The Role Of IL-6 In Subchondral Bone Abnormalities In Osteoarthritis Mice And Its Mechanism

Part I Disease phenotype of cartilage and subchondral bone in osteoarthritis miceObjective: Build a model of osteoarthritis(OA)in the mice by surgically transecting anterior cruciate ligament(ACLT).Histological analysis of the knee joint was made to investigate tibial cartilage and subchondral bone of OA in the mice.Materials and Methods: The anterior cruciate ligament of the knee of the 8-week,male C57BL/6J mice was transected.A paraffin section of the affected knee joint was made 8 weeks after the surgery.The sections were stained with Alcian blue/hematoxylin & orange G(AB/H&OG)and studied microscopically by histology.The severity of gross cartilage and subchondral bone damage was assessed with OARSI scoring system.The area of tibial cartilage was calculated with software Image J.The depth of tibial cartilage was observed microscopically and was measured with bone histomorphometric software Bioquant.Results: After surgical for eight weeks:(1)A severe tibial cartilage loss of late-stage OA in the mice was noted as compared to the sham operation control group.The hyaline cartilage was severely damaged,and subchondral bone was exposed.OARSI score of the ACLT group was significantly higher than the control group.(2)The remaining area of tibial cartilage of late-stage OA in the mice was much smaller than the sham operation group(P<0.05).(3)The average subchondral bone plate thickness of late-stage OA in the mice was significantly higher than that of the sham operation group.Conclusion: These results demonstrate that ACLT is effective in induction of OA model in the mice.The pathological changes as measured histologically are similar to that of human knee OA in terminal stage.Part II Disease phenotype of subchondral bone marrow mesenchymal stem cells in osteoarthritis miceObjective: ACLT OA model was created,bone marrow stromal cells(BMSCs)in tibial subchondral bone of the mice were isolated and cultured in vitro.The osteogenetic differential potential,capacity of mineralization and collagen synthesis of BMSCs in tibial subchondral bone of the mice were measured after operation.Materials and Methods:A model of osteoarthritis(OA)in an 8-week,male C57BL/6J mouse by ACLT was built.The proximal tibia was collected and BMSCs were isolated from subchondral bone.BMSCs were:(1)inoculated in 10-cm dishes and osteoblastic induced in vitro for 21 days.Alizarin red staining was used,then eluted and quantitatively analyzed with the spectrophotometry;(2)The proportion of Nestin and Osterix positive cells was measured by flow cytomertry;(3)the total RNA and protein were extracted.The expression of m RNA and protein of Col1A1,Col1A2 were measured by quantitative RT-PCR and western-blot,respectively;the ratio of Col1A1/Col1A2 was calculated.(4)The knees of the mice were collected and paraffin sections were made.Immunohistochemical staining for Nestin and Osterix was performed and pictured under the microscope.Results:(1)The expression of Nestin and Osterix in BMSCs in tibial subchondral bone of late-stage OA in the mice significantly increased as compared to the sham operation group(P<0.05).(2)The number of mineral node stained with alizarin red in tibial subchondral bone of late-stage OA in the mice significantly decreased as compared to the sham operation group(P<0.05).(3)The expression of m RNA and proteins of Col1A1,Col1A2 significantly increased as compared to the sham operation group(P<0.05).The ratio of Col1A1/Col1A2 significantly increased as well.Conclusion: In a mouse model of late-stage OA,(1)The capacity of osteoblast mineralization of BMSCs in tibial subchondral bone decreases.(2)The expression of Nestin and Osterix in BMSCs in tibial subchondral bone increases.(3)The number of BMSCs and pre-osteoblast increases.(4)The capacity of type I collagen synthesis of BMSCs in tibial subchondral bone increases.However,the ratio of Col1A1/Col1A2 increases as well,which leads to defects in mineralization of tibial subchondral bone.Part III Role of IL-6 in the establishment of subchondral bone disease phenotype in osteoarthritis miceObjective: The expression of IL-6 in BMSCs of tibia subchondral bone of OA mice was detected,and the effect of IL-6/STAT3 signal on osteogenic differentiation of subchondral bone BMSCs in osteoarthritis mice was observed.Materials and Methods: A model of osteoarthritis(OA)in an 8-week,male C57BL/6J mouse by ACLT was built.The proximal tibia was collected and BMSCs were isolated from subchondral bone.BMSCs were collected.(1)the knees of the mice were collected and paraffin sections were made.Immunohistochemical staining for IL-6 and p-STAT3 was performed and pictured under the microscope.