朱麗華，熊御云，夏琳，芮棵，陰晴

(江蘇大學(xué)附屬醫(yī)院，江蘇鎮(zhèn)江212001)

積雪草酸預(yù)處理對(duì)膿毒癥小鼠急性腎損傷的影響及機(jī)制

朱麗華，熊御云，夏琳，芮棵，陰晴

(江蘇大學(xué)附屬醫(yī)院，江蘇鎮(zhèn)江212001)

目的 探討積雪草酸(AA)預(yù)處理對(duì)膿毒癥小鼠急性腎損傷的作用及機(jī)制。方法 選擇雄性BALB/c小鼠24只，隨機(jī)分為空白對(duì)照組、脂多糖(LPS)組、AA低劑量組、AA高劑量組各6只。AA低劑量組、AA高劑量組每天分別以AA 10、30 mg/kg灌胃預(yù)處理，空白對(duì)照組、LPS組每天以等量的0.5%羧甲基纖維素鈉灌胃。第3天灌胃2 h后，空白對(duì)照組腹腔注射PBS，其余三組腹腔注射LPS 10 mg/kg建立小鼠膿毒癥模型。各組于第3天腹腔注射4 h后，采用二乙酰肟比色法檢測(cè)血清尿素氮(BUN)水平；取腎組織，行HE染色觀察腎組織病理變化；采用real-time PCR法檢測(cè)炎癥因子(TNF-α、IL-1β、IL-6)及Notch信號(hào)通路相關(guān)基因(Notch1～4、Dll4 、Jag2)表達(dá)。結(jié)果 LPS組、AA低劑量組、AA高劑量組血清BUN水平均高于空白對(duì)照組，AA低劑量組、AA高劑量組血清BUN水平均低于LPS組(P均<0.05)。HE染色可見正常對(duì)照組小鼠腎組織正常；LPS組表現(xiàn)為腎小管壞死，內(nèi)皮細(xì)胞腫脹以及炎性細(xì)胞浸潤(rùn)；AA低劑量組、AA高劑量組表現(xiàn)為內(nèi)皮細(xì)胞腫脹減輕、炎癥細(xì)胞浸潤(rùn)減少，病理?yè)p傷較LPS組明顯減輕。LPS組TNF-α、IL-1β、IL-6 mRNA表達(dá)均高于空白對(duì)照組(P均<0.05)，AA低劑量組、AA高劑量組TNF-α、IL-1β、IL-6 mRNA表達(dá)均低于LPS組(P均<0.05)。LPS組Notch信號(hào)相關(guān)基因Notch1、Notch2、Notch3、Notch4、Dll4及 Jag2 mRNA表達(dá)均低于空白對(duì)照組(P均<0.05)，AA低劑量組、AA高劑量組Notch1、Notch2、Notch3、Notch4、Dll4及 Jag2 mRNA表達(dá)均高于LPS組(P均<0.05)。結(jié)論 AA預(yù)處理能夠明顯減輕膿毒癥小鼠的腎損傷，其機(jī)制可能與調(diào)控Notch信號(hào)通路發(fā)揮抗炎作用有關(guān)。

積雪草酸；腎臟；脂多糖；Notch信號(hào)通路；炎癥因子；膿毒癥；急性腎損傷

嚴(yán)重膿毒癥或膿毒性休克是導(dǎo)致嚴(yán)重腎損傷(AKI)的主要誘因，其發(fā)病率逐年上升，每年在全球范圍內(nèi)造成超過400萬人死亡。因此，有效防治感染繼發(fā)的腎功能障礙是降低全身性感染患者病死率的重要手段。Notch信號(hào)通路是一種高度保守的細(xì)胞內(nèi)信號(hào)機(jī)制，在決定細(xì)胞生死、分化、腫瘤、神經(jīng)退變病及炎癥反應(yīng)中具有重要意義[1,2]。細(xì)胞表面的Notch受體與配體結(jié)合后，可在蛋白水解的作用下釋放胞內(nèi)段，轉(zhuǎn)而募集轉(zhuǎn)錄共激活物，活化下游靶基因的轉(zhuǎn)錄表達(dá)，參與細(xì)胞內(nèi)的各種生理活動(dòng)。積雪草酸(AA)是中草藥積雪草提取物中五環(huán)三萜烯類組分的五環(huán)三萜類化合物，具有抗氧化、抗炎、神經(jīng)保護(hù)及抗腫瘤等多種藥理活性[3～6]，但其對(duì)發(fā)生膿毒癥時(shí)AKI的預(yù)防作用尚不清楚。2016年3～12月，我們通過腹腔注射脂多糖(LPS)建立小鼠膿毒癥模型，觀察AA對(duì)AKI的影響。

