Effect And Mechanism Of BFGF On The Repair Of Primary Cilia Promoting Articular Cartilage Repair

ObjectiveThis study was to investigate the effect of bFGF on articular cartilage repair by in vivo animal experiments.To investigate whether bFGF regulates the formation and maintenance of cilia by in vitro cell experiments,and to explore whether the bFGF can regulate the expression of IFT88 and primary cilia formation In vitro chondrocytes,for the repair of articular cartilage opened up a new direction.Method(1)Sprague-Dawley rats were randomly divided into control group,model group,bFGF group.Knee joint tissues were collected and the appearance of knee cartilage in each group was observed at 4,8,and 12 weeks after operation respectively.HE staining sections were made to observe the knee Pathological changes of articular cartilage.TUNEL method was used to detect the apoptosis of articular cartilage in each group.The expressions of type I collagen,type II collagen,MMP-3 and MMP-9 were detected by immunohistochemistry.(2)The expressions of IFT88 mRNA and protein in ATDC5 cells were detected after treated with different concentrations of bFGF respectively.The expressions of IFT88 mRNA and protein in ATDC5 cells were detected after treated with bFGF for different intervention times respectively.ATDC5 cells were treated with bFGF,ERK inhibitor PD0325901 and bFGF+PD0325901 respectively,and the expressions of P-ERK,ERK and IFT88 protein were detected.ATDC5 cells were treated with bFGF and FGFR inhibitor BGJ398 respectively,and the expression of P-ERK,ERK and IFT88 protein were detected.The transfected siIFT88 method was used to establish the IFT88 expression inhibitory cell line,and the expression of IFT88,P-ERK and ERK protein was detected in the transfected cells.Immunofluorescence assay to detect the growth of primary cilia after treated with bFGF in ATDC5 cells.Result(1)With the passage of time,there was no obvious tissue filling in the articular cartilage defect area of the model group,and the cartilage defect could not be repaired.The tissue filling of the bFGF group was significantly better than that of the model group.At the 4th,8th and 12th postoperative week,compared with the control group,the apoptotic rate of knee joint cartilage cells in the model group and the bFGF group was significantly higher;At the 8th week and 12th week,the apoptosis rate in the bFGF groups were significantly lower than that in the model group.The results of HE staining showed that there was a large amount of fibrous tissue in the cartilage defect area of the knee joint in the model group after operation.The thickness of the surrounding cartilage was reduced,and the cartilage surface was obviously uneven.In the bFGF group,there was only a small amount of fibrous tissue around the injury area,the chondrocytes showed clumps and the surface was slightly uneven;while the cartilage defect still existed in the bFGF group after operation,the number and thickness of cartilage were significantly better than those in the model group.Immunohistochemistry was used to detect the expression and distribution of type ? collagen in the cartilage of the knee joints of rats in each group.The results showed that the immunohistochemical staining of type ? collagen was negative in control rats at each time point;The model group showed signs of fibrous tissue repair postoperatively,and the number of chondrocytes in the defect area decreased,and the cartilage matrix and most of the cells stained positively.In the bFGF group,a little fibrous tissue was repaired on the defect surface,and both the extracellular matrix and the cartilage cell mass were deeply stained,there was a clear demarcation between the adjacent normal cartilage tissue,and the hypertrophic chondrocytes increase,and the cytoplasm staining is positive.Immunohistochemistry was used to detect the expression and distribution of collagen type ? in the cartilage of knee joints of rats in each group.The results showed that the staining results of type ? collagen in the cartilage tissue of the control group were positive at each time point;The expression of type ? collagen in cartilage cells and surrounding stroma was significantly decreased at each time point,and no sign of articular cartilage repair was observed at any time point;bFGF group stained weakly positively postoperative,and most of the cytoplasm was cytoplasmic,the extracellular matrix staining was positive and the deep chondrocytes stained positively.The expression and distribution of MMP-3 protein in the cartilage tissue of the knee joints of the rats in each group were detected by immunohistochemistry.The result showed that at the 4th,8th,and 12th weeks of the control group,the normal cartilage tissues were scattered with weakly stained chondrocytes,the remaining chondrocytes stained negative for MMP-3;In the model group,MMP-3 staining was strongly positive in full-thickness chondrocytes and their surrounding stroma at all time points.In the bFGF group,MMP-3 staining was positive in the cartilage matrix and some chondrocytes,however,the positive area and degree of MMP-3 staining were significantly lower in the three time points than in the model group.Immunohistochemistry was used to detect the expression and distribution of MMP-9 protein in the cartilage tissue of the knee joints of the rats in each group.The results showed that only a small amount of chondrocytes staining positively stained chondrocytes were found in the deep layer of cartilage,but no extracellular matrix was positively stained in the control group at the 4th,8th,and 12th weeks postoperatively.In the model group,MMP-9 protein was seen in the extracellular matrix of chondrocytes,staining of extracellular matrix of cartilage was deepened in some area,and positive cells were mainly concentrated in deep cartilage;The degree of cartilage matrix staining was lower in the bFGF group than that in the model group at at each time point,the number of chondrocytes was significantly increased compared with the model group,the cytoplasm of cartilage cells stained positively,mainly in the deep layers of cartilage,but the degree of staining was less than that of the model group.(2)The effect of bFGF on the expression of IFT88 in ATDC5 cells The results showed that the expression levels of IFT88 mRNA and protein in IFT88 cells were increased to some extent,the expression level of IFT88 mRNA and protein peaked when the concentration of bFGF was 5 ng/mL,and then dropped back.Compared with group 0,the expression levels of IFT88 mRNA and protein in IFT88 cells increased to different extents at 12,24,36 and 48 h,and the levels of IFT88 mRNA and protein peaked at 24 h after bFGF treatment.The effect of bFGF on the expression of P-ERK,ERK and IFT88 after ERK inhibitor treatment showed that the expressions of P-ERK and IFT88 protein in bFGF and bFGF+PD0325901 groups were significantly increased compared with control group.The expression of P-ERK and IFT88 in bFGF group was significantly higher than that in bFGF+PD0325901 group.Compared with control group,the expressions of P-ERK and IFT88 in PD0325901 group were significantly decreased.There was no significant difference in the expression level of ERK protein in each group.The effect of BGJ398 on the expression of IFT88,P-ERK and ERK protein showed that the expression levels of P-ERK and IFT88 in bFGF group were significantly higher than those in control group,while those in BGJ398 group P-ERK and IFT88 protein expression levels were significantly reduced.There was no significant difference in the expression level of ERK protein in each group.The results showed that the expression of IFT88 mRNA and protein in siIFT88 group was significantly lower than that in negative control group,suggesting that the IFT88-inhibited ATDC5 cell line was constructed successfully,Which could be used in the next experimental study.The expression level of P-ERK protein in siIFT88 group was significantly lower than that in negative control group,while the expression level of ERK protein did not change significantly.The results of bFGF on ciliary growth on ATDC5 cells showed that the positive staining for acetylated ?-tubulin contained in primary cilia was red after staining with anti-acTub antibody.The number of primary cilia in the bFGF group was significantly higher than that in the control group.Conclusion(1)bFGF promotes the proliferation of chondrocytes after articular cartilage injury,and has an inhibitory effect on the proliferation of fibrous tissue at the cartilage defect.(2)bFGF can up-regulate the expression of IFT88 mRNA and protein,thereby promoting primary cilia formation in chondrocytes.