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HO-1/CO信號(hào)通路對(duì)切口痛大鼠炎癥因子的調(diào)控作用

2016-10-20 03:19王云濤單世民劉曉智
天津醫(yī)藥 2016年9期
關(guān)鍵詞:血紅素脊髓切口

王云濤,單世民△,劉曉智

實(shí)驗(yàn)研究

HO-1/CO信號(hào)通路對(duì)切口痛大鼠炎癥因子的調(diào)控作用

王云濤1,單世民1△,劉曉智2

目的觀察脊髓血紅素氧合酶-1(HO-1)/CO信號(hào)通路對(duì)切口痛大鼠炎癥因子的調(diào)節(jié)作用及可能機(jī)制。方法切口痛模型大鼠36只在實(shí)驗(yàn)即刻(0 h),術(shù)后1、4、8、12及24 h各處死6只,取患側(cè)脊髓腰膨大處,Western blot法檢測HO-1蛋白表達(dá),酶聯(lián)免疫吸附試驗(yàn)(ELISA)法檢測炎癥因子腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素(IL)-1β、IL-6和高遷移率族蛋白1(HMGB1)。另外,對(duì)照(C)組36只大鼠不做切口痛模型;切口痛模型大鼠144只按處理方式不同分為切口痛(IP)組,切口痛+HO-1誘導(dǎo)劑(IP+hemin)組,術(shù)前給予腹腔注射大鼠100 mg/kg血紅素(hemin);切口痛+HO-1抑制劑(IP+Znpp-IX)組,術(shù)前給予腹腔注射大鼠45 μmoL/kg鋅原卟啉(Znpp)-IX;切口痛+ CO釋放劑(IP+CORM-2)組,于術(shù)前經(jīng)腹腔注射給予10 mg/kg一氧化碳釋放分子(CORM)-2。各組于實(shí)驗(yàn)即刻(0 h),術(shù)后1、4、8、12及24 h行機(jī)械縮足閾值(PWMT)和熱縮足潛伏期(PWTL)的檢測,應(yīng)用ELISA法檢測各組TNF-α、IL-1β、IL-6和HMGB1的表達(dá)情況。結(jié)果與0 h比較,術(shù)后1、4、8、12及24 h HO-1蛋白表達(dá)水平和TNF-α、IL-1β、IL-6和HMGB1均增高(P<0.05)。與C組比較,其余組大鼠各時(shí)間點(diǎn)PWMT值和PWTL值減少,TNF-α、IL-1β、IL-6和HMGB1表達(dá)增加(P<0.05);與IP組比較,IP+hemin組和IP+CORM-2組大鼠1、4、8、12及24 h時(shí)PWMT值和PWTL值均增高,而TNF-α、IL-1β、IL-6和HMGB1表達(dá)減少,IP+Znpp-IX組PWMT值和PWTL值降低,但TNF-α、IL-1β、IL-6和HMGB1表達(dá)增加(P<0.05)。結(jié)論切口痛可誘發(fā)大鼠HO-1表達(dá)增加,而HO-1/CO信號(hào)通路在調(diào)控切口痛大鼠炎癥因子的釋放方面發(fā)揮重要作用。

疼痛,手術(shù)后;炎癥;疾病模型,動(dòng)物;腫瘤壞死因子α;白細(xì)胞介素1β;高遷移率族蛋白1;血紅素氧合酶-1;HO-1/CO信號(hào)通路

術(shù)后疼痛是手術(shù)后即刻發(fā)生的最常見的急性傷害性疼痛,直接影響患者術(shù)后的康復(fù),但持續(xù)時(shí)間一般少于7 d,目前尚無有效治療措施[1]。手術(shù)可對(duì)患者神經(jīng)末梢產(chǎn)生機(jī)械性損傷,引起傷害性感受及周圍和中樞神經(jīng)系統(tǒng)敏感性改變,而受損的神經(jīng)組織釋放的炎性致痛物質(zhì),可引起炎癥反應(yīng),炎癥反應(yīng)釋放的細(xì)胞因子是聯(lián)系神經(jīng)-免疫反應(yīng)的關(guān)鍵介質(zhì),可造成周圍神經(jīng)活化和敏感化,進(jìn)而加重疼痛[2]。研究表明,炎性痛引起的脊髓血紅素氧合酶-1(HO-1)的增多可能在痛的感覺和痛覺過敏產(chǎn)生中發(fā)揮了重要作用[3]。HO-1是催化血紅素分解成一氧化碳(CO)、Fe2+和膽紅素分解的限速酶,HO-1能發(fā)揮有力的鎮(zhèn)痛作用,且HO-1或CO的上調(diào)對(duì)神經(jīng)病理性疼痛炎癥因子的釋放發(fā)揮重要的調(diào)控作用[4]。因此,本實(shí)驗(yàn)通過大鼠切口痛模型復(fù)制人體術(shù)后急性疼痛,觀察HO-1和相關(guān)炎癥因子在切口痛大鼠中的表達(dá)變化,探討HO-1/CO信號(hào)通路對(duì)切口痛炎癥因子的可能作用機(jī)制。

