Effects of tribulus terrestris saponins on interleukin-1β-induced chondrocyte apoptosis and inflammatory factor secretion
Objective:To observe the effects of tribulus terrestris saponins(TTS)on interleukin-1β(IL-1β)-induced chondrocyte apop-tosis and inflammatory cytokine secretion,and to explore its underlying mechanism.Methods:①The mouse chondrocytes(ATDC5 cells)were harvested and cultured in the Dulbecco's Modified Eagle's Medium(DMEM).After 24-hour culture,the ATDC5 cells were transfect-ed with pcDNA-circ＿0045714,pcDNA,si-circ＿0045714,and si-NC,respectively,by using transfection reagents.②The ATDC5 cells were inoculated onto the 96-well plates,with one group cultured in normal DMEM(normal group),one group in DMEM containing 10 ng/mL IL-1β(IL-1β group),and the rest in DMEM adding with10 ng/mL IL-1β and TTS with concentration of 25,50,100,200,and400 μg/mL,re-spectively.After 24-hour culture,the survival rate of chondrocytes in each group was calculated,based on which the concentration of TTS used in subsequent experiments was determined.③The ATDC5 cells were divided into control group,IL-1β group,IL-1β+low-dose TTS(L-TTS)group,IL-1β+medium-dose TTS(M-TTS)group,and IL-1β+high-dose TTS(H-TTS)group.The ATDC5 cells in control group were cultured in conventional DMEM,the ones in IL-1β group in DMEM with 10 ng/mL IL-1β,and the ones in the other 3 groups in DMEM adding with 10 ng/mL IL-1β and TTS with concentration of 50,100,and 200 μg/mL,respectively.The pcDNA-circ＿0045714-and pcDNA-transfected ATDC5 cells were cultured with the same method as IL-1β group,and were labeled as IL-1β+pcDNA-circ＿0045714 group and IL-1β+pcDNA group,respectively.The si-circ＿0045714-and si-NC-transfected ATDC5 cells were cultured with the same meth-od as IL-1β+H-TTS group,and were labeled as IL-1β+H-TTS+si-circ＿0045714 group and IL-1β+H-TTS+si-NC group,respectively.After 24-hour culture,the proliferation inhibition rate of the chondrocytes in each group was calculated.④The ATDC5 cells and the pcDNA-circ＿0045714-,pcDNA-,si-circ＿0045714-and si-NC-transfected ATDC5 cells were inoculated onto the 6-well plates.After 12-hour culture,the apoptosis rate of chondrocytes in each group was detected by using flow cytometry.⑤The culture medium of chondrocytes was collected from each group,and the levels of tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6)in the supernatant were detected.⑥The total RNA was extracted from chondrocytes in each group for synthesizing the cDNA by reverse transcription,followed by amplification for calcu-lating the expression level of circ＿0045714 in chondrocytes.Results:①The survival rate of chondrocytes was lower in IL-1β group com-pared to normal group(LSD-t =15.396,P =0.000),and lower compared with that of IL-1β+50 μg/mL TTS group,IL-1β+100 μg/mL TTS group,IL-1β+200 μg/mL TTS group and IL-1β+400 μg/mL TTS group(LSD-t =4.879,P =0.047;LSD-t =7.686,P =0.001;LSD-t =10.657,P =0.000;LSD-t =10.073,P =0.000);while the differences between IL-1β group and IL-1β+25 μg/mL TTS group,and between IL-1β+200 μg/mL TTS group and IL-1β+400 μg/mL TTS group were not statistically significant(LSD-t =1.555,P = 0.918;LSD-t =0.584,P =0.999).Therefore,the TTS with concentrations of 50,100 and200 μg/mL were selected for the further experi-ments and were labeled as L-TTS group,M-TTS group,and H-TTS group,respectively,based on the concentrations.