常山酮通過CD14/NF-κB通路調(diào)控LPS誘導(dǎo)的大鼠急性肺損傷免疫系統(tǒng)紊亂①

章小山

(河南省南陽醫(yī)學(xué)高等?？茖W(xué)校第一附屬醫(yī)院，南陽 473000 )

急性肺損傷(Acute lung injury,ALI)是由感染、創(chuàng)傷、休克等原因引起的以呼吸窘迫和低氧血癥為特征的臨床綜合征[1]?，F(xiàn)代研究表明，免疫系統(tǒng)紊亂引起的血-氣屏障破壞和肺水腫是導(dǎo)致呼吸窘迫的主要原因[2]。同時(shí)，炎癥反應(yīng)在ALI發(fā)生和急劇惡化的過程中發(fā)揮著重要作用[3-5]。因此，有效調(diào)控免疫系統(tǒng)紊亂、抑制炎癥反應(yīng)是治療ALI的關(guān)鍵。常山酮(Halofuginone,HF，圖1)是中藥常山的主要活性成分[6]?，F(xiàn)代研究表明，HF具有較強(qiáng)的免疫調(diào)節(jié)功能，能夠減輕多種疾病的炎癥反應(yīng)，抑制細(xì)胞凋亡[7-9]。但HF是否能夠調(diào)控脂多糖(Lipopolysa-ccharide,LPS)誘導(dǎo)的ALI免疫系統(tǒng)紊亂還未見報(bào)道。因此，本文將通過復(fù)制大鼠ALI模型，探討HF對(duì)大鼠ALI的作用及作用機(jī)制。

1 材料與方法

1.1試劑與儀器 LPS購自美國Sigma公司，HF購自中國上海晶都生物科技公司。流式細(xì)胞儀購自美國ThermoFisher公司。白介素-1 beta(Interleukin-1β,IL-1β)、IL-6和IL-18 ELISA試劑盒購自美國Millipore公司。CD14、Toll樣受體4(Toll-like receptor 4,TLR4)和核轉(zhuǎn)錄因子-κB p65(Nuclear factor of kappa B p56,NF-κB p65)一抗均購自美國Sigma公司，二抗購自美國Proteintech公司。

1.2方法

1.2.1復(fù)制大鼠ALI模型 參照文獻(xiàn)[10]報(bào)道的方法復(fù)制大鼠ALI模型。雄性Wistar大鼠購自北京實(shí)驗(yàn)動(dòng)物中心。將大鼠適應(yīng)性飼養(yǎng)3 d后將其隨機(jī)分為正常對(duì)照組、LPS組、LPS+HF (1 mg/kg BW)組、LPS+HF (5 mg/kg BW)組和LPS+HF (10 mg/kg BW)組。實(shí)驗(yàn)前12 h禁食不禁水。除正常對(duì)照組外，其余各組大鼠腹腔注射LPS 10 mg/kg，正常對(duì)照組則腹腔注射等量的生理鹽水。隨后各受試物組大鼠即刻腹腔注射給予不同濃度的HF，連續(xù)7 d，對(duì)照組和LPS組給予等量溶媒。7 d后處死大鼠，取肺進(jìn)行后續(xù)實(shí)驗(yàn)。

1.2.2蘇木精-伊紅(Hematoxylin and eosin,HE)染色 制作大鼠肺冰凍切片。用PBS清洗3次后，將冰凍切片放入蘇木精溶液中染色5 min，于1%鹽酸乙醇液中褪色。用自來水沖洗后，用乙醇進(jìn)行梯度脫水。隨后滴加0.5%伊紅復(fù)染，無水乙醇脫水。最后用二甲苯透明，封片，于顯微鏡下觀察記錄。

1.2.3TUNEL檢測(cè)凋亡小體 制作大鼠肺組織冰凍切片。用4%多聚甲醛室溫固定組織切片 30 min，滴加0.5% TritonX-100透膜15 min。用2%H2O2室溫封閉10 min后，加入TUNEL溶液37℃孵育 1 h，最后滴加DAB染色液復(fù)染10 min，于顯微鏡下每片隨機(jī)選取5個(gè)視野進(jìn)行統(tǒng)計(jì)分析。

