| Osteoarthritis(OA)is a chronic orthopedic disease characterized by articular cartilage degeneration with high incidence and heavy clinical burden.The main clinical manifestations of OA are chronic pain and joint mobility disorders,which seriously affect the quality of life of elderly patients.With the increasing aging of the social population,the high prevalence of OA will cause heavy economic pressure to the country and society.In addition,the current clinical non-surgical treatment for OA is palliative treatment that mainly alleviates symptoms,and there is still a lack of treatment methods to reverse cartilage degeneration and intervene disease progression in a real sense.Therefore,it is the priority of current clinical treatment to develop new therapeutic drugs to delay or even reverse cartilage degeneration in the early stage of osteoarthritis.In recent years,with the in-depth study of OA,it has been found that the pathogenesis of OA is not only related to articular cartilage,but also related to subchondral bone remodeling.Osteoclasts are the main contributors to subchondral bone remodeling.Excessive activation of osteoclasts leads to the release of various factors in bone matrix that regulate chondrocyte metabolism and participate in cartilage degeneration.Therefore,inhibition of osteoclast differentiation to restore abnormal subchondral bone remodeling has become a new target for the treatment of OA.Natural compounds are more easily accepted by patients due to their high safety and stability,and have gradually become a research hotspot in recent years.Previous studies have shown that the plant-derived drug Amentoflavone(AF)has shown potent anti-osteoclast differentiation and anti-inflammatory properties.However,there is no report on the specific regulatory function of AF in the pathological process of OA.In view of the close relationship between inflammation and osteoclast differentiation and homeostasis between extracellular matrix(ECM),we speculate that AF has significant therapeutic effect and unlimited potential in the treatment of OA.To verify the above hypothesis,we mainly planned to carry out the following studies:(1)to explore the effect of AF on il-1β-induced inflammatory response and ECM homeostasis in mouse chondrocytes and its molecular mechanism;(2)To explore the effect of AF on RANKL-induced osteoclast differentiation in mice and its molecular mechanism;(3)To explore whether AF has therapeutic effect on osteoarthritis in mice.This article can be divided into following parts:The first part: Study of effects and mechanisms of AF on inflammatory responseand ECM homeostasis in mouse chondrocytes in vitroObjective: To investigate the effects and mechanisms of AF on IL-1β-induced inflammatory response and ECM homeostasis in mouse chondrocytes.Methods:(1)Cell counting KIT-8(CCK-8)assay was used to detect the effect of different concentrations of AF on the proliferation of mouse chondrocytes in the presence or absence of IL-1β(10ng/m L).(2)Il-1β stimulated primary mouse chondrocytes to construct an in vitro model of OA.The effect of AF on ECM homeostasis was detected by high-density chondrocyte culture and toluidine blue staining.(3)The effect of AF on the expression of inflammatory mediators(i NOS,COX-2,IL-6 and TNF-α)was detected by real time PCR,Western blot and Elisa.(4)The effect of AF on the expression of ECM metabolites(Col2a1,SOX9,MMP13,ADAMTS5,etc.)was detected by real time PCR and Western blot.The effect of AF on ECM homeostasis was further verified by immunofluorescence assay(Aggrecan and Collagen-Ⅱ).(5)Western blot was used to detect the effect of AF on NF-κB/JNK/ERK signaling pathway in mouse chondrocytes.At the same time,the effect of AF on IL-1β induced p65 entry into the nucleus was detected by immunofluorescence.Results:(1)AF had no effect on the proliferation activity of primary mouse chondrocytes in the concentration range of 0-10μm.AF attenuated the inhibitory effect of IL-1β on chondrocyte activity in a dose-dependent manner in the safe concentration range(0-10μm).(2)Toluidine blue staining results showed that the blue staining intensity of chondrocytes decreased under IL-1β stimulation,and the blue staining intensity gradually increased after AF treatment,and the degree of deepening was positively correlated with the concentration of AF.(3)AF treatment significantly inhibited the expression of inflammatory mediators such as i NOS,COX-2,IL-6 and TNF-α in chondrocytes after IL-1βstimulation.