Effect Of Cartilage Conditional Knockout Of KDM5C On Knee Osteoarthritis And The Treatment Of Ginsenoside Rb2

BackgroundOsteoarthritis(OA)is a common chronic disease whose prevalence and incidence increases with age.Restoring the integrity and function of articular cartilage is essential to stop or reverse the development of osteoarthritis.Therefore,it is of great practical importance to explore new OA prevention and treatment strategies and pathological mechanisms.Ginsenoside Rb2 is one of the most abundant saponins in ginseng.As a phytoactive component,ginsenoside Rb2 has antioxidant,anti-inflammatory,anti-cancer and tumor growth inhibitory effects,but the study of Rb2 in cartilage degradation has also not been confirmed.In addition,KDM5C as an X-chromosome linked gene,and recent studies have found that mutations in KDM5C cause phenotypes such as short stature and mental retardation.Our previous study found that KDM5C knockdown not only inhibited osteogenic differentiation,but also had a significant inhibitory effect on chondrocyte differentiation.However,we still lack insight into the role of KDM5C in cartilage development and cartilage injury repair,especially the development of OA,and related mechanisms.Objective1.To investigate in depth the role of ginsenoside Rb2 in chondrocyte and cartilage metabolism and to confirm the effect of Rb2 on the development of knee osteoarthritis,in order to be able to find an effective and safe natural drug component for OA prevention and treatment.2.It is important to study in depth the effect of specific knockdown of KDM5C gene in postnatal cartilage on articular cartilage development and OA progression,to further enrich the development of the role of KDM5C in cartilage defect repair and OA progression,and to provide new ideas and targets for cartilage regeneration and prevention and treatment of OA.MethodsIn this study,we investigated the role of ginsenoside Rb2 in the prevention and treatment of osteoarthritis by constructing a mouse chondrocyte inflammation model,a mouse medial meniscus instability(DMM)-induced knee osteoarthritis model,a KDM5C systemic knockout mouse model(abbreviated:KDM5C KO)and an endochondral KDM5C knockout mouse model(abbreviated:KDM5C cKO)to investigate the role of KDM5C in cartilage repair and osteoarthritis.This study was divided into four main parts:Part Ⅰ:In vitro experimental study of Rb2 promoting chondrocyte anabolism to slow down cartilage degradation1.Primary isolated and cultured 1-week-old C57BL/6 murine knee chondrocytes were identified by immunofluorescence staining for type Ⅱ collagen(COL2)as well as toluidine blue staining.Chondrocyte activity assay was performed using CCK-8 kit under the intervention of different concentrations of ginsenoside Rb2 to assess whether different concentrations of Rb2 are toxic to chondrocytes.2.Cells were intervened with different concentrations of Rb2(1 μM,10 μM)and the effect of Rb2 on chondrogenesis-related markers was assessed by qRT-PCR and immunofluorescence staining to detect the expression of COL2,Acan,and SOX9.3.Chondrocytes were added to LPS(2 μg/ml)for inflammation induction,and the anti-inflammatory protective effect of Rb2 was assessed by chondrocyte activity assay using CCK-8 kit under the intervention of different concentrations of ginsenoside Rb2.4.The addition of ginsenoside Rb2(10 μM)while cultured under the stimulation of LPS(2 μg/ml)was followed by qRT-PCR assay and immunofluorescence staining for inflammation-related indicators IL-β and MMP13,and immunofluorescence staining was used to detect the phosphorylation level and nucleation of p-65 as a way to assess whether Rb2 regulates inflammation levels through the NF-κB pathway.Part Ⅱ:In vivo experimental study of ginsenoside Rb2 to improve OA progressionMale C57BL/6 mice of 20g-25g were selected and randomly divided into sham-operated group(sham group),model group(DMM+PBS group)and drug intervention group(DMM+Rb2 group)by numbers.A model of medial meniscal destabilization(DMM)-induced