Based On IL-1β/MAPKs Pathways To Investigate The Impact Of Total Saponins Of Clematis On Osteoarthritis Chondrocytes Apoptosis

ObjectiveEstablish rabbit knee osteoarthritis models and a culture system of primary chondrocytes in vitro.Observe the impact of Total Saponins of Clematis(TSC)on chondrocytes apoptosis, to investigate the intervention mechanism of TSC on osteoarthritis chondrocytes apoptosis through IL-1β/MAPKs Pathways.Methods1 The impact of TSC on rabbit osteoarthritis IL-1β/MAPKs pathways and chondrocytes apoptosis through experiments in vivo48 healthy New Zealand white rabbits were randomly divided into blank group(n=8) and model group(n=40). The left hind limb of animals in model group were immobilized in extension position by cast immobilization.After successful modeling, the rabbits in model group were randomly divided into five groups: model control group,the low dose group of TSC,the middle dose group of TSC,the high dose group of TSC,the group of celecoxib,8 for each group.The blank group and the model control group were received an equivalent amount of saline only(10ml/day),the low、middle、high dose group of TSC were respectively given the TSC(25mg/Kg/d、50mg/Kg/d、100mg/Kg/d),the group of celecoxib were received a oral dose of celecoxib(10mg/Kg/d).All the rabbits were sacrificed after6 weeks,knee joint cartilage specimens were fixed by paraformaldehyde,paraffin-embedded,the morphological changes were observed after staining with HE,the concentrations of IL-1β in cartilage specimens were detected by ELISA.The m RNA expressions of Bcl-2、Bax、Caspase-3、Caspase-9 were detected by RT-PCR assay.The protein expressions of p-P38、p-ERK-1 and p-ERK-2were deteced by Western-blot.2 The impact of TSC on chondrocytes apoptosis and MAPKs pathways of IL-1β-affected articular cartilage cells through experiments in vitro(1) Knee joint cartilage was isolated from 20 healthy and four-weeks-old New Zealand white rabbits,and used to establish a culture system of primary chondrocytes in vitro, chondrocytes were identifide with toluidine blue stain.The morphosis of chondrocytes were observed by microscope.(2) The 3rd generation chondrocytes were randomly divided into normal group、IL-1β group、IL-1β+TSC 1 group(25μg/m L)、IL-1β+TSC 2 group(50μg/m L)、IL-1β+TSC 3 group(100μg/m L)、IL-1β+TSC 4 group(200μg/m L)、IL-1β+TSC 5 group(400μg/m L),Every group was added IL-1β(the concentration 10ng/m L)except normal group,cultured for 24 h,48h,72 h,96h,the activity of chondrocytes by MTT assay to determine the best intervention time and concentration of TSC.(3)The 3rd generation chondrocytes were randomly divided into blank group、IL-1β group、IL-1β+TSC group,the last two groups were added IL-1β(the concentration:10ng/m L),meanwhile, IL-1β+TSC group was added the best concentration of TSC,cultured for the best intervention time determined by the above experiments.Chondrocytes apoptosis was detected by flow cytometry.The m RNA expressions of Bcl-2、Bax、Caspase-3、Caspase-9 were detected by RT-PCR assay.The protein expressions of p-P38、p-ERK-1 and p-ERK-2were deteced by Western-blot.Results1 The impact of TSC on rabbit osteoarthritis IL-1β/MAPKs pathways and chondrocytes apoptosis through experiments in vivo(1)The knee in model group X-ray showed that knee joint space became narrow,the artichlar surface was not smooth,knee joint appeared bone hyperplasia and sclerosis,appeared osteophyte formation.After 6 weeks,HE staining showed TSC and celecoxib could promote osteoarthritis cartilage repair,the best treatment effect was high dose group of TSC. The IL-1βlevel of model control group was respectively higher than blank group(P<0.05), the low、middle、high dose group of TSC were respectively lower than model control group(P<0.05),the IL-1β level decreased with the elevation of the TSC dose(P<0.05), the high group were respectively lower than the celecoxib group(P<0.05).