基于Toll样受体4/核因子κB信号通路探究苦参碱对类风湿关节炎风湿热痹证的治疗作用及机制

The the rapeutic effects and mechanism of matrine on rhe umatoid arthritis with wind-dampness-heat arthromyodynia via Toll-like receptor 4/nuclear factor-κB signaling pathway

目的:基于Toll样受体4(Toll-like receptor4,TLR4)/核因子κB(nuclear factor-κB,NF-κB)信号通路探究苦参碱对类风湿关节炎(rheumatoid arthritis,RA)风湿热痹证的治疗作用及机制。方法:将60只8周龄SPF级雄性SD大鼠随机分为正常组、模型组、甲氨蝶呤组及苦参碱低、中、高剂量组,每组10只。除正常组外,其余5组均采用牛Ⅱ型胶原诱导联合人工气候箱干预建立RA风湿热痹证模型。甲氨蝶呤组大鼠按照1.0mg·kg^(-1)以甲氨蝶呤灌胃,苦参碱低、中、高剂量组大鼠分别按照30mg·kg^(-1)、60mg·kg^(-1)、120mg·kg^(-1)以苦参碱灌胃,正常组和模型组大鼠则以等体积蒸馏水灌胃。药物干预均每周1次,共干预4次。观察大鼠的一般情况,测定足趾肿胀度、关节炎指数,以ELISA法检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)含量,HE染色观察膝关节滑膜组织病理学改变,以实时定量PCR法检测膝关节滑膜组织BaxmRNA、Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表达量,以WesternBlot法检测膝关节滑膜组织Cleaved caspase-3蛋白、TLR4蛋白、NF-κBp65蛋白、磷酸化核因子κBp65(phosphorylated nuclear factor-κBp65,p-NF-κB p65)蛋白表达量。结果:①大鼠一般情况。正常组大鼠毛发顺滑有光泽,饮食、饮水正常,足趾无肿胀;造模后模型组、甲氨蝶呤组及苦参碱低、中、高剂量组大鼠均出现毛发干燥无光泽,摄水量增加,易激惹、攻击性强,足趾肿胀、红热、蜷缩、僵硬等表现;与模型组相比,药物干预后甲氨蝶呤组及苦参碱低、中、高剂量组大鼠毛发干燥无光泽、足趾肿胀等情况有所改善。②足趾肿胀度。6组大鼠的足趾肿胀度比较,差异有统计学意义(0.08±0.01,0.51±0.07,0.24±0.04,0.44±0.06,0.37±0.04,0.28±0.06,F=118.983,P=0.000)。模型组大鼠的足趾肿胀度高于其余5组(P=0.000;P=0.000;P=0.000;P=0.000;P=0.000),苦参碱低剂量组大鼠的足趾肿胀度高于甲氨蝶呤组和苦参碱中、高剂量组(P=0.007;P=0.000;P=0.000),苦参碱中剂量组大鼠的足趾肿胀度高于甲氨蝶呤组和苦参碱高剂量组(P=0.001;P=0.000),甲氨蝶呤组和苦参碱高剂量组大鼠足趾肿胀度的差异无统计学意义(P=0.096)。③关节炎指数。正常组大鼠足部未见异常,关节炎指数为0分;其余5组大鼠关节炎指数比较,差异有统计学意义[(7.02±0.24)分,(4.36±0.12)分,(6.32±0.16)分,(5.58±0.20)分,(4.48±0.14)分,F=422.684,P=0.000]。模型组大鼠的关节炎指数高于甲氨蝶呤组和苦参碱低、中、高剂量组(P=0.000;P=0.000;P=0.000;P=0.000),苦参碱低剂量组大鼠的关节炎指数高于甲氨蝶呤组和苦参碱中、高剂量组(P=0.000;P=0.000;P=0.000),苦参碱中剂量组大鼠的关节炎指数高于甲氨蝶呤组和苦参碱高剂量组(P=0.000;P=0.000),甲氨蝶呤组和苦参碱高剂量组大鼠关节炎指数的差异无统计学意义(P=0.054)。④血清TNF-α、IL-1β含量。6组大鼠的血清TNF-α、IL-1β含量比较,组间差异均有统计学意义[TNF-α:(48.09±4.88)pg·mL^(-1),(351.49±32.39)pg·mL^(-1),(143.05±10.02)pg·mL^(-1),(281.51±26.50)pg·mL^(-1),(207.63±17.00)pg·mL^(-1),(154.40±14.23)pg·mL^(-1),F=311.253,P=0.000;IL-1β:(30.71±4.60)pg·mL^(-1),(258.14±20.85)pg·mL^(-1),(105.27±10.38)pg·mL^(-1),(201.57±16.51)pg·mL^(-1),(158.97±16.18)pg·mL^(-1),(114.37±10.48)pg·mL^(-1),F=337.119,P=0.000]。模型组大鼠的血清TNF-α、IL-1β含量均高于其余5组(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000),苦参碱低剂量组大鼠的血清TNF-α、IL-1β含量均高于甲氨蝶呤组和苦参碱中、高剂量组(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000),苦参碱中剂量组大鼠的血清TNF-α、IL-1β含量均高于甲氨蝶呤组和苦参碱高剂量组(P=0.000,P=0.000;P=0.000,P=0.000),甲氨蝶呤组和苦参碱高剂量组大鼠血清TNF-α、IL-1β含量的差异均无统计学意义(P=0.054;P=0.067)。⑤膝关节滑膜组织病理学观察结果。HE染色结果显示,正常组大鼠膝关节滑膜组织结构完整,细胞排列整齐,无炎症细胞浸润;与正常组相比,模型组大鼠滑膜组织增生明显,有大量炎症细胞浸润,滑膜细胞排列紊乱,边界模糊不清;与模型组相比,甲氨蝶呤组和苦参碱低、中、高剂量组滑膜组织增生程度减轻,滑膜细胞排列较整齐,炎症细胞浸润情况均得到不同程度改善。⑥膝关节滑膜组织Bax mRNA、Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表达量。6组大鼠膝关节滑膜组织BaxmRNA、Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表达量比较,组间差异均有统计学意义(Bax mRNA:0.80±0.07,0.40±0.03,0.72±0.08,0.53±0.05,0.63±0.05,0.71±0.06,F=69.870,P=0.000;Bcl-2 mRNA:0.19±0.02,0.78±0.06,0.33±0.03,0.67±0.05,0.54±0.06,0.36±0.04,F=258.197,P=0.000;TLR4 mRNA:0.13±0.01,0.61±0.07,0.25±0.02,0.54±0.05,0.45±0.04,0.27±0.03,F=206.811,P=0.000;NF-κB p65 mRNA:0.17±0.01,0.56±0.04,0.26±0.02,0.46±0.04,0.34±0.04,0.28±0.03,F=220.358,P=0.000)。