1.2.4ELISA 用組織勻漿機(jī)將大鼠肺組織勻漿后，以4 000 r/min離心10 min，取上清液，根據(jù)ELISA試劑盒說明書檢測(cè)上清液中IL-1β、IL-18和IL-6的含量。

1.2.5流式細(xì)胞術(shù) 給予受試物7 d后，用10%多聚甲醛麻醉大鼠，心臟取血，并加入肝素抗凝。將獲得的血液用Ficoll溶液進(jìn)行梯度離心獲得單核細(xì)胞，加入CD14抗體室溫孵育30 min，用染色緩沖液終止染色，并離心重懸細(xì)胞，將細(xì)胞密度調(diào)整至1×107個(gè)/ml，用流式細(xì)胞儀進(jìn)行分選。

1.2.6Western blot 將所獲得的肺組織剪碎至1 mm3，用組織裂解液提取各組大鼠組織總蛋白。BCA試劑盒檢測(cè)蛋白濃度后，將各組蛋白濃度調(diào)平。用SDS-PAGE分離組織蛋白并轉(zhuǎn)移蛋白至PVDF膜。5%脫脂奶粉室溫封閉PVDF膜2 h，加入適當(dāng)濃度的一抗(CD14,1∶1 100;TLR4,1∶1 000;NF-κB p65,1∶800)4℃封閉過夜。次日棄去一抗，用TBST清洗3次后，加入對(duì)應(yīng)二抗室溫孵育1 h，滴加ECL顯色液進(jìn)行曝光顯影。

2 結(jié)果

2.1HF對(duì)ALI大鼠肺病理損傷的影響 為了探究HF對(duì)LPS誘導(dǎo)的ALI大鼠肺損傷的作用，我們用HE染色法檢測(cè)了肺組織病理變化情況。如圖1B所示，與正常對(duì)照組比較，LPS組肺泡腔內(nèi)可見大量炎性細(xì)胞滲出，病灶內(nèi)肺泡萎陷，肺纖維化程度加重；與LPS組比較，LPS+HF (1 mg/kg SW)組、LPS+HF (5 mg/kg SW)組和LPS+HF (10 mg/kg SW)組肺泡腔內(nèi)炎性細(xì)胞減少，肺泡壁變薄，肺纖維化程度減輕，并呈現(xiàn)出量效關(guān)系。

2.2HF對(duì)ALI大鼠肺細(xì)胞凋亡的影響 為了探究HF對(duì)ALI大鼠肺細(xì)胞凋亡的影響，我們用TUNEL法檢測(cè)了凋亡小體的形成情況。實(shí)驗(yàn)結(jié)果表明，LPS可誘導(dǎo)肺組織凋亡小體的形成(P<0.05，圖2)；HF能顯著抑制LPS誘導(dǎo)的凋亡小體形成，并隨劑量的增大，作用逐漸增強(qiáng)(P<0.05，圖2)。

2.3HF對(duì)ALI大鼠炎癥反應(yīng)的影響 ELISA實(shí)驗(yàn)結(jié)果表明，與正常對(duì)照組比較，LPS組肺組織炎癥因子IL-1β、IL-6和IL-18的含量明顯增多(P<0.05，圖3)；與LPS組比較，各受試物組大鼠肺組織IL-1β、IL-6和IL-18的含量明顯減少(P<0.05，圖3)，并呈現(xiàn)出量效關(guān)系。

圖1 HF對(duì)ALI大鼠肺病理損傷的影響Fig.1 Influence of HF on pulmonary injury in ALI ratsNote: A.The chemical structure of HF;B.Rats were injected with LPS and followed with HF for 7 days.The pathological lesion was measured by HE staining.

圖2 HF對(duì)ALI大鼠肺細(xì)胞凋亡的影響Fig.2 Influence of HF on apoptosis in lungNote: The apoptosis was calculated by TUNEL assay,*.P<0.05 vs healthy control，#.P<0.05 vs LPS.

圖3 HF對(duì)ALI大鼠炎癥反應(yīng)的影響Fig.3 Influence of HF on inflammation in ALI ratsNote: The levels of IL-1β，IL-6 and IL-18 in lung tissue were measured by ELISA kits.*.P<0.05 vs Healthy control；#.P<0.05 vs LPS.