osteoarthritis(OA)was established for the model and drug intervention groups,and a 4-week post-surgical joint cavity injection of Rb2(400 μM,10μl)or an equal volume of PBS was performed.we evaluated the therapeutic effect of Rb2 using the mouse DMM model,and toluidine blue-solid green staining and saffron O-solid green staining were used to observe the joint changes in wear and tear,and immunohistochemical staining was used to detect the altered expression of COL2 and NF-κB.Part Ⅲ:Mechanisms of KDM5C regulation of cartilage degeneration1.In order to investigate the potential role of KDM5C in cartilage differentiation,we analyzed the changes of KDM5C expression level by qRT-PCR and Western Blot during the first 14 days of chondrogenesis induction in mouse bone marrow MSCs,and also detected the changes of SOX9,Acan,and COL2.2.Establishing a KDM5C conventional knockout(KDM5C KO)mouse model,bone marrow mesenchymal stem cells(BMSCs)from 8-week-old mice were primary isolated and cultured,and chondrogenic differentiation was induced in KDM5C KO and WT mouse BMSCs.The effect of KDM5C knockout on chondrogenic differentiation was assessed by qRT-PCR and Alisin blue staining.3.Conditional KDM5C knockout(KDM5C cKO)mice were constructed,and the differences in the thickness of the knee growth plate and articular surface and the differences in the expression of the cartilage metabolic markers Acan,COL2,and MMP13 between 4-week-old KDM5C cKO and WT mice were assessed by staining with senna O-fix green and immunohistochemical staining,and microCT was performed on the knee joints of 12-month-old mice.We also performed microCT reconstruction and staining of the knee joints of 12-month-old mice with saffron O-solid green to observe the wear and tear of the articular surfaces and the production of bone redundancy in KDM5C cKO and WT mice.4.8-week-old KDM5C cKO and WT mice were selected to construct cartilage defect models,and the cartilage defect models were charged at 4 and 8 weeks post-operatively,and the knee joints were stained with Safranin O/Fast green(SFO)and histologically scored quantitatively to assess the effect of intracellular knockdown of KDM5C chondrocytes on cartilage defect repair.5.Rib cartilage was isolated from KDM5C cKO mice and littermate WT control mice(both injected with tamoxifen),and altered gene transcriptome levels of KDM5C-deficient chondrocytes were detected using RNA-seq analysis.6.Primary isolation and culture of chondrocytes from 2-week-old KDM5C cKO and WT mice to detect differences in the expression of MitoTracker Red,LC3B,ROS,and active caspase3 and to assess the effects of knockdown in KDM5C chondrocytes on mitochondrial dysfunction,mitochondrial autophagy,reactive oxygen species,and apoptosis7.To validate the protein interaction between KDM5C and HIF1α in chondrocytes of mice using CO-IP and molecular docking methods,and to select immunofluorescence co-localization to detect the difference of KDM5C and HIF1α interaction in chondrocytes of KDM5C cKO and WT mice.Part IV:The effect of KDM5C deletion on OA progression and the interventional role of Rb2.Eight-week-old KDM5C cKO and WT mice were selected to construct a DMM model,and the DMM model was intervened by postoperative joint cavity injection of PBS or Rb2(400 μM,10 μl),and the knee joints were collected 4 weeks later to assess changes in joint wear in the knee joints of KDM5C chondrocyte knockout mice using staining with Safranin O/Fast green staining and quantitative histological scoring of OARSI.Immunohistochemical staining was used to detect changes in p-p65 expression and to assess the effect on NF-κB expression levels after KDM5C deletion,as well as changes after Rb2 intervention.Results1.In a mouse chondrocyte model,experimental findings showed that Rb2 was not toxic to mouse chondrocytes at concentrations between 0 μM and 50 μM,and promoted the production of chondrogenesis-related markers such as COL2,Acan,and SOX9 at concentrations of 1 μM and 10 μM,thereby reducing the