模型组大鼠的膝关节滑膜组织Bax mRNA表达量低于其余5组(P=0.000;P=0.000;P=0.000;P=0.000;P=0.000),Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表达量均高于其余5组(P=0.000,P=0.000,P=0.000;P=0.000,P=0.019,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000);苦参碱低剂量组大鼠的膝关节滑膜组织BaxmRNA表达量低于甲氨蝶呤组和苦参碱中、高剂量组(P=0.000;P=0.000;P=0.000),Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表达量均高于甲氨蝶呤组及苦参碱中、高剂量组(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000);苦参碱中剂量组大鼠的膝关节滑膜组织Bax mRNA表达量低于甲氨蝶呤组和苦参碱高剂量组(P=0.000;P=0.000),Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表达量均高于甲氨蝶呤组和苦参碱高剂量组(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.001);甲氨蝶呤组和苦参碱高剂量组大鼠膝关节滑膜组织Bax mRNA、Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表达量的组间差异均无统计学意义(P=0.755,P=0.074,P=0.096,P=0.096)。⑦膝关节滑膜组织Cleaved caspase-3蛋白、TLR4蛋白、NF-κBp65蛋白、p-NF-κBp65蛋白表达量。6组大鼠的Cleaved caspase-3蛋白、TLR4蛋白表达量及p-NF-κB p65/NF-κB p65蛋白表达量比值比较,组间差异均有统计学意义(Cleaved caspase-3蛋白:0.74±0.06,0.32±0.03,0.62±0.05,0.39±0.04,0.47±0.05,0.58±0.05,F=127.351,P=0.001;TLR4蛋白:0.17±0.02,0.67±0.06,0.25±0.03,0.43±0.05,0.35±0.03,0.27±0.02,F=216.610,P=0.001;p-NF-κB p65/NF-κB p65蛋白表达量比值:0.24±0.02,0.64±0.07,0.27±0.03,0.49±0.05,0.36±0.04,0.29±0.03,F=129.880,P=0.001)。模型组大鼠的Cleaved caspase-3蛋白表达量低于其余5组(P=0.000;P=0.000;P=0.000;P=0.000;P=0.000),TLR4蛋白表达量及p-NF-κB p65/NF-κB p65蛋白表达量比值均高于其余5组(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000);苦参碱低剂量组大鼠的Cleaved caspase-3蛋白表达量低于甲氨蝶呤组和苦参碱中、高剂量组(P=0.000;P=0.001;P=0.000),TLR4蛋白表达量及p-NF-κB p65/NF-κB p65蛋白表达量比值均高于甲氨蝶呤组和苦参碱中、高剂量组(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000);苦参碱中剂量组大鼠的Cleaved caspase-3蛋白表达量低于甲氨蝶呤组和苦参碱高剂量组(P=0.000;P=0.000),TLR4蛋白表达量及p-NF-κBp65/NF-κB p65蛋白表达量比值均高于甲氨蝶呤组和苦参碱高剂量组(P=0.000,P=0.000;P=0.000,P=0.000);甲氨蝶呤组和苦参碱高剂量组大鼠Cleaved caspase-3蛋白、TLR4蛋白表达量及p-NF-κB p65/NF-κB p65蛋白表达量比值的组间差异均无统计学意义(P=0.090,P=0.096,P=0.153)。结论:苦参碱可有效减轻RA风湿热痹证大鼠的症状和体征,且疗效存在剂量依赖性;其作用机制可能是通过抑制TLR4/NF-κB信号通路,减轻炎症反应、促进滑膜细胞凋亡。

Objective:To investigate the therapeutic effects and mechanism of matrine on rheumatoid arthritis(RA)with wind-dampness-heat arthromyodynia via Toll-like receptor 4(TLR4)/nuclear factor κB(NF-κB)signaling pathway.Methods:Sixty 8-week-old specific pathogen-free(SPF)-grade male Sprague-Dawley(SD)rats were randomly assigned into normal group, RA model group, methotrexate group, matrine low-dose group, matrine medium-dose group and matrine high-dose group, 10 cases in each group.The rats in RA model group, methotrexate group, matrine low-dose group, matrine medium-dose group and matrine high-dose group were intervened by bovine typeⅡcollagen in artificial climate chamber for inducing RA with wind-dampness-heat arthromyodynia.After successful modeling, the rats in methotrexate group were intragastric administrated