利福昔明對(duì)SIBO+腹瀉型腸易激綜合征患者NF-κB及炎性因子的影響

劉治宏，崔立紅

小腸細(xì)菌過度生長(zhǎng)(small intestinal bacterial overgrowth，SIBO)是一種腸道微生物失調(diào)的表現(xiàn)，具體以近端小腸分泌物菌落數(shù)≥105cfu/ml，小腸細(xì)菌過量或菌群失衡為特征[1-2]。腸易激綜合征(irritable bowel syndrome，IBS)是一種慢性胃腸道疾病[3]，其特征是在沒有已知器官病理性改變的情況下出現(xiàn)排便習(xí)慣紊亂和腹痛等癥狀，在西方國(guó)家發(fā)病率約為10%[4-5]。按照患者癥狀，IBS可分為便秘型(IBS-C)、腹瀉型(IBS-D)、混合型(IBS-A)和未定型(IBS-U)，其中以IBS-D最常見，約占40%[6-8]。超過25%的排便為布里斯托分類的6或7型，并且少于25%的排便為1或2型[9]可診斷為IBS-D。SIBO與IBS-D關(guān)系密切，IBS患者有更高的SIBO患病率[10]。本課題組前期已證明TLR4/NF-κB通路在IBS-D大鼠模型中的作用，其具體機(jī)制可能是通過促進(jìn)炎性因子的釋放而影響腸道菌群。本研究觀察利福昔明治療后IBS-D患者核因子κB(nuclear factor κB，NF-κB)及炎性因子的變化，以期進(jìn)一步明確IBS-D的發(fā)病機(jī)制。

1 資料與方法

1.1 一般資料 選取2016年6月－2017年12月于海軍總醫(yī)院進(jìn)行甲烷和氫氣呼氣試驗(yàn)(SIBO陽性)的IBS-D患者(滿足羅馬Ⅲ標(biāo)準(zhǔn))78例，隨機(jī)分成試驗(yàn)組與對(duì)照組(n=39)。試驗(yàn)組男18例，女21例，年齡37.7±9.8歲；對(duì)照組男16例，女23例(后脫失2例)，年齡38.0±9.5歲。兩組男女比例、年齡等差異均無統(tǒng)計(jì)學(xué)意義(P＞0.05)。排除標(biāo)準(zhǔn)：①肝臟病變、急性感染、其他慢性炎性疾病及妊娠患者；②在試驗(yàn)開始前1個(gè)月內(nèi)使用抗生素和益生菌的患者；③糖尿病患者[11]；④長(zhǎng)期使用質(zhì)子泵抑制劑、慢性萎縮性胃炎的患者[12]；⑤肥胖患者[13]。本研究經(jīng)海軍總醫(yī)院倫理委員會(huì)審核通過，所有患者均簽署知情同意書。

1.2 甲烷和氫氣呼氣試驗(yàn) 本試驗(yàn)采用甲烷和氫氣呼氣試驗(yàn)[14]檢測(cè)SIBO。受檢者口服底物前漱口，而后向采氣袋內(nèi)吹入氣體，30min后服用乳果糖10ml。每隔30min重復(fù)上述步驟采氣，直至收集完8個(gè)采氣袋。檢測(cè)由甲烷和氫氣檢測(cè)工作站(美國(guó)Quintron公司)完成。檢查注意事項(xiàng)：空腹8h以上接受檢查，整個(gè)過程禁食，可飲水；晨起后禁止吸煙；檢測(cè)過程中不做劇烈運(yùn)動(dòng)。

1.3 SIBO診斷標(biāo)準(zhǔn) ①氫氣濃度＞基礎(chǔ)值20ppm；②甲烷濃度升高＞基礎(chǔ)值12ppm；③甲烷和氫氣濃度之和升高＞基礎(chǔ)值15ppm；④整個(gè)檢查過程中出現(xiàn)雙峰曲線。

1.4 分組及治療 所有參與試驗(yàn)患者均被要求試驗(yàn)前1個(gè)月及實(shí)驗(yàn)過程中不服用任何已知的可以改變胃腸功能、緩解疼痛的藥物以及精神類藥品。試驗(yàn)開始前完善癥狀評(píng)分并檢測(cè)白細(xì)胞介素-8(interleukin-8，IL-8)、腫瘤壞死因子α(tumour necrosis factor-α，TNF-α)、NF-κB等指標(biāo)。試驗(yàn)組給予利福昔明治療(0.2g/次，4次/d，共2周，意大利ALFA WASSERMANN制藥公司)；對(duì)照組給予安慰劑治療2周。再次進(jìn)行癥狀評(píng)分并檢測(cè)上述指標(biāo)。治療前對(duì)患者進(jìn)行飲食、生活方式等的指導(dǎo)。試驗(yàn)流程如圖1所示。