1 資料與方法

1.1一般資料(1)實(shí)驗(yàn)動(dòng)物。清潔級(jí)雄性SD大鼠216只,體質(zhì)量180~250 g,8~10周齡,購自軍事醫(yī)學(xué)科學(xué)院。大鼠入室后分籠飼養(yǎng),自由飲食水,適應(yīng)環(huán)境7 d。(2)主要儀器與試劑。血紅素(hemin)、鋅原卟啉(Znpp)-IX、一氧化碳釋放分子(CORM)-2、HO-1多克隆抗體和β-actin抗體購自美國Sigma公司,辣根過氧化物酶標(biāo)記的二抗購自北京中杉金橋生物技術(shù)有限公司,腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素(IL)-1β、IL-6和高遷移率族蛋白1(HMGB1)試劑盒均購自美國R&D公司,Von Frey購自美國Stoelting公司,熱輻射刺激儀購自意大利Ugo Basile公司。

1.2大鼠切口痛模型制作及分組參考文獻(xiàn)[5]的方法,取180只大鼠做切口痛模型。腹腔注射水合氯醛麻醉大鼠后,將大鼠固定在手術(shù)臺(tái)上,用10%的碘伏消毒左后肢皮膚,鋪無菌洞巾,用手術(shù)刀片從足底近端0.5 cm處縱行做一長約1 cm的切口,切開皮膚和筋膜,用眼科鑷挑起足底肌肉并縱向切割,保持肌肉的起止和附著完整,用紗布按壓止血后,用5-0尼龍絲線褥式縫合皮膚。切口覆蓋紅霉素軟膏抗感染。術(shù)后手術(shù)側(cè)后足無運(yùn)動(dòng)障礙,且術(shù)后表現(xiàn)出手術(shù)側(cè)后足不愿著地及舔咬等現(xiàn)象,表明模型復(fù)制成功。造模成功大鼠180只,不做模型的36只大鼠為對(duì)照(C)組。

1.3切口痛大鼠HO-1蛋白和炎癥因子TNF-α、IL-1β、IL-6及HMGB1隨時(shí)間變化情況

1.3.1分組取切口痛模型大鼠36只,在實(shí)驗(yàn)即刻(0 h),術(shù)后1、4、8、12及24 h各處死6只,取患側(cè)脊髓腰膨大處,用于檢測HO-1蛋白和炎癥因子TNF-α、IL-1β、IL-6和HMGB1表達(dá)水平。

1.3.2Western blot法檢測HO-1蛋白表達(dá)取患側(cè)脊髓腰膨大處,用細(xì)胞裂解液提取蛋白,BCA法測定蛋白濃度。取50 μg蛋白樣品,經(jīng)10%十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分離后,轉(zhuǎn)移到NC膜上。室溫封閉2 h;TBST漂洗3次×5 min,加入兔抗鼠HO-1多克隆抗體(稀釋度為1∶1 000)和內(nèi)參β-actin抗體(稀釋度1∶5 000),4℃過夜。TBST漂洗3次×5 min,辣根過氧化物酶標(biāo)記的二抗(稀釋度1∶2 000),室溫下?lián)u蕩孵育1 h。TBST漂洗3次×5 min,ECL發(fā)光試劑盒暗室發(fā)光、顯影、定影。Bio-Rad凝膠成像系統(tǒng)采集圖像,用Bio Imaging system(Gene Genius)對(duì)圖像進(jìn)行灰度掃描分析。以目的蛋白條帶灰度值與β-actin條帶灰度值的比值反映目的蛋白的表達(dá)水平。

1.3.3酶聯(lián)免疫吸附試驗(yàn)(ELISA)法檢測各炎癥因子的表達(dá)收集脊髓腰膨大處,將組織勻漿后,參照說明書檢測TNF-α、IL-1β、IL-6和HMGB1的濃度。