with methotrexate in dosage of 1.0 mg/kg, the ones in matrine low-,medium-and high-dose groups with matrine in dosages of 30 mg/kg, 60 mg/kg and 120 mg/kg respectively, and the ones in normal group and RA model group with the same dosage of distilled water, once a week for consecutive 4 times.The general condition of the rats was observed, the toe swelling degree and arthritis index were detected, and the synovial tissues of rat knee joints were stained with hematoxylin-eosin(HE)for observing the pathological changes.Furthermore, the serum levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by using enzyme linked immunosorbent assay(ELISA),the mRNA expression levels of Bax, Bcl-2,TLR4 and NF-κB p65 as well as the protein expression levels of Cleaved caspase-3,TLR4,NF-κB p65 and phosphorylated nuclear factor-κB p65(p-NF-κB p65)in rat knee synovial tissues were detected by using real-time quantitative PCR(RT-qPCR)and Western-blot assays respectively.Results:(1)Rats with smooth and shiny furs, normal eating and drinking as well as healthy toes were observed in normal group.After modeling, the rats with such symptoms as dry and lusterless furs, polydipsia, irritability, aggression and the abnormal toes, manifesting as swelling, red-heat, curled up and stiffness were observed in RA model group, methotrexate group, matrine low-dose group, matrine medium-dose group and matrine high-dose group, however, compared to RA model group, the symptoms of dry and lusterless furs as well as swollen toes were improved after drug intervention in rats of methotrexate group, matrine low-dose group, matrine medium-dose group and matrine high-dose group.(2)There was statistical difference in toe swelling degree among the 6 groups(0.08±0.01,0.51±0.07,0.24±0.04,0.44±0.06,0.37±0.04,0.28±0.06,F=118.983,P=0.000).The degree of toe swelling in rats was higher in RA model group compared to the other five groups(P=0.000;P=0.000;P=0.000;P=0.000;P=0.000),and it was higher in the matrine low-dose group compared to methotrexate group, matrine medium-dose group and matrine high-dose group(P=0.007;P=0.000;P=0.000),and was higher in matrine medium-dose group compared to methotrexate group and matrine high-dose group(P=0.001;P=0.000),however, the difference was not significant between methotrexate group and matrine high-dose group(P=0.096).(3)No abnormality was found in the toes of rats in the normal group with arthritis index evaluated as 0 point, while there was statistical difference in arthritis index among the other 5 groups(7.02±0.24,4.36±0.12,6.32±0.16,5.58±0.20,4.48±0.14 points, F=422.684,P=0.000).The arthritis index was higher in RA model group compared to methotrexate group, matrine low-dose group, matrine medium-dose group and matrine high-dose group(P=0.000;P=0.000;P=0.000;P=0.000),and was higher in matrine low-dose group compared to methotrexate group, matrine medium-dose group and matrine high-dose group(P=0.000;P=0.000;P=0.000),and was higher in matrine medium-dose group compared to methotrexate group and matrine high-dose group(P=0.000;P=0.000),however, the difference was not significant between methotrexate group and matrine high-dose group(P=0.054).