1.5 IBS-D癥狀評(píng)分 治療前及治療后1周，分別記錄兩組如表1[15]所示的癥狀及頻率，并計(jì)算每日評(píng)分，最后計(jì)算治療前及治療后1周的平均評(píng)分。大便性狀評(píng)分參考Bristol分型[16]。

圖1 試驗(yàn)流程圖Fig.1 Experimental flow graph

表1 IBS-D癥狀評(píng)分表[15]Tab.1 IBS-D symptom score table[15]

1.6 血清IL-8、TNF-α、NF-κB等指標(biāo)檢測(cè) 采集清晨空腹12h靜脈血3ml，離心后存放于–80℃冰箱備用。檢測(cè)試劑盒由北京億鳴生物有限公司提供。

1.7 統(tǒng)計(jì)學(xué)處理 采用SPSS 22.0軟件進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料以±s表示，組間及治療前后比較采用t檢驗(yàn)。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 兩組IBS-D患者治療前后癥狀評(píng)分比較 對(duì)照組給予安慰劑，并進(jìn)行健康飲食生活宣教，治療前后癥狀評(píng)分比較差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。試驗(yàn)組治療前后評(píng)分比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。試驗(yàn)組IBS各癥狀改善在治療后優(yōu)于對(duì)照組(P＜0.05，表2)。

2.2 兩組IBS-D患者治療前后IL-8、TNF-α、NF-κB比較 對(duì)照組治療前后各指標(biāo)差異均無統(tǒng)計(jì)學(xué)意義(P＞0.05)。試驗(yàn)組治療后IL-8、TNF-α、NF-κB較治療前明顯下降，差異均有統(tǒng)計(jì)學(xué)意義(表3)。

3 討 論

IBS-D是一種常見的功能性腸病，其具體發(fā)病機(jī)制尚不清楚，目前臨床上主要采用對(duì)癥治療。有文獻(xiàn)報(bào)道在IBS的大鼠模型中，TLR4通過NF-κB信號(hào)通路對(duì)內(nèi)臟敏感性造成影響[17]。本課題組前期通過IBS-D大鼠模型發(fā)現(xiàn)TLR4/NF-κB信號(hào)通路在IBS-D的發(fā)病中發(fā)揮作用。

表2 兩組IBS-D患者治療前后癥狀評(píng)分比較(±s)Tab. 2 Comparison of symptom scores before and after treatment between two groups (±s)

表2 兩組IBS-D患者治療前后癥狀評(píng)分比較(±s)Tab. 2 Comparison of symptom scores before and after treatment between two groups (±s)

(1)P＜0.05 compared with pre-treatment; (2)P＜0.05 compared with control group

Item Control group (n=37) Experimental group (n=39)Pre-treatment Post-treatment Pre-treatment Post-treatment Abdominal pain time (h/d) 1.65±0.63 1.57±0.60 1.67±0.66 0.69±0.52(1)(2)Abdominal distention time (h/d) 1.95±0.74 1.84±0.73 1.97±0.78 1.41±0.75(1)(2)Stool character 1.92±0.76 1.81±0.74 1.95±0.76 1.36±0.63(1)(2)Defecation frequency (times/d) 1.65±0.63 1.57±0.55 1.54±0.60 0.97±0.67(1)(2)

表3 兩組IBS-D患者治療前后NF-κB、炎性因子比較(±s)Tab. 3 Comparison of NF-κB and inflammatory factors before and after treatment between two groups (±s)

表3 兩組IBS-D患者治療前后NF-κB、炎性因子比較(±s)Tab. 3 Comparison of NF-κB and inflammatory factors before and after treatment between two groups (±s)

(1)P＜0.05 compared with pre-treatment; (2)P＜0.05 compared with control group

Group TNF-α(pg/ml) NF-κB(ng/ml) IL-8(pg/ml)Pre-treatment Post-treatment Pre-treatment Post-treatment Pre-treatment Post-treatment Control group 10.33±3.03 10.18±3.08 1.17±0.23 1.12±0.24 18.49±8.08 17.55±7.89 Experimental group 10.13±2.93 8.22±2.20(1)(2) 1.12±0.19 0.98±0.15(1)(2) 17.25±7.04 11.12±4.61(1)(2)