1.4切口痛大鼠和正常大鼠疼痛行為學(xué)變化

1.4.1分組C組36只不做切口痛模型;切口痛模型大鼠144只按術(shù)前處理方式的不同分為切口痛(IP)組;切口痛+ HO-1誘導(dǎo)劑(IP+hemin)組,術(shù)前給予腹腔注射100 mg/kg hemin;切口痛+HO-1抑制劑(IP+Znpp-IX)組,于術(shù)前給予腹腔注射45 μmoL/kg Znpp-IX;切口痛+CO釋放劑(IP+CORM-2)組,于術(shù)前給予腹腔注射10 mg/kg的CORM-2。各組于實(shí)驗(yàn)即刻(0 h),術(shù)后1、4、8、12及24 h各取6只大鼠行機(jī)械痛敏和熱痛敏的檢測,每個(gè)時(shí)間點(diǎn)檢測行為學(xué)后處死大鼠,并取患側(cè)脊髓腰膨大處,應(yīng)用ELISA法檢測炎癥因子TNF-α、IL-1β、IL-6和HMGB1,檢測方法同1.3.3。

1.4.2各組大鼠疼痛行為學(xué)檢測(1)機(jī)械縮足閾值(PWMT)。將大鼠置于有機(jī)玻璃箱內(nèi),玻璃箱底部是鐵絲網(wǎng)格,大鼠適應(yīng)環(huán)境30 min,以不同力度的Von Frey纖毛刺激大鼠足底,以纖毛稍稍彎曲作為完全受力標(biāo)準(zhǔn),大鼠無縮足反應(yīng)時(shí)繼續(xù)加大力度,直到出現(xiàn)縮足反應(yīng),大鼠縮足的同時(shí)自動(dòng)記錄到1個(gè)數(shù)值,每次至少間隔20 s,PWMT值的最大值設(shè)定為50 g,大于此力度的大鼠剔除本實(shí)驗(yàn),取3次的平均值作為大鼠的PWMT。(2)熱縮足潛伏期(PWTL)。將大鼠放入有機(jī)玻璃箱內(nèi),適應(yīng)環(huán)境30 min,用熱輻射刺激儀照射小鼠足底中部,記錄從照射開始至小鼠出現(xiàn)抬腿回避終止的時(shí)間,共計(jì)5次,每次5 min,熱刺激強(qiáng)度在整個(gè)實(shí)驗(yàn)過程中維持一致,自動(dòng)切斷時(shí)間為30 s,以防止皮膚被燙傷,若超過此上限的大鼠剔除本實(shí)驗(yàn),取后3次平均值計(jì)為PWTL值。

1.5統(tǒng)計(jì)學(xué)方法采用SPSS 13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行分析。符合正態(tài)分布的計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示,多組均數(shù)間比較采用單因素方差分析(ANOVA),組間多重比較采用LSD-t檢驗(yàn),P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

Tab.1Changes of TNF-α,IL-1β,IL-6 and HMGB1 in different time points in the rat model of incisional pain表1 切口痛模型大鼠TNF-α、IL-1β、IL-6、HMGB1隨時(shí)間變化情況(n=6,)

Tab.1Changes of TNF-α,IL-1β,IL-6 and HMGB1 in different time points in the rat model of incisional pain表1 切口痛模型大鼠TNF-α、IL-1β、IL-6、HMGB1隨時(shí)間變化情況(n=6,)

**P<0.01;a與0 h比較,P<0.05

炎癥因子TNF-α(ng/g)IL-1β(ng/g)IL-6(ng/g)HMGB1(μg/g)0 h 12.10±3.64 6.85±1.09 5.30±0.85 1.20±0.26 1 h 50.62±10.98a 33.61±7.22a 23.56±3.31a 3.40±0.32a 4 h 116.55±19.80a 70.70±10.02a 55.71±8.15a 5.13±0.37a 8 h 71.67±10.73a 49.71±7.91a 49.05±5.53a 5.83±0.54a 12 h 46.03±8.83a 40.80±12.49a 35.15±6.56a 8.43±0.58a 24 h 28.13±5.73a 28.02±5.37a 22.55±5.02a 7.15±0.82a F 62.100**41.350**70.640**152.300**

