壯醫(yī)針挑對(duì)哮喘小鼠miRNA-126、IL-4、IFN-g及IgE的影響

唐漢慶,竇錫彬,李克明,鄭建宇,李曉華,朱曉瑩

?

壯醫(yī)針挑對(duì)哮喘小鼠miRNA-126、IL-4、IFN-g及IgE的影響

唐漢慶1,竇錫彬1,李克明1,鄭建宇2,李曉華1,朱曉瑩1

(1.右江民族醫(yī)學(xué)院,百色 533000;2.右江民族醫(yī)學(xué)院附屬醫(yī)院,百色 533000)

觀察壯醫(yī)針挑療法對(duì)哮喘模型小鼠肺組織miRNA-126和血清IL-4、IFN-g及IgE的影響,探討其起效的可能機(jī)制。將30只6～8周齡SPF級(jí)雌性BALB/c小鼠隨機(jī)分為對(duì)照組、模型組、針挑組,每組10只。模型組、針挑組采用卵清蛋白致敏及激發(fā)制備哮喘小鼠模型,針挑組在此基礎(chǔ)上采用壯醫(yī)針挑療法,針挑大椎、肺俞、定喘、風(fēng)門、腎俞、脾俞,每日1次,7 d為1個(gè)療程。療程結(jié)束后取材,RT-PCR法檢測(cè)肺組織miRNA-126表達(dá),ELISA法檢測(cè)血清IL-4、IFN-g及IgE水平。模型組miRNA-126表達(dá)水平顯著高于對(duì)照組(＜0.01)。針挑組miRNA-126表達(dá)水平顯著低于模型組(＜0.01)。與對(duì)照組比較,模型組血清IL-4和IgE水平均顯著升高(＜0.01),而IFN-g水平顯著降低(＜0.01)。與模型組比較,針挑組血清IL-4和IgE水平均顯著降低(＜0.01),而IFN-g水平顯著升高(＜0.01)。壯醫(yī)針挑療法能夠下調(diào)肺組織miRNA-126表達(dá),降低血清IL-4及IgE水平,上調(diào)血清IFN-g水平,恢復(fù)IL-4、IFN-g水平,糾正Th1/Th2失衡狀態(tài)可能是其起效環(huán)節(jié)之一。

壯醫(yī)藥學(xué);針刺;哮喘;挑治;小鼠

支氣管哮喘是Th1和Th2細(xì)胞亞群細(xì)胞因子、各種免疫活性細(xì)胞綜合作用的結(jié)果,以嗜酸性粒細(xì)胞浸潤(rùn)、氣道高反應(yīng)性(AHR)和血清IgE升高為特征[1]。研究[2]已經(jīng)證實(shí)Th1/Th2細(xì)胞亞群的失衡是哮喘發(fā)病的核心環(huán)節(jié)。IL-4是促進(jìn)Th0向Th2細(xì)胞分化的關(guān)鍵細(xì)胞因子[3],IL-4還可誘導(dǎo)B細(xì)胞產(chǎn)生IgE,在過(guò)敏性疾病炎癥反應(yīng)中起著重要作用[4],g干擾素(interferon- gamma,IFN-g)則是促進(jìn)Th0細(xì)胞向Th1方向分化,并抑制向Th2方向克隆分化的主要細(xì)胞因子[5],能夠顯著抑制IL-4誘導(dǎo)的IgE的合成過(guò)程,從而減輕哮喘的炎癥反應(yīng)[6],推測(cè)IL-4和IFN-g之間的平衡對(duì)于Th1/Th2平衡有調(diào)節(jié)意義。微型RNA(Micro RNA,miRNA)參與細(xì)胞增殖、分化等功能的調(diào)節(jié),近來(lái)在哮喘發(fā)病機(jī)制及作用受到關(guān)注[7-8],更早些的研究表明選擇性抑制其中miRNA-126可以下調(diào)Th2細(xì)胞因子表達(dá),減輕哮喘癥狀,是基因?qū)用嬲{(diào)控哮喘的一個(gè)重要分子[9]。本實(shí)驗(yàn)建立哮喘模型小鼠,觀察壯醫(yī)針挑療法對(duì)哮喘模型小鼠肺組織miRNA-126和血清IL-4、IFN-g及IgE的影響,探討壯醫(yī)針挑療法治療哮喘的作用機(jī)制。

