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[1]周婷婷,楊文廣,胡艷婷,等.基于Toll樣受體4/核因子κB信號(hào)通路探究苦參堿對(duì)類風(fēng)濕關(guān)節(jié)炎風(fēng)濕熱痹證的治療作用及機(jī)制[J].中醫(yī)正骨,2022,34(10):1-9,26.
　ZHOU Tingting,YANG Wenguang,HU Yanting,et al.The therapeutic effects and mechanism of matrine on rheumatoid arthritis with wind-dampness-heat arthromyodynia via Toll-like receptor 4/nuclear factor-κB signaling pathway[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2022,34(10):1-9,26.
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基于Toll樣受體4/核因子κB信號(hào)通路探究苦參堿對(duì)類風(fēng)濕關(guān)節(jié)炎風(fēng)濕熱痹證的治療作用及機(jī)制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第34卷
期數(shù):
2022年10期
頁(yè)碼:
1-9,26
欄目:
基礎(chǔ)研究
出版日期:
2022-10-20

文章信息/Info

Title:
The therapeutic effects and mechanism of matrine on rheumatoid arthritis with wind-dampness-heat arthromyodynia via Toll-like receptor 4/nuclear factor-κB signaling pathway
作者:
周婷婷1; 楊文廣1; 胡艷婷1; 馬俊福2
(1.商丘市中醫(yī)院,河南 商丘 476000; 2.河南省中醫(yī)院,河南 鄭州 450002)
Author(s):
ZHOU Tingting1; YANG Wenguang1; HU Yanting1; MA Junfu2
1.Shangqiu Hospital of Traditional Chinese Medicine,Shangqiu 476000,Henan,China 2.Henan Provincial Hospital of TCM,Zhengzhou 450002,Henan,China
關(guān)鍵詞:
關(guān)節(jié)炎; 類風(fēng)濕;  風(fēng)濕;  熱痹;  苦參堿;  Toll樣受體4;  NF-κB
Keywords:
arthritis; rheumatoid;  wind dampness;  arthralgia due to heattoxicity;  matrine;  Toll-like receptor 4;  NF-kappa B
摘要:
目的:基于Toll樣受體4(Toll-like receptor 4,TLR4)/核因子κB(nuclear factor-κB,NF-κB)信號(hào)通路探究苦參堿對(duì)類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis,RA)風(fēng)濕熱痹證的治療作用及機(jī)制。方法:將60只8周齡SPF級(jí)雄性SD大鼠隨機(jī)分為正常組、模型組、甲氨蝶呤組及苦參堿低、中、高劑量組,每組10只。除正常組外,其余5組均采用牛Ⅱ型膠原誘導(dǎo)聯(lián)合人工氣候箱干預(yù)建立RA風(fēng)濕熱痹證模型。甲氨蝶呤組大鼠按照1.0 mg·kg-1以甲氨蝶呤灌胃,苦參堿低、中、高劑量組大鼠分別按照30 mg·kg-1、60 mg·kg-1、120 mg·kg-1以苦參堿灌胃,正常組和模型組大鼠則以等體積蒸餾水灌胃。藥物干預(yù)均每周1次,共干預(yù)4次。觀察大鼠的一般情況,測(cè)定足趾腫脹度、關(guān)節(jié)炎指數(shù),以ELISA法檢測(cè)血清腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白細(xì)胞介素-1β(interleukin-1β,IL-1β)含量,HE染色觀察膝關(guān)節(jié)滑膜組織病理學(xué)改變,以實(shí)時(shí)定量PCR法檢測(cè)膝關(guān)節(jié)滑膜組織Bax mRNA、Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表達(dá)量,以Western Blot法檢測(cè)膝關(guān)節(jié)滑膜組織Cleaved caspase-3蛋白、TLR4蛋白、NF-κB p65蛋白、磷酸化核因子κB p65(phosphorylated nuclear factor-κB p65,p-NF-κB p65)蛋白表達(dá)量。結(jié)果:①大鼠一般情況。正常組大鼠毛發(fā)順滑有光澤,飲食、飲水正常,足趾無(wú)腫脹; 造模后模型組、甲氨蝶呤組及苦參堿低、中、高劑量組大鼠均出現(xiàn)毛發(fā)干燥無(wú)光澤,攝水量增加,易激惹、攻擊性強(qiáng),足趾腫脹、紅熱、蜷縮、僵硬等表現(xiàn); 與模型組相比,藥物干預(yù)后甲氨蝶呤組及苦參堿低、中、高劑量組大鼠毛發(fā)干燥無(wú)光澤、足趾腫脹等情況有所改善。