Tab.2The behavioral changes of PWMT and PWTL in different time points of five groups表2 各組不同時(shí)間點(diǎn)疼痛行為學(xué)PWMT和PWTL的變化(n=6,)

Tab.2The behavioral changes of PWMT and PWTL in different time points of five groups表2 各組不同時(shí)間點(diǎn)疼痛行為學(xué)PWMT和PWTL的變化(n=6,)

**P<0.01;a與C組比較,b與IP組比較,P<0.05;IP+hemin組、IP+Znpp-IX組和IP+CORM-2組之間不做比較;表3同

組別C組IP組IP+hemin組IP+Znpp-IX組IP+CORM-2組F 0 h 41.70±7.70 42.02±8.37 41.55±8.66 41.58±7.33 41.75±7.71 0.003 1 h 41.73±7.02 23.97±4.98a 36.23±6.20ab 13.02±3.02ab 34.80±6.61ab 23.780**PWMT(g)4 h 41.52±7.45 22.15±4.58a 32.22±5.79ab 12.23±2.84ab 32.69±6.24ab 24.140**8 h 42.08±7.27 20.90±4.57a 29.85±4.28ab 11.35±3.14ab 29.90±4.13ab 33.150**12 h 42.08±7.54 19.20±3.91a 28.92±5.06ab 9.88±2.98ab 28.47±5.44ab 31.870**24 h 42.33±7.61 20.70±4.16a 31.93±5.30ab 11.23±3.26ab 32.65±5.95ab 28.960**組別C組IP組IP+hemin組IP+Znpp-IX組IP+CORM-2組F 0 h 14.55±0.75 14.83±0.93 14.97±0.89 14.92±0.92 14.88±1.07 0.193 1 h 15.3±0.81 6.55±0.73a 12.17±1.23ab 3.10±0.28ab 12.22±1.04ab 190.600**PWTL(s)4 h 14.85±0.74 6.13±0.72a 11.80±1.14ab 2.93±0.24ab 11.90±1.20ab 182.900**8 h 15.37±1.30 5.75±0.80a 11.40±1.15ab 2.77±0.31ab 11.33±1.30ab 139.300**12 h 14.77±1.21 5.40±0.80a 10.72±0.99ab 2.55±0.29ab 10.85±1.01ab 168.600**24 h 15.08±1.07 5.80±0.72a 11.42±0.86ab 2.80±0.34ab 11.47±0.87ab 212.400**

2.1切口痛大鼠HO-1蛋白的表達(dá)變化與0 h(0.050±0.004)比較,術(shù)后1、4、8、12及24 h HO-1蛋白表達(dá)水平均增高,分別為0.245±0.016、0.467± 0.029、0.562±0.036、0.340±0.022及0.285±0.019(F= 59.16,P<0.05)。

2.2切口痛大鼠TNF-α、IL-1β、IL-6及HMGB1隨時(shí)間變化情況與0 h比較,術(shù)后1、4、8、12及24 h大鼠TNF-α、IL-1β、IL-6和HMGB1表達(dá)增加(P<0.05);在4 h時(shí)TNF-α、IL-1β及IL-6表達(dá)達(dá)到峰值,在12 h時(shí)HMGB1表達(dá)達(dá)到峰值,見表1。

2.3各組疼痛行為學(xué)檢測結(jié)果比較與C組比較,其余組1、4、8、12及24 h時(shí)PWMT值和PWTL值均降低(P<0.05);與IP組比較,IP+hemin組和IP+ CORM-2組大鼠1、4、8、12及24 h時(shí)PWMT值和PWTL值均增高,而IP+Znpp-IX組PWMT值和PWTL值降低(P<0.05),見表2。

2.4HO-1/CO對(duì)切口痛大鼠炎癥因子的影響與C組相比,其余組大鼠1、4、8、12及24 h時(shí)TNF-α、IL-1β、IL-6和HMGB1表達(dá)增加(P<0.05);與IP組相比,IP+hemin組和IP+CORM-2組大鼠1、4、8、12及24 h時(shí)炎癥因子TNF-α、IL-1β、IL-6和HMGB1表達(dá)減少,而IP+Znpp-IX組TNF-α、IL-1β、IL-6和HMGB1表達(dá)增加(P<0.05),見表3。