1 材料與方法

1.1 動(dòng)物與試劑 雄性BALB/c小鼠24只，6～8周齡，體質(zhì)量18～22 g，由揚(yáng)州大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。AA購(gòu)自Sigma公司；BUN試劑盒購(gòu)自南京建成生物工程有限公司；反轉(zhuǎn)錄、PCR試劑盒購(gòu)自Takara公司；PCR反應(yīng)引物由南京金斯瑞有限公司提供；其余試劑均為國(guó)產(chǎn)分析純。

1.2 分組與干預(yù)方法 小鼠適應(yīng)性飼養(yǎng)3 d后，隨機(jī)分為空白對(duì)照組、LPS組、AA低劑量組、AA高劑量組各6只。AA低劑量組、AA高劑量組每天分別灌胃AA 10、30 mg/kg，空白對(duì)照組、LPS組每天灌胃等量的0.5%羧甲基纖維素鈉(CMC-Na)。第3天灌胃2 h后，LPS組、AA低劑量組、AA高劑量組腹腔注射LPS 10 mg/kg建立小鼠膿毒癥模型，空白對(duì)照組腹腔注射PBS。

1.3 血清BUN檢測(cè) 各組于第3天腹腔注射LPS 4 h后，眼眶后緣靜脈取血，收集血清，采用二乙酰肟比色法檢測(cè)血清BUN水平。

1.4 腎臟組織病理學(xué)觀察 取血后麻醉大鼠，取出腎組織，腎組織標(biāo)本經(jīng)10%甲醛固定、脫水、石蠟包埋處理后，行HE染色觀察腎組織病理變化。

1.5 腎臟組織中炎癥因子及Notch信號(hào)通路相關(guān)基因檢測(cè) 采用real-time PCR法檢測(cè)炎癥因子(TNF-α、IL-1β、IL-6)及Notch信號(hào)通路相關(guān)基因(Notch1～4、Dll4、Jag2)mRNA表達(dá)。小鼠處死后，剪下雙腎組織，立即置于液氮中-180 ℃ 保存。腎組織總RNA的提取應(yīng)用TRIzol試劑的方法進(jìn)行。取1 μg RNA，應(yīng)用Takara PrimeScriptTMRT-PCR試劑盒的操作步驟，進(jìn)行逆轉(zhuǎn)錄反應(yīng)合成cDNA。采用Takara公司SYBR Premix Ex TaqTM試劑盒進(jìn)行real-time PCR操作。引物由南京金斯瑞有限公司合成，炎癥因子、Notch信號(hào)通路相關(guān)基因及內(nèi)參引物序列見表1。用2-ΔΔCt法計(jì)算目標(biāo)基因與內(nèi)標(biāo)基因熒光強(qiáng)度比值，以其表示目標(biāo)基因表達(dá)量。

表1 炎癥因子、Notch信號(hào)通路相關(guān)基因引物序列

2 結(jié)果

2.1 各組血清BUN水平比較 空白對(duì)照組、LPS組、AA低劑量組、AA高劑量組血清BUN水平分別為(7.79±0.69)、(20.15±1.42)、(12.66±1.26)、(11.55±1.48)mmol/L。LPS組、AA低劑量組、AA高劑量組血清BUN水平均高于空白對(duì)照組(P均<0.05)，AA低劑量組、AA高劑量組血清BUN水平均低于LPS組(P均<0.05)。

2.2 各組腎臟病理表現(xiàn) HE染色可見正常對(duì)照組小鼠腎單位結(jié)構(gòu)正常，無水腫和炎細(xì)胞浸潤(rùn)。LPS組則表現(xiàn)為腎小管壞死，內(nèi)皮細(xì)胞腫脹以及炎性細(xì)胞浸潤(rùn)的增加。AA低劑量組、AA高劑量組表現(xiàn)為內(nèi)皮細(xì)胞腫脹減輕、炎癥細(xì)胞浸潤(rùn)減少，病理?yè)p傷較LPS組明顯減輕。

2.3 各組腎臟組織炎癥因子mRNA表達(dá)比較 LPS組TNF-α、IL-1β、IL-6 mRNA表達(dá)均高于空白對(duì)照組(P均<0.05)，AA低劑量組、AA高劑量組TNF-α、IL-1β、IL-6 mRNA表達(dá)均低于LPS組(P均<0.05)。見表1。

