DL-3-N-Butylphthalide Regulating The Progression Of Osteoarthritis Through PI3K/AKT/FoxO3a Pathway And Its Underlying Mechanism

Osteoarthritis(OA)is a chronic degenerative joint disease characterized by cartilage destruction,subchondral osteosclerosis,synovitis and osteophyte formation.It is reported that OA is the main cause of pain and disability for the elderly in the world,which has seriously affected their quality of life and health.The main strategy of early OA is anti-inflammatory and analgesic treatment.However,the existing drugs can only alleviate pain,and have no significant clinical effect on preventing or improving OA disease.Articular cartilage is a kind of non-vascular tissue,which is composed of chondrocytes and their extracellular matrix(ECM).The ECM maintains the mechanical properties and joint lubrication function of articular cartilage.Chondrocytes are the only resident cells in cartilage and have been found to play an important role in regulating ECM synthesis and maintaining cartilage structure.Therefore,the state of chondrocytes directly affects the function of articular cartilage.The disorder of cell apoptosis can lead to pathological conditions of the body,such as cancer,dysplasia and degenerative diseases.Existing studies have confirmed that chondrocyte apoptosis is significantly correlated with articular cartilage destruction and matrix degradation,and is positively correlated with the severity of OA.In addition,oxidative stress plays an important role in the occurrence and development of OA.Excessive production of reactive oxygen species in articular cartilage will lead to oxidative damage of chondrocytes.This leads to chondrocyte apoptosis and ECM degradation,thus causing irreversible damage to articular cartilage.Therefore,drugs that regulate apoptosis and oxidative stress may be a potential treatment for OA.In the forkhead box O(FoxO)subfamily,FoxO3 a plays a key role in oxidative stress,cell apoptosis,proliferation and DNA damage.Some studies have shown that up-regulation of FoxO3 a or inhibition of its degradation can inhibit cell apoptosis.TRIM33 protects osteoblasts from oxidative stress-induced apoptosis by inhibiting the ubiquitination and degradation of FoxO3 a.DL-3-n-Butylphthalide(NBP)is a small molecular compound extracted from celery seeds.So far,a large number of studies have found that NBP has extensive therapeutic benefits and plays an important role in many neurodegenerative diseases.In general,its mechanisms mainly include anti-apoptosis,anti-oxidation,anti-inflammatory and angiogenesis.NBP has been shown to have anti-apoptotic effects in a variety of cells,including neurons,cardiomyocytes and bone marrow stem cells.NBP has the characteristics of multi-target treatment,but there is no relevant report on the protective and therapeutic effects of NBP on OA.The purpose of this study was to explore the effect of NBP on the synthesis of ECM components,such as type II collagen(COL2A1),aggregate proteoglycan(ACAN),proteoglycan 4(PRG4)and sex determining region Y-box 9(SRY-box 9,SOX9).The protective effect was verified by inhibiting the progression of OA in rats and the related molecular mechanism was explored.Part I: DL-3-N-Butylphthalide retards the progression of osteoarthritis in ratsObjectives: To investigate the effect of NBP injection on delaying the progression of osteoarthritis in rats.Methods: 30 male SD rats weighing about 200 g were selected,and the OA model was established by destroying the medial meniscus and anterior cruciate ligament.The rats were randomly divided into 5 groups,including sham operation group,normal saline group and NBP intervention group(30,60 and 90 mg/kg).The rats were injected with100 μl of normal saline or NBP twice a week for 8 weeks.The hot plate analgesia test and static load test were used to evaluate the degree of knee joint pain,and then the degree of OA was evaluated.The degree of knee joint disease was evaluated by the Osteoarthritis Research Society International(OARSI)grading system.Hematoxylin eosin(HE)staining,safranin O fast green staining and collagen II immunohistochemistry were used to evaluate the degree of knee joint degeneration and the content of cartilage and collagen.HE staining of liver,kidney,spleen,heart and lung was performed to evaluate the drug safety of NBP.Results: 1.All rats in the experiment survived without death or infection.2.After the intervention of NBP,the OARSI score of rats was significantly decreased,the reaction time of lower limbs to hot plate was significantly increased,and the difference in weight bearing between lower limbs was significantly reduced.3.After NBP drug intervention,the degree of damage and cartilage erosion of the knee joint surface of rats were significantly reduced,the