3 討論

研究認(rèn)為,HO-1具有抗炎、抗凋亡、抗增生等效應(yīng),并且在動(dòng)脈粥樣硬化、腦缺血、器官移植排斥反應(yīng)中具有細(xì)胞保護(hù)作用,當(dāng)出現(xiàn)HO-1表達(dá)降低,細(xì)胞更易受各種外界有害刺激的破壞[6]。HO-1是血紅素分解的起始酶和限速酶,將血紅素分解為膽紅素、CO和Fe2+,分解產(chǎn)物具有抗炎和抗氧化等作用[7]。內(nèi)源性CO作為一種新型的神經(jīng)遞質(zhì)或者神經(jīng)調(diào)質(zhì),在疼痛疾病的發(fā)生發(fā)展中發(fā)揮著重要的作用[8]。研究顯示,福爾馬林誘發(fā)的炎性痛小鼠HO-1表達(dá)上調(diào),并發(fā)揮了重要的抗炎和鎮(zhèn)痛作用[9]。Castany等[10]研究表明,HO-1的增加對(duì)糖尿病神經(jīng)病理性疼痛大鼠具有治療作用,可改善疼痛行為學(xué)的變化,通過調(diào)控δ阿片受體調(diào)控鎮(zhèn)痛作用。本實(shí)驗(yàn)結(jié)果顯示,與0 h比較,切口痛大鼠術(shù)后1、4、8、12及24 h脊髓組織中HO-1蛋白表達(dá)水平均增高,可能是通過調(diào)節(jié)切口痛大鼠炎癥反應(yīng)或者氧化損傷實(shí)現(xiàn)的,從而發(fā)揮對(duì)切口痛大鼠的保護(hù)作用。

Tab.3The changes of TNF-α,IL-1β,IL-6 and HMGB1 in different time points of five groups表3 各組不同時(shí)間點(diǎn)TNF-α、IL-1β、IL-6和HMGB1表達(dá)的變化(n=6,)

Tab.3The changes of TNF-α,IL-1β,IL-6 and HMGB1 in different time points of five groups表3 各組不同時(shí)間點(diǎn)TNF-α、IL-1β、IL-6和HMGB1表達(dá)的變化(n=6,)

組別C組IP組IP+hemin組IP+Znpp-IX組IP+CORM-2組F 0 h 9.7±2.8 10.4±2.5 9.5±2.8 9.3±2.4 9.6±2.8 0.148 1 h 9.5±2.7 64.8±8.4a 35.6±5.9ab 85.5±10.3ab 35.1±6.0ab 102.200**TNF-α(ng/g)4 h 9.5±2.8 110.7±17.9a 69.8±7.4ab 139.2±7.8ab 69.9±7.7ab 143.400**8 h 9.6±2.8 85.5±7.4a 41.1±6.2ab 104.8±13.7ab 47.3±9.0ab 115.400**12 h 9.6±2.9 70.9±12.2a 37.1±5.5ab 92.6±10.4ab 37.8±5.7ab 95.950**24 h 9.5±2.9 60.9±9.3a 33.0±5.1ab 77.4±13.7ab 34.4±6.5ab 59.850**IL-1β(ng/g)組別C組IP組IP+hemin組IP+Znpp-IX組IP+CORM-2組F 0 h 7.3±0.9 7.6±0.8 7.4±0.7 7.2±0.7 7.1±0.7 0.380 1 h 7.5±0.7 56.7±6.1a 23.8±3.5ab 76.7±8.2ab 24.5±2.5ab 192.500**4 h 7.5±1.0 87.8±9.0a 58.7±6.0ab 107.8±8.6ab 52.8±3.9ab 210.900**8 h 7.6±0.9 68.3±5.5a 42.4±6.5ab 87.4±6.3ab 44.1±5.2ab 194.100**12 h 7.5±1.1 54.4±5.6a 36.5±3.6ab 73.4±8.0ab 36.9±4.2ab 140.700**24 h 7.4±0.7 46.0±5.6a 32.3±3.7ab 66.8±7.5ab 31.4±3.7ab 123.300**組別C組IP組IP+hemin組IP+Znpp-IX組IP+CORM-2組F IL-6(ng/g)0 h 5.7±0.7 5.7±0.7 5.8±0.4 5.7±0.7 5.9±0.6 0.121 1 h 5.0±0.9 25.5±6.2a 14.4±2.5ab 39.2±4.1ab 13.8±1.9ab 78.820**4 h 5.8±0.3 51.7±8.4a 37.5±4.6ab 78.2±5.9ab 37.3±4.3ab 142.500**8 h 5.8±0.4 47.9±5.4a 32.9±3.6ab 71.4±4.1ab 32.2±3.8ab 235.600**12 h 5.5±0.6 39.2±4.4a 26.8±3.3ab 55.3±5.5ab 29.1±3.3ab 138.900**24 h 5.9±0.5 27.6±3.9a 19.3±2.3ab 42.9±4.5ab 18.3±3.2ab 109.100組別C組IP組IP+hemin組IP+Znpp-IX組IP+CORM-2組F HMGB1(μg/g)0 h 1.1±0.2 0.8±0.2 1.0±0.2 1.1±0.2 1.1±0.2 2.550**1 h 0.9±0.2 2.1±0.3a 1.9±0.2a 2.7±0.3a 1.9±0.2a 42.000**4 h 1.0±0.2 6.5±0.5a 4.0±0.4ab 8.3±0.6ab 4.2±0.4ab 236.400**8 h 1.0±0.1 8.3±0.5a 5.8±0.4ab 10.7±2.2ab 5.5±0.6ab 69.840**12 h 1.0±0.2 10.4±0.6a 6.1±0.7ab 12.1±1.3ab 6.0±0.7ab 184.000**24 h 0.9±0.2 7.7±0.8a 4.9±0.6ab 10.2±1.6ab 5.0±0.4ab 96.570**

