姜黃素對周圍神經(jīng)再生和S100表達的研究

李亞龍，李亞芳，趙一安，楊舒婷，劉國民，李秋菊

(1.吉林大學第二醫(yī)院 骨科，吉林 長春130041；2.鄭州大學第一附屬醫(yī)院 口腔科，河南 鄭州450001；3.吉林大學第二醫(yī)院 口腔科 ，吉林 長春130041)

姜黃素對周圍神經(jīng)再生和S100表達的研究

李亞龍1*，李亞芳2，趙一安3，楊舒婷3，劉國民1，李秋菊1*

(1.吉林大學第二醫(yī)院 骨科，吉林 長春130041；2.鄭州大學第一附屬醫(yī)院 口腔科，河南 鄭州450001；3.吉林大學第二醫(yī)院 口腔科 ，吉林 長春130041)

目的 探討周圍神經(jīng)橫斷損傷后，姜黃素對周圍神經(jīng)再生的影響及其機制。方法 將BALB/c小鼠(N=200)隨機分為正常對照組(0 mg/kg/天)、模型對照組(0 mg/kg/天)、低劑量組(10 mg/kg/天)、中劑量組(20 mg/kg/天)、高劑量組(40 mg/kg/天)，每組40只，后4組建立坐骨神經(jīng)橫斷損傷模型，姜黃素胃內(nèi)給藥1周。在姜黃素給藥后的第1、2、4、8周，每組均進行電神經(jīng)生理檢測、免疫組織化學染色、Luxol Fast Blue (LFB)染色、實時定量聚合酶鏈反應(qPCR)、Western Blotting檢測。結果 電神經(jīng)生理檢測顯示，每組動作電位幅度和運動神經(jīng)傳導速度(MNCV)從第1周到第8周呈遞增趨勢，每周中劑量組和高劑量組的增幅大于低劑量組和模型對照組(P<0.05)，高劑量組和低劑量組之間、低劑量組和模型對照組之間電生理特征差異無統(tǒng)計學意義(P>0.05)；免疫組織化學染色顯示，中劑量組和高劑量組的染色強度明顯強于低劑量組和模型對照組；LFB染色顯示，中劑量組和高劑量組重生有髓纖維的直徑和數(shù)量明顯多于低劑量組和模型對照組；qPCR和Western Blotting檢測L4-6脊髓節(jié)段S100的表達水平，結果顯示中劑量組和高劑量組S100的表達水平明顯強于低劑量組和模型對照組(P<0.05)。結論 本研究結果表明，姜黃素可通過上調(diào)S100的表達，以濃度依賴性方式，促進橫斷周圍神經(jīng)的再生。本研究的結果有助于開發(fā)利用姜黃素治療周圍神經(jīng)損傷。

姜黃素；周圍神經(jīng)損傷；坐骨神經(jīng)；S100；神經(jīng)再生

(ChinJLabDiagn,2017,21:1405)

姜黃素是姜科植物姜黃根莖的多酚提取物，呈黃色，在亞洲許多國家常用作烹飪調(diào)料[1]。有研究表明，姜黃素具有抗氧化、抗炎、抗病毒、抗癌等作用[2]，因此也常用于治療某些疾病[3，4]。姜黃素在中樞神經(jīng)系統(tǒng)中具有廣泛的靶點，通過調(diào)節(jié)HPA軸、影響腦神經(jīng)遞質(zhì)、改善神經(jīng)因子和神經(jīng)再生、抑制神經(jīng)元凋亡等多種途徑，起到保護神經(jīng)的作用[5-9]，也可改善老年人的認知和情緒[10]。

姜黃素能夠預防帕金森病和阿爾茨海默病[11-15]，也能改善化療藥物誘發(fā)的神經(jīng)病變[16,17]。外周神經(jīng)損傷可導致相應神經(jīng)元的死亡，姜黃素雖可促進坐骨神經(jīng)擠壓傷后的神經(jīng)再生[18]，但對周圍神經(jīng)橫斷損傷后功能恢復的影響及其機制的研究較少。