(4)At the same time,AF treatment significantly inhibited the expression of MMP13 and ADAMTS5,and up-regulated the expression of Col2a1 and SOX9 in chondrocytes stimulated by IL-1β.The immunofluorescence experiments of Aggrecan and Collagen-Ⅱ showed that the fluorescence staining of chondrocytes stimulated only by IL-1β was very light,while the immunofluorescence staining of both chondrocytes was significantly enhanced after the co-treatment with AF and IL-1β.(5)AF significantly inhibited the degradation of IκBα and the phosphorylation of p65,JNK and ENK in mouse chondrocytes stimulated by IL-1β.The translocation of p65 into the nucleus was also significantly inhibited by AF.Conclusion:AF stabilizes the inflammatory response and ECM homeostasis of chondrocytes stimulated by IL-1β by inhibiting NF-κB/JNK/ERK signaling pathway.The second part: Study of effects and mechanisms of AF on differentiation ofmouse osteoclasts in vitroObjective: To investigate the effects and mechanisms of AF on osteoclast differentiation induced by RANKL.Methods:(1)Cell counting Kit-8(CCK-8)assay was used to detect the effects of different concentrations of AF on the proliferation of mouse BMMs cells and Raw264.7 cells.(2)Osteoclast differentiation was induced by RANKL in vitro.TRAP staining and actin ring assay were used to detect the effect of AF on osteoclast formation.(3)The effect of AF on the expression of osteoclast specific markers(c-Fos,NFATc1,TRAP,DC-STAMP,Calcitonin receptor,V-ATPase D2,etc.)was detected by real time PCR and Western blot.(4)Western blot was used to detect the effect of AF on NF-κB/JNK/ERK signaling pathway in mouse osteoclasts.At the same time,the effect of AF on the nucleation of p65 during osteoclast differentiation was detected by immunofluorescence.Results :(1)At the concentration of 0-10μm,AF had no effect on the proliferation activity of mouse BMMs cells and RAW264.7 cells.(2)Upon co-stimulation with M-CSF and RANKL cytokines,mouse BMMs cells were induced to mature multinucleated osteoclasts,and the number of F-actin rings was also significantly increased.However,the number and area of TRAP positive multinucleated osteoclasts and the number of actin rings decreased in a dose-dependent manner after treatment with different concentrations of AF.(3)Real time PCR results showed that AF significantly inhibited the expression of osteoclast specific genes c-fos,NFATc1,TRAP,DC-STAMP,Calcitonin receptor and V-ATPase d2.The results were further verified by Western Blot analysis.AF significantly decreased the protein expression of c-fos and NFATc1.(4)AF significantly inhibited the degradation of IκBα and the phosphorylation of JNK and ENK during osteoclast formation induced by RANKL.The translocation of p65 was also significantly inhibited by AF.Conclusion:AF inhibits RANKL-induced osteoclast cell formation by inhibiting NF-κB/JNK/ERK signaling pathway.The third part: Effect of AF on DMM induced osteoarthritis in miceObjective: To explore whether AF has therapeutic effect on osteoarthritis in mice in vivo.Methods:(1)In vivo DMM model of osteoarthritis mice was established by dissecting the medial meniscus of mice.(2)One week after successful model construction,AF was intraperitoneally injected into the experimental group(DMM+AF group),and PBS was injected into the control group(DMM group)and Sham operation group(Sham group).Injections were administered every 2 days for 8 weeks,after which mouse knee specimens were collected.(3)The degree of cartilage degeneration was evaluated by HE staining,Safranin O-Fast Green Staining and OARSI score of articular cartilage sections.The effect of AF on ECM homeostasis during OA was further verified by immunohistochemical assay(MMP13 and COL2a1).(4)TRAP staining was used to detect the effect of AF on the remodeling status of subchondral bone in OA mice,and immunofluorescence was used to detect the expression of p-p65.Results:(1)The results of HE staining and Safranin O-Fast Green Staining showed that a large amount of proteoglycan loss and cartilage erosion were observed in the DMM group compared with the Sham group.Compared with DMM group,AF injection significantly inhibited proteoglycan loss and cartilage erosion.Similarly,knee OARSI scores yielded consistent results.In addition,the expression of MMP13 and COL2a1 was inhibited by AF treatment.(2)After AF treatment,the number and area of TRAP-positive multinucleated cells in subchondral bone decreased.(3)Immunofluorescence assay of tissue sections showed that the expression of p-p65 was inhibited after AF treatment.Conclusion:AF has dual protective effects on articular cartilage degeneration and subchondral bone loss in mice with osteoarthritis. |
PDF Full Text Request