外周組織損傷或炎癥產(chǎn)生對(duì)傷害性刺激產(chǎn)生過強(qiáng)的反應(yīng)可導(dǎo)致術(shù)后痛覺過敏。手術(shù)切口可誘發(fā)小膠質(zhì)細(xì)胞等激活Toll樣受體(TLR)4的表達(dá),促進(jìn)其下游細(xì)胞因子IL-1β、TNF-α、IL-6等分泌和合成[11-12];TLR能識(shí)別凋亡細(xì)胞釋放或者應(yīng)激細(xì)胞分泌內(nèi)源性分子,如內(nèi)源性晚期細(xì)胞炎癥因子HMGB1等分子,激發(fā)炎癥反應(yīng)信號(hào)。有研究顯示,神經(jīng)病理性疼痛刺激脊髓和背根神經(jīng)節(jié),誘發(fā)神經(jīng)痛大鼠的炎癥反應(yīng)發(fā)生,釋放了TNF-α和HMGB1等大量的炎癥介質(zhì)[8]。本研究結(jié)果顯示,與0 h時(shí)間點(diǎn)比較,術(shù)后1、4、8、12及24 h大鼠TNF-α、IL-1β、IL-6和HMGB1表達(dá)增加,表明切口痛術(shù)后脊髓組織可能釋放了過量的炎癥因子,提示疼痛對(duì)中樞系統(tǒng)的有害刺激,可能會(huì)激活膠質(zhì)細(xì)胞和巨噬細(xì)胞等免疫細(xì)胞,從而激活炎癥反應(yīng)發(fā)生,釋放炎癥介質(zhì),后者又可進(jìn)一步刺激免疫系統(tǒng)激活免疫細(xì)胞,進(jìn)一步釋放炎癥介質(zhì),形成惡性循環(huán)。因此,從減少炎癥介質(zhì)的釋放這一切入點(diǎn)出發(fā),探討炎癥反應(yīng)與疼痛發(fā)生、發(fā)展及疼痛誘發(fā)的炎癥反應(yīng)的關(guān)系,至關(guān)重要。研究表明,HO-1催化產(chǎn)生的CO具有抑制炎癥誘發(fā)的痛覺過敏作用[13]。本研究結(jié)果顯示,與IP組比較,IP+hemin組和IP+CORM-2組大鼠1、4、8、12及24 h時(shí)PWMT值和PWTL值均增高,而TNF-α、IL-1β、IL-6和HMGB1表達(dá)減少;IP+Znpp-IX組PWMT值和PWTL值降低,但TNF-α、IL-1β、IL-6和HMGB1表達(dá)增加,表明與切口痛大鼠相比,給予HO-1的抑制劑(Znpp-IX)后大鼠機(jī)械痛敏和熱痛敏值降低、炎癥介質(zhì)的釋放增加,而給予誘發(fā)劑(Hemin)和CO的釋放劑(CORM-2)處理后,大鼠的機(jī)械痛敏和熱痛敏值增加,而炎癥因子的表達(dá)明顯減少。研究報(bào)道,IL-1β、TNF-α、IL-6和HMGB1等這些炎癥介質(zhì)在不同的動(dòng)物模型中都被證實(shí)能夠提高痛覺過敏癥狀[14-15]。Chen等[8]在神經(jīng)病理性疼痛的動(dòng)物模型中發(fā)現(xiàn),HO-1/CO信號(hào)通路可調(diào)控大鼠脊髓組織炎癥介質(zhì)的釋放,Hemin和CORM-2可抑制CCI誘發(fā)的神經(jīng)痛理性疼痛大鼠IL-1β、TNF-α和HMGB1的釋放,與本實(shí)驗(yàn)結(jié)果一致。結(jié)合本研究和相關(guān)研究結(jié)果,筆者認(rèn)為在切口痛大鼠中保護(hù)性蛋白HO-1表達(dá)增加,可能是由于切口痛應(yīng)激性的激活體內(nèi)應(yīng)激系統(tǒng)釋放大量保護(hù)性蛋白所致;外源性HO-1的表達(dá)增加,CO釋放增加,可進(jìn)一步發(fā)揮其保護(hù)作用,減輕炎癥因子的釋放,減少炎癥反應(yīng)的破壞作用,發(fā)揮細(xì)胞的保護(hù)作用,這可能是切口痛對(duì)炎癥反應(yīng)的一個(gè)保護(hù)性機(jī)制。

