杨梅素体外抗EV71病毒作用的研究

目的初步探究杨梅素抗肠道病毒71型(EV71)的活性及其机制。方法采用EV71感染非洲绿猴肾细胞(Vero细胞)模型,采用致细胞病变(CPE)法和空斑实验观察杨梅素的抗病毒效果。以不同浓度的杨梅素处理细胞,结合结晶紫染色的方法检测杨梅素的细胞毒性。以免疫印迹法检测杨梅素对VP1蛋白表达的影响。应用RT-PCR的方法评价杨梅素对VP1基因表达的影响。结果杨梅素在2.5~20μmol·L^-1的浓度下预处理病毒能够显著抑制EV71感染诱导的细胞死亡,且这种抑制作用具有剂量依赖性,IC50为5.6μmol·L^-1。与病毒组相比,杨梅素在2.5~20μmol·L^-1的浓度下还能够显著降低病毒滴度。免疫印迹和RT-PCR的结果显示,杨梅素还能够明显降低病毒衣壳蛋白VP1的基因和蛋白表达。结论杨梅素具有显著的体外抗EV71病毒活性。     

Ozone exposure in the culture medium inhibits enterovirus 71 virus replication and modulates cytokine production in rhabdomyosarcoma cells

In the present study, the effects of ozone exposure on enterovirus 71 (EV71) replication and related cytokine production were investigated. Rhabdomyosarcoma cells (RD) were exposed to 0.5, 1, 1.5 and 2 ppm ozone or filtered air under different exposure regimens before or after infection for 1 or 2 h. The results revealed that at a proper concentration of ozone, e.g., 1.5 or 2 ppm, ozone exposure restricted virus production, prolonged survival time of cells and modulated cytokine production related to EV71 infection. Upon exposure of non-infected cells to ozone at 1.5 or 2 ppm for 1h, the production of IL-1beta, IL-6 and TNF-alpha was primed and boosted by the subsequent EV71 infection, generating an inhibitory effect on EV71 replication during the post-infection period of 48 h. While infected cells were exposed to ozone for 2 h at 1.5 or 2 ppm, ozone did not affect cytokine production by RD cells in response to EV71 infection. The data showed that ozone effect on induction of cytokine was only found in uninfected cells. The ozone-induced cytokines produced prior to the onset of EV71 infection generated antiviral effects, which proved beneficial in suppressing the subsequent EV71 infection.     

Antiviral Activity of Chrysin Derivatives against Coxsackievirus B3 in vitro and in vivo

Chrysin is a 5,7-dihydroxyflavone and was recently shown to potently inhibit enterovirus 71 (EV71) by suppressing viral 3C protease (3C^pro) activity. In the current study, we investigated whether chrysin also shows antiviral activity against coxsackievirus B3 (CVB3), which belongs to the same genus (Enterovirus) as EV71, and assessed its ability to prevent the resulting acute pancreatitis and myocarditis. We found that chrysin showed antiviral activity against CVB3 at 10 μM, but exhibited mild cellular cytotoxicity at 50 μM, prompting us to synthesize derivatives of chrysin to increase the antiviral activity and reduce its cytotoxicity. Among four 4-substituted benzyl derivatives derived from C(5) benzyl-protected derivatives 7, 9–11 had significant antiviral activity and showed the most potent activity against CVB3 with low cytotoxicity in Vero cells. Intraperitoneal injection of CVB3 in BALB/c mice with 1×10⁶ TCID₅₀ (50% tissue culture infective dose) of CVB3 induced acute pancreatitis with ablation of acinar cells and increased serum CXCL1 levels, whereas the daily administration of 9 for 5 days significantly alleviated the pancreatic inflammation and reduced the elevation in serum CXCL1 levels. Collectively, we assessed the anti-CVB3 activities of chrysin and its derivatives, and found that among 4-substituted benzyl derivatives, 9 exhibited the highest activity against CVB3 in vivo, and protected mice from CVB3-induced pancreatic damage, simultaneously lowering serum CXCL1 levels.     