圖4 HF對(duì)ALI大鼠外周血CD14表達(dá)的影響Fig.4 The influence of HF on CD14+ cells in bloodNote: A.The CD14+ cells were selected by fluorescence-activated cell sorting;B.Quantification of Fig.4A.*.P<0.05 vs Healthy control，#.P<0.05 vs LPS.

2.4HF對(duì)ALI大鼠外周血CD14表達(dá)的影響 CD14是LPS共受體，能與LPS結(jié)合誘導(dǎo)炎癥反應(yīng)[11]。實(shí)驗(yàn)結(jié)果表明，LPS能顯著誘導(dǎo)CD14在ALI大鼠外周血單核細(xì)胞中的表達(dá)(P<0.05，圖4)；HF可明顯減少ALI大鼠外周血中CD14+細(xì)胞數(shù)(P<0.05，圖4)，并呈現(xiàn)量效關(guān)系。

圖5 HF對(duì)CD14/NF-κB信號(hào)通路的影響Fig.5 Influence of HF on CD14/NF-κB signa-ling pathwayNote: A.The protein levels of CD14，TLR4 and NF-κB p56 were measured by western blot；B.Quantification of Fig.5A.*.P<0.05 vs Healthy control，#.P<0.05 vs LPS,GAPDH was used as loading control.

2.5HF對(duì)CD14/NF-κB信號(hào)通路的影響 為了探討HF調(diào)控ALI免疫系統(tǒng)紊亂的作用機(jī)制，我們檢測(cè)了CD14/NF-κB信號(hào)通路蛋白的表達(dá)情況。實(shí)驗(yàn)結(jié)果表明，與正常對(duì)照組比較，LPS組CD14、TLR4和NF-κB p65的表達(dá)水平明顯升高(P<0.05，圖5)；與LPS組比較，LPS+HF (1 mg/kg BW)組TLR4和NF-κB p65的表達(dá)水平明顯降低(P<0.05，圖5)；LPS+HF (5 mg/kg BW)組和LPS+HF(10 mg/kg BW)組CD14、TLR4和NF-κB p65的表達(dá)水平也均明顯降低(P<0.05，圖5)，且具有量效關(guān)系。

3 討論

常山是草本植物常山的干燥根，中醫(yī)常用其治療痰飲停聚、胸膈痞塞和瘧疾[12]。HF是常山的主要活性成分之一，現(xiàn)代研究發(fā)現(xiàn)HF具有抗炎、免疫調(diào)節(jié)、抗癌等藥理學(xué)活性[8,13,14]。HF可通過調(diào)控Th17和Treg細(xì)胞之間的平衡緩解自身免疫性關(guān)節(jié)炎的發(fā)展，表明其具有較強(qiáng)的免疫調(diào)節(jié)功能[8]。大量研究表明，HF可通過抑制TGF-β的表達(dá)和膠原蛋白的合成抑制肺纖維化[15-17]。Calik等[18]發(fā)現(xiàn)，HF可明顯改善輻射誘導(dǎo)的ALI，減輕肺組織炎性細(xì)胞的浸潤(rùn)及肺纖維化。本文研究表明，HF可明顯改善LPS誘導(dǎo)的ALI，可減輕炎性細(xì)胞浸潤(rùn)及肺纖維化程度、改善肺泡結(jié)構(gòu)。同時(shí)，HF還可明顯減弱LPS誘導(dǎo)凋亡小體形成的作用，表明其可通過減輕肺纖維化、抑制細(xì)胞凋亡發(fā)揮肺保護(hù)作用。