S100基因表達的S100蛋白含有兩個鈣結合位點，同時有高度保守的氨基酸序列[19]，在細胞內(nèi)和細胞外有多種功能[20]，可誘導施萬細胞的增殖促進神經(jīng)再生[21]。本研究在此基礎上，探討了姜黃素在周圍神經(jīng)(坐骨神經(jīng))橫斷損傷BLAB/c小鼠模型中對周圍神經(jīng)再生的作用及其相關機制。

1 材料和方法

1.1 材料

姜黃素(St.Louis,MO,USA)購自Sigma-Aldrich，分子量368.39Da；雄性BALB/c小鼠(北京華阜康生物科技有限公司)200只，體重25±2 g，質(zhì)量檢測單位：中國醫(yī)學科學院醫(yī)學實驗動物研究所，許可證號：SCXK(京)2014-0004，飼養(yǎng)于吉林大學基礎醫(yī)學院動物實驗中心。動物實驗方案已獲得吉林大學動物倫理委員會批準。

1.2 動物管理

1.2.1 坐骨神經(jīng)橫斷損傷模型的建立 將小鼠(N=200)分為正常對照組、模型對照組、低劑量姜黃素組(10 mg/kg/天)、中劑量姜黃素組(20 mg/kg/天)、高劑量姜黃素組(40 mg/kg/天)，每組40只；后4組建立坐骨神經(jīng)橫斷損傷模型：室溫下向小鼠腹腔內(nèi)注射3%氯胺酮(100 mg/kg/天)進行麻醉，俯臥位固定，在單側后大腿做一長約2 cm的縱向切口，暴露梨狀肌，將坐骨神經(jīng)分離，在坐骨結節(jié)下方0.5 cm處將其切斷，然后立即行顯微手術將斷端吻合，縫合肌肉和皮膚。

1.2.2 給藥劑量分別取姜黃素50 mg、25 mg、12.5 mg、0 mg，各溶于25 μl的二甲基亞砜(DMSO)中，然后分別加0.9%NaCl溶液至50 ml，使DMSO在0.9%NaCl溶液中的最終體積濃度為0.05%，形成濃度為1 mg/ml的高劑量組、0.5 mg/ml的中劑量組、0.25 mg/ml的低劑量組、0 mg/ml的模型對照組。對各組小鼠進行灌胃治療，1 ml/天，持續(xù)1周。未手術、無藥物治療的小鼠作為正常對照組；已手術，給予無姜黃素、含DMSO的0.9%NaCl溶液的小鼠作為模型組；已手術、給予姜黃素治療的小鼠作為實驗組。

1.3 電生理檢測

姜黃素給藥后的第1、2、4、8周，每組各取10只小鼠，分別在腹腔內(nèi)注射3%氯胺酮(100 mg/kg/天)進行麻醉，室溫下暴露坐骨神經(jīng)，放置肌電圖誘發(fā)電位儀(Keypoint○R,Medtronic A/C Inc,Skovlunde,Denmark)，給予10 mA單電流刺激后，檢測電極記錄動作電位幅度和運動神經(jīng)傳導速度(MNCV=兩個刺激電極之間的距離/動作電位潛伏期差異)。

1.4 L4-6脊髓節(jié)段取樣

電生理檢測完畢后，經(jīng)后椎體中線切口打開椎管，將損傷的坐骨神經(jīng)所連接的L4-6脊髓節(jié)段摘除，并立即儲存在液氮中。

1.5 免疫組織化學染色

將姜黃素給藥后第1、2、4、8周摘取的坐骨神經(jīng)在10%中性甲醛中固定3天。將脊髓切片置于H2O2中浸泡10 min以阻斷內(nèi)源性過氧化物酶，并在檸檬酸鈉溶液(0.01 mol/L，pH=6.0)中煮沸10 min。用0.01 mol/L的PBST(pH=7.4)沖洗切片，將切片用抗物種血清封閉處理15 min，加入兔抗小鼠S100多克隆抗體(1∶500，中國江蘇省南京市碧云天生物技術研究所)后4℃條件下培養(yǎng)整夜。在37℃下加入生物?；纳窖蚩雇肐gG(1∶1 000，中國湖北省武漢市博士德生物科技有限公司)處理20 min，然后加入鏈霉親和素和Horseradish過氧化物酶處理20 min。利用3,3-二氨基聯(lián)苯胺進行標記，棕色染色代表陽性蛋白。兩名病理學專家利用顯微鏡(Olympus PM-10A0，中國北京市奧林巴斯有限公司)進行盲檢，使用圖像分析軟件Image-Pro○RPlus 6.0 (MediaCybernetics Inc,Silver Spring,MD,USA)掃描染色區(qū)域來分析免疫組織化學光密度(IOD)。

