PPARy mRNA expression level and protein level have statistical sense. (P<0.05); (3) In the chondrocytes were treated with anti-RAGE(5μg/ml)+AGEs, compared with the group treated with AGEs (100μg/ml), the mRNA expression level of PPARy increased significantly (P<0.05). (4) In the chondrocytes were treated with inhibitors of P38-MAPK(SB203580) and AGEs or inhibitors of JNK-MAPK(SP600125) and AGEs, compared with the group treated with AGEs alone, the mRNA expression level and protein level of PPARy increased significantly (P<0.05), the higher the concentration of inhibitors, the higher the mRNA expression level and protein level of PPARγ; There was no significant difference between the group treated with AGEs alone and the group treated with inhibitors of ERK-MAPK (PD98059) and AGEs.Conclusion 1. AGEs time-depondently and concentration- dependently decreased PPARγexpression in chondrocytes; 2. The downregulation effect of AGEs on PPARγexpression is RAGE-mediated (AGEs-RAGE-MAPK); 3. The JNK and p38- MAPK, but not ERK, regulated pathway mediate the AGEs-induced downregulation of PPARy expression.PartⅢThe effects and related mechanism of PPARγagonist on the expression of TNF-a and MMP-13 in cultured rabbit chondrocytesObjectives In the present study, we investigated the effects and related mechanism of pioglitazone, the PPARγagonist, on AGEs-induced production of TNF-αand MMP-13 in cultured rabbit chondrocytes, and further studied on the role, mechanism and meaning of the PPARγdownregulation in AGEs-induced Osteoarthritis.Methods The primary cultured rabbit chondrocytes were used in the present study. The chondrocytes were incubated with different concentrations of pioglitazone (1,10,50μM) for 1h, then exposed to AGEs at a concentration of 100μg/ml for 48h. (1) the mRNA expression level of r TNF-a and MMP-13 was determined using RT-PCR (2) The level of CAT, activity of SOD and MDA were determined by Kits. (3) The level of ROS was tested by pyrene fluorescence probe method. (4) The transport of NF-κB-p65 Subunit was tested using immunoflurescene staining method and the protein level of NF-κB were determined using Western blot.Results (1) The mRNA expression level of TNF-a and MMP-13 showed a marked decline in the samples treated with different concentrations of pioglitazone (1,10,50μM) and AGEs, compared with samples treated with AGEs (P<0.05). The higher the concentration of pioglitazone, the lower the mRNA expression level of TNF-a and MMP-13, the difference was not statistically significant between the samples treated with 50μM pioglitazone and AGEs or treated with 50μM pioglitazone alone and the normal control sample. (2) After the chondrocytes were incubated with different concentrations of pioglitazone (1,10,50μM) for 1h, pioglitazone dose-dependently counteracted the decreasing level of CAT, activity of SOD and the increasing level of MDA,ROS induced by AGEs (P<0.05). It is also to be noted that the maximum suppression was observed in samples treated with 50μmol/l pioglitazone. (3) The chondrocytes were incubated with different concentrations of pioglitazone (1,10,50μM) for1h, then exposed to AGEs at a concentration of 100μg/ml for 48h. The transport of NF-κB p65 Subunit suppressed dose-dependently. The suppressive effect increased significantly in the sample treated with 100μg/ml AGEs alone, compared with the normal control sample (P<0.05). The protein level of NF-κB increased significantly in samples treated with 100μg/ml AGEs alone compared with the normal control sample, and decreased significantly in the samples treated with different concentrations of pioglitazone (1,10,50μM) and AGEs compared with samples treated with 100μg/ml AGEs alone.Conclusion 1. There exist a intracellular signal pathway in the chondrocytes:AGEs→RAGE→oxygen free radicals↑→activition of MAPK (JNK and P38-MAPK→downregulation of PPARγ→activition of NF-κB→TNF-a, MMP-13↑, by which AGEs can stimulate the TNF-a and MMP-13 high-expression in the chondrocytes; 2. Downregulation of PPARy has a pivotal role in the high-expression of TNF-a and MMP-13 induced by AGEs in the chondrocytes; 3. PPARy agonist pioglitazone can inhibit high-expression of TNF-αand MMP-13 induced by AGEs in the chondrocytes significantly; 4. Pioglitazone counteracted the damage of chondrocytes induced by AGEs by inhibiting the RAGE/ROS/NF-κB Signal Pathway; 5. AGEs-mediated lesion in OA may exist the following intracellular signal pathway:AGEs Combined with RAGEs→oxygen free radicals↑→activition of MAPK→downregulation of PPARγ→activition of NF-κB by intracellular Signaling transduction→the expression of inflammatory and injury factors→cartilage damage.
|
PDF Full Text Request