郭 昆 李洪偉
(1.徐州醫(yī)學院研究生學院,江蘇徐州221004;2.徐州醫(yī)學院附屬醫(yī)院骨科,江蘇徐州221002)
?基礎研究?
咖啡酸苯乙酯對RAW264.7細胞向破骨細胞分化的影響
郭昆1李洪偉2*
(1.徐州醫(yī)學院研究生學院,江蘇徐州221004;2.徐州醫(yī)學院附屬醫(yī)院骨科,江蘇徐州221002)
背景:觀察天然蜂膠提取物咖啡酸苯乙酯(CAPE)對核因子(NF)-κB受體活化因子配體(RANKL)誘導的小鼠單核/巨噬細胞株RAW264.7向破骨細胞分化的影響。方法:近年來,骨質疏松癥、磨損顆粒介導的骨溶解等骨吸收類疾病的發(fā)生率越來越高。目前臨床上治療骨溶解性疾病的藥物以西藥為主,而中草藥成分的研制還處于起步階段。目的:利用不同濃度的CAPE與RANKL單獨或共同處理RAW264.7細胞。CCK-8法測定細胞增殖活性,抗酒石酸酸性磷酸酶(TRAP)染色法觀察TRAP陽性多核細胞。逆轉錄-聚合酶鏈(RT-PCR)測定破骨細胞TRAP、MMP-9基因mRNA的含量。Western blot檢測NF-κB p65及核轉錄因子κB抑制蛋白(IκB)-α表達水平。結果:CCK-8結果表明75、150、300 nmol/LCAPE對RAW.264.7細胞增殖能力無影響。CAPE可抑制RANKL誘導的TRAP陽性多核細胞形成,下調RANKL誘導的TRAP和MMP-9基因mRNA表達。Western blot檢測顯示,CAPE能下調RANKL誘導的NF-κB表達水平。結論:CAPE能有效地抑制RANKL誘導的RAW264.7向破骨細胞分化。
破骨細胞;核因子-κB;受體活化因子配體
【Abstract】Background:In recent years,the incidence of bone resorptive diseases such as osteoporosis,wear particle mediated osteolysis is getting higher and higher.Drugs currently used in clinical treatment of bone resorptive diseases are western medicine,and the development of Chinese herbal medicine components is still in the emerging stage.Objective:To observe the effect of caffeic acid phenethyl ester(CAPE,a propolis extract)on receptor activator of NF-κB ligand(RANKL)induced osteoclast differentiation using the monocyte-macrophage cell line RAW264.7.Methods:Mouse mononuclear cell line RAW264.7 were cultured.CAPE and/or RANKL were added.Cell Counting Kit-8(CCK-8)assay was used to analyze the viability of RAW264.7 cells which were exposed to different concentrations of CAPE(75,150 and 300 nmol/L).Tartrate-resistant acid phosphatase(TRAP)staining was used to observe the formation of osteoclasts.RT-PCR was used to measure the mRNA expression of TRAP and matrix metalloproteinase 9(MMP-9).Western blot was used to determine the nuclear factor κB(NF-κB)pathway and IκB-α.Results:Different concentrations of CAPE did not affect RAW264.7 cell viability.Mature osteoclasts were obtained from RAW264.7 cells stimulated with RANKL for 6 days and detected by TRAP staining.CAPE could inhibit the formation of TRAP-positive cells and result in an obvious reduction of TRAP and MMP-9 mRNA expression.Western blot showed that CAPE suppressed RANKL-induced IκB-α degradation and NF-κB nuclear translocation in RAW264.7 cells.Conclusions:CAPE can inhibit the ososteoclast differentiation of mature osteoclasts from RAW264.7 induced with RANKL.