②足趾腫脹度。6組大鼠的足趾腫脹度比較,差異有統(tǒng)計(jì)學(xué)意義(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)。模型組大鼠的足趾腫脹度高于其余5組(P=0.000; P=0.000; P=0.000; P=0.000; P=0.000),苦參堿低劑量組大鼠的足趾腫脹度高于甲氨蝶呤組和苦參堿中、高劑量組(P=0.007; P=0.000; P=0.000),苦參堿中劑量組大鼠的足趾腫脹度高于甲氨蝶呤組和苦參堿高劑量組(P=0.001; P=0.000),甲氨蝶呤組和苦參堿高劑量組大鼠足趾腫脹度的差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.096)。③關(guān)節(jié)炎指數(shù)。正常組大鼠足部未見(jiàn)異常,關(guān)節(jié)炎指數(shù)為0分; 其余5組大鼠關(guān)節(jié)炎指數(shù)比較,差異有統(tǒng)計(jì)學(xué)意義[(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]。模型組大鼠的關(guān)節(jié)炎指數(shù)高于甲氨蝶呤組和苦參堿低、中、高劑量組(P=0.000; P=0.000; P=0.000; P=0.000),苦參堿低劑量組大鼠的關(guān)節(jié)炎指數(shù)高于甲氨蝶呤組和苦參堿中、高劑量組(P=0.000; P=0.000; P=0.000),苦參堿中劑量組大鼠的關(guān)節(jié)炎指數(shù)高于甲氨蝶呤組和苦參堿高劑量組(P=0.000; P=0.000),甲氨蝶呤組和苦參堿高劑量組大鼠關(guān)節(jié)炎指數(shù)的差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.054)。④血清TNF-α、IL-1β含量。6組大鼠的血清TNF-α、IL-1β含量比較,組間差異均有統(tǒng)計(jì)學(xué)意義[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]。模型組大鼠的血清TNF-α、IL-1β含量均高于其余5組(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),苦參堿低劑量組大鼠的血清TNF-α、IL-1β含量均高于甲氨蝶呤組和苦參堿中、高劑量組(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.000),苦參堿中劑量組大鼠的血清TNF-α、IL-1β含量均高于甲氨蝶呤組和苦參堿高劑量組(P=0.000,P=0.000; P=0.000,P=0.000),甲氨蝶呤組和苦參堿高劑量組大鼠血清TNF-α、IL-1β含量的差異均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.054; P=0.067)。⑤膝關(guān)節(jié)滑膜組織病理學(xué)觀察結(jié)果。HE染色結(jié)果顯示,正常組大鼠膝關(guān)節(jié)滑膜組織結(jié)構(gòu)完整,細(xì)胞排列整齊,無(wú)炎癥細(xì)胞浸潤(rùn); 與正常組相比,模型組大鼠滑膜組織增生明顯,有大量炎癥細(xì)胞浸潤(rùn),滑膜細(xì)胞排列紊亂,邊界模糊不清; 與模型組相比,甲氨蝶呤組和苦參堿低、中、高劑量組滑膜組織增生程度減輕,滑膜細(xì)胞排列較整齊,炎癥細(xì)胞浸潤(rùn)情況均得到不同程度改善。⑥膝關(guān)節(jié)滑膜組織Bax mRNA、Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表達(dá)量。6組大鼠膝關(guān)節(jié)滑膜組織Bax mRNA、Bcl-2 mRNA、TLR4 mRNA、NF-κB p65 mRNA表達(dá)量比較,組間差異均有統(tǒng)計(jì)學(xué)意義(Bax mRNA:0.80±0.07,0.40±0.03,0.72±0.08,0.53±0.05,0.63±0.05,0.71±
Abstract:
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:①Rats 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.②There 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).③No 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).④There 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).⑤The 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.⑥There 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).⑦There 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.

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備注/Memo

備注/Memo:
基金項(xiàng)目:2022年度河南省中醫(yī)藥科學(xué)研究專項(xiàng)課題(2022ZY1062)
更新日期/Last Update: 1900-01-01