content of cartilage on the joint surface was increased,and the expression level of COLII was significantly increased.4.After NBP drug intervention,there were no significant changes in liver,kidney,spleen,heart and lung compared with the sham operation group.Conclusion: 1.NBP injection can inhibit the progression of OA,relieve knee pain,and reduce the loss of extracellular matrix components in rats.2.NBP injection into the knee joint of rats is safe.Part II: Effects of DL-3-N-Butylphthalide on extracellular matrix component production,apoptosis and oxidative stress in human OA cartilageObjective: To investigate the effects of NBP on extracellular matrix component production,apoptosis and oxidative stress in human OA cartilage.Methods: Firstly,we extracted chondrocytes from discarded cartilage of knee arthroplasty patients and identified them as human chondrocytes by alcian blue staining and collagen II immunofluorescence.Human OA chondrocytes were treated with different concentrations of NBP(0,0.01,0.1,1,10 and 100 μM),and the cell viability was detected by cell counting Kit-8(CCK-8)method to screen the drug concentrations for our subsequent experiments.Human OA chondrocytes were treated with different concentrations of NBP(0,0.01,0.1,1 and 10 μM)for 24 h.Western blot was used to detect the effect of different concentrations of NBP on the protein expression levels of COL2A1 and ACAN in human OA chondrocytes.The m RNA expression levels of extracellular matrix related components were detected by RT-PCR,and the expression levels of COLII and ACAN in human OA chondrocytes were detected by immunofluorescence staining.Secondly,we used NBP at different concentrations(0,0.01,0.1 and 1 μM)for 2weeks to treat the discarded cartilage from knee arthroplasty patients.The content of proteoglycans and type II collagen in cartilage blocks was detected by safranine staining and immunohistochemistry staining.DMMB COLORIMETRY and ds DNA HS kit were used to quantify the content of glycosaminoglycan(GAG)and DNA in cartilage blocks.RT-PCR was used to detect the m RNA expression levels of extracellular matrix-related components in cartilage blocks.Next,flow cytometry and TUNEL staining were used to detect the apoptosis level of human OA chondrocytes.The protein expression levels of apoptosis-related proteins Caspase-3,Bcl-2 and Bax in human OA chondrocytes were detected by Western blot,and the m RNA expression levels of apoptosis-related genes Bcl-2 and Bax in human OA chondrocytes were detected by RT-PCR.Finally,the ROS expression level in human OA chondrocytes was detected by reactive oxygen species detection kit,and the expression levels of SOD,MDA and GSH in human OA chondrocytes were detected by oxidative stress kit.Western blot was used to detect the protein expression levels of SOD2 and CAT in human OA chondrocytes,and RT-PCR was used to detect the m RNA expression levels of SOD2 and CAT in human OA chondrocytes.Results: 1.Aggrecan in chondrocytes was stained blue by alcian blue,and collagen II was stained red by immunofluorescence.2.CCK-8 assay showed that NBP had no inhibitory effect on the viability of human OA chondrocytes at the concentration of 0-10μM.3.After treatment with NBP(0.01-10 μM),Western blot analysis showed that the protein expression of COL2A1 and ACAN was significantly increased,and the m RNA level of ECM-related genes was up-regulated.Immunofluorescence also showed that after treatment with NBP(0.1 μM),the protein expression of ACAN was significantly increased.The expression levels of COLII and ACAN were significantly upregulated in human OA chondrocytes.4.After NBP intervention,safranin staining and collagen II immunohistochemical staining showed that the content of aggrecan and collagen II in cartilage explants was significantly increased.Quantitative analysis showed that NBP increased GAG content and DNA content in OA cartilage explants.RT-PCR results showed that NBP increased the m RNA expression level of ECM related components.5.After NBP intervention,flow cytometry analysis and TUNEL staining showed that the level of apoptosis in human OA chondrocytes was decreased.RT-PCR and Western blot showed that the expression levels of apoptosis-related genes Bax and Caspase-3 were decreased,and the expression level of Bcl-2 was increased.6.After NBP intervention,the expression levels of ROS and MDA in human OA chondrocytes were decreased,and the expression levels of GSH and SOD were increased.RT-PCR and Western blot showed that the expression levels of antioxidant enzymes SOD2 and CAT were increased.Conclusion: 1.NBP can promote the production of extracellular matrix components in human OA cartilage.2.NBP could promote the production of extracellular matrix components in cartilage explants in