Asian Journal of Biomedical and Pharmaceutical Sciences

Research Article - Asian Journal of Biomedical and Pharmaceutical Sciences (2017) Volume 7, Issue 60

Tumor Necrosis Factor (TNF-?)-Induced Upregulation of Cartilage Degradation-Associated Genes by Chondrocytes Requires Epithelial Sodium Channel Activity

Purpose: Chronic inflammation due to increased TNF-α is the main cause of Rheumatoid Arthritis cartilage destruction. We investigated the effects of hTNF-α on chondrocyte and its relationship with water salt metabolism.

Methods: Chondrocytes were cultured from the knee cartilage of ICR mouse pups. Various concentrations of hTNF-α (0, 25, 50, 100 ng/ml) were used to treat chondrocytes and apoptosis was measured by flow cytometry. hTNF-α 25 ng/ml was used in TNF-α, Amiloride (Ami), TNF-α+Ami and control groups. MMPs, IL-1α, BAX and caspase 3 mRNA levels were examined by Real-time PCR. The length and growth rate of metatarsus were measured by stereo microscope. Histological changes were observed by H&E staining and Toluidine blue staining. TUNEL staining determined the cartilage apoptosis on the tissue level.

Results: Our result further conformed that hTNF-αin three concentrations significantly enhanced apoptosis compared to the control group (p<0.05). Also hTNF-α significantly increased MMP9, MMP13, IL-1α, BAX and caspase 3 mRNA expression (p<0.05) compared with control group, and added Ami on hTNF-αgroup can reduce the above indicators and partially neutralized the effect of hTNF-α on MMP9, MMP13, IL-1α (p<0.05) and almost neutralized the effect of hTNF-α on BAX and caspase 3 in vitro. In metatarsals ex vivo, TNF-α inhibited metatarsal growth (P<0.01) while Ami had no significant effect compared to the control group. However, metatarsal growth rate in Ami+TNF-α group was lower than control and higher than TNF-α-treated group (P<0.01). HE and Toluidine blue staining showed that the proliferative area of chondrocytes in hTNF-α group was significantly decreased with chaotic alignment in chondrocyte layer. Ami showed no changes and hTNF-α+Ami treatment showed some improvement compared to hTNF-α group but was worse than Ami group. TUNEL staining showed that hTNF-α led to significant chondrocytic apoptosis while Ami did not compared to the control. hTNF-α+Amiloride showed milder apoptosis compared to hTNF-α group.

Conclusion: In Summary, hTNF-αimpacts chondrocytes by promoting MMPs and apoptosis. Amiloride could partially neutralize the above indicators in vitro and metatarsals ex vivo. It was speculated that there existed a relation in the effect of TNFa on chondrocytes with ENaC, a key protein in water salt metabolism.

Author(s): Min Li, Ge Li, Lu Tang, Xingyan Lu, Jun Chen, Qingnan Li, Guozhu Yang, Huanhuan Jia, Yu?e Wu, Yu Zhang

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