1.6 Luxol Fast Blue(LFB)染色

將姜黃素給藥后第1、2、4、8周摘取的坐骨神經(jīng)在10%中性甲醛中固定3天，60℃下將坐骨神經(jīng)切片在LFB溶液中染色12 h。將切片在95%乙醇中培養(yǎng)5 min，在0.05%碳酸鋰溶液里浸泡15 s，再用70%乙醇和蒸餾水洗滌。將切片脫水，并清理干凈，加蓋蓋玻片。每個切片中，隨機選擇5個區(qū)域在40×的倍數(shù)下進行顯微鏡觀察。使用Image-Pro○RPlus 6.0圖像分析軟件計算有髓纖維的直徑和數(shù)量。

1.7 實時定量聚合酶鏈反應(qPCR)

使用TRIzol試劑法，從姜黃素給藥后第1、2、4、8周摘除的L4-6脊髓節(jié)段樣品中提取出總RNA，以其為模板，通過逆轉(zhuǎn)錄酶合成cDNA，以cDNA為模板，通過qPCR技術，定量合成S100 mRNA。S100和甘油醛-3-磷酸脫氫酶(GAPDH)的引物(表1)由軟件Beacon Designer 7(Premier Biosoft Inc,PaloAlto,CA,USA)設計，由生工生物工程(上海)股份有限公司合成。每個反應體系中，都有一對GAPDH引物作為內(nèi)部對照，在95℃下反應30 s、58℃下反應60 s、72℃反應60 s，循環(huán)40次，獲得循環(huán)閾值(Ct)。使用2-ΔΔCt法計算感興趣區(qū)/GAPDH表達的mRNA。

表1 由Beacon Designer 7軟件設計的S100和GAPDH引物

GAPDH:Glyceraldehyde-3-phosphate dehydrogenase.

1.8 Western Blotting檢測

將姜黃素給藥后第1、2、4、8周摘取的L4-6脊髓樣品低溫放在RIPA裂解緩沖液(碧云天生物技術研究所)中裂解。十二烷基硫酸鈉-聚丙烯酰胺凝膠(12%)電泳分離蛋白質(zhì)，將蛋白質(zhì)電聚到聚偏二氟乙烯膜上。兔抗鼠S100多克隆抗體用含有1%牛血清白蛋白的PBS(碧云天生物技術研究所)按照1∶5 000稀釋，將聚偏二氟乙烯膜置于其中，在4℃下培養(yǎng)整夜。再將其與Horseradish過氧化物酶標記的山羊抗兔IgG 在37℃下培養(yǎng)1 h，并用Western Blotting 3,3’-二氨基聯(lián)苯胺試劑盒(碧云天生物技術研究所)對其染色，進行X射線熒光掃描和分析。感興趣區(qū)與GAPDH蛋白質(zhì)相對灰度值的比值表示蛋白質(zhì)水平，用軟件Image-Pro○RPlus 6.0對蛋白質(zhì)水平進行分析。

1.9 統(tǒng)計分析

用SPSS17.0(SPSS Inc,Chicago,IL,USA)統(tǒng)計分析數(shù)據(jù)的平均值±SD，用單因素方差分析法和Dunnett-t檢驗比較組間差異，P<0.05為有統(tǒng)計學意義。

2 結果

2.1 電生理檢測

為觀測神經(jīng)的功能情況，本研究用肌電圖誘發(fā)電位儀(Keypoint○R,Medtronic A/C Inc,Skovlunde,Denmark)檢測記錄了坐骨神經(jīng)動作電位幅度和MNCV(圖1)。給予姜黃素治療后，每組動作電位幅度和MNCV從第1周到第8周呈遞增趨勢。每周高劑量組和中劑量組的增幅大于低劑量組和模型對照組(P<0.05)。高劑量組第8周時動作電位幅度和MNCV增加到正常水平，分別為低劑量組的1.67倍和1.22倍。高劑量組和低劑量組之間、低劑量組和模型對照組之間電生理特征差異無統(tǒng)計學意義(P>0.05)。這些結果表明，姜黃素可促進小鼠坐骨神經(jīng)損傷的功能恢復。

