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[1]何鏡,王艷杰,陳卓玥,等.基于調(diào)節(jié)性T細(xì)胞/輔助性T細(xì)胞17平衡探討低強(qiáng)度脈沖聚焦超聲治療小鼠類風(fēng)濕關(guān)節(jié)炎的作用機(jī)制[J].中醫(yī)正骨,2025,37(04):30-40.
　HE Jing,WANG Yanjie,CHEN Zhuoyue,et al.Exploring the mechanism of focused low-intensity pulsed ultrasound in treating rheumatoid arthritis in mice based on the balance between regulatory T cell and T helper cell 17[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(04):30-40.
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基于調(diào)節(jié)性T細(xì)胞/輔助性T細(xì)胞17平衡探討低強(qiáng)度脈沖聚焦超聲治療小鼠類風(fēng)濕關(guān)節(jié)炎的作用機(jī)制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期數(shù):
2025年04期
頁(yè)碼:
30-40
欄目:
基礎(chǔ)研究
出版日期:
2025-04-20

文章信息/Info

Title:
Exploring the mechanism of focused low-intensity pulsed ultrasound in treating rheumatoid arthritis in mice based on the balance between regulatory T cell and T helper cell 17
作者:
何鏡1; 王艷杰2; 陳卓玥1; 崔博2; 藍(lán)泉博3; 周文智4
1.成都體育學(xué)院,四川 成都 641418; 2.四川省骨科醫(yī)院,四川 成都 610041; 3.紐約大學(xué),紐約 10012; 4.成都市婦女兒童中心醫(yī)院,四川 成都 610091
Author(s):
HE Jing1; WANG Yanjie2; CHEN Zhuoyue1; CUI Bo2; LAN Quanbo3; ZHOU Wenzhi4
1.Chengdu Sport University,Chengdu 641418,Sichuan,China 2.Sichuan Province Orthopedic Hospital,Chengdu 610041,Sichuan,China 3.New York University,New York 10012,USA 4.Chengdu Women's and Children's Central Hospital,Chengdu 610091,Sichuan,China
關(guān)鍵詞:
關(guān)節(jié)炎; 類風(fēng)濕;  膠原誘導(dǎo)性關(guān)節(jié)炎;  瘢痕灸療法;  超聲療法;  T-淋巴細(xì)胞; 調(diào)控;  Th17細(xì)胞;  小鼠
Keywords:
arthritis; rheumatoid;  collagen-induced arthritis;  scarring moxibustion therapy;  ultrasonic therapy;  T-lymphocytes; regulatory;  Th17 cells;  mice
摘要:
目的:觀察低強(qiáng)度脈沖聚焦超聲治療小鼠類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis,RA)的效果,并基于調(diào)節(jié)性T細(xì)胞(regulatory T cell,Treg)/輔助性T細(xì)胞17(helper T cell 17,Th17)平衡探討其作用機(jī)制。方法:將36只雄性DBA/1J小鼠隨機(jī)分為正常組、模型組、不治療組、安慰劑組、瘢痕灸組和聚焦超聲組,每組6只。除正常組外的各組小鼠均采用Ⅱ型膠原誘導(dǎo)法建立膠原誘導(dǎo)性關(guān)節(jié)炎模型。在造模結(jié)束時(shí),采用關(guān)節(jié)炎指數(shù)評(píng)分評(píng)價(jià)小鼠雙側(cè)踝關(guān)節(jié)關(guān)節(jié)炎情況,判斷造模是否成功。在造模成功后,模型組小鼠直接處死取材,聚焦超聲組在小鼠腎俞、足三里穴位采用低強(qiáng)度脈沖聚焦超聲治療,瘢痕灸組在相同穴位采用艾柱直接灸,安慰劑組在相同部位采用棉花直接灸,正常組、不治療組不采取任何治療措施。聚焦超聲組、瘢痕灸組、安慰劑組每天治療1次,治療6 d為1個(gè)療程,共治療2個(gè)療程。在治療1個(gè)療程時(shí)、治療結(jié)束時(shí),記錄小鼠雙側(cè)踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分。治療結(jié)束后,采用HE染色觀察各組小鼠踝關(guān)節(jié)軟骨和滑膜組織病變情況,采用流式細(xì)胞術(shù)檢測(cè)各組小鼠脾臟組織中Th17、Treg的百分比,分別采用實(shí)時(shí)定量PCR和Western Blot法檢測(cè)各組小鼠脾臟組織中叉頭框蛋白3(forkhead box protein 3,FOXP3)、維甲酸受體相關(guān)孤兒受體(retinoid acid receptor-related orphan receptor,ROR)γt、核因子-κB(nuclear factor-κB,NF-κB)的mRNA表達(dá)水平和蛋白表達(dá)水平。結(jié)果:①小鼠踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分結(jié)果。不治療組和安慰劑組小鼠踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分隨時(shí)間變化均呈上升趨勢(shì)(F=4.903,P=0.019; F=4.189,P=0.031),瘢痕灸組小鼠踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分隨時(shí)間變化基本保持不變(F=1.683,P=0.212),聚焦超聲組小鼠踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分隨時(shí)間變化呈下降趨勢(shì)(F=11.354,P=0.000)。