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

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;20μg/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、p-NF-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(110.63±0.55比90.93±0.60)、IFN-γ(64.5±0.82比48.1±1.25)及细胞存活率(179.03±0.74比120.07±0.60)均显著降低;敲减TRIM21显著抑制薏苡附子败酱散中浓度对损伤细胞的治疗作用且显著升高TLR4(0.45±0.06比0.61±0.02)、p-NF-κB p65(0.49±0.02比0.56±0.02)、p-IκB-α(0.38±0.01比0.62±0.01)的蛋白表达。结论薏苡附子败酱散增强TNF-α诱导的MH7A细胞的生存能力,其作用机制与上调TRIM21抑制TLR4-NF-κB信号通路活性有关。

Objective To observe the effect of different concentrations of Yiyi Fuzi Baijiang San on TNF-αof MH7A cell proliferation,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;20μg/L TNF-αTreated MH7A cells are recorded as TNF-αGroup,TNF-α+MH7A cells treated with drug containing serum were recorded 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-I κ B- 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, IL- 1β 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.