2.2 免疫組織化學染色

姜黃素給藥后的第1、2、4、8周，對S100陽性蛋白的密度進行半定量檢測，發(fā)現(xiàn)IOD值和陽性表達強度呈正相關(圖2)，同時，高劑量組的IOD值是低劑量組的1.79倍。從第4周開始，陽性細胞開始降低它的染色強度。每個時間段內(nèi)，高劑量組和中劑量組的染色強度明顯強于低劑量組和模型對照組。這些結果表明，姜黃素能夠促進S100的表達。

A:Action potential amplitude;B:motor nerve conductive velocity.Data are expressed as the mean±SD,n=5.One-way analysis of variance and Dunnett’s test were used to analyze the differences among groups.*P< 0.05,vs.model group;#P<0.05,vs.low-dose curcumin group.

圖1 姜黃素給藥后第1、2、4、8周的動作電位幅度(mV)和運動神經(jīng)傳導速度(m/s)

A:Immunohistochemical staining of S100 protein 2 weeks following curcumin administration at 40× magnification.Scale bar:200 μm.Arrows indicate positive immunoreactivity for S100,which is seen as fine brown particles in the cytoplasm.B: Staining intensity.The staining intensity in the high- and moderate-dose curcumin groups is stronger than that in the low-dose curcumin and model groups.Data are expressed as the mean± SD (n=5).One-way analysis of variance and Dunnett’s tests were used to analyze the difference among groups.*P<0.05,vs.model group;#P<0.05,vs.low-dose curcumin group.

圖2 姜黃素給藥8周后L4-6脊髓節(jié)段切片上的S100免疫反應

2.3 LFB染色

經(jīng)LFB染色(圖3)發(fā)現(xiàn)，姜黃素給藥后的第1、2、4周，坐骨神經(jīng)的髓鞘結構在各組間并無明顯差異，第8周進行LFB染色后，軸突呈白色，髓磷脂呈藍色。高劑量組和中劑量組中的髓磷脂形狀規(guī)則，厚度均勻，邊界清晰，髓鞘周圍增生少；低劑量組中的髓磷脂規(guī)則不整，厚度不均，邊界較清晰，有纖維結締組織呈間質(zhì)性增生；模型對照組髓磷脂情況最差。利用Image-Pro○RPlus 6.0測量有髓神經(jīng)纖維數(shù)量和直徑發(fā)現(xiàn)，高劑量組和中劑量組有髓神經(jīng)纖維的數(shù)量和直徑均大于低劑量組和模型對照組，高劑量組有髓神經(jīng)纖維的數(shù)量和直徑甚至分別是低劑量組的1.49倍和1.35倍。這些結果表明，高劑量組和低劑量組姜黃素可明顯促進受損坐骨神經(jīng)髓鞘的恢復。

A:Luxol fast blue staining of transverse sections of sciatic nerve myelin.Images were obtained at 40× magnification;scale bar is 200 μm.Arrowsindicate the sciatic nerve myelin sheath dyed blue.Myelin in the high- and moderate-dose curcumin groups appears regular and uniform.However,myelin in the low-dose curcumin and model groups appears irregular and exhibits fibrous connective tissues.B:The number (n/mm2)and diameter (μm) of myelinated fibers in the high- and moderate-dose curcumin groups are larger than those in the low-dose curcumin andmodel groups.Data are expressed as the mean ±SD,n=5.One-way analysis of variance and Dunnett’s test were used to analyze the differencesamong groups.*P<0.05,vs.model group;#P<0.05,vs.low-dose curcumin group.