【Key words】Osteoclast;NF-κB;Receptor activator of NF-κB ligand
破骨細胞活性改變是引起各種代謝性骨病的主要原因,也是研究熱點[1]。破骨細胞數量和活性的增加與多種骨質流失的臨床疾病密切相關,它還主導骨移植物的骨吸收作用和炎癥性骨丟失等,這些疾病及其引發(fā)的骨折將嚴重影響患者的生活質量和生存時間[2]。傳統(tǒng)中藥蜂膠中的活性物質咖啡酸苯乙酯(caffeic acid phenethyl ester,CAPE)含有與NF-κB抑制劑相似的化學結構。本研究旨在探討CAPE對RANKL誘導RAW264.7細胞分化為成熟破骨細胞的影響及機制。
1.1細胞培養(yǎng)
小鼠單核/巨噬細胞株RAW264.7(ATCC號:TIB-71)用含10%胎牛血清(四季青公司)的α-MEM培養(yǎng)基培養(yǎng),其中含有1%青霉素、1%鏈霉素。
1.2CCK-8檢測細胞增殖
細胞接種于96孔板,在培養(yǎng)箱預培養(yǎng)24 h。加入不同濃度的CAPE(75、150、300 nmol/L,上海紫一試劑)刺激12、24、48 h后,每孔加入10 μl CCK-8溶液(日本同仁化學)孵育2 h,在酶標儀上測定在450 nm處的吸光度。
1.3TRAP 染色檢測成熟破骨細胞
RAW264.7細胞以4×103/孔接種于48孔板,培養(yǎng)過夜后用50 ng/L RANKL(美國Peprotech公司)和不同濃度的CAPE誘導,細胞每2天換液1次。第6天TRAP染色(美國Sigma-Aldri-ch公司,387-A),細胞用多聚甲醛固定20 min,在以萘酚AS-BI磷酸鹽作為底物的孵育液中37°C孵育1 h,蒸餾水洗3次,蘇木精復染3 min,PBS沖洗3次,在倒置相差顯微鏡下觀察及拍片。
1.4RT-PCR
在RAW264.7培養(yǎng)基中分別加入RANKL和不同濃度的CAPE,培養(yǎng)6 d后,采用TRIzol(Tiangen公司)試劑,按操作說明提取細胞內總RNA。逆轉錄采用Tiangen公司的反轉錄試劑盒按說明書進行,合成cDNA,再行PCR擴增TRAP、MMP-9,以GAPDH為管家基因,引物由上海生工生物工程公司設計合成,引物序列如下:TRAP:正向引物:5'-CCCTGTGATTTGGCTCTTCT-3',反向引物:5'-GTGGCATTGGGTCTTCTCA-3'。MMP-9:正向引物:5'-GACTACGATAAGGACGGCAAA-3',反向引物:5'-AGGGCAGAAGCCATACAGTT-3'。GAPDH:正向引物:5'-ACCCTTAAGAGGGATGCTGC-3',反向引物:5'-GAAGGGGCGGAGATGATGAC-3'。PCR產物用1.2%瓊脂糖電泳,溴化乙錠染色后于紫外燈下拍照。
1.5Western blot
收集處理后的細胞,用蛋白提取試劑盒分別提取細胞總蛋白及核蛋白,BCA法進行蛋白定量。取50 μg蛋白質進行10SDA-PAGE分離,蛋白質電轉移至PVDF膜上,BSA室溫封閉2 h,加入NF-κB p65、IκB-α一抗(1∶1000,Cell Signaling公司),4°C搖床孵育過夜,堿性磷酸酶標記二抗(1∶500,中杉金橋公司)搖床孵育2 h,TBST洗膜30 min,堿性磷酸酶顯色試劑顯影。
1.6統(tǒng)計學方法
采用SPSS 13.0統(tǒng)計軟件分析,計量資料以均數±標準差表示,采用單因素方差分析(one-way ANOVA)檢驗,P<0.05為差異有統(tǒng)計學意義。
2.1CAPE 對細胞活性的影響
CCK-8檢測結果顯示,0~300 nmol/L CAPE對RAW264.7細胞增殖無明顯影響(P>0.05,表1)。
表1 CAPE對RAW264.7細胞增殖的影響()
表1 CAPE對RAW264.7細胞增殖的影響()
CAPE(nmol/L)0 75 150 300 12 h 1.07±0.04 1.06±0.07 1.04±0.08 1.06±0.07 24 h 1.02±0.07 1.04±0.14 1.04±0.13 0.98±0.11 48 h 1.14±0.09 1.12±1.13 1.19±0.13 1.12±0.11
