| Objective: Connexin43(Cx43) is essential for maintenance of metabolic chondrocytes homeostasis. PGE2 is the major prostaglandins synthesized by chondrocytes and is responsible for mediating the imbalance between anabolic and catabolic metabolism in cartilage. This study was undertaken in order to determine the relationship between PGE2 and Cx43 in chondrocytes. To demonstrate if PGE2 could direct influence express and function of Cx43Methods: Twenty-four week-old Spraguee-Dawley rats were taken, then were divided into six groups at random. To obtain primary chondrocytes with enzyme digestion method.Cells were onto 75 cm2 culture bottle.Chondrocytes were cultured in Dulbecco’s modified Eagle’s medium supplemented with 100 mg/m L Primocin and 15% fetal calf serum.Cells were grown to approximately 90% confluence for Protein expression assays and gene expression assays with immunocytochemistry,quantitative Real-time PCR and western blot. Before the test,in time-dependent group chondrocytes were treated to 5u M PGE2 for 2h, 8h and 16 h, in dose-dependent group chondrocytes were exposed to 1u M, 5u M and 10 u M PGE2 for 16 h. chondrocytes were exposed by vehicle treated in control group.Results: Rat primary chondrocytes express high levels of Cx43 by immunocytochemistry assays. To further confirm the expression level of Cx43, their expression was evaluate by the western blot. The OD values of Cx43 in vehicle treated control group were 11.89±4.09 and the OD values of Cx43 in chondrocytes treated for 2, 8 and 16 hours with 5u M PGE2 were 27.98±8.22,32.83±9.49,42.09±11.52, respectively. A 3.5 fold increase in Cx43 protein abundance was observed at 16 hours treaed with 5u M PGE2.There was statistical significant difference between the group as cells treated for 16 hours with 5u M PGE2 and vehicle control group(P<0.05). increase in Cx43 protein abundance was observed after 2 and 8 hours of treatment with 5u M PGE2, indicates no statistical significant difference from the vehicle control(P>0.05). The OD values of Cx43 in chondrocytes treated with 1, 5 and 10 u M PGE2 for 16 hours were 17.07±5.47,42.09±11.52,48.29±11.56, respectively. analysis indicate increases in Cx43 expression of 3.5- and 4.1-fold relative to vehicle treated controls after treatment with 5 and 10 u M PGE2 for 16 hours, respectively. The increase of the Cx43 expression had statistical significant difference from vehicle control group(P<0.05).There was no statistical significant difference between the group as cells treated for 1u M PGE2 with16 hours and vehicle control group(P>0.05). The number of gap junctions between the chondrocytes were counted by oil immersion lens. The number of gap junctions in vehicle treated control group were 1.00±0.04. It in chondrocytes treated for 2, 8 and 16 hours with 5u M PGE2 were 3.84±0.07, 4.06±0.02, 6.83±0.05, respectively. 3.8-,4.1-and 6.8-fold increases in the number of gap junctions relative to vehicle treated controls, respectively.There were statistical significant difference from vehicle control group( P < 0.05). The number of gap junctions in chondrocytes treated with 1, 5 and 10 u M PGE2 for 16 hours were 3.69±0.02, 6.83±0.05, 7.95±0.03, respectively. analysis indicate increases in the number of gap junctions of 3.7- of 6.8- and 8.0-fold relative to vehicle controls group after 16 hours of treatment with 1,5 and 10 u M PGE2, respectively. There were statistical significant difference from vehicle control group(P<0.05). level of Cx43 m RNA was evaluate by quantitative Real-time PCR,The 2-ΔΔCT of Cx43 m RNA in vehicle treated control group were 1±0. The 2-ΔΔCT of Cx43 m RNA in chondrocytes treated for 2, 8 and 16 hours with 5u M PGE2 were 2.34±0.26,2.49±0.64,3.06±0.44, respectively. There was no statistical significant difference between the group as cells treated for 2h with 5u M PGE2 and vehicle control group(p=0.05). 2.5- and 3.1- fold increase in Cx43 m RNA abundance was observed after 8 and 16 hours of treatment with 5u M PGE2,respectively.There was statistical significant difference from vehicle control group(P<0.05). The 2-ΔΔCT of Cx43 m RNA in chondrocytes treated with 1, 5 and 10 u M PGE2 for 16 hours were 1.57±0.28,3.06±0.44,3.85±0.37, respectively. analysis indicate increases in Cx43 m RNA expression of 3.1- and 3.9-fold relative to vehicle treated controls at 16 hours treatment with 5 and 10 u M PGE2, respectively.There were statistical significant difference from vehicle control group(P<0.05). There was no statistical significant difference between the group as cells treated for 16 with 1u M PGE2 and vehicle control group(P>0.05).The study revealed that PGE2 could induce overexpression and/or loss of localization of Cx43 with increased of number of gap junction. PGE2 also changed the Cx43 m RNA level.(For multiple comparisons of three groups or more than three groups, one-way ANOVA was used. The statistical significance difference was defined as P < 0.05.)Conclusion: The present research provide the first evidence that PGE2 direct influenced the Cx43 protein expression in cultured rats Chondrocytes with in a time-dependent and(or) in a dose-dependent and alter localization of Cx43 protein. PGE2 also changed the Cx43 m RNA level. |
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