Effects Of Histone Deacetylase 4 And Resveratrol On Differentiation Of Chondrocytes And Its Mechanism

Objective:Articular cartilage related diseases are of the high incidence in clinical. The reasons of the common morbidity are due to external trauma and inner cartilage degradation. The self-repairing capacity of cartilage after injury is very limited. Even a minor lesion of the cartilage might lead to progressive damage and articular degeneration. The poor spontaneous heal capability can be explained by the fact that articular cartilage is avascular tissue and lack of cytokines and sufficient reparative cells. In recent years, the tissue engineering of cartilage has made tremendous achievements. Mesenchymal stem cells(MSCs) are widely used in restoring cartilage, because they are comparatively easy to isolation, proliferation and differentiation. The major challenge of cartilage repair in clinical now is how to induce MSCs differentiate into chondrocytes and keep phenotype of chondrocytes from terminal differentiating into hypotrophy chondrocytes. Thus, there is a need to understand the process of chondrocyte differentiation, and its regulation in field of tissue engineering.The first part of the study was focused on the role of HDAC4 in chondrogenesis and hypertrophic differentiation by using an inducing model in vitro. The results provided a theoretical basis for cartilage tissue engineering. The second part of the study was based on a hypertrophic inducing model. In this model, we tested the effect of resveratrol in hypertrophic process, and provided a potential regulator in subchondral bone regeneration in vitro. Method and Contents: The study consists of two parts. Different regulators were tested in different differentiation processes. 1. HDAC4 promotes MSCs pre-chondrocyte differentiation by direct interaction with HIF1α(1) The construction of cell lines and the establishment of chondrocytic induction systemMSCs stably expressing HDAC4 were generated by lentivirus infection at different MOIs. HDAC4 si RNA MSCs were generated in the same way as Lv.HDAC4 MSCs. CCK8 was chosen to test the cell viability after transfection. Different groups were cultured in induction media which contained TGF-β3 for 14 days. The chondrocytic-related genes(Col II、Sox9) were detected by q PCR.(2) Effect of overexpression or interference expression of HDAC4 on cartilage differentiationIn the induction system, the m RNA and protein were collected for detecting the chondrocytic and hypertrophic marker gene expression. And toluidine blue staining analysis was used to detect GAG in ECM.(3) The mechanism of HDAC4-mediated promotion in chondrogenesisThe expression of HIF1α was detected by q PCR and western blot in all groups. The interaction of HIF1α and HDAC4 was demonstrated by using Duolink-PLA. To explore how the interaction impact on chondrogenesis, 2-Me2OE2 or IOX2 was used in inhibiting or activating HIF1α. The shuttle of HDAC4 was inhibited by using LB-100. 2. The effects and mechanisms of resveratrol on chondrocyte hypertrophy(1) The establishment of hypertrophic induction system and the determination of safety resveratrol concentrationATDC5 cells were treated with induction culture medium for 14 days. The expression of hypertrophy-related factors including Runx2, MMP13 and Col X were detected using q RT-PCR and western blot. The cells were cultured with basal culture medium for 24 h and 48 h with different concentrations of RES(0, 0.1, 0.5, 1, 5, 10, 20μM) followed by CCK-8 assays.(2) The effect of resveratrol on the hypertrophic differentiationATDC5 cells were divided into control group(0μM) and RES group(1μM) on hypertrophy induced system for 14 days. The expression of hypertrophy-related factors including Runx2, MMP13 and Col X were detected using q RT-PCR and western blot. Toluidine blue staining for GAG and immunohistochemistry for Col X and Runx2 were performed.(3) The mechanism of resveratrol-mediated promotion in hypertrophic differentiationExpression of altered transcriptional factors and key molecules in canonical chondrogenic pathways including WNT, IHH and FGF were examined by q RT-PCR. Results: 1. HDAC4 promotes MSCs pre-chondrocyte differentiation by direct interaction with HIF1α(1) Establish transfection cell lines and the chondrocytic induction system in vitroThe positive efficiency increased with the enhancement of MOI. Considering the infection efficiency and cell viability, lentiviral HDAC4 delivery at MOI 500 was chosen in this research. The expression of HDAC4 in Lv.HDAC4 was higher than Lv.Control, which indicated the overexpression cell line was successfully constructed. The interference cell line was also well constructed by detecting the loss of HDAC4. The expression of chondrocytic marker genes were increased in chondrocytic induction system, which indicated the chondrocyte hypertrophy model was well established.(2) The study of the effects on cartilage differentiation with or without HDAC4 expressionThe marker genes of chondrocytes including Sox9 and Col II dramatically increased in Lv.HDAC4 MSCs. More extensive staining by toluidine blue was observed at day 14 in Lv.HDAC4 MSCs, reflecting high contents of proteoglycans. The Inhibition of HDAC4 reduced the Col II and Sox9 expression, but increased Runx2, Mef2 c and collagen type X(Col X), markers of chondrocyte hypertrophy, as well.(3) The research on the mechanism of HDAC4-mediated promotion in chondrogenesisCompare HIF1α expression pattern between Lv.HDAC4 group and the si RNA HADC4 group, HIF1α was notably increased by using Western blot and immunofluorescence staining. Duolink-PLA staining red points demonstrated the interaction of two proteins in MSCs. The expression of Col II and Sox9 m RNA were significantly decreased in Lv.HDAC4 MSCs with 2-Me2OE2 treatment. And the chondrocytes marker gene expression of Sox9 and Col II was dramatically increased with IOX2 treatment. After treatment of LB-100, less accumulation of HIF1α were observed in nuclei in Lv.HDAC4 infected MSCs. 2. The effects and mechanisms of resveratrol on chondrocyte hypertrophy(1) Establish of hypertrophic induction system and determine the safety resveratrol concentrationAfter induction by hypertrophy medium with T3 for 14 days, ATDC5 was considered into hypertrophy stage. The concentration of RES below 1μM had no toxic effect in this experiment.(2) The study on the effect of the hypertrophic differentiation with resveratrolAfter induction by hypertrophy medium for 14 days, toluidine blue staining intensity was increased following RES treatment. Treated ATDC5 with RES showed a significant increase both in the m RNA and protein of hypertrophy-related genes including Runx2, MMP13 and Col X on day 3, 7, 14. And the staining by immunohistochemistry shown that the Col X and Runx2 increased in RES group.(3) The study on the mechanism of resveratrol-mediated promotion in hypertrophic differentiationThe expression of β-catenin and GLI2 in RES group was increased and GLI3 was decreased significantly. Expression of PI3 K was not changed in RES treatment group. Conclusion:In our chondrogenesis induction system, overexpression of HDAC4, via direct interaction with HIF1α, accounts for the MSCs pre-chondrocyte differentiation. Conversely, loss of HDAC4 promotes chondrocyte hypertrophy via up regulation of hypertrophic markers. We also show that HDAC4 nuclear import contributes to HIF1α nuclear accumulation. These results indicated that HDAC4 might be a potential target to maintain chondrocyte phenotype and to regenerate articular cartilage. Moreover, in chondrocyte hypertrophy induction system, we found RES promotes ATDC5 hypertrophy by up-regulating β-catenin and GLI2, but down-regulating GLI3. And these results demonstrated that resveratrol might be a hopeful molecular in entochondrostosis-based bone regeneration.