SIBO與IBS-D關(guān)系密切。研究顯示，SIBO可導(dǎo)致營(yíng)養(yǎng)吸收不良和腸道炎癥[18]，腸道的低度炎癥也是IBS的發(fā)病機(jī)制之一[19-20]。Ahn等[21]發(fā)現(xiàn)，IBS-D患者腸黏膜免疫細(xì)胞計(jì)數(shù)較高。Peralta等[22]發(fā)現(xiàn)，根除SIBO后有75%的IBS患者癥狀緩解。Mizutani等[23]指出，輕微的炎癥也可以導(dǎo)致胃腸道神經(jīng)、平滑肌功能的持續(xù)變化。Rhee等[24]的研究顯示，腸道菌群可調(diào)節(jié)腸道運(yùn)動(dòng)、分泌，反之，腸道運(yùn)動(dòng)又可能影響菌群。SIBO不僅僅是腸道蠕動(dòng)改變的結(jié)果，也會(huì)導(dǎo)致腸道蠕動(dòng)發(fā)生改變[25]。腸道炎癥和SIBO激活的免疫應(yīng)答均可引起細(xì)胞因子失衡[23]。David等[26]通過檢測(cè)鈣衛(wèi)蛋白明確腸道炎癥情況，發(fā)現(xiàn)SIBO陽性的IBS患者比陰性患者有更高的腸道炎癥。IBS/SIBO與腸道炎癥的關(guān)系如圖2所示。

圖2 IBS/SIBO與腸道炎癥的關(guān)系圖Fig.2 The relationship between IBS/SIBO and intestinal inflammation

利福昔明是一種不易吸收的口服抗生素，具有良好的安全性，對(duì)于旅行者腹瀉、功能性腹脹、腸易激綜合征和SIBO等都有較好的療效[27]。利福昔明降低IL-8、TNF-α、NF-κB的機(jī)制可能是直接的殺菌、抑菌作用，減弱局部炎癥[28]。Hoffman等[29]認(rèn)為利福昔明能夠降低SIBO患者的腸道通透性，抑制細(xì)菌穿透黏膜，進(jìn)而穩(wěn)定腸道菌群。Giuseppe等[30]發(fā)現(xiàn)在Caco-2細(xì)胞中，利福昔明可通過孕烷X受體(pregnane X receptor，PXR)抑制TLR4/MyD88/NF-κB，進(jìn)而抑制炎癥。另外也有文獻(xiàn)報(bào)道，在結(jié)腸炎模型中利福昔明可作用于人PXR，進(jìn)而抑制NF-κB的表達(dá)[31]。Mencarelli等[32]認(rèn)為在腸上皮細(xì)胞中利福昔明無法阻止TNF-α抑制PXR表達(dá)的過程，也不能使腸道活檢的炎性組織PXR表達(dá)上調(diào)，所以利福昔明在炎癥反應(yīng)中是增強(qiáng)PXR的活性而不是增加其表達(dá)。

本研究應(yīng)用利福昔明治療SIBO+的IBS-D患者，發(fā)現(xiàn)根除SIBO后，患者NF-κB、IL-8、TNF-α等均下降(IL-8、TNF-α均為促炎性細(xì)胞因子，在炎癥中起關(guān)鍵作用[33])，部分IBS-D患者癥狀明顯改善，從而提示SIBO可能導(dǎo)致NF-κB升高，進(jìn)而加重IBS-D癥狀。但是本試驗(yàn)也存在一些不足之處，如未進(jìn)一步檢測(cè)黏膜的炎性因子，未設(shè)置SIBO-對(duì)照組等，下一步須進(jìn)一步完善實(shí)驗(yàn)。

總之，本研究結(jié)果表明，SIBO陽性的IBS-D患者經(jīng)利福昔明治療后，血清IL-8、TNF-α和NF-κB水平以及IBS癥狀評(píng)分顯著下降，提示SIBO可能通過NF-κB影響炎性因子及IBS-D患者的癥狀，進(jìn)一步闡明了SIBO與IBS-D的關(guān)系及IBS-D發(fā)病機(jī)制。另外利福昔明作為一種新型抗生素，對(duì)IBS-D合并SIBO陽性患者有較好療效。

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