表1 各組腎臟組織炎癥因子基因表達(dá)比較±s)

注：與空白對(duì)照組比較，*P<0.05；與LPS組比較，#P<0.05。

2.4 各組腎臟組織Notch信號(hào)通路相關(guān)基因表達(dá)比較 LPS組Notch1、Notch2、Notch3、Notch4、Dll 4及 Jag2 mRNA表達(dá)均低于空白對(duì)照組(P均<0.05)，AA低劑量組、AA高劑量組Notch1、Notch2、Notch3、Notch4、Dll 4及 Jag2 mRNA表達(dá)均高于LPS組(P均<0.05)。見表2。

表2 各組腎臟組織Notch信號(hào)通路相關(guān)基因表達(dá)比較±s)

注：與空白對(duì)照組比較，*P<0.05；與LPS組比較，#P<0.05。

3 討論

膿毒癥時(shí)全身性的炎癥會(huì)導(dǎo)致器官的受損，其中腎臟是首要的靶器官。膿毒癥時(shí)過度的炎癥反應(yīng)可誘導(dǎo)AKI的發(fā)生，并導(dǎo)致腎功能的惡化，炎癥因子在這一過程中發(fā)揮了重要作用[7]。因此，抑制炎癥因子的產(chǎn)生成為膿毒癥治療中腎臟保護(hù)的重要策略[8]。AA是從常用中草藥積雪草中提取的一種五環(huán)三萜類的化合物，具有抗炎作用[4]。但對(duì)于AA在膿毒癥時(shí)腎損傷中的作用尚未見報(bào)道。

膿毒癥是AKI產(chǎn)生的常見病因，能夠明顯提高AKI發(fā)病率，誘發(fā)慢性腎臟疾病，并使病死率提高6～8倍。膿毒癥所致AKI的機(jī)制主要與微血管損傷、炎癥及代謝異常引起的內(nèi)皮細(xì)胞損傷有關(guān)[9]，LPS可明顯誘導(dǎo)炎癥因子釋放的增加，從而導(dǎo)致AKI[10]。大量的研究表明，炎癥因子如TNF-α、IL-1β和IL-6等參與了LPS誘導(dǎo)的腎損傷，其原因可能是由于腎臟的高血流量使其易于受到循環(huán)中炎癥因子的刺激[11]。本研究發(fā)現(xiàn)，LPS組血清BUN水平明顯高于空白對(duì)照組，腎組織HE染色發(fā)現(xiàn)腎小管壞死、內(nèi)皮細(xì)胞腫脹以及炎性細(xì)胞浸潤(rùn)的增加，腎組織中炎癥因子TNF-α、IL-1β和IL-6 mRNA表達(dá)均顯著增加，提示炎癥因子在膿毒癥后AKI的發(fā)病中具有重要意義。

已有文獻(xiàn)報(bào)道，AA具有多種藥理活性，如抗氧化、抗炎和抗腫瘤等[3,5,6]。AA的抗炎作用主要與其抑制炎癥因子iNOS、COX-2、IL-6和IL-1β等有關(guān)，可以通過抑制線粒體介導(dǎo)的NLRP3炎癥小體的激活，從而減輕糖酐酯鈉誘導(dǎo)的小鼠腸炎[12]。同時(shí)在LPS誘導(dǎo)的小鼠急性肺損傷模型中也發(fā)現(xiàn)，AA可以對(duì)抗Toll樣受體4(TLR4)/NF-κB通路介導(dǎo)的多種炎癥因子的增加[4, 13]。本研究發(fā)現(xiàn)，AA低劑量組、AA高劑量組血清BUN水平均低于LPS組，腎組織內(nèi)皮細(xì)胞腫脹減輕、炎性細(xì)胞浸潤(rùn)減少，病理?yè)p傷較LPS組明顯減輕，腎組織TNF-α、IL-1β、IL-6 mRNA表達(dá)均低于LPS組，提示AA可以減輕LPS誘導(dǎo)的小鼠腎臟損傷，發(fā)揮腎臟保護(hù)作用，可能與抑制炎癥因子表達(dá)有關(guān)。