1 材料與方法

1.1 材料

1.1.1 動(dòng)物與分組

6～8周齡SPF級(jí)雌性BALB/c小鼠30只,體質(zhì)量(24.68±8.14)g,合格證編號(hào)為SCXK(京)2013-0002。由本院科學(xué)實(shí)驗(yàn)中心購(gòu)買并提供。小鼠按數(shù)字隨機(jī)表法分3組,即對(duì)照組、模型組、針挑組,每組10只。實(shí)驗(yàn)中對(duì)動(dòng)物的處理遵循相關(guān)動(dòng)物使用倫理學(xué)原則。

1.1.2 主要試劑和儀器

烏拉坦(國(guó)藥集團(tuán)化學(xué)試劑有限公司,批號(hào)20080610),氯仿(北京化學(xué)試劑公司,批號(hào)20110322),異丙醇(北京化學(xué)試劑公司,批號(hào)20100811),卵清蛋白(OVA)(Sigma,USA),IL-4檢測(cè)試劑盒(上海森雄,批號(hào)20130021),IgE檢測(cè)試劑盒(R&B公司,USA), IFN-g檢測(cè)試劑盒(武漢博士德,批號(hào)000254),miRNA- 126引物、內(nèi)參引物(Genecopia,USA),RNA逆轉(zhuǎn)錄kit (Gibcobrl公司),酶標(biāo)儀(MK3型,Thermo Labsystem公司),凝膠成像系統(tǒng)(chemidoc XRS型), PCR儀(7900HT型,ABI公司),紫外分光光度儀(DU530型,Beckman公司),超低溫冰箱(MDF-U72V型,日本三洋公司)。

1.2 方法

1.2.1 造模與各組處理

造模方法參考Perrier等報(bào)道[10],模型組、針挑組第l、4、7、10、13天,每日1次,每次l mL(50mg/L)分別腹腔內(nèi)注射OVA混懸液(含氫氧化鋁400mg和OVA100mg)進(jìn)行基礎(chǔ)致敏,對(duì)照組以生理鹽水代替。第14天將小鼠置于自制有機(jī)玻璃盒中,給予霧化吸入OVA(0.1 mol/LPBS稀釋OVA1 mg),每次30 min,每日1次,連續(xù)l星期激發(fā)。哮喘模型制備評(píng)價(jià)指標(biāo)參考李美容等[11]報(bào)道。

對(duì)照組以生理鹽水霧化代替。針挑組第14天起,激發(fā)后1 h按照李忠仁主編的《實(shí)驗(yàn)針灸學(xué)》取穴大椎、肺俞、定喘、風(fēng)門、腎俞、脾俞。腧穴皮膚常規(guī)消毒,采用消毒大號(hào)縫衣針將上述穴位作為針挑點(diǎn),挑出皮下一些白色纖維組織,用消毒銳利陶瓷片割裂纖維(一般不出血),然后用針在該點(diǎn)垂直快速點(diǎn)刺,刺入皮下1 mm,點(diǎn)刺后以5%碘酒、75%乙醇消毒。每日1次,7 d為1個(gè)療程。模型組和對(duì)照組不作特殊處理。共進(jìn)行1個(gè)療程針挑治療。

1.2.2 取材

療程結(jié)束后第1天,按5 mL/kgip20%烏拉坦麻醉并固定。眼球取血1.0 mL,離心(3000 r/min, 10 min)后收集并取右肺濾紙吸干,均﹣70℃保存待檢。