治療結(jié)束時(shí),瘢痕灸組和聚焦超聲組小鼠踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分均低于不治療組和安慰劑組(不治療組:P=0.000,P=0.000; 安慰劑組:P=0.000,P=0.000),不治療組和安慰劑組小鼠踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分的差異無(wú)統(tǒng)計(jì)學(xué)意義(P=1.000),聚焦超聲組小鼠踝關(guān)節(jié)關(guān)節(jié)炎指數(shù)評(píng)分低于瘢痕灸組(P=0.000)。②小鼠踝關(guān)節(jié)組織病理學(xué)觀察結(jié)果。模型組小鼠軟骨層變薄、軟骨面粗糙不平、軟骨細(xì)胞分布不均勻,滑膜組織細(xì)胞排列紊亂,有增生及炎癥細(xì)胞浸潤(rùn); 與模型組相比,不治療組和安慰劑組小鼠踝關(guān)節(jié)組織病變略有加重,瘢痕灸組小鼠踝關(guān)節(jié)組織病變略有減輕,聚焦超聲組小鼠踝關(guān)節(jié)組織病變明顯減輕。③脾臟組織中Th17、Treg的百分比檢測(cè)結(jié)果。不治療組和安慰劑組小鼠脾臟組織中Th17的百分比均高于模型組(P=0.000,P=0.000),Treg的百分比均低于模型組(P=0.000,P=0.000); 瘢痕灸組、聚焦超聲組小鼠脾臟組織中Th17的百分比均低于模型組(P=0.002,P=0.000),瘢痕灸組小鼠脾臟組織中Treg的百分比低于模型組(P=0.000),聚焦超聲組小鼠脾臟組織中Treg的百分比高于模型組(P=0.000)。聚焦超聲組小鼠脾臟組織中Th17的百分比低于瘢痕灸組(P=0.000),Treg的百分比高于瘢痕灸組(P=0.000)。④小鼠脾臟組織中FOXP3、RORγt、NF-κB的mRNA表達(dá)水平檢測(cè)結(jié)果。不治療組、安慰劑組小鼠脾臟組織中FOXP3的mRNA相對(duì)表達(dá)量均低于模型組(P=0.001,P=0.001),RORγt、NF-κB的mRNA相對(duì)表達(dá)量均高于模型組(RORγt:P=0.001,P=0.019; NF-κB:P=0.000,P=0.000); 瘢痕灸組小鼠脾臟組織中FOXP3、RORγt、NF-κB的mRNA相對(duì)表達(dá)量與模型組的組間差異均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.999,P=0.052,P=0.995); 聚焦超聲組小鼠脾臟組織中FOXP3的mRNA相對(duì)表達(dá)量均高于模型組(P=0.000),RORγt、NF-κB的mRNA相對(duì)表達(dá)量均低于模型組(P=0.000,P=0.000); 聚焦超聲組小鼠脾臟組織中FOXP3的mRNA相對(duì)表達(dá)量均高于瘢痕灸組(P=0.000),RORγt、NF-κB的mRNA相對(duì)表達(dá)量均低于瘢痕灸組(P=0.000,P=0.000)。⑤小鼠脾臟組織中FOXP3、RORγt、NF-κB的蛋白表達(dá)水平檢測(cè)結(jié)果。不治療組、安慰劑組小鼠脾臟組織中FOXP3的蛋白相對(duì)表達(dá)量均低于模型組(P=0.001,P=0.000),RORγt、NF-κB的蛋白相對(duì)表達(dá)量均高于模型組(RORγt:P=0.010,P=0.013; NF-κB:P=0.014,P=0.001); 瘢痕灸組、聚焦超聲組小鼠脾臟組織中FOXP3的蛋白相對(duì)表達(dá)量與模型組的差異均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.573,P=0.304); 瘢痕灸組小鼠脾臟組織中RORγt、NF-κB的蛋白相對(duì)表達(dá)量與模型組的組間差異均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.999,P=0.999),聚焦超聲組小鼠脾臟組織中RORγt、NF-κB的蛋白相對(duì)表達(dá)量均低于模型組(P=0.017,P=0.010); 聚焦超聲組小鼠脾臟組織中FOXP3的蛋白相對(duì)表達(dá)量高于瘢痕灸組(P=0.021),RORγt、NF-κB的蛋白相對(duì)表達(dá)量均低于瘢痕灸組(P=0.016,P=0.010)。結(jié)論:RA在不治療的情況下病情會(huì)不斷加重,瘢痕灸和低強(qiáng)度脈沖聚焦超聲均能夠遏制RA病情進(jìn)展,且低強(qiáng)度脈沖聚焦超聲的效果優(yōu)于瘢痕灸; 其作用機(jī)制可能與調(diào)節(jié)FOXP3、RORγt、NF-κB的表達(dá)從而影響Treg/Th17平衡有關(guān)。
Abstract:
Objective:To observe the therapeutic effects of focused low-intensity pulsed ultrasound(FLIPUS)on rheumatoid arthritis(RA)in mice,and to explore its underlying mechanism based on the balance between regulatory T cell(Treg)and helper T cell 17(Th17).Methods:Thirty-six male DBA/1J mice were randomized into normal group,model group,untreated group,placebo group,scar moxibustion group,and FLIPUS group,with 6 cases in each group.All mice but the ones in normal group were modeled by typeⅡcollagen for inducing collagen-induced arthritis(CIA).At the end of the modeling,the arthritis of bilateral ankles were assessed using the