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(2016-04-07收稿2016-05-27修回)

(本文編輯陸榮展)

The regulatory effect of HO-1/CO pathway on inflammatory cytokines in a rat model of incisional pain

WANG Yuntao1,SHAN Shimin1△,LIU Xiaozhi2
1 Department of Anesthesiology,2 Central Laboratory,Tianjin Fifth Central Hospital,Tianjin 300450,China△

E-mail:15022171377@163.com

ObjectiveTo investigate the effects of HO/CO pathway on inflammation cytokines in a rat model of incisional pain.MethodsThirty-six rats were executed to collect ipsilateral spinal cord tissues for HO-1 detection by Western blot assay,and cytokines tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6 and high mobility group box(HMGB)1 were detected by ELISA before and at 1,4,8,12 and 24 h after establishing incisional pain model.Additionally,36 rats without establishment of incisional pain model were used as control group.A total of 144 model rats of incisional pain were divided into incisional pain(IP)group,IP+hemin group(100 mg/kg hemin was injected by i.p.before operation),IP+ Znpp-IX group(45 μmoL/kg Znpp-IX was injected by i.p.before operation)and IP+CORM-2 group(10 mg/kg CORM-2 was injected by i.p.before operation).Values of paw withdrawal mechanical threshold(PWMT)and paw withdrawal thermal latency(PWTL)were detected,and expressions of TNF-α,IL-1 β,IL-6 and HMGB1 were measured by ELISA before and at 1,4,8,12 and 24 h after operation.ResultsCompared with pre-operation of incisional pain in rats,expression levels of HO-1 protein and cytokines TNF-α,IL-1 β,IL-6 and HMGB1 were increased at 1,4,8,12 and 24 h after operation(P<0.05).Compared with control group,values of PWMT and PWTL were obviously decreased,and expression levels of IL-1β,TNF-α,IL-6 and HMGB1 were increased at 1,4,8,12 and 24 h after operation in IP groups(P<0.05).Compared with IP groups,values of PWMT and PWTL were significantly increased and cytokines TNF-α,IL-1 β,IL-6 and HMGB1 were decreased at 1,4,8,12 and 24 h after operation in IP+hemin group and IP+CORM-2 group(P<0.05).Values of PWMT andPWTL were decreased and cytokines TNF-α,IL-1β,IL-6 and HMGB1 were increased in IP+Znpp-IX group(P<0.05). ConclusionIncisional pain can increase the expression of HO-1,and HO-1/CO pathway exists the regulatory effect on inflammatory cytokines in the rat model of incisional pain.

pain,postoperation;inflammation;disease models,animal;tumor necrosis factor-alpha;interleukin-1beta;high mobility group protein 1;heme oxygenase 1;HO-1/CO signal path

R614.4,R441.1

A

10.11958/20160268

天津市衛(wèi)生局科技基金資助項(xiàng)目(2014KZ016)

1天津第五中心醫(yī)院麻醉科(郵編300450),2中心實(shí)驗(yàn)室

王云濤(1974),男,學(xué)士學(xué)位,主要從事急慢性疼痛的診療方面研究△

E-mail:15022171377@163.com

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