圖3 姜黃素給藥8周后，LFB染色顯示的坐骨神經(jīng)橫斷面有髓纖維的直徑和數(shù)量

2.4 qPCR

在正常小鼠的坐骨神經(jīng)中，幾乎檢測不到S100 mRNA。給予姜黃素治療后，L4-6脊髓中S100 mRNA水平升高(圖4)。第1周時，高劑量組和中劑量組S100 mRNA水平達到峰值，顯著高于低劑量組和模型對照組，差異有統(tǒng)計學意義(P<0.05)，其中高劑量組S100 mRNA水平比低劑量組約高2.15倍。低劑量組和模型對照組S100 mRNA水平則在第2周達到峰值。第8周時，S100 mRNA水平在各組間的差異無統(tǒng)計學意義(P>0.05)。這些結果表明，高劑量組和中劑量組姜黃素可明顯促進S100表達。

2.5 Western-blotting檢測

如圖5所示，姜黃素給藥后第2周，各組S100蛋白水平均上調(diào)，達到峰值。高劑量組和中劑量組明顯高于低劑量組和模型組(P<0.05)，其中高劑量組比低劑量組高1.4倍。這些結果表明，高劑量組和中劑量組對S100的表達有明顯的促進作用。第8周時，各組間S100蛋白水平差異無統(tǒng)計學意義(P>0.05)。

Data are expressed as the mean±SD(n=5).One-way analysis of variance and Dunnett’s test were used to analyze the difference among groups.*P< 0.05,vs.model group;#P< 0.05,vs.low-dose curcumin group.

圖4 姜黃素給藥后1、2、4、8周L4-6脊髓節(jié)段中的S100 mRNA的表達

A:Western blot assay results for the S100 protein; B: Grayscale ratio of S100/GAPDH protein.S100 protein levels peak at the second week after curcumin administration in each curcumin group,and are dose-dependent for each time period.Data were expressed as the mean±SD(n=5).One-way analysis of variance and Dunnett’s test were used to analyze the difference among groups.*P<0.05,vs.model group;#P<0.05,vs.low-dose curcumin group.

圖5 姜黃素給藥后第1、2、4、8周L4-6脊髓節(jié)段中的S100 蛋白的表達

3 討論

姜黃素對中樞神經(jīng)系統(tǒng)具有保護作用[22-24]，但其能否促進橫斷周圍神經(jīng)再生的相關研究較少，本研究對此進行了相關探究。用電神經(jīng)生理檢測觀察坐骨神經(jīng)的功能恢復情況，發(fā)現(xiàn)姜黃素以40 mg/kg/天(高劑量組)和20 mg/kg/天(中劑量組)的劑量給藥一周后，橫斷的坐骨神經(jīng)的功能有明顯恢復。LFB染色實驗顯示，高劑量組和中劑量組再生坐骨神經(jīng)髓鞘的數(shù)量和直徑均大于低劑量組(10 mg/kg/天)和模型對照組，為姜黃素能夠促進受損坐骨神經(jīng)功能恢復提供了直接證據(jù)，同時表明姜黃素以濃度依賴性方式促進神經(jīng)再生，也有相關研究證實了這一結果[18,25]。

本研究中，姜黃素20 mg/kg/天和40 mg/kg/天的給藥劑量可有效促進小鼠受損神經(jīng)的功能恢復，但當給藥劑量為10 mg/kg/天時，小鼠受損坐骨神經(jīng)功能恢復效果不明顯，因此推測，姜黃素20 mg/kg/天的給藥劑量可能是促進小鼠受損周圍神經(jīng)功能恢復的最低有效劑量。

為探究姜黃素促進坐骨神經(jīng)再生的機制，本研究通過免疫組織化學染色、實時qPCR和Western blotting檢測了S100 mRNA和蛋白質(zhì)的表達水平。研究結果顯示，S100 mRNA水平在姜黃素給藥1周后達到峰值，S100蛋白質(zhì)在姜黃素給藥后第2周達到峰值，比S100 mRNA延遲1周，這是因為mRNA在蛋白質(zhì)積累的同時也在快速降解。累積的S100蛋白質(zhì)誘導坐骨神經(jīng)Schwann細胞增殖，增殖的Schwann細胞促進坐骨神經(jīng)的持續(xù)增生和功能恢復，因此S100常被作為神經(jīng)細胞增殖的標志[26-28]。

綜上所述，姜黃素以濃度依賴性方式，在20 mg/kg/天和40 mg/kg/天的給藥劑量下，通過上調(diào)S100的表達，促進受損坐骨神經(jīng)再生。本研究結果有助于開發(fā)利用姜黃素促進周圍神經(jīng)損傷的恢復。

[1]Mahmood K,Zia KM,Zuber M,et al.Recent developments in curcumin and curcumin based polymeric materials for biomedical applications:A review [J].Int J Biol Macromol,2015,81:877.