近年來，關(guān)于Notch信號(hào)轉(zhuǎn)導(dǎo)通路在炎癥中作用的研究日益受到關(guān)注。哺乳動(dòng)物表達(dá)4個(gè)Notch受體(Notch1～4)及5個(gè)Notch配體(Jag1、Jag2、Dll1、Dll3、Dll4)。細(xì)胞接受胞外信號(hào)后，Notch受體與配體結(jié)合，經(jīng)水解酶切割后釋放Notch胞內(nèi)段。這個(gè)過程觸發(fā)了γ 分泌酶依賴性的Notch胞內(nèi)段的水解，誘導(dǎo)胞內(nèi)段的核定位，導(dǎo)致了CSL家族復(fù)合物的形成。Notch胞內(nèi)段替代了共抑制因子，招募共激活因子復(fù)合物，激活CSL依賴性的轉(zhuǎn)錄[14]。Notch信號(hào)通路參與了自身免疫和炎癥反應(yīng)過程，在類風(fēng)濕關(guān)節(jié)炎、系統(tǒng)性紅斑狼瘡和細(xì)菌病毒感染等疾病中均具有重要意義[15～17]。研究[18]發(fā)現(xiàn)，LPS可以通過直接調(diào)控Notch的酪氨酸殘基的硝化作用及抑制Notch胞內(nèi)段的轉(zhuǎn)錄活性，從而抑制Notch信號(hào)通路。本研究發(fā)現(xiàn)，LPS組Notch信號(hào)相關(guān)基因Notch1、Notch2、Notch3、Notch4、Dll4及 Jag2 mRNA表達(dá)均低于空白對(duì)照組，提示Notch信號(hào)參與了LPS導(dǎo)致的腎損傷；AA低劑量組、AA高劑量組Notch1、Notch2、Notch3、Notch4、Dll4及 Jag2 mRNA表達(dá)均高于LPS組，提示AA可能通過激活Notch信號(hào)通路，提高Notch受體1～4及配體Dll4、Jag2轉(zhuǎn)錄水平的表達(dá)，從而發(fā)揮抗炎作用，進(jìn)而發(fā)揮腎臟保護(hù)作用。

綜上所述，AA預(yù)處理可通過激活Notch信號(hào)通路，抑制LPS引起的小鼠腎臟中促炎細(xì)胞因子的生成，從而減輕小鼠腎功能損傷的發(fā)生發(fā)展。我們的研究為AA抗炎作用的機(jī)制提供了新的方向及思路。

[1] Hurlbut GD, Kankel MW, Lake RJ, et al. Crossing paths with Notch in the hyper-network [J]. Curr Opin Cell Biol, 2007,19(2):166-175.

[2] Bray SJ. Notch signalling: a simple pathway becomes complex [J]. Nat Rev Mol Cell Biol, 2006,7(9):678-689.

[3] Lee MK, Kim SR, Sung SH, et al. Asiatic acid derivatives protect cultured cortical neurons from glutamate-induced excitotoxicity [J]. Res Commun Mol Pathol Pharmacol, 2000,108(1-2):75-86.

[4] Yun KJ, Kim JY, Kim JB, et al. Inhibition of LPS-induced NO and PGE(2) production by asiatic acid via NF-kappa B inactivation in RAW 264.7 macrophages: possible involvement of the IKK and MAPK pathways[J]. Int Immunopharmacol, 2008,8(3):431-441.

[5] Park BC, Bosire KO, Lee ES, et al. Asiatic acid induces apoptosis in SK-MEL-2 human melanoma cells[J]. Cancer Lett, 2005, 218(1): 81-90.

[6] Xiong Y, Ding H, Xu M, et al. Protective effects of asiatic acid on rotenone- or H2O2-induced injury in SH-SY5Y cells [J]. Neurochem Res, 2009,34(4):746-754.

[7] Sen V, Uluca U, Ece A, et al. Role of ankaferd on bacterial translocation and inflammatory response in an experimental rat model of intestinal obstruction[J]. Int J Clin Exp Med, 2014,7(9):2677-2686.

[8] Cohen J. The immunopathogenesis of sepsis [J]. Nature, 2002,420(6917):885-891.

[9] Gomez H, Kellum JA. Sepsis-induced acute kidney injury [J]. Curr Opin Crit Care, 2016,22(6):546-553.

[10] Doi K, Leelahavanichkul A, Yuen PS, et al. Animal models of sepsis and sepsis-induced kidney injury [J]. J Clin Invest, 2009, 119(10): 2868-2878.

[11] Zhang L, Sun DD, Bao Y, et al. Nerolidol protects against LPS-induced acute kidney injury via inhibiting TLR4/NF-kappa B signaling [J]. Phytother Res, 2017,31(3):459-465.

[12] Guo WJ, Liu W, Jin B, et al. Asiatic acid ameliorates dextran sulfate sodium-induced murine experimental colitis via suppressing mitochondria-mediated NLRP3 inflammasome activation [J]. Int Immunopharmacol, 2015,24(2):232-238.

