Study On Prdx5 Regulates The Proliferation And Apoptosis Of Human Osteoarthritis Chondrocytes And Its Mechanisms

Part oneExpression of Prdx5 in human osteoarthritis chondrocytesObjective:To examine the expression of Prdx5 in human OA articular cartilage.Methods:1. The expression of Prdx5 in human OA articular cartilage specimens and t normal articular cartilage tissues was analyzed by Western blot and qRT-PCR.2. The expression level of MMP-13 in human OA articular cartilage specimens and normal articular cartilage tissues was analyzed by Western blot.Results:1. The mRNA and protein expression levels of Prdx5 in human OA articular cartilage specimens were significantly elevated compared with the normal articular cartilage tissues (P< 0.05). The mRNA expression level of Prdx5 was overexpressed by 3.5 ± 0.21 times in human OA articular cartilage specimens compared to normal articular cartilage tissues, and the protein expression level of Prdx5 in human OA articular cartilage specimens were 2.31 ± 0.18 times higher than in normal articular cartilage tissues (P< 0.05).2. The protein expression of MMP-13 in human OA articular cartilage specimens was significantly increased compared to the normal articular cartilage tissues (P< 0.05).Conclusion:Prdx5 and MMP-13 are highly expressed in human OA articular cartilage specimens. And the expression of Prdx5 and MMP-13 was may be closely related to the occurrence and development of osteoarthritis.Part twoEffect of Prdx5 on the proliferation and apoptosis in human osteoarthritis chondrocytesObjective:To investigate the effect of silenced Prdx5 on proliferation and apoptosis in osteoarthritis chondrocytes.Methods:1. The effect of silenced Prdx5 on the proliferation and apoptosis in osteoarthritis chondrocytes were detected using MTT and flow cytometry assays.2. A ROS assay kit was used to measure intracellular ROS in osteoarthritis chondrocytes after Prdx5 knockdown.3. The effect of NAC on the proliferation and apoptosis in silenced Prdx5 osteoarthritis chondrocytes were detected by MTT and flow cytometry assays.Results:1. It was showed that the proliferation of silenced Prdx5 osteoarthritis chondrocytes was significantly decreased compared to controls by MTT assays. And flow cytometry analysis showed that the apoptosis in the silenced Prdx-5 osteoarthritis chondrocytes was increased significantly compared to controls (P< 0.05).2. It was showed that the content of ROS in silenced Prdx5 osteoarthritis chondrocytes was significantly increased compared with controls (P< 0.05).3. After treatment of silenced Prdx5 osteoarthritis chondrocytes with NAC for 24 h, the results showed that the proliferation was increased and the apoptosis was decreased significantly in NAC-treated silenced Prdx5 osteoarthritis chondrocytes compared to untreated silenced Prdx5 osteoarthritis chondrocytes (P< 0.05).Conclusion:Prdx5 knockdown suppressed the proliferation and activated the apoptosis in osteoarthritis chondrocytes, and may be due to an increase in ROS.Part threeThe mechanisms of Prdx5 modulate the proliferation and apoptosis of osteoarthritis chondrocytesObjective:To investigate the effect of Prdx5 knockdown on expression of Wnt/β-catenin pathway related genes, reveal the mechanisms of Prdx5 modulate the proliferation and apoptosis of osteoarthritis chondrocytes.Methods:1. The expression of nuclear β-catenin and cytoplasmic β-catenin, a critical factor of Wnt/p-catenin pathway, other proteins associated with the Wnt signaling pathway such as Wnt-4, Frizzled-2, GSK-3β, p-GSK-3βser9, p-β-cateninser33/37 and two Wnt target gene CyclinD1, MMP-13 in Prdx5 silenced osteoarthritis chondrocytes and controls by Western blot assays. The mRNA expression levels of CyclinD1， MMP-13 were also analyzed by qRT-PCR.2. An alteration of intracellular distribution of β-catenin in Prdx5 silenced osteoarthritis chondrocytes and control osteoarthritis chondrocytes were detected by immunofluorescence staining.3. The transcriptionactivity of β-catenin in Prdx5 silenced osteoarthritis chondrocytes was detected by TOPflash luciferase report gene system.4. The expression of nuclear β-catenin and cytoplasmic β-catenin, a critical factor of Wnt/β-catenin pathway, other proteins associated with the Wnt signaling pathway such as GSK-3β, p-β-cateninser33/37 in Prdx5 silenced normal chondrocytes and controls by Western blot assays. 5. A ROS assay kit was used to measure intracellular ROS in silenced Prdx5 osteoarthritis chondrocytes after treatment of Wnt/β-catenin pathway inhibitor XAV-939.Results:1. The level of intranuclear β-catenin was significantly increased in silenced Prdx5 osteoarthritis chondrocytes, whereas the level of cytoplasmic β-catenin was significantly decreased in silenced Prdx5 osteoarthritis chondrocytes (P<0.05). In addition, the level of phosphorylated β-catenin (Ser33/37) was significantly decreased in silenced Prdx5 osteoarthritis chondrocytes (P<0.05). However, the total expression level of β-catenin was largely unchanged. The level of GSK-3β was significantly decreased in silenced Prdx5 osteoarthritis chondrocytes, whereas the level of phosphorylated GSK-3βSer9 was significantly increased in silenced Prdx5 osteoarthritis chondrocytes (P<0.05). Furthermore, the levels of Wnt-4, Frizzled-2, CyclinD1, MMP-13 were significantly increased in silenced Prdx5 osteoarthritis chondrocytes, and the up-regulation of CyclinD1 and MMP-13 mRNA expression levels in silenced Prdx5 osteoarthritis chondrocytes were determinated by qRT-PCR assays.2. It was showed that β-catenin was observed to translocate from its location in the cytoplasm in mock osteoarthritis chondrocytes to the nucleus in silenced Prdx5 osteoarthritis chondrocytes by immunofluorescence staining.3. It was showed that the β-catenin/Tcf-dependent transcriptional activity was significantly enhanced in osteoarthritic chondrocytes following Prdx5 silencing by TOPflash luciferase report gene system (P＜0.05).4. The Wnt/β-catenin signaling was not significantly affected in normal chondrocytes after transfection with Prdx5shRNA by Western blot assays.5. XAV-939 alone reduced endogenous production of ROS in osteoarthritic chondrocytes. However, treatment with XAV-939 in osteoarthritic chondrocytes with Prdx5 knockdown did not further reduce the endogenous production of ROS compared to osteoarthritic chondrocytes treated with XAV-939 alone.Conclusion:Prdx5 silencing upregulated the Wnt/β-catenin pathway and it plays a critical role in the proliferation and apoptosis of human osteoarthritic chondrocytes possibly through modulating the Wnt/β-catenin pathway.