[3]Bimonte S,Barbieri A,Leongito M,et al.Curcumin AntiCancer Studies in Pancreatic Cancer[J].Nutrients,2016,8(7):433.

[4]Sarkar A,De R,Mukhopadhyay AK.Curcumin as a potential therapeutic candidate for Helicobacter pylori associated diseases[J].World J Gastroenterol,2016,22(9):2736.

[5]Daily JW,Yang M,Park S.Efficacy of Turmeric Extracts and Curcumin for Alleviating the Symptoms of Joint Arthritis: A Systematic Review and Meta-Analysis of Randomized Clinical Trials[J].J Med Food,2016,19(8):717.

[6]Jaisin Y,Thampithak A,Meesarapee B,et al.Curcumin I protects the dopaminergic cell line SH-SY5Y from 6-hydroxydopamine-induced neurotoxicity through attenuation of p53-mediated apoptosis[J].Neurosci Lett,2011,489(3):192.

[7]Patzkó A,Bai Y,Saporta MA,et al.Curcumin derivatives promote Schwann cell differentiation and improve neuropathy in R98C CMT1B mice[J].Brain,2012,135(12):3551.

[8]Cao H,Zheng JW,Li JJ,et al.Effects of curcumin on pain threshold and on the expression of nuclear factor κ B and CX3C receptor 1 after sciatic nerve chronic constrictive injury in rats[J].Chin J Integr Med,2014,20(11):850.

[9]Stankowska DL,Krishnamoorthy VR,Ellis DZ,et al.Neuroprotective effects of curcumin on endothelin-1 mediated cell death in hippocampal neurons[J].Nutr Neurosci,2015:1.

[10]Cox KH,Pipingas A,Scholey AB.Investigation of the effects of solid lipid curcumin on cognition and mood in a healthy older population[J].J Psychopharmacol,2015,29(5):642.

[11]Yang J,Song S,Li J,et al.Neuroprotective effect of curcumin on hippocampal injury in 6-OHDA-induced Parkinson's disease rat[J].Pathol Res Pract,2014,210(6):357.

[12]Venigalla M,Sonego S,Gyengesi E,et al.Novel promising therapeutics against chronic neuroinflammation and neurodegeneration in Alzheimer's disease[J].Neurochem Int,2016,95:63.

[13]He Y,Wang P,Wei P,et al.Effects of curcumin on synapses in APPswe/PS1dE9 mice[J].Int J Immunopathol Pharmacol,2016,29(2):217.

[14]Song S,Nie Q,Li Z,et al.Curcumin?improves neurofunctions of 6-OHDA-induced parkinsonian rats[J].Pathol Res Pract,2016,212(4):247.

[15]Huang HC,Zheng BW,Guo Y,et al.Antioxidative and neuroprotective effects of curcumin in an Alzheimer's disease rat model co-treated with intracerebroventricular streptozotocin and subcutaneous D-galactose[J].J Alzheimers Dis,2016,52(3):899.

[16]Babu A,Prasanth KG,Balaji B.Effect of curcumin in mice model of vincristine-induced neuropathy [J].Pharm Biol,2015,53(6):838.

[17]Agthong S,Kaewsema A,Charoensub T.Curcumin ameliorates functional and structural abnormalities in cisplatin-induced neuropathy[J].Exp Neurobiol,2015,24(2):139.

[18]Ma J,Yu H,Liu J,et al.Curcumin promotes nerve regeneration and functional recovery after sciatic nerve crush injury in diabetic rats[J].Neurosci Lett,2016,610:139.

[19]Bresnick AR,Weber DJ,Zimmer DB.S100 proteins in cancer[J].Nat Rev Cancer,2015，15(2):96.

[20]Donato R,Cannon BR,Sorci G,et al.Functions of S100 proteins[J].Curr Mol Med,2013,13(1):24.

[21]Wolf H,Frantal S,Pajenda G,et al.Analysis of S100 calcium binding protein B serum levels in different types of traumatic intracranial lesions[J].J Neurotrauma,2015,32(1): 23.