1.2.3 RT-PCR法檢測(cè)miRNA-126表達(dá)

取100mL肺組織勻漿,用Trizol試劑提取總RNA,氯仿和異丙醇抽提。紫外分光光度儀和瓊脂糖電泳鑒定RNA的濃度和純度,取10mL根據(jù)逆轉(zhuǎn)錄試劑盒說(shuō)明逆轉(zhuǎn)錄合成cDNA,﹣70℃凍存?zhèn)溆?。?mL逆轉(zhuǎn)錄產(chǎn)物進(jìn)行PCR擴(kuò)增反應(yīng),以通用引物為內(nèi)對(duì)照miRNA-126引物(mmu-miRNA-126:F 5′-TGGAACCAGGAAGGCGGCACD-3,R5′-AACGTCCGTGGCCCCTGT-3′);has-miRNA-126:F5′-GTCCAGGAAGGCGGCACG-3,R5′-AGCGTCCGTGGCCCTTGCG-3′;內(nèi)參引物: (5′-GCCGCTTCACGAATTTGCTGGTCGGACGC-3′,has-miRNA-126:5′-CGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGG-3′,mmu-miRNA-126:5′GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGA-3′)。各樣品目的miRNA和內(nèi)參分別進(jìn)行real-time PCR反應(yīng)。

1.2.4 IL-4、IFN-g和IgE的檢測(cè)

ELISA法檢測(cè),按照試劑盒說(shuō)明書進(jìn)行操作。

1.2.5 統(tǒng)計(jì)學(xué)方法

應(yīng)用SPSS12.0統(tǒng)計(jì)軟件,計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差表示,miRNA-126表達(dá)數(shù)據(jù)采用REST軟件進(jìn)行分析。組間比較采用單因素方差分析和檢驗(yàn),＜0.05為有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 miRNA-126表達(dá)

模型組miRNA-126表達(dá)水平顯著高于對(duì)照組(＜0.01)。針挑組miRNA-126表達(dá)水平顯著低于模型組(＜0.01),趨近于對(duì)照組,與對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(＞0.05)。詳見(jiàn)表1。

表1 miRNA-126表達(dá)倍數(shù)比較 (±s)

注:與對(duì)照組比較1)＜0.01;與模型組比較2)＜0.01

2.2 血清IL-4、IFN-g和IgE水平

與對(duì)照組比較,模型組血清IL-4和IgE水平均顯著升高(＜0.01),而IFN-g水平顯著降低(＜0.01)。與模型組比較,針挑組血清IL-4和IgE水平均顯著降低(＜0.01),而IFN-g水平顯著升高(＜0.01)。與對(duì)照組比較,針挑組IL-4、IFN-g和IgE水平均有趨近趨勢(shì),差異沒(méi)有統(tǒng)計(jì)學(xué)意義(＞0.05)。詳見(jiàn)表2。

表2 IL- 4、IFN-g和IgE水平比較 (±s)

注:與對(duì)照組比較1)＜0.01;與模型組比較2)＜0.01

3 討論

哮喘是一種常見(jiàn)的慢性氣道炎癥性疾病,其臨床特點(diǎn)是反復(fù)發(fā)作性喘息、胸悶、咳嗽等,緩解和發(fā)作交替,并有組織形態(tài)學(xué)上的氣道梗阻和氣道重塑表現(xiàn)[1],哮喘的病理特征是嗜酸粒細(xì)胞、T細(xì)胞、肥大細(xì)胞、中性粒細(xì)胞等長(zhǎng)期浸潤(rùn)以及Th2細(xì)胞優(yōu)勢(shì)分化的免疫應(yīng)答,突出表現(xiàn)為Th2型細(xì)胞因子如IL-4、IL-5、IL-13與IgE的大量釋放和水平升高[12]。

哮喘的治療首選激素治療,但在臨床上我們觀察到有部分患者對(duì)激素治療無(wú)反應(yīng)性,提示針對(duì)Th2細(xì)胞優(yōu)勢(shì)分化的免疫應(yīng)答治療需要在Th2細(xì)胞優(yōu)勢(shì)分化的上游調(diào)控機(jī)制中找到靶點(diǎn)從而找到新的治療對(duì)策[13],在哮喘發(fā)病的調(diào)控機(jī)制中,miRNA的調(diào)控受到關(guān)注,miRNA的主要功能是在轉(zhuǎn)錄后水平調(diào)控與機(jī)體生長(zhǎng)、發(fā)育、疾病發(fā)生相關(guān)基因的表達(dá)水平[14],miRNA-126是調(diào)控氣道及肺組織相關(guān)基因表達(dá)的miRNA,研究證實(shí)抑制miRNA-126可以下調(diào)Th2細(xì)胞因子表達(dá),減輕哮喘癥狀[9]。IL-4是最重要的Th2型細(xì)胞因子,對(duì)IgE的合成及釋放均有關(guān)鍵作用,調(diào)控哮喘的炎癥反應(yīng)程度[6],IFN-g則是抑制向Th2方向克隆分化、減輕哮喘炎癥反應(yīng)的主要細(xì)胞因子[5],因此,IL-4和IFN-g可視為Th2和Th1最具有代表性的細(xì)胞因子,Th1/Th2平衡與IL-4和IFN-g的水平應(yīng)有密切關(guān)系。

