| 刘冬梅,刘润萍,曾芳馨.薏苡附子败酱散通过抗三结构域蛋白 21-Toll样受体 4-t-核因子 -κB信号通路对类风湿关节炎 MH7A细胞的影响[J].安徽医药,2024,28(8):1512-1517. |
| 薏苡附子败酱散通过抗三结构域蛋白 21-Toll样受体 4-t-核因子 -κB信号通路对类风湿关节炎 MH7A细胞的影响 |
| Experimental study on the treatment of rheumatoid arthritis by Yiyi Fuzi Baijiang San regulating TRIM21-TLR4-NF-κB signal pathway |
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| DOI：10.3969/j.issn.1009-6469.2024.08.006 |
| 中文关键词: 关节炎，类风湿 败酱草 薏苡仁 附子 薏苡附子败酱散 抗三结构域蛋白 21 Toll样受体 4-NF-κB信号通路 |
| 英文关键词: Arthritis, rheumatoid Atrina glass Coix seed Prepared common monkshood branched root Yiyi Fuzi Baijiang San TRIM21 TLR4-NF-κB signal path |
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| 中文摘要: |
| 目的观察不同浓度薏苡附子败酱散对肿瘤坏死因子 -α(TNF-α)诱导的类风湿关节炎成纤维细胞( MH7A)增殖、凋亡及炎症反应的影响，探究其作用机制。方法 2022年 2―8月，大鼠用不同浓度薏苡附子败酱散及蒸馏水灌胃制备含药血清； 20 μg/L的 TNF-α处理的 MH7A细胞记为 TNF-α组， TNF-α+含药血清处理的 MH7A细胞记为薏苡附子败酱散低浓度组、薏苡附子败酱散中浓度组、薏苡附子败酱散高浓度组，正常培养的 MH7A细胞标记为对照组。细胞计数试剂盒( CCK8)、流式细胞术、酶联免疫吸附测定(ELISA)检测细胞的存活、凋亡及白细胞介素( IL)-1β、干扰素( IFN)-γ；蛋白质印迹法( WB)检测基质金属蛋白酶( MMP)-3、MMP -9、抗三结构域蛋白 21(TRIM21)、 Toll样受体 4(TLR4)、 t-核因子( NF)-κB p65、磷酸化( p)-NF-κB p65、pNF-κB抑制因子( IκB)-α和 t-IκB-α蛋白表达。 pcDNA 3.1组(转染空载体)、 pcDNA+TRIM21组(转染 pcDNA 3.1+TRIM21)、薏苡附子败酱散中浓度 +si-con组(转染 si-con+4.70 g/kg含药血清)、薏苡附子败酱散中浓度 +si-TRIM21组(转染 si-TRIM21+4.70 g/kg含药血清)。结果与对照组相比， TNF-α组细胞存活率( 105.07±1.90比 185.67±3.06)、 TRIM21(0.56±0.02比 0.68±0.04)、 MMP-3(0.18±0.00比 0.63±0.02)、 MMP-9(0.39±0.01比 0.82±0.01)、 TLR4(0.25±0.02比 0.68±0.07)、 p-NF-κB p65(0.24±0.01比 0.68±0.06)、 p-IκB-α(0.22±0.01比 0.55±0.04)蛋白表达和 IL-1β(31.65±0.66比 101.93±0.60)、 IFN-γ(8.53±0.16比 63.76±1.35)含量均显著升高，凋亡率( 6.54±0.06比 1.17±0.08)均显著降低；与 TNF-α组相比，薏苡附子败酱散低浓度组( 185.67±3.06比 153.77±3.09；0.68±0.04比 0.37±0.02；0.63±0.02比 0.47±0.02；0.82±0.01比 0.73±0.01；103.2±0.7比 92.93±0.85；66.3±1.45比 54.47±1.46；0.68±0.07比 0.53±0.05；0.68±0.06比 0.53±0.06；0.55±0.04比 0.47±0.01；1.84±0.07比 6.67±0.28)、薏苡附子败酱散中浓度组( 185.67±3.06比 136.23±1.57；0.68±0.04比 0.57±0.02；0.63±0.02比 0.37±0.02；0.82±0.01比 0.57±0.00；103.2±0.7比 86.4± 0.75；66.3±1.45比 38.1±0.92；0.68±0.07比 0.43±0.07；0.68±0.06比 0.59±0.01；0.55±0.04比 0.40±0.03；1.84±0.07比 9.59±0.29)、薏苡附子败酱散高浓度组( 185.67±3.06比 143.47±1.50；0.68±0.04比 0.47±0.02；0.63±0.02比 0.41±0.05；0.82±0.01比 0.62±0.00；103.2±0.7比 89.63±0.42；66.3±1.45比 40.17±0.90；0.68±0.07比 0.51±0.06；0.68±0.06比 0.69±0.07；0.55±0.04比 0.41±0.02；1.84±0.07比 8.89±0.08)细胞存活率、 TRIM21、MMP-3、MMP -9蛋白和 IL-1β、IFN-γ含量及 TLR4、p-NF-κB p65、p-IκB-α蛋白表达均显著降低，凋亡率均显著升高( P<0.05)；与 pcDNA组相比， pcDNA+TRIM21组细胞 TRIM21蛋白表达( 0.24±0.03比 0.51±0.04)、细胞凋亡率( 1.61±0.11比 12.43±0.61)均显著升高， MMP-3(0.64±0.02比 0.41±0.04)、 MMP -9(0.82±0.03比 0.45±0.02)、 IL-1β |
| 英文摘要: |
| Objective To observe the effect of different concentrations of Yiyi Fuzi Baijiang San on TNF-α of MH7A cell prolifera- tion, apoptosis and inflammation, and explore the mechanism. Methods This study was conducted from February 2022 to August 2022 Rats were perfused with different concentrations of Yiyi Fuzi Baijiang San and distilled water to prepare drug containing serum; 20 μg/L TNF-α Treated MH7A cells are recorded as TNF-α Group, TNF-α+ MH7A cells treated with drug containing serum were re-corded as low concentration group, medium concentration group and high concentration group of Yiyi Fuzi Baijiang San, and normal cultured MH7A cells were marked as control group. Cell counting Kit (CCK8), flow cytometry and enzyme linked immunosorbent assay(ELISA) were used to detect cell survival, apoptosis and interleukin (IL) -1 β, Interferon (IFN)-γ, Matrix metalloproteinase (MMP) -3, MMP-9, tridomain protein 21 (TRIM21), toll like receptor 4 (TLR4), t-nuclear factor (NF)-κ B p65, phosphorylated (p) -NF-κ B p65, p-NF-κ B suppressor (I κ B)-α and t-IκB-α protein expression were detected by Western blotting (WB) assay. PcDNA 3.1 group(transfected empty vector), pcDNA + TRIM21 group (transfected pcDNA 3.1 + TRIM21), Yiyi Fuzi Baijiang San medium concentration+ si-con group (transfected si-con + 4.70 g/kg medicated serum), Yiyi Fuzi Baijiang San medium concentration + si-trim21 group (trans- fected with si-trim21+4.70 g/kg medicated serum).Results Compared with the control group, The cell survival rate of TNF-α group (105.07±1.90 vs. 185.67±3.06), TRIM21 (0.56±0.02 vs. 0.68±0.04), MMP-3 (0.18±0.00 vs. 0.63±0.02), MMP-9 (0.39±0.01 vs. 0.82± 0.01), TLR4 (0.25±0.02 vs. 0.68±0.07), p-NF-κB p65 (0.24±0.01 vs. 0.68±0.06), p-IκB-α (0.22±0.01 vs. 0.55±0.04) and the contents of IL-1β (31.65±0.66 vs. 101.93±0.60) and IFN-γ (8.53±0.16 vs. 63.76±1.35) were significantly increased, while the apoptosis rate (6.54±0.06 vs. 1.17±0.08) was significantly decreased. Compared with TNF-α group, low concentration of Yiyi Fuzi Baijiang San(185.67±3.06 vs. 153.77±3.09; 0.68±0.04 vs. 0.37±0.02; 0.63±0.02 vs. 0.47±0.02; 0.82±0.01 vs. 0.73±0.01; 103.2±0.7 vs. 92.93±0.85; 66.3±1.45 vs. 54.47±1.46; 0.68±0.07 vs. 0.53±0.05; 0.68±0.06 vs. 0.53±0.06; 0.55±0.04 vs. 0.47±0.01; 1.84±0.07 vs. 6.67±0.28), medium concentra- tion of Yiyi Fuzi Baijiang San (185.67±3.06 vs. 136.23±1.57; 0.68±0.04 vs. 0.57±0.02; 0.63±0.02 vs. 0.37±0.02; 0.82±0.01 vs. 0.57± 0.00; 103.2±0.7 vs. 86.4±0.75; 66.3±1.45 vs. 38.1±0.92; 0.68±0.07 vs. 0.43±0.07; 0.68±0.06 vs. 0.59±0.01; 0.55±0.04 vs. 0.40±0.03; 1.84±0.07 vs. 9.59±0.29), Yiyi Fuzi Baijiang San high concentration group (185.67±3.06 vs. 143.47±1.50; 0.68±0.04 vs. 0.47±0.02; 0.63±0.02 vs. 0.41±0.05; 0.82±0.01 vs. 0.62±0.00; 103.2±0.7 vs. 89.63±0.42; 66.3±1.45 vs. 40.17±0.90; 0.68±0.07 vs. 0.51±0.06; 0.68±0.06 vs. 0.69±0.07; 0.55±0.04 vs. 0.41±0.02; 1.84±0.07 vs. 8.89±0.08), the levels of TRIM21, MMP-3 and MMP-9 proteins, IL1β and IFN-γ, and the expressions of TLR4, p-Nf-κb p65 and p-IκB-α proteins were significantly decreased, and the apoptosis rate was significantly increased (P<0.05). Compared with pcDNA group, the expression of TRIM21 protein (0.24±0.03 vs. 0.51±0.04) and apoptosis rate (1.61±0.11 vs. 12.43±0.61) in pcDNA+TRIM21 group were significantly increased. MMP-3 (0.64±0.02 vs. 0.41±0.04), MMP-9 (0.82±0.03 vs. 0.45±0.02), IL-1β (110.63±0.55 vs. 90.93±0.60), IFN-γ (64.5±0.82 vs. 48.1±1.25) and cell viability (179.03±0.74 vs. 120.07±0.60) were significantly decreased. TRIM21 knockdown significantly inhibited the therapeutic effect of Yiyi Fuzi Baiji- ang San and increased TLR4 (0.45±0.06 vs. 0.61±0.02), p-NF-κB p65 (0.49±0.02 vs. 0.56±0.02), p-IκB-α (0.38±0.01 vs. 0.62±0.01). Conclusion Yiyi Fuzi Baijiang San can enhance the viability of MH7A cells induced by TNF-α, which may be related to the inhibi-tion of TLR4-NF-κB signaling pathway by upregulating TRIM21 |
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