Histone Deacetylase 4 Ameliorates Osteoarthritis Articular Cartilage Degeneration By Inhibiting ER Stress Induced Chondrocytes Apoptosis

Background:The pathogenesis of osteoarthritis?OA?is unclear,and there is no effective way to delay or block the progression of OA.Chondrocytes are the only cell type in articular cartilage and play an important role in maintaining the balance of anabolism and catabolism of cartilage.The apoptosis of chondrocytes mediated by endoplasmic reticulum stress?ERS?plays an important role in the pathogenesis of OA.Chondrocytes apoptosis can lead to the imbalance of anabolism and catabolism of cartilage,and this change can promote the apoptosis of chondrocytes,thus accelerating the pathological process of OA.ERS mediated apoptosis is mainly regulated by PERK-eIF2?-ATF4 pathway.Activation transcription factor 4?ATF4?is one of the key molecules.Its overexpression can activate the expression of downstream apoptosis promoting genes,thus causing apoptosis.However,the regulation of ATF4 expression in OA and its role in promoting apoptosis are still unclear.To clarify the role of ERS mediated by ATF4 in OA articular cartilage degeneration and its expression regulation is of positive significance to clarify the pathogenesis of OA and take effective measures to ameliorates or block OA articular cartilage degeneration.Objective:To investigate the role of ERS mediated by ATF4 in the pathogenesis of OA,and to investigate whether the reduced histone deacetylase 4?HDAC4?in cartilage is associated with the activation of ATF4 and the apoptosis of chondrocytes during OA,and then establish an anterior cruciate ligament transection?ACLT?rat OA model to investigate whether HDAC4 supplementation can reduce the apoptosis of chondrocytes by inhibiting ATF4 mediated ER stress,so as to delay the degeneration of OA articular cartilage.Methods:1.To observe whether there are ER stress and chondrocyte apoptosis mediated by ATF4 in human OA articular cartilage and the change of HDAC4 content in OA:The articular cartilage samples were obtained from OA patients who performed total knee replacement,and the relatively normal articular cartilage?Outerbridge I?and OA cartilage tissue?Outerbridge I?were taken.The expression of HDAC4 and ERS related indexes ATF4,CHOP,GRP78 and Caspase12 at protein and mRNA levels were detected by immunohistochemistry,western blot and real-time qPCR.2.To study the regulation mechanism of HDAC4 on the expression ATF4:?1?Experiment 1:rat costal chondrocytes were diveded into two groups.The experimental group was treated with H₂O₂ at the concentration of 300?m for 12 hours,while the control group was not treated.Then the expression of ERS indexes such as ATF4,CHOP,GRP78and caspase12 was detected by real-time qPCR.?2?Experiment 2:on the basis of Experiment 1,HDAC4 plasmid and HDAC4 siRNA were transfected in the experimental group to up regulate and down regulate HDAC4 in chondrocytes,while negative control plasmid and siRNA were transfected in the control group.After 24 hours of transfection,ERS were induced in vitro by adding H₂O₂ with concentration of 300?m for 12 hours.The expression of ATF4,CHOP,GRP78 and Caaspase12 in mRNA and protein level was detected by real-time qPCR and Western blot.The apotosis of chondrocytes were detected by flow cytometry.3.In order to observe whether HDAC4 ameliorates the degeneration of OA articular cartilage by inhibiting ATF4 mediated apoptosis of chondrocytes,eight-week-old male SD rats?n=52?were randomly divided into three groups:ACLT+Ad-GFP group?henceforth,Ad-GFP;n=17?,ACLT+Ad-HDAC4-GFP group?henceforth,Ad-HDAC4;n=20?,sham-operated+Ad-GFP group?henceforth,sham-operated;n=15?.Ad-HDAC4-GFP and Ad-GFP were administered by intra-articular injection 48 h after surgery,and every 3weeks thereafter.Three rats from the Ad-HDAC4 group were sacrificed at 3 days,2 weeks,and 3 weeks after the initial adenovirus injection for histological examination of 5-?m sections to determine the location and expression of HDAC4 in the rat knee and tibial plateau using fluorescence microscopy.Two rats from the Ad-GFP group and 2 rats from the Ad-HDAC4 group were sacrificed 2 weeks after the initial adenovirus injection;the articular cartilage