②The proliferation inhi-bition rate,the apoptosis rate,and the levels of TNF-α and IL-6 in the chondrocytes were higher in IL-1β group compared with those of con-trol group,IL-1β+L-TTS group,IL-1β+M-TTS group,and IL-1β+H-TTS group(the proliferation inhibition rate:LSD-t =55.829,P = 0.000;LSD-t =8.879,P =0.001;LSD-t =20.507,P =0.000;LSD-t =30.315,P =0.000;the apoptosis rate:LSD-t =27.508,P =0.000;LSD-t =5.076,P =0.032;LSD-t =11.689,P =0.000;LSD-t =21.284,P =0.000;the level of TNF-α in the chondrocytes:LSD-t = 29.990,P =0.000;LSD-t =7.720,P =0.002;LSD-t =17.182,P =0.000;LSD-t =24.615,P =0.000;the level of IL-6 in the chondro-cytes:LSD-t =33.441,P =0.000;LSD-t =6.324,P =0.008;LSD-t =15.440,P =0.000;LSD-t =25.096,P =0.000),and they were higher in IL-1β+L-TTS group compared to IL-1β+M-TTS group and IL-1β+H-TTS group(the proliferation inhibition rate:LSD-t = 11.627,P =0.000;LSD-t =21.436,P =0.000;the apoptosis rate:LSD-t =6.613,P =0.006;LSD-t =16.209,P =0.000;the level of TNF-α in the chondrocytes:LSD-t =9.463,P =0.000;LSD-t =16.895,P =0.000;the level of IL-6 in the chondrocytes:LSD-t =9.117,P =0.001;LSD-t = 18.773,P = 0.000),furthermore,they were higher in IL-1β+M-TTS group compared to IL-1β+H-TTS group(LSD-t =9.808,P =0.000;LSD-t =9.595,P =0.000;LSD-t =7.432,P =0.003;LSD-t =9.656,P =0.000).③The expression level of circ＿0045714 in the chondrocytes was lower in IL-1β group compared to control group,IL-1β+L-TTS group,IL-1β+M-TTS group,and IL-1β+H-TTS group(LSD-t =43.218,P =0.000;LSD-t =9.487,P =0.000;LSD-t =22.136,P =0.000;LSD-t =34.785,P =0.000),and was lower in IL-1β+L-TTS group compared to IL-1β+M-TTS group and IL-1β+H-TTS group(LSD-t =12.649,P =0.000;LSD-t = 25.298,P =0.000),moreover,it was lowly expressed in IL-1β+M-TTS group compared to IL-1β+H-TTS group(LSD-t =12.649,P = 0.000).④The expression level of circ＿0045714 showed a significant difference in the chondrocytes transfected with pcDNA and pcDNA-circ＿0045714(1.00±0.00,4.18±0.14,t =39.342,P =0.000),which indicated the chondrocytes with overexpression of circ＿0045714 were successfully constructed.The difference in the expression level of circ＿0045714 among the chondrocytes transfected with si-NC and si-circ＿0045714 indicated the successful silencing of circ-0045714 in chondrocytes(1.00±0.00,0.28±0.04,t =31.177,P =0.000).⑤The IL-1β+pcDNA-circ＿0045714 group showed lower proliferation inhibition rate,apoptosis rate,and TNF-α and IL-6 levels in the chondrocytes compared to IL-1β+pcDNA group(t =16.290,P =0.000;t =11.359,P =0.000;t =11.988,P =0.000;t =12.266,P = 0.000).⑥The IL-1β+H-TTS+si-circ＿0045714 group exhibited higher proliferation inhibition rate,apoptosis rate,and TNF-α and IL-6 levels in the chondrocytes compared to IL-1β+H-TTS+si-NC group(t =9.586,P =0.001;t =9.120,P =0.001;t =7.069,P =0.002;t =10.548,P =0.001).Conclusion:The TTS can inhibit IL-1β-induced chondrocyte apoptosis and the expression of inflammatory cyto-kine,which has potential value in treatment of osteoarthritis.It may exert the effects by upregulating the expression of circ＿0045714 in the chondrocytes,and the effects are stronger at the concentration of 200 μg/mL.
NETL
NSTL
万方数据
维普
