Kaempferol Induces Cell Apoptosis And Cycle Arrest In Rheumatoid Arthritis Fibroblast-like Synoviocytes

BackgroundRheumatoid arthritis(RA)refers to chronic and systemic autoimmune diseases characterized by chronic inflammation in synovial joints.Fibrobla st-like synoviocytes(FLSs)play an important role in the destruction of cartilage.Activated RA FLSs and tumor cells have a variety of biological properties and play an important role in angiogenesis by actively proliferating and Anti-apoptosis,eventually leading to the destruction of joint bones and cartilage.Therefore,induction of cell apoptosis and cycle arrest plays a therapeutic role in RA.Duan Teng Yi Mu Decoction(DTYMD)is one of our decoctions to treat RA.Howerver,its potential mechanism has not been elucidated).UFLC-Q-TOF-MS/MS combined with serum drug chemistry method was used to analyze the active constituents of DTYMD in the early stage of the research group,and Kaempferol was selected as one of the possible active constituents.Kaempferol is a kind of flavonoids.Previous studies showed that it has various pharmacological effects such as anti-cancer,anti-inflammation,anti-oxidation and immunosuppression.While,the effects of Kaempferol on RA FLSs' apoptosis,cycle arrest,migration and invasion have not been reported.Based on its antibacterial activity and regulatory roles in inflammation and immunology in tumor and osteoarthritis,we hypothesize that kaempferol also regulates the abnormal biological behavior of RA.Therefore,we choose Kaempferon which has a better anti-inflammatory activity in DTYMD for further study,thus,drug action is elucidated and the target of drug action is identified.Our work not only provides theoretical support and experimental evidence for kaempferol treatment of RA,but also integrates multi components,multi targets,multi pathways mechanism for its clinical application,provides a scientific basis for the modernization of traditional Chinese medicine compound research,and lay the foundation for the development of RA innovative drugs.Part ? Kaempferol induces cell apoptosis and cycle arrest in rheumatoid arthritis fibroblast-like synoviocytesObjective1.To culture and identify RA FLSs.2.To explore the roles of Kaempferol in regulating the growth morphology,activation,proliferation of RA FLSs.3.To explore the effects of kaempferol on RA FLSs apoptosis and cycle arrest.MethodsNine patients with diagnostic criteria of American ACR in 1987 were recruited and synovial tissues were obtained from synovectomy or knee arthroplasty by knee arthroscopy.The synovial tissue was further isolated and cultured to generate RA FLSs.The morphology of cells were observed with light microscope.The vimentin protein which specifically expressed in FLS was identified by immunofluorescence.After treated with different concentrations of kaempferol,CCK-8 assay was used to determine the effect of kaempferol on the cell viability.The morphological changes of cells were observed under inverted microscope.The cell proliferation rate was detected by EdU assay.The apoptosis rate and cycle arrest were detected by Western blotting.The expressions of Bcl-2,Bax,Cleaved Caspase 3,Cleaved Caspase 9,CyclinDl,CDK4 level,and PI3K/Akt pathway related protein expression levels were determined by western blottinResultsPrimary cells were observed under optical microscope crawling out from the edge of the tissues in 5-7d after seeding.The 70%to 80%of bottom was covered with FLSs in 3 to 4 weeks.After 3 generations,the cell types were gradually purified,the cells were long-spindle type,the nucleus was oval-shaped and the morphology was consistent with FLSs.Immunofluorescence experiments showed that the cells expressed vimentin highly,thus,the cells can be identified as RA FLSs.The results of CCK-8 showed that the viability of RA FLSs treated with 50 u M kaempferol,100 u M kaempferol,200 u M kaempferol and 20 ?M MTX for 24 hours decreased.Compared with the blank control group,the cell viability of RA FLSs in the kaempferol groups(100 ?M,200 ?M)and MTX group were significantly decreased(P<0.05 or P<0.01).Kaempferol and MTX could inhibit the activity of RA FLSs in a dose-dependent way.So the 100?M kaempferol,200 ?M kaempferol and 20 ?M MTX were used for further study.The results of EdU assay showed that the proliferation rate of RA FLSs decreased significantly(P<0.05 or P<0.01)after being treated with kaempferol and MTX in a concentration-dependent manner,suggesting that kaempferol and MTX could inhibit the proliferation of RA FLSs.The apoptotic test results showed that kaempferol and MTX significantly induced apoptosis of RA FLSs(P<0.05 or P<0.01).The results of cell cycle test showed that compared with the blank control group,cell cycle arrest was significantly induced in RA FLSs with kaempferol or MTX,the proportion of cells in G0/G1 phase increased(P<0.05 or P<0.01),the proportion of cells in G2/M phase was significantly decreased(P<0.05 or P<0.01).Cells in G0/G1 phase increased nearly 34%and decreased 32%in G2/M phase with 200 ?M kaempferol.It suggests that kaempferol and MTX can induce the growth retardation of RA FLSs in G0/G1 phase.ConclusionKaempferol inhibits the proliferation of rheumatoid arthritis fibroblast-like synoviocytes;kaempferol may induce cell apoptosis and cycle arrest in RA FLSs and may therefore have therapeutic potential for the treatment of