我們?cè)谂R床上對(duì)哮喘患者采用壯醫(yī)針挑療法治療,對(duì)治療進(jìn)行效果評(píng)價(jià),主要觀察患者喘息、咳嗽、咯痰、胸悶等癥狀和肺功能的改善,具體的療效判定標(biāo)準(zhǔn)依據(jù)中華醫(yī)學(xué)會(huì)呼吸病學(xué)分會(huì)哮喘學(xué)組支氣管哮喘防治指南[15],實(shí)踐中我們注意到對(duì)其中部分對(duì)激素治療無(wú)反應(yīng)的患者采用針挑療法有一定的作用,結(jié)合上述關(guān)于miRNA的研究進(jìn)展,我們推測(cè)miRNA-126對(duì)IL-4和IFN-g有調(diào)節(jié)作用并由此調(diào)控Th2分化和免疫應(yīng)答。在本實(shí)驗(yàn)工作中,我們觀察到哮喘模型,經(jīng)過(guò)針挑治療處理后,miRNA- 126表達(dá)水平顯著降低,IL-4和IgE水平也均顯著降低,而IFN-g水平顯著升高,均接近對(duì)照組水平,提示miRNA-126有可能通過(guò)調(diào)節(jié)IL-4/IFN-g進(jìn)而調(diào)控Th2分化和免疫應(yīng)答。

壯醫(yī)針挑療法對(duì)哮喘、頭痛、失眠等一些慢性病及疑難癥有良好的療效[16]。壯醫(yī)針挑療法以壯醫(yī)理論為指導(dǎo),壯醫(yī)名家黃漢儒[17]認(rèn)為挑刺可以激發(fā)人體正氣,將郁滯體內(nèi)的有形或無(wú)形之毒從體表針挑點(diǎn)驅(qū)出,疏通“龍路”、“火路”,使“水道”、“氣道”、“谷道”暢通,天地人三氣同步。如能從分子機(jī)制上闡明壯醫(yī)針挑療法治療哮喘的機(jī)制,將會(huì)有助于這一治法的發(fā)揚(yáng)和推廣交流。

[1] Rautajoki KJ, Kylaniemi MK, Raghav SK,. An insight into mo- lecular mechanisms of human T helper cell differentiation[J]. Ann Med, 2008,40(5):322-335.

[2] Ray A, Cohn L. Altering the Th1/Th2 balance as a therapeutic strategy in asthmatic diseases[J]. Curr Opin Investig Drugs, 2000,1 (4):442-448.

[3] Takabayashi A, Ihara K, Sasaki Y,. Childhood atopic asthma: positive association with a polymorphism of IL-4 receptor alpha gene but not with that of IL-4 promoter or Fc epsilon receptor I beta gene[J]. Exp Clin Immunogenet, 2000,17(2):63-70.

[4] Al-Shami A, Spolski R, Kelly J,. A role for TSLP in the development of inflammation in an asthma model[J]. J Exp Med, 2005,202 (6):829-839.

[5] Teixeira LK, Fonseca BP, Barboza BA,. The role of interferon- gamma on immune and allergic responses[J]. Mem Inst Oswaldo Cruz, 2005,100(suppl 1):137-144.

[6] Deichmann KA, Heinzmann A, Forster J,. Linkage and allelic association of atopy and markers flanking the IL-4 receptor gene[J]. Clin Exp Allergy, 1998,28(2):151-155.

[7] Foster PS, Plank M, Collison A,. The emerging role ofmicro- RNAsin regulating immune and inflammatory responses in the lung[J]. Immunol Rev, 2013,253(1):198-215.