(2)the total RNA and proteins were extracted.The expression of m RNA and protein of IL-6 and p-STAT3 were measured by quantitative RT-PCR,Western blot and ELISA,respectively.(3)BMSCs were cultured in vitro.Cells mineralization was induced by BMP-2.IL-6 neutralization antibody,IL-6 si RNA and AG490 inhibitor were added to the medium.The cells were stained with alizarin red 4 weeks later and pictured under microscope.The staining was eluted and quantitatively analyzed with the spectrophotometry.(4)BMSCs were cultured in vitro.Cells mineralization was induced by BMP-2.IL-6 neutralization antibody,IL-6 si RNA and AG490 inhibitor were added to the medium.medium.The expression of m RNA of Col1A1,Col1A2 was measured with quantitative RT-PCR.The ratio of Col1A1/Col1A2 was calculated.(5)BMSCs were cultured in vitro.Cells mineralization was induced by BMP-2.IL-6 neutralization antibody,IL-6 si RNA and AG490 inhibitor were added to the medium.The expression of proteins of Col1A1,Col1A2 was measured with western blot.The ratio of Col1A1/Col1A2 was calculated.Results:(1)The expression of m RNA and proteins of IL-6 and p-STAT3 in BMSCs in tibial subchondral bone of the OA mice significantly increased as compared to the sham operation group(P<0.05).(2)The number of mineral node stained with alizarin red in tibial subchondral bone of the OA mice significantly increased after administration of IL-6 neutralization antibody,IL-6 si RNA or AG490 inhibitor,as compared to non-administrated control group.(3)The expression of proteins of Col1A1 and Col1A2 in BMSCs in tibial subchondral bone of the OA mice significantly decreased after administration of IL-6 neutralization antibody,IL-6 si RNA or AG490 inhibitor,as compared to non-administrated control group;the ratio of Col1A1/Col1A2 decreased as well.(m RNA and protein levels)Conclusion:(1)The expression levels of IL-6 and p-STAT3 in subchondral bone marrow mesenchymal stem cells of the OA mice were significantly increased.(2)Over-activated IL-6/STAT3 signaling is associated with decreased mineralization capacity of subchondral bone marrow mesenchymal stem cells in the OA mice.(3)Over-activated IL-6/STAT3 signaling is associated with abnormal secretion of type I collagen in the subchondral bone marrow mesenchymal stem cells in the OA mice.Part IV The Molecular Mechanism of ICA on Bone Marrow Stromal Cells in tibial subchondral bone of OA in the miceObjective: OA in the mice was treated with early administration of ICA.The molecular mechanism of ICA on bone marrow stromal cells in tibial subchondral bone of OA in the mice was explored.Materials and Methods: Anterior cruciate ligament transaction(ACLT)was performed in 8-week-old male C57BL/6 mice to induce OA.In a first assay,mice received intraperitoneal injections weekly of an IL-6 antibody or an Ig G isotype control for 8 weeks from the day after the ACLT.In a second assay,the mice were treated daily with an intraperitoneal injection of AG490 or DMSO vehicle control for 8 weeks from the day after the ACLT.Paraffin sections were stained with Alcian blue/hematoxylin & orange(GB/H&OG),the severity of gross cartilage and subchondral bone damage was assessed with OARSI scoring system.The area of tibial cartilage was calculated with software Image J.The depth of tibial cartilage was observed microscopically and was measured with bone histomorphometric software Bioquant.Immunohistochemical staining for Nestin and Osterix was performed and pictured.The proportion of Nestin and Osterix positive cells was measured by flow cytomertry.Results:(1)Sectional morphological observation showed that intraperitoneal injection of IL-6 neutralizing antibody or AG490 was more effective in protecting articular cartilage in the knee joint of the osteoarthritis mouse.(2)By intraperitoneal injection of IL-6 neutralizing antibody or AG490,the OARSI score of the knee joint of osteoarthritis mice can be improved.(3)By intraperitoneal injection of IL-6 neutralizing antibody or AG490,the knee cartilage area of osteoarthritis mice can be increased.(4)By intraperitoneal injection of IL-6 neutralizing antibody or AG490,the thickness of the subchondral bone of the knee joint of osteoarthritis mice can be increased.(5)By intraperitoneal injection of IL-6 neutralizing antibody or AG490,the number of subchondral bone marrow mesenchymal stem cells and pre-osteoblasts in osteoarthritic mice can be reduced.Conclusion:(1)Blocking IL-6/STAT3 signal can attenuate the degree of destruction of articular cartilage in osteoarthritis mice.(2)Blocking IL-6/STAT3 signal can reduce the occurrence of subchondral bone sclerosis in mice with osteoarthritis.(3)Blocking IL-6/STAT3 signaling can reduce the number of subchondral bone marrow mesenchymal stem cells and pre-osteoblasts in osteoarthritic mice.