(2)Expressions of Bcl-2、Bax、Caspase-3 and Caspase-9 m RNA in joint cartilage of each group after 6 weeks: expression of Bcl-2m RNA,model control group was respectively lower than blank group(P<0.05),the low、middle、high dose group of TSC were respectively higher than model control group(P<0.05),the middle dose group of TSC was respectively higher than the low dose group of TSC(P<0.05),the high dose group of TSC was respectively higher than the middle dose group of TSC(P < 0.05), the high dose group of TSC was respectively higher than the celecoxib group(P<0.05);expression of Baxm RNA,model control group was respectively higher than blank group(P<0.05), there was no significant difference between the low、middle dose group of TSC and model control group(P>0.05),the high dose group of TSC and the celecoxib group were respectively lower than model control group(P<0.05), there was no significant difference between the high dose group of TSC and the celecoxib group(P>0.05);expressions of caspase-3 、caspase-9 m RNA,model control group was respectively higher than blank group(P <0.05),the low、middle、high dose group of TSC and the celecoxib group were respectively lower than model control group(P<0.05);expression of caspase-3 m RNA, there was no significant difference in the low、middle dose group and the celecoxib group(P>0.05),but the high dose group was respectively lower than the low、middle、dose group and the celecoxib group(P < 0.05); expression of caspase-9 m RNA, there was significant difference in the low、middle、high dose group(P<0.05),but there was no significant difference between the high dose group and the celecoxib group(P>0.05).(3)The protein expressions of p-P38、p-ERK-1 and p-ERK-2 in joint cartilage of each group after 6 weeks:the protein expressions of p-P38、p-ERK-1 and p-ERK-2,model control group was respectively higher than blank group(P<0.05),the low、middle、high dose group of TSC and the celecoxib group were respectively lower than model control group,the high dose group group was the lowest(P<0.05);the protein expressions of p-ERK-1 and p-ERK-2 decreased with the elevation of the TSC dose(P < 0.05);the protein expression of p-P38,there was no significant difference between the low dose group of TSC and the middle dose group of TSC(P>0.05),the high dose group of TSC was respectively lower than the middle dose group of TSC(P<0.05).2 The impact of TSC on chondrocytes apoptosis and MAPKs pathways of IL-1β-affected articular cartilage cells through experiments in vitro(1)The growing status of the original generation、2nd generation and 3rd generation chondrocytes were good.The identification with toluidine blue staining, the original generation chondrocytes were metachromic.(2)The best intervention time and concentration of TSC on IL-1β-affected articular cartilage cells were 72 h and 200μg/m L by MTT assay.(3)The chondrocytes apoptosis rate of each group: IL-1βgroup was respectively higher than blank group(P<0.05), IL-1β+TSC group was respectively lower than IL-1βgroup(P<0.05).(4)Expressions of Bcl-2、Bax、caspase-3 and caspase-9 m RNA after intervened the 3rd generation chondrocytes for 72h:expression of bcl-2 m RNA, IL-1βgroup was respectively lower than blank group(P<0.05), IL-1β+TSC group was respectively higher than IL-1βgroup(P<0.05); expressions of bax、caspase-3、caspase-9m RNA, IL-1βgroup was respectively higher than blank group(P<0.05), IL-1β+TSC group was respectively lower than IL-1βgroup(P<0.05).(5)The protein expressions of p-P38、p-ERK-1 and p-ERK-2 after intervened the 3rd generation chondrocytes for 72h: IL-1βgroup was respectively higher than blank group(P<0.05), IL-1β+TSC group was respectively lower than IL-1βgroup(P<0.05).Conclusions1 According to research in vivo and vitro,knee osteoarthritis and chondrocyte apoptosis were related to high activation of IL-1β/MAPKs pathways 、high expression of Caspase-3、Caspase-9、Bax and low expression of Bcl-2。2 According to research in vivo and vitro, TSC can repair cartilage and inhibit the apoptosis of cartilage cells.It can regulate IL-1β/MAPKs pathways and expression of chondrocyte apoptosis gene, including Caspase-3、Caspase-9、Bax、Bcl-2.This may be one of the mechanisms of TSC in the prevention and treatment of osteoarthritis. Moreover,there was a certain relationship between the effect and the concentration of TSC.3 According to research in vivo,high dose TSC in the inhibition IL-1β/MAPKs pathways and up-regulation expression of bcl-2,down-regulation expression of Caspase-3 was better than celecoxib;there was no significant difference between the high dose of TSC and celecoxib in down-regulation expression of Bax and caspase-9.