was harvested,and the expression of HDAC4 was examined by RT-qPCR.All other animals were euthanized 8 weeks after surgery.?1?X-ray was used to observe the formation of osteophyte;?2?the destruction of articular cartilage and the loss of proteoglycan were detected by India ink staining and Safranin O/Fast green staining;?4?the expression of ATF4,CHOP,GRP78 and collagen II?Col II?was detected by immunohistochemistry and real-time qPCR;?5?TUNEL staining was used to detect the apoptosis of chondrocytes.Results:1.The relatively normal articular surface of human cartilage was smooth,the proteoglycan in the superficial layer of cartilage was slightly lost,the chondrocytes were arranged orderly,while the surface of OA articular cartilage was damaged,the proteoglycan in cartilage was largely lost,and the chondrocytes were accumulated in clusters.TUNEL staining showed that the apoptosis rate of OA chondrocytes was significantly higher than that of relatively normal cartilage?P<0.05?.The results of immunohistochemistry showed that compared with relatively normal cartilage,the expression of ATF4 in OA articular cartilage increased,while the expression of HDAC4decreased,and the results of real-time qPCR were consistent with that of immunohistochemistry;Western blot further confirmed the results of immunohistochemistry and real-time qPCR.Immunohistochemistry showed that the protein of CHOP,GRP78 and Caspase12 in OA cartilage was significantly higher than that in the relatively normal cartilage.Similarly,the mRNA expression of CHOP,GRP78 and Caspase12 in OA articular cartilage was higher than that in normal cartilage?P<0.05?.2.The expression of ATF4,CHOP,GRP78 and Caspase12 mRNA in the experimental group was higher than that in the control group?P<0.05?,indicating that the ERS model was successfully constructed in vitro.The results of real-time qPCR showed that upregulation of HDAC4 reduce the expression of ATF4,CHOP,GRP78 and Caspase12 mRNA,while downregulation of HDAC4 expression by siRNA increased the expression of these factors,which was statistically significant?P<0.05?.The results of western blot were consistent with real-time qPCR.The results of flow cytometry showed that under the condition of ERS,up regulation of HDAC4 could reduce the apoptosis of chondrocytes,while down regulation of HDAC4 could increase the apoptosis of chondrocytes?P<0.05?.3.Injection of Ad-HDAC4-GFP into articular cavity can effectively infect articular cartilage.The expression of green fluorescence was found in articular cartilage at 3 days after injection,reached the peak at 2 weeks,and weakened at 3 weeks.The results of real-time qPCR showed that the level of HDAC4 mRNA in Ad-HDAC4 group was8.87-fold higher than that in Ad-GFP group.At 8 weeks after operation,the X-ray results showed that there were less osteophytes in Ad-HDAC4 group,Indian ink staining showed that there were less Indian ink residues on the cartilage surface of Ad-HDAC4 group;safranin O staining showed that the articular cartilage surface of Ad-HDAC4 group was smooth,and the proteoglycan loss was less;immunohistochemistry showed that the ATF4,CHOP,GRP78 protein expression of Ad-HDAC4 group was less than that of Ad-GFP group,while the expression of Col II was higher than that of Ad-GFP group,and the results of real-time qPCR were consistent with those of immunohistochemistry.TUNEL staining showed that the apoptosis rate of chondrocytes in Ad-HDAC4 group was lower than that in Ad-GFP group?P<0.05?.Conclusion:The endoplasmic reticulum stress mediated by ATF4 is activated continuously in OA,which leads to the increase of chondrocyte apoptosis and participates in the pathological process of OA articular cartilage degeneration.In the pathological process of OA,the decreased HDAC4 in articular cartilage released the inhibition of ATF4,which led to the continuous activation of ATF4 endoplasmic reticulum stress signal pathway and the increase of chondrocyte apoptosis,thus promoting the degeneration of OA articular cartilage.HDAC4 attenuated the degeneration of OA articular cartilage by inhibiting the expression of ATF4 and reduce the apoptosis of chondrocytes caused by endoplasmic reticulum stress and promote the anabolism of cartilage in rats.