(4)There was statistical difference in serum levels of TNF-α and IL-1β among the 6 groups(TNF-α:48.09±4.88,351.49±32.39,143.05±10.02,281.51±26.50,207.63±17.00,154.40±14.23 pg/mL,F=311.253,P=0.000;IL-1β:30.71±4.60,258.14±20.85,105.27±10.38,201.57±16.51,158.97±16.18,114.37±10.48 pg/mL,F=337.119,P=0.000).The serum levels of TNF-α and IL-1β were higher in RA model group compared to the other 5 groups(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000),and were higher in matrine low-dose group compared to methotrexate group, matrine medium-dose group and matrine high-dose group(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000),and were higher in matrine medium-dose group compared to methotrexate group and matrine high-dose group(P=0.000,P=0.000;P=0.000,P=0.000),however, the difference was not significant between methotrexate group and matrine high-dose group(P=0.054;P=0.067).(5)The HE staining results showed that the complete structure and orderly arranged cells without infiltration by inflammatory cells were observed in knee synovial tissues of rats from normal group;compared to normal group, the obvious proliferation and disorderly arranged cells infiltrated by a large number of inflammatory cells with smeared-out boundary were observed in knee synovial tissues of rats from RA model group;compared to RA model group, the reduced proliferation and the relatively well-arranged cells with improved inflammatory cell infiltration in varying degrees were observed in knee synovial tissues of rats from methotrexate group, matrine low-dose group, matrine medium-dose group and matrine high-dose group.(6)There was statistical difference in mRNA expression levels of Bax, Bcl-2,TLR4 and NF-κB p65 in rat knee synovial tissues among the 6 groups(Bax mRNA:0.80±0.07,0.40±0.03,0.72±0.08,0.53±0.05,0.63±0.05,0.71±0.06,F=69.870,P=0.000;Bcl-2 mRNA:0.19±0.02,0.78±0.06,0.33±0.03,0.67±0.05,0.54±0.06,0.36±0.04,F=258.197,P=0.000;TLR4 mRNA:0.13±0.01,0.61±0.07,0.25±0.02,0.54±0.05,0.45±0.04,0.27±0.03,F=206.811,P=0.000;NF-κB p65 mRNA:0.17±0.01,0.56±0.04,0.26±0.02,0.46±0.04,0.34±0.04,0.28±0.03,F=220.358,P=0.000).The mRNA expression level of Bax in rat knee synovial tissues was lower, while the mRNA expression levels of Bcl-2,TLR4 and NF-κB p65 in rat knee synovial tissues were higher in RA model group compared to the other 5 groups(P=0.000;P=0.000;P=0.000;P=0.000;P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.019,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000).The mRNA expression level of Bax in rat knee synovial tissues was lower, while the mRNA expression levels of Bcl-2,TLR4 and NF-κB p65 in rat knee synovial