[13] Li ZL, Xiao XZ, Yang MS. Asiatic acid inhibits lipopolysaccharide-induced acute lung injury in mice [J]. Inflammation, 2016, 39(5): 1642-1648.

[14] Mumm JS, Schroeter EH, Saxena MT, et al. A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1 [J]. Mol Cell, 2000,5(2):197-206.

[15] Park JS, Kim SH, Kim K, et al. Inhibition of Notch signalling ameliorates experimental inflammatory arthritis [J]. Ann Rheum Dis, 2015, 74(1):267-274.

[16] Zhang WJ, Xu W, Xiong SD. Blockade of Notch1 signaling alleviates murine lupus via blunting macrophage activation and M2b polarization [J]. J Immunol, 2010,184(11):6465-6478.

[17] Ito T, Allen RM, Carson WF, et al. The critical role of notch ligand delta-like 1 in the pathogenesis of influenza A virus (H1N1) infection [J]. PLoS Pathog, 2011, 7(11).

[18] Kim MY, Park JH, Mo JS, et al. Downregulation by lipopolysaccharide of Notch signaling, via nitric oxide [J]. J Cell Sci, 2008,121(9):1466-1476.

Effects of pretreatment of asiatic acid on acute renal injury of mice with sepsis

ZHULihua,XIONGYuyun,XIALin,RUIKe,YINQing,

(TheAffiliatedHospitalofJiangsuUniversity,Zhenjiang212001,China)

Objective To investigate the protective effects of pretreatment of asiatic acid (AA) on acute renal injury of mice with sepsis. Methods Twenty-four male BALB/c mice were randomly divided into the blank control group, lipopolysaccharide (LPS) group, low-dose AA group, and high-dose AA group with 6 mice in each. Mice in the low-dose AA group and high-dose AA group were administered 10 and 30 mg/kg AA for 3 consecutive days, meanwhile, mice in the blank control and LPS group were intragastrically administered equal volume of 0.5% sodium carboxyl methyl cellulose (CMC-Na). At 2 h after the last administration, mice in the blank control group were intraperitoneally injected with PBS, while mice in the other three groups were injected with 10 mg/kg LPS to make the sepsis models. After 4-hour LPS injection on day 3, the level of serum urea nitrogen (BUN) was measured by diacetylmonoxime colorimetric assay. The pathological change of kidney was investigated by HE staining. The mRNA expression levels of cytokines (TNF-α, IL-1β and IL-6) and Notch signaling pathway-related genes (Notch1-4, Dll4, and Jag2) were measured by real-time PCR. Results Compared with the blank control group, the level of BUN in the LPS, low-dose and high-dose AA groups was higher. Meanwhile, the level of BUN in the low-dose AA and high-dose AA groups was lower than that of the LPS group (allP<0.05). The histological examination showed LPS treatment resulted in neutrophil infiltration, tubular necrosis and endothelial cell swelling. Against these changes, the low-dose and high-dose AA groups showed significantly alleviated neutrophil infiltration, endothelial cell swelling and pathological injury as compared with that of the LPS group. The mRNA levels of TNF-α, IL-1β, and IL-6 in the LPS group were higher than those of the blank control group (allP<0.05), at the same time, the mRNA levels of TNF-α, IL-1β, and IL-6 in the low-dose and high-dose AA groups were lower than those of the LPS group (allP<0.05). The mRNA levels of Notch related genes including Notch1, Notch2, Notch3, Notch4, Dll 4 and Jag2 in the LPS group were lower than those of the blank control group (allP<0.05), meanwhile, the mRNA levels in the low-dose and high-dose AA groups were higher than those of the LPS group (allP<0.05). Conclusion AA pretreatment may significantly alleviate the acute renal injury of mice with sepsis by regulating Notch signaling pathway to develop anti-inflammatory effect.

asiatic acid; kidney; lipopolysaccharide; Notch signaling pathway; inflammation; sepsis; acute kidney injury

國(guó)家自然科學(xué)基金資助項(xiàng)目(81301657)；江蘇省自然科學(xué)基金資助項(xiàng)目(BK20130476)。

朱麗華(1989-)，女，技師，研究方向?yàn)槟摱景Y急性腎損傷。E-mail: 18344711862@163.com。

熊御云(1983-)，女，博士，主管技師，研究方向?yàn)槎嗯K器功能衰竭的治療。E-mail: 191853184@qq.com。

10.3969/j.issn.1002-266X.2017.30.003

R967

A

1002-266X(2017)30-0010-04

2017-02-19)