arthritis index(AI)score to evaluate whether the modeling was successful.After successful modeling,the mice in model group were sacrificed for collecting their spleens and bilateral ankle joints.The ones in FLIPUS group were treated with FLIPUS at Shenshu(BL23)and Zusanli(ST36),the ones in scar moxibustion group with moxa cone moxibustion at the same acupoints as FLIPUS group,the ones in placebo group with cotton moxibustion at the same sites,and the ones in normal group and untreated group were not given any treatment.The mice in FLIPUS group,scar moxibustion group,and placebo group were treated once a day for consecutive 2 courses of treatment,6 days for each course.The AI scores of bilateral ankle joints were recorded after 6-day treatment and at the end of treatment,respectively.After the end of treatment,all mice but the ones in model group were sacrificed for harvesting their spleens and bilateral ankle joints,then the collected ankle cartilages and synovial tissues were stained with HE for observing the histopathological changes,and the percentages of Th17 and Treg in spleen tissues were detected by flow cytometry.Moreover,the mRNA and protein expression levels of forkhead box protein 3(FOXP3),retinoid acid receptor-rela-ted orphan receptor(ROR)γt,and nuclear factor-κB(NF-κB)in the spleen tissues of mice in each group were detected by real-time quantitative PCR(RT-qPCR)and Western Blot,respectively.Results:①The ankle AI score.The ankle AI score presented an upward trend over time in untreated group and placebo group(F=4.903,P=0.019; F=4.189,P=0.031),remained essentially unchanged over time in scar moxibustion group(F=1.683,P=0.212),and presented an downward trend over time in FLIPUS group(F=11.354,P=0.000).At the end of treatment,the ankle AI score was lower in scar moxibustion group and FLIPUS group compared to untreated group and placebo group(untreated group:P=0.000,P=0.000; placebo group:P=0.000,P=0.000),with no significant difference between untreated group and placebo group(P=1.000),and a much lower score in FLIPUS group compared to scar moxibustion group(P=0.000).②The pathological changes in ankle tissues.The changes,manifesting as thinned cartilage layer,roughened and uneven cartilage surface,and irregular distribution of chondrocyte,accompanied by disorderedly arranged synovial tissue architecture with hyperplasia and inflammatory cell infiltration,were observed in the ankle joint tissues of mice in model group; while,compared with that of model group,the ankle joint pathology slightly progressed in mice of untreated group and placebo group,whereas,which was moderately attenuated in scar moxibustion group,and marked attenuated in FLIPUS group.