degradation of the extracellular matrix.2.The expression of IL-1β and MMP13 increased after LPS intervention in mouse chondrocytes,and Rb2 had anti-inflammatory protective effects at concentrations between 0.5 μM and 50 μM,and the expression of IL-1β and MMP13 decreased after Rb2(10 μM)intervention.entry into the nucleus was achieved.3.In a mouse DMM model,staining with Safranin O/Fast green green and toluidine blue showed that ginsenoside Rb2 injection into the joint cavity of mice reduced joint surface wear and improved cartilage proteoglycan deficiency.Immunohistochemical staining showed that ginsenoside Rb2 intervention increased COL2 expression and decreased NF-κB expression compared to the DMM group,thus alleviating the degradation of articular cartilage.4.During the chondrogenic differentiation of mouse bone marrow mesenchymal stem cells(mBMSCs),KDM5C showed an upregulation trend during these 14 days of chondrogenesis induction compared to day0,which was consistent with the trend of chondrogenesis-related markers COL2,Aggrecan and SOX9.5.Experimental results on chondrogenic differentiation of mBMSCs isolated and cultured from KDM5C systemic knockout mice showed that KDM5C knockout severely inhibited chondrogenic differentiation of MSCs and reduced the expression of COL2,SOX9 and Aggrecan at the transcriptional level.6.KDM5C chondrocyte-specific knockout mice showed thinner knee growth plates and articular surfaces,reduced expression of KDM5C,COL2 and Acan,and increased expression of MMP13 at 4 weeks of age compared with WT mice in the same litter;in addition,the experimental results showed that the articular surfaces of knee joints in 12-month-old KDM5C cKO mice showed early cartilage wear and bone formation.7.The results at 4 and 8 weeks after cartilage defect surgery showed that KDM5C intrachondral specific knockdown delayed cartilage repair.8.RNA-seq sequencing results were performed showing altered gene transcription in the HIF1 pathway,mitochondrial TCA cycle and oxidative phosphorylation in KDM5C-deficient chondrocytes,and that KDM5C knockdown decreased chondrocyte ATP production.9.KDM5C chondrocyte-specific knockdown induced mitochondrial dysfunction in chondrocytes,with reduced mitochondrial fragmentation and increased tubular structures in KDM5C knockdown cells and significantly increased mitochondrial number and fluorescence intensity in KDM5C cKO chondrocytes.10.LC3B levels were elevated in KDM5C cKO chondrocytes compared to WT chondrocytes.By double immunofluorescence staining with LC3B and Tom20,we observed that deletion of KDM5C increased LC3B-positive autophagic vesicles co-localized with mitochondria.We assessed reactive oxygen species(ROS)levels and found that H2DCFH-DA staining revealed elevated ROS levels in KDM5C chondrocytes.In addition,IF analysis showed increased levels of active caspase3 in KDM5C-deficient chondrocytes compared to WT chondrocytes.11.CO-IP showed a protein interaction between KDM5C and HIF1α,suggesting that KDM5C may mitigate cartilage degradation by stabilizing HIF1α.12.At 4 weeks after DMM,the articular surface wear was more severe in cartilage conditional knockout of KDM5C mice compared to WT mice;in KDM5C cKO mice,Rb2 intervention alleviated articular surface wear and downregulated p-p65 expression.Conclusion1.Rb2 is able to slow cartilage degradation by promoting chondrocyte anabolism and in LPS-induced cartilage cell inflammation by blocking the NF-κB pathway and improving the development of OA in mice.cellular inflammation by blocking the NF-κB pathway and reducing the level of inflammation and improving the development of OA in mice.2.KDM5C deficiency delayed cartilage damage repair and accelerated the development of OA.3.KDM5C deficiency induces mitochondrial dysfunction,mitochondrial autophagy and apoptosis in chondrocytes.4.KDM5C may alleviate cartilage degeneration by stabilizing HIF1α.5.Ginsenoside Rb2 ameliorates OA progression exacerbated by KDM5C knockdown by inhibiting NF-κB pathway and thereby.