RA.Part ? The molecular mechanism of kaempferol-induced RA FLSs cell apoptosis and cycle arrestObjectiveTo explore the signaling mechanisms that induced in the regulation of RA FLSs cell apoptosis and cycle arrest by kaempferol.MethodsCells from the 3rd to 5th passage(P3 to P5)were identified and used to the experiment.Western blotting was used to detect the expression of Cyclin D1,Bcl-2,Bax,Cleaved Caspase 3,Cleaved Caspase 9,PI3K,Akt and phosphorylation levels.The effect of PI3K inhibitor(LY294002)on the expression of cycle related proteins and apoptosis related proteins in RA FLSs was detected by western blotting.Flow cytometry was used to detect the effects of signal pathway inhibitor on cell apoptosis and cycle arrest.ResultsWestern blotting showed that phosphorylation of Bcl-2,PI3K and Akt in kaempferol intervention group was significantly decreased(P<0.05 or P<0.01),The expression of Bax,Cleaved Caspase 3 and Cleaved Caspase 9 increased significantly(P<0.05 or P<O.01)),in a dose-dependent manner.PI3K inhibitor LY294002 enhanced the effect of kaempferol on RA FLSs:compared with kaempferol,the cell viability and apoptosis rate of LY294002 combined with kaempferol group were decreased;the phosphorylation of Bcl-2 and Akt protein were significantly increased(P<0.05).The expression of Bax,Cleaved Caspase 3 and Cleaved Caspase 9 increased(P<0.05).Western blotting showed that the expression of Cyclin D1 and CDK4 were decreased in kaempferol intervention group(P<0.05 or P<0.01).PI3K inhibitor(LY294002)enhanced the effect of kaempferol on RA FLSs:Compared with kaempferol group,the proportion of cells in LY294002 combined with kaempferol group or G0/G1 phase increased while the proportion of cells in G2/M phase was significant decreased(P<0.05).The expressions of Cyclin D1 and CDK4 decreased(P<0.05).Conclusionkaempferol induces cell apoptosis and cycle arrest in RA FLSs and that these effects are mediated by PI3K/Akt signaling pathway.Part ? Therapeutic effects of Kaempferol on CIA miceObjectiveTo evaluate the effects of kaempferol on severity of arthritis in mice with collagen-induced arthritis.Methods48 SPF DBA/1 male mice,7-8 weeks old,were divided in experimental group,Model,kaempferol(High,Medium and Low dose),Methotrexate(MTX)and Normal control group.The normal mice as normal control group,MTX as positive control group.Each group has 8 mice.CIA,The classic RA model,Collagen Induced Arthritis(CIA)were prepared)After the secondary immunization in the 21th day of this experiment,treatment measures were taken on the mice.Equal volume menstrum were given to CIA model group and normal group;Kaempferol(High,Medium and Low dose)groups with Kaempferol(200 mg/kg,100 mg/kg,50 mg/kg);MTX group with MTX(2 mg/Kg).All the interventions were given by intragastric administration per day for 28 days,but MTX group was given by intragastric administration every 3 days for 9 times.The mice were observed for general conditions every 4 days,and scored for arthritis.The index of the spleen index was calculated.The ankle joints was observed by HE staining and the apoptosis of synovial cells was detected by fluorescent Tunel method.The expression of IL-1?,IL-6 and TNF-? were detected by ELISA.ResultsThe severity of arthritis in mice was dynamically evaluated.Compared with the model group,the arthritis scores of the kaempferol high-dose group and MTX group decreased significantly(P<0.05 or P<0.01)9 d after administration.Arthritis score decreased significantly in medium-dose group and MTX group(P<0.05).The arthritis scores of mice in low dose of kaempferol showed a decreasing trend,but there was no statistical significance(P>0.05).Spleen index:compared with the normal group,the spleen index of model group increased(P<0.05),while treatment group mice decreased especially kaempferol group and MTX group(P<0.05).The changes of serum inflammatory cytokines in mice:After modeling,the expression of IL-1?,IL-6 and TNF-a in model group were significantly increased(P<0.05 or P<0.01).Compared with the model group,the expression of IL-1?,IL-6 and TNF-? decreased in all treatment groups,and the levels of IL-1?,IL-6 and TNF-? decreased significantly in high and medium dose groups of kaempferol and MTX group(P<0.05 or P<0.01).The pathology of ankle joints in experimental groups were as following:synovial cell hyperplasia,disorderly arrangement,infiltration of a large number of inflammatory cells,destruction of articular cartilage and bone in the model group.Synovial hyperplasia alleviated,Infiltration decreased,the scope of bone destruction narrowed,some of the joint structure has been restored in treatment group.Compared with the model group,the pathological score of mice in each treatment group showed a decreasing trend.The pathological scores of kaempferol high,middle dose groups and MTX group decreased significantly(P<0.05).The pathological changes of TUNEL staining in the ankle joints of mice in each group:Compared with the model group,the apoptosis rate in treatment groups showed an upregulated trend,while the apoptosis rate in the high and middle doses of kaempferol and MTX group increased statistically Difference(P<0.05 or P<0.01)ConclusionKaempferol and MTX can effectively alleviate CIA mice arthritis symptoms,inhibit synovitis and articular cartilage and bone destruction.Kaempferol exhibits a potential therapeutic benefit in RA.