炎癥反應(yīng)是ALI的主要病理表現(xiàn)，也是導(dǎo)致ALI惡化的主要原因[3]。ALI可由多種原因誘發(fā)，如內(nèi)毒素、酸中毒、補(bǔ)體激活等[1]。LPS是內(nèi)毒素的主要組成部分，是導(dǎo)致肺損傷最常見的原因之一[19]。LPS可導(dǎo)致肺組織炎性細(xì)胞浸潤(rùn)，誘導(dǎo)大量促炎癥因子的釋放而誘發(fā)肺組織的炎癥反應(yīng)，從而導(dǎo)致細(xì)胞凋亡[20，21]。IL-1β、IL-18和IL-6等是ALI炎性過程的主要特征因子，其參與了ALI炎癥反應(yīng)的整個(gè)過程[7,22]。其中，IL-1β可促進(jìn)IL-6、IL-18的生成[23]。IL-6可誘導(dǎo)黏附分子和其他炎癥因子的表達(dá)，還能促進(jìn)巨噬細(xì)胞的分化和浸潤(rùn)[24]。研究表明，IL-1β、IL-18和IL-6在ALI中表達(dá)明顯增多[25-27]。HF具有較強(qiáng)的抗炎活性，可抑制TNF-α、IL-1β和IL-6等炎癥因子的表達(dá)[7,22]。Sun等[9]研究發(fā)現(xiàn)，HF可通過調(diào)控IL-1β、IL-6、IL-10和TNF-α等前炎癥因子的表達(dá)影響病毒性心肌炎的發(fā)展。本文研究發(fā)現(xiàn)，HF可明顯抑制LPS誘導(dǎo)的ALI炎癥因子IL-1β、IL-6和IL-18的表達(dá)，并隨濃度升高，作用逐漸增強(qiáng)，表明其能夠?qū)笰LI炎癥反應(yīng)，從而抑制肺細(xì)胞的凋亡。

CD14是位于巨噬細(xì)胞和單核細(xì)胞上的LPS共受體，LPS誘導(dǎo)炎癥反應(yīng)依賴于與CD14的結(jié)合[11]。本文研究發(fā)現(xiàn)，HF能明顯抑制LPS誘導(dǎo)的CD14表達(dá)，并具有量效關(guān)系，表明其可能是通過減弱LPS與CD14結(jié)合抑制下游炎癥反應(yīng)相關(guān)通路的激活。CD14分為可溶性CD14和膜結(jié)合CD14兩種類型[28]。研究表明，CD14與LPS結(jié)合后可將LPS傳遞給TLR4使其活化，活化的TLR4又激活TLR/MyD88從而誘導(dǎo)NF-κB p65激活，使其轉(zhuǎn)移至細(xì)胞核，并與DNA結(jié)合，從而促進(jìn)IL-1β、IL-18和IL-6等炎癥因子的表達(dá)，誘導(dǎo)炎癥反應(yīng)發(fā)生[5,29-31]。已有研究表明，LPS誘導(dǎo)ALI與調(diào)控其下游CD14/NF-κB信號(hào)通路有關(guān)[32,33]，但HF調(diào)控LPS誘導(dǎo)的ALI免疫系統(tǒng)紊亂是否與CD14/NF-κB信號(hào)通路有關(guān)還未見報(bào)道。LPS明顯上調(diào)了ALI大鼠肺組織CD14和TLR4的表達(dá)水平，并促進(jìn)肺細(xì)胞核蛋白NF-κB p65的表達(dá)，其中，NF-κB p65是NF-κB的一個(gè)重要亞型，其轉(zhuǎn)移入核是調(diào)控NF-κB活化的關(guān)鍵[34]。結(jié)合實(shí)驗(yàn)結(jié)果表明LPS可激活CD14/NF-κB信號(hào)通路。同時(shí)，HF可顯著降低CD14、TLR4和NF-κB p65的蛋白表達(dá)水平，表明其可抑制LPS誘導(dǎo)的CD14/NF-κB通路的激活，并且HF可能通過調(diào)控CD14/NF-κB信號(hào)通路抑制ALI大鼠的炎癥反應(yīng)及肺細(xì)胞的凋亡。

綜上所述，HF可通過抑制炎癥因子IL-1β、IL-18和IL-6的釋放明顯改善LPS誘導(dǎo)的ALI大鼠肺組織的病變，抑制肺纖維化及細(xì)胞凋亡，并且其機(jī)制與抑制CD14/NF-κB信號(hào)通路激活有關(guān)。本文可能為ALI的治療提供了一種新的潛力藥物，但HF對(duì)ALI其他病理環(huán)節(jié)的作用還有待進(jìn)一步探討。

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