[22]Srivastava P,Yadav RS,Chandravanshi LP,et al.Unraveling the mechanism of neuroprotection of curcumin in arsenic induced cholinergic dysfunctions in rats[J].Toxicol Appl Pharmacol,2014,279(3):428.

[23]Noorafshan A,Asadi-Golshan R,Abdollahifar MA,et al.Protective role of curcumin against sulfite-induced structural changes in rats' medial prefrontal cortex[J].Nutr Neurosci,2015,18(6):248.

[24]Ji MH,Qiu LL,Yang JJ,et al.Pre-administration of curcumin prevents neonatal sevoflurane exposure-induced neurobehavioral abnormalities in mice[J].Neurotoxicology,2015,46:155.

[25]Da Silva Morrone M,Schnorr CE,Behr GA,et al.Oral administration of curcumin relieves behavioral alterations and oxidative stress in the frontal cortex,hippocampus,and striatum of ovariectomized Wistar rats[J].J Nutr Biochem,2016,32:181.

[27]Liu Z,Jin YQ,Chen L,et al.Specific marker expression and cell state of Schwann cells during culture in vitro[J].PLoS One,2015,10(4):e0123278.

[28]Jiang X,Ma J,Wei Q,et al.Effect of Frankincense Extract on Nerve Recovery in the Rat Sciatic Nerve Damage Model[J].Evid Based Complement Alternat Med,2016,2016:3617216.

Effect of curcumin on peripheral nerve regeneration and S100 expression

LIYa-long1,LIYa-fang2,ZHAOYi-an3,etal.

(1.DepartmentofOrthopedics,theSecondHospitalofJilinUniversity,Changchun130041,China;2.DepartmentofStomatology,theFirstAffiliatedHospitalofZhengzhouUniversity,Zhengzhou450001,China;3.DepartmentofStomatology,theSecondHospitalofJilinUniversity,Changchun130041,China)

Objective To investigate the effect of curcumin on peripheral nerve regeneration after peripheral nerve transection and its mechanism.Methods BALB / c mice (N=200) were randomly divided into normal control group (0 mg/kg/day),model control group (0mg/kg/day),low dose group (10 mg/kg/day),middle dose group (20 mg/kg/day),high dose group (40 mg/kg/day),and each group of 40.BALB/c mice underwent complete sciatic nerve amputation,and followed by an immediate epineurium anastomosis.Mice were intragastrically administered curcumin at doses of 40 (high),20 (moderate),and 10 mg/kg/d (low) for 1 week.Following the 1st,2nd,4th and 8th week after curcumin administration were electroneurophysiological tests,immunohistochemical staining,Luxol Fast Blue (LFB) staining,real-time quantitative polymerase chain reaction (qPCR) and Western Blotting assay each group.Results Electroneurophysiological tests showed that the amplitude of each action potential and motor nerve conduction velocity (MNCV) were increasing from the first week to the eighth week.The increase of the middle dose group and the high dose group was higher than that of the low dose group and the model control group (P<0.05).There was no significant difference in electrophysiological characteristics between high dose group and low dose group or low dose group and model control group (P>0.05).Immunohistochemical staining showed that the staining intensity of the middle dose group and the high dose group was significantly higher than that of the low dose group and the model control group.Luxol Fast Blue (LFB) staining showed that the diameter of the myelinated fibers in the middle and high dose groups.QPCR and Western Blotting were used to detect the expression of S100 in L4-6 spinal cord segment.The results showed that the expression level of S100 in middle dose group and high dose group was significantly higher than that in low dose group and model control group (P<0.05).Conclusion The results of this study show that curcumin can promote the regeneration of peripheral nerves by up-regulating the expression of S100 in a concentration-dependent manner.The results of this study may help to develop the use of curcumin in the treatment of peripheral nerve injury.

Curcumin Peripheral nerve injury Sciatic nerve S100 Nerve regeneration

吉林省科技廳重大科技攻關項目(20150311038YY)

1007-4287(2017)08-1405-07

R651.3

A

李亞龍(1993-)，男，河南省安陽市人，在讀碩士研究生，主要從事神經(jīng)再生方面的研究。

2017-03-25)

*通訊作者