[8] Greene CM, Gaughan KP. microRNAs in asthma: potential therapeu- tic targets[J].Curr Opin Pulm Med, 2013,19(1):66-72.

[9] Mattes J, Collison A, Plank M,. Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease[J]. Proc Natl Acad Sci USA, 2009,106(44): 18704-18709.

[10] Perrier C, Thierry AC, Mercenier A,. Allergen-specific antibody and cytokine responses, mast cell reactivity and intestinal permeability upon oral challenge of sensitized and tolerized mice[J]. Clin Exp Allergy, 2010,40(1):153-162.

[11] 李美容,王敏.哮喘小鼠模型的建立與評(píng)價(jià)[J].臨床肺科雜志, 2011,16(5):664-665.

[12] Wills-Karp M. Immunologic basis of antigen induced airway hyperres- ponsiveness[J]. Annu Rev Immunol, 1999,17:255-281.

[13] Séguin RM, Ferrari N. Emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease[J]. Expert Opin Investig Drugs, 2009,18(10):1505-1517.

[14] Kim VN. MicroRNA biogenesis: coordinated cropping and dicing[J]. Nat Rev Mol Cell Biol, 2005,6(5):376-385.

[15] 中華醫(yī)學(xué)會(huì)呼吸病學(xué)分會(huì)哮喘學(xué)組.支氣管哮喘防治指南(支氣管哮喘的定義、診斷、治療和管理方案)[J].中華哮喘雜志(電子版), 2008,2(1):3-13.

[16] 林辰,蔣桂江,陳攀,等.壯醫(yī)針刺研究的新進(jìn)展[J].中國(guó)民族醫(yī)藥雜志,2011,17(5):65-67.

[17] 黃漢儒.壯醫(yī)理論體系概述[J].中國(guó)中醫(yī)基礎(chǔ)醫(yī)學(xué)雜志,1996,2 (6):3-7.

Effect of Zhuang Nationality Medical Picking Therapy on miRNA-126, IL-4, IFN-gand IgE in Asthma Mice

-1,-1,-1,-2,-1,-1.

1.,533000,; 2.,533000,

To investigate the effect of Zhuang nationality medical picking therapy on lung miRNA-126 and serum IL-4, IFN-gand IgE in a mouse model of asthma and explore the possible mechanism by which it produces the effect on asthma.Thirty female SPF BALB/c mice aged 6-8 weeks were randomly allocated to control, model and picking therapy groups, 10 mice each. A mouse model of asthma was made by ovalbumin sensitization and challenge in the model and picking therapy groups. In addition, the picking therapy group received needle picking out treatment at Dazhui (GV14), Feishu (BL13), Dingchuan (Ex-B1), Fengmen (BL12), Shenshu (BL23) and Pishu (BL20) once daily, seven times as a course. The materials were taken after the end of treatment course. Lung expression of miRNA-126 was examined by RT-PCR. Serum IL-4, IFN-gand IgE levels were measured by ELISA.The expression of miRNA-126 was significantly higher in the model group than in the control group (＜0.01) and significantly lower in the picking therapy group than in the model group (＜0.01). Compared with the control group, serum IL-4 and IgE levels increased significantly and IFN-glevels decreased significantly in the model group (both＜0.01). Compared with the model group, serum IL-4 and IgE levels decreased significantly and IFN-glevels increased significantly in the picking therapy group (both＜0.01).Zhuang nationality medical picking therapy can down-regulate lung expression of miRNA-126, decrease serum IL-4 and IgE levels, up-regulate serum IFN-glevels and restore IL-4 and IFN-glevels. Correction of Th1/Th2 imbalance may be one of the links via which it produces the effect.

Zhuang nationality medicine; Acupuncture; Asthma; Picking therapy; Mice

1005-0957(2014)08-0769-03

R2-03

A

10.13460/j.issn.1005-0957.2014.08.0769

2014-02-20

廣西高等學(xué)校優(yōu)秀人才資助計(jì)劃[桂教人(2011)40號(hào)]

唐漢慶(1976 - ),男,副教授,博士

竇錫彬(1978 - ),男,主治醫(yī)師,碩士