③The percentages of Th17 and Treg in spleen tissues.The percentage of Th17 in the spleen tissue was higher,while that of Treg was lower in untreated group and placebo group compared to model group(P=0.000,P=0.000; P=0.000,P=0.000).The percentage of Th17 in spleen tissue was lower in scar moxibustion group and FLIPUS group compared to model group(P=0.002,P=0.000),while that of Treg was lower in scar moxibustion group compared to model group,but was higher in FLIPUS group compared to model group(P=0.000,P=0.000).Furthermore,the percentage of Th17 in the spleen tissue was lower,while that of Treg was higher in FLIPUS group compared to scar moxibustion group(P=0.000,P=0.000).④The mRNA expression levels of FOXP3,RORγt,and NF-κB in the spleen tissues.The relative mRNA expression level of FOXP3 in the spleen tissues was lower,while that of RORγt and NF-κB were higher in untreated group and placebo group compared to model group(FOXP3:P=0.001,P=0.001; RORγt:P=0.001,P=0.019; NF-κB:P=0.000,P=0.000).The relative mRNA expression levels of FOXP3,RORγt,and NF-κB in the spleen tissues were not significantly different from each other between scar moxibustion group and model group(P=0.999,P=0.052,P=0.995).The relative mRNA expression level of FOXP3 in the spleen tissues was higher,while that of RORγt and NF-κB were lower in FLIPUS group compared to model group(P=0.000,P=0.000,P=0.000).Furthermore,the relative mRNA expression level of FOXP3 in the spleen tissues was higher,while that of RORγt and NF-κB were lower in FLIPUS group compared to scar moxibustion group(P=0.000,P=0.000,P=0.000).⑤The protein expression levels of FOXP3,RORγt,and NF-κB in the spleen tissues.The relative protein expression level of FOXP3 in the spleen tissues was lower,while that of RORγt and NF-κB were higher in untreated group and placebo group compared to model group(FOXP3:P=0.001,P=0.000; RORγt:P=0.010,P=0.013; NF-κB:P=0.014,P=0.001).The relative protein expression level of FOXP3 in the spleen tissues was not significantly different from each other between scar moxibustion group and model group,and between FLIPUS group and model group(P=0.573,P=0.304).The relative protein expression levels of RORγt and NF-κB in the spleen tissues were not significantly different from each other between scar moxibustion group and model group(P=0.999,P=0.999),while,were lower in FLIPUS group compared to model group(P=0.017,P=0.010).Furthermore,the relative protein expression level of FOXP3 in the spleen tissues was higher,while that of RORγt and NF-κB were lower in FLIPUS group compared to scar moxibustion group(P=0.021,P=0.016,P=0.010).Conclusion:The RA will continuously worsen without treatment.Both scar moxibustion and FLIPUS can slow the progression of RA,with FLIPUS demonstrating superior efficacy to scar moxibustion.It may work by influencing the balance of Treg/Th17 via modulating the expression of FOXP3,RORγt,and NF-κB.

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備注/Memo

備注/Memo:
基金項(xiàng)目:四川省中醫(yī)藥管理局科學(xué)技術(shù)研究專項(xiàng)課題(2023MS611)
通訊作者:王艷杰 E-mail:[email protected]
更新日期/Last Update: 1900-01-01