2.2CAPE對RANKL誘導的RAW264.7形成破骨細胞的影響
RANKL誘導6 d后,CAPE誘導組TRAP染色陽性細胞數較空白組顯著增多,而隨著CAPE濃度的增加,TRAP染色陽性細胞數逐漸減少(圖1)。
圖1 不同濃度CAPE對RANKL誘導的RAW264.7形成破骨細胞的影響
2.3CAPE下調TRAP、MMP-9基因mRNA表達
RAW264.7細胞受RANKL刺激后,TRAP、MMP-9 mRNA的表達均明顯增加,且隨著CAPE濃度的增加,TRAP和MMP-9基因的表達量逐漸減少(圖2),差異有統(tǒng)計學意義(P<0.05)。
2.4CAPE對RANKL誘導的RAW264.7細胞NF-κB核轉位及IκB-α降解的影響
300 nmol/L的CAPE預處理RAW264.7細胞2 h,加入或不加入50 ng/L RANKL處理8 h,收集細胞并提取蛋白,采用Western blot檢測細胞中IKB-α的表達及細胞核內NF-κB p65的表達。結果表明,與RANKL處理組相比,CAPE處理組細胞核內NF-κB p65蛋白的表達明顯減少(圖3)。而且,CAPE可抑制RANKL誘導的RAW264.7細胞IKB-α降解。同時,分別用75、150、300 nmol/L的CAPE預處理RAW64.7細胞2 h后,用50 ng/L RANKL處理8 h,結果顯示,隨著CAPE濃度的增加,RAW264.7細胞核內NF-κB p65蛋白表達逐漸下降,且RAW264.7細胞IκB-α降解明顯減少(圖4)。
圖2 CAPE對RANKL誘導的RAW264.7細胞TRAP、MMP-9 mRNA表達的影響
RAW264.7細胞在RANKL刺激下能夠分化為多核破骨細胞,在大量對破骨細胞的研究中,RAW264.7細胞被視為破骨細胞前體細胞而廣泛應用[3,4]。
CAPE是一種從蜂膠中提取的中藥單體,具有多種藥理作用。研究表明,CAPE有顯著的抗氧化、抗炎和抗腫瘤作用,CAPE對胸膜炎、耐藥性化膿性眼內膜炎、大腸桿菌性腎盂腎炎等都有明顯療效[5-7],國內外研究均已證實CAPE可抑制NF-κB活化[8,9]。本研究以RAW264.7為研究對象,RANKL誘導6 d后TRAP染色提示形成大量破骨細胞;藥物組OC形成數量減少,且隨CAPE濃度增加OC數量越來越少,表明CAPE能抑制RANKL誘導的RAW264.7分化為破骨細胞。
圖3 CAPE對RANKL誘導的RAW264.7NF-κB p65蛋白表達和IκB-α蛋白降解的影響
圖4 不同濃度CAPE對RANKL誘導的RAW264.7NF-κB p65蛋白表達和IκB-α蛋白降解的影響
破骨細胞分化自破骨細胞前體細胞(osteoclastprecurosor,OCP),來源于血液中單核-吞噬細胞系,是承擔體內骨質吸收功能的一類細胞[10]。其最終分化的表現(xiàn)形式有特征性標志物基因(TRAP、CTSK、MMP-9及CTR)的表達、形態(tài)轉換成大的多核細胞、形成骨吸收陷窩[11-14]。本實驗的結果顯示CAPE在RANKL誘導的RAW264.7細胞中降低了TRAP、MMP-9基因mRNA的表達,表現(xiàn)出抑制OC分化的效果。
NF-κB被認為是RANKL誘導的破骨細胞生成的關鍵轉錄因子[15]。在未受刺激的細胞中,NF-κB由P65和P50組成的異源二聚體,存在于細胞胞漿中,其p50、p65亞基與它的抑制蛋白IKB結合后,IκB磷酸化后發(fā)生降解,導致NF-κB活化并從細胞質易位至細胞核內[16]。本研究發(fā)現(xiàn)CAPE可抑制RANKL誘導的RAW264.7細胞NF-κB核轉位和IκB-α降解,這說明CAPE對RANKL誘導的RAW264.7細胞中NF-κB激活具有抑制作用。
綜上所述,CAPE能夠抑制RANKL誘導小鼠單核/巨噬細胞株RAW264.7細胞向破骨細胞分化,其機制可能與抑制NF-κB激活有關。因此,CAPE有望成為治療骨質疏松癥、假體周圍骨溶解等骨吸收類疾病新的治療藥物。
[1]Teitelbaum SL.Osteoclasts:what do they do and how do they do it?Am J Pathol,2007,170(2):427-435.