tissues were higher in matrine low-dose group compared to methotrexate group, matrine medium-dose group and matrine high-dose groups(P=0.000;P=0.000;P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000).The mRNA expression level of Bax in rat knee synovial tissues was lower, while the mRNA expression levels of Bcl-2,TLR4 and NF-κB p65 in rat knee synovial tissues were higher in matrine medium-dose group compared to methotrexate group and matrine high-dose group(P=0.000;P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.001).However, there was no statistical difference in the mRNA expression levels of Bax, Bcl-2,TLR4 and NF-κB p65 in rat knee synovial tissues between methotrexate group and matrine high-dose group(P=0.755,P=0.074,P=0.096,P=0.096).(7)There was statistical difference in protein expression levels of Cleaved caspase-3 and TLR4 as well as the ratio of protein expression level of p-NF-κB p65 to NF-κB p65 among the 6 group(Cleaved caspase-3 protein: 0.74±0.06,0.32±0.03,0.62±0.05,0.39±0.04,0.47±0.05,0.58±0.05,F=127.351,P=0.001;TLR4 protein: 0.17±0.02,0.67±0.06,0.25±0.03,0.43±0.05,0.35±0.03,0.27±0.02,F=216.610,P=0.001;the ratio of protein expression level of p-NF-κB p65 to NF-κB p65:0.24±0.02,0.64±0.07,0.27±0.03,0.49±0.05,0.36±0.04,0.29±0.03,F=129.880,P=0.001).The protein expression level of Cleaved caspase-3 was lower, while the protein expression level of TLR4 and the ratio of protein expression level of p-NF-κB p65 to NF-κB p65 were higher in RA model group compared to the other 5 groups(P=0.000;P=0.000;P=0.000;P=0.000;P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000).The protein expression level of Cleaved caspase-3 was lower, while the protein expression level of TLR4 and the ratio of protein expression level of p-NF-κB p65 to NF-κB p65 were higher in matrine low-dose group compared to methotrexate group, matrine medium-dose group and matrine high-dose group(P=0.000;P=0.001;P=0.000;P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000).The protein expression level of Cleaved caspase-3 was lower, while the protein expression level of TLR4 and the ratio of protein expression level of p-NF-κB p65 to NF-κB p65 were higher in matrine medium-dose group compared to methotrexate group and matrine high-dose group(P=0.000;P=0.000;P=0.000,P=0.000;P=0.000,P=0.000).However, there was no statistical difference in the protein expression levels of Cleaved caspase-3 and TLR4 as well as the ratio of protein expression level of p-NF-κB p65 to NF-κB p65 between methotrexate group and matrine high-dose group(P=0.090,P=0.096,P=0.153).Conclusion:The matrine can effectively improve the symptoms and physical signs in RA rat models with wind-dampness-heat arthromyodynia, and it exhibits dose-dependence in the efficacy.Its mechanisms may be that it can reduce inflammatory reaction and promote synovial cell apoptosis through inhibiting TLR4/Nf-κb signaling pathway.