vitro.3.NBP can reduce the level of apoptosis in human OA chondrocytes.4.NBP could attenuate oxidative stress in human OA chondrocytes.Part III: DL-3-N-Butylphthalide regulates the function of human OA chondrocytes through PI3K/AKT/FoxO3 a pathwayObjective: To investigate the effect of NBP on the function of human OA chondrocytes through PI3K/AKT/FoxO3 a pathway.Methods: 1.The chemical structure of NBP was downloaded from Pub Chem database,and its potential therapeutic targets were predicted by Pharmapper.OA disease gene targets were collected from OMIM and Genecards,and the overlap between them was screened.2.Human OA chondrocytes were cultured and treated with 0.1 μM NBP for24 h.RNA was extracted and transcriptome sequencing was performed,and the differential genes were analyzed by GO analysis and KEGG pathway enrichment analysis.3.Human OA chondrocytes were extracted and treated with different concentrations of NBP(0,0.01,0.1,1 and 10 μM)for 24 h.Western blot was used to detect the protein expression levels of PI3 K,AKT,p-PI3 K and p-AKT in human OA chondrocytes.The protein expression levels of FoxO-related proteins FoxO1,FoxO3 a,FoxO4 and p-FoxO3 a in human OA chondrocytes were detected.RT-PCR was used to detect the m RNA expression levels of FoxO1,FoxO3 a and FoxO4 in human OA chondrocytes.Immunofluorescence was used to detect the expression level of FoxO3 a in human OA chondrocytes.4.FoxO3 a knockdown lentivirus was constructed and transfected into human OA chondrocytes.The expression of FoxO3 a was evaluated by RT-PCR and Western blot analysis.After successful lentiviral transfection,NBP was used to intervene human OA chondrocytes.The protein expression levels of COL2A1 and ACAN were detected by Western blot,the m RNA expression levels of COL2A1,ACAN,PRG4 and SOX9 were detected by RT-PCR,and the expression level of COLII was detected by immunofluorescence.5.Human OA chondrocytes were cultured and treated with 0.1 μM NBP for 24 h.RNA was extracted and the expression levels of FoxO downstream genes were detected by RT-PCR.Human OA chondrocytes were transfected with si FoxO3 a lentivirus and treated with NBP.Western blot was used to detect the protein expression levels of apoptosis-related proteins Caspase-3,Bcl-2 and Bax.RT-PCR was used to detect the m RNA expression levels of apoptosis-related genes Bcl-2 and Bax.Results:1.A total of 108 potential targets were found by network pharmacology.GO functional analysis showed that the extracellular matrix containing collagen was enriched,and KEGG enrichment analysis showed that PI3K-AKT signaling pathway,apoptosis and FoxO signaling pathway were enriched.2.Among the differentially expressed genes in the transcriptome,FoxO3 a was significantly up-regulated,GO analysis showed that extracellular matrix was significantly enriched,and FoxO signaling pathway,apoptosis and PI3K-AKT signaling pathway were found in KEGG pathway.3.Western blot analysis showed that the protein expression levels of p-PI3 K and p-AKT were significantly decreased after NBP intervention in human OA chondrocytes,while the expression levels of PI3 K and AKT were not significantly different.4.After NBP intervention in human OA chondrocytes,Western blot analysis showed that the protein expression level of FoxO3 a was increased,while the protein expression level of p-FoxO3 a was decreased.RT-PCR results showed that FoxO3 a m RNA level was significantly increased.Immunofluorescence also confirmed that NBP increased the expression level of FoxO3 a.5.Lv-FoxO3 a significantly reduced the protein and m RNA expression levels of FoxO3 a in human OA chondrocytes.Western blot analysis showed that Lv-FoxO3 significantly down-regulated the protein expression of COL2A1 and ACAN increased by NBP.The immunofluorescence staining of COLII was consistent with Western blot analysis.Lv-FoxO3 a significantly down-regulated the m RNA levels of ECM-related genes with increased NBP.6.RT-PCR results showed that FoxO downstream genes TNFSF10,FASL and BCL2L11 were significantly down-regulated,while BCL-6 was significantly up-regulated after NBP intervention in human OA chondrocytes.7.When chondrocytes were treated with NBP(0.1 μM)and/or Lv-FoxO3 a,Western blot and RT-PCR showed that NBP treatment inhibited the m RNA and protein expression of Bax and Caspase-3,while increasing the expression of Bcl-2.After Lv-FoxO3 a transfection into chondrocytes,the inhibitory effect of NBP on apoptosis was partially weakened.Conclusion: 1.NBP can regulate PI3K/AKT/FoxO3 a pathway in human OA chondrocytes.2.NBP can promote the expression of FoxO3 a in human OA chondrocytes.3.NBP increased ECM metabolism in human OA chondrocytes by up-regulating FoxO3 a.4.NBP inhibits the apoptosis of human OA chondrocytes by up-regulating FoxO3 a.