[2]Nakashima T,Hayashi M,Takayanagi H.New insights into osteoclastogenic signaling mechanisms.Trends Endocrinol Metab,2012,23(11):582-590.
[3]Kagiya T,Nakamura S.Expression profiling of micro-RNAs in RAW264.7 cells treated with a combination of tumor necrosis factor alpha and RANKL during osteoclast differentiation.J Periodontal Res,2013,48(3):373-385.
[4]Spolski R,Leonard WJ.Interleukin-21:basic biology and implications for cancer and autoimmunity.Annu Rev Immunol,2008,26:57-79.
[5]Yildirim O,Yilmaz A,Oz O,et al.Effect of caffeic acid phenethyl ester on treatment of experimentally induced methicillin-resistantStaphylococcusepidermidisendophthalmitis in a rabbit model.Cell Biochem Funct,2007,25(6):693-700.
[6]Celik S,Gorur S,Aslantas O,et al.Caffeic acid phenethyl ester suppresses oxidative stress in Escherichia coli-induced pyelonephritis in rats.Mol Cell Biochem,2007,297(1-2):131-138.
[7]Usia T,Banskota AH,Tezuka Y,et al.Constituents of Chinese propolis and their antiproliferative activities.J Nat Prod,2002,65(5):673-676.
[8]Song JJ,Lim HW,Kim K,et al.Effect of caffeic acid phenethyl ester(CAPE)on H2O2induced oxidative and inflammatory responses in human middle ear epithelial cells. Int J Pediatr Otorhinolaryngol,2012,76(5):675-679.
[9]Khan M,Elango C,Ansari MA,et al.Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focalcerebral ischemia.J Neurochem,2007,102(2):365-377.
[10]徐煒,董啟榕.假體周圍骨溶解的機制研究及治療進展.中華骨與關節(jié)外科雜志,2009,2(3):256-259.
[11]Anusaksathien O,Laplace C,Li X,et al.Tissue-specific and ubiquitous promoters direct the expression of alternatively spliced transcripts from the calcitonin receptor gene. J Biol Chem,2001,276(25):22663-22674.
[12]Motyckova G,Weilbaecher KN,Horstmann M,et al.Linking osteopetrosis and pycnodysostosis:regulation of cathepsin K expression by the microphthalmiatranscription factor family.Proc NatlAcad Sci U SA,2001,98(10):5798-5803.
[13]Reddy SV,Hundley JE,Windle JJ,et al.Characterization of the mouse tartrate-resistant acid phosphatase(TRAP)gene promoter.J Bone Miner Res,1995,10(4):601-606.
[14]Choi HJ,Park YR,Nepal M,et al.Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappaB signalingpathways.Eur J Pharmacol,2010,636(1-3):28-35.
[15]Bharti AC,Takada Y,Aggarwal BB.Curcumin(diferuloylmethane)inhibits receptor activator of NF-kappa B ligandinduced NF-kappa B activation inosteoclast precursors and suppresses osteoclastogenesis.J Immunol,2004,172(10):5940-5947.
[16]Karin M,Yamamoto Y,Wang QM.The IKK NF-kappa B system:a treasure trove for drug development.Nat Rev Drug Discov,2004,3(1):17-26.
Effect of caffeic acid phenethyl esteron osteoclast differentiation in RAW264.7 macrophages
GUO Kun1,LI Hongwei2*
(1.Graduate School,Xuzhou Medical College,Xuzhou 221004,Jiangsu;2.Department of Orthopedics,Affiliated Hospital of Xuzhou Medical College,Xuzhou 221002,Jiangsu,China)
2095-9958(2016)04-0161-04
10.3969/j.issn.2095-9958.2016.02-15
李洪偉,E-mail:lihongwei2000@126.com