Ozone exposure in the culture medium inhibits enterovirus 71 virus replication and modulates cytokine production in rhabdomyosarcoma cells

In the present study, the effects of ozone exposure on enterovirus 71 (EV71) replication and related cytokine production were investigated. Rhabdomyosarcoma cells (RD) were exposed to 0.5, 1, 1.5 and 2 ppm ozone or filtered air under different exposure regimens before or after infection for 1 or 2 h. The results revealed that at a proper concentration of ozone, e.g., 1.5 or 2 ppm, ozone exposure restricted virus production, prolonged survival time of cells and modulated cytokine production related to EV71 infection. Upon exposure of non-infected cells to ozone at 1.5 or 2 ppm for 1 h, the production of IL-1β, IL-6 and TNF-α was primed and boosted by the subsequent EV71 infection, generating an inhibitory effect on EV71 replication during the post-infection period of 48 h. While infected cells were exposed to ozone for 2 h at 1.5 or 2 ppm, ozone did not affect cytokine production by RD cells in response to EV71 infection. The data showed that ozone effect on induction of cytokine was only found in uninfected cells. The ozone-induced cytokines produced prior to the onset of EV71 infection generated antiviral effects, which proved beneficial in suppressing the subsequent EV71 infection.     

Inhibition of enterovirus 71 replication by chrysosplenetin and penduletin

In recent years, enterovirus 71 (EV71) infections have caused an increasing epidemic in young children, accompanying with more severe nervous system disease and more deaths. Unfortunately, there is no specific medication for it so far. Here we investigated the anti-EV71 activity of chrysosplenetin and penduletin, two o-methylated flavonols isolated from the leaves of Laggera pterodonta. These two compounds were found to have strong activity in vitro against EV71 with low cytotoxicity. In the cytopathic effect (CPE) inhibition assays, both plaque reduction assay and virus yield inhibition assay, the compounds showed a similar 50% inhibitory concentration (IC(50)) value of about 0.20 μM. The selectivity indices (SI) of chrysosplenetin and penduletin were 107.5 and 655.6 in African green monkey kidney (Vero) cells, and 69.5 and 200.5 in human rhabdomyosarcoma (RD) cells, accordingly. The preliminary mechanism analysis indicates that they function not through blocking virus entry or inactivating virus directly but inhibiting viral RNA replication. In the time-of-addition assay, both compounds inhibited progeny virus production and RNA replication by nearly 100% when introduced within 4h post infection. In addition to EV71, both compounds inhibited several other human enteroviruses with similar efficacy. These findings provide a significant lead for the discovery of anti-EV71 drug.     

MEK1-ERKs signal cascade is required for the replication of Enterovirus 71 (EV71)

The role of the MEK1-ERK signaling cascade in the replication cycle of Enterovirus 71 (EV71), the primary cause of hand, foot and mouth disease (HFMD), has been analyzed. In vitro infection with EV71 induced a biphasic activation of ERK. The two phases of activation appeared to be triggered by different mechanisms, with the first phase being activated by the binding of viral particles to the membrane receptor of host cells and the second probably being in response to the production of new virus particles. Inhibition of ERK activation by U0126 was found to severely impair virus production. A similar reduction in EV71 replication was also observed when MEK1 expression was subject to knockdown using specific siRNAs. By contrast knockdown of MEK2 expression showed that it was dispensable for virus replication cycle, despite both MEK isoforms being activated and translocated to the nucleus equally well in response to virus infection. Overall, this study suggests distinct functions of the two isoforms of MEK in EV71 replication cycle, with an essential role for MEK1 in stimulating the ERK signaling cascade to promote virus replication. Taken together with our previous work on herpes simplex virus type 2 (HSV2) this study highlights MEK1 as a potential broad antiviral molecular target.     

Prevention of human enterovirus 71 infection by kappa carrageenan

Enterovirus 71 (EV 71), the newest member of Enteroviridae, is notorious for its etiological role in epidemics of the hand-foot-and-mouth disease, particularly in association with fatal neurological complications in young children. Searching for new and more effective agents against EV 71 infections has never relented as corresponding vaccines or antiviral drugs remain unavailable. Sulfated polysaccharides from seaweed are known to possess a broad range of biological activities across anti-virus, anti-tumor, immunomodulation, anti-coagulation, etc. In this study, we report kappa carrageenan also has a strong and effective anti-EV 71 activity able to reduce plaque formation, prevent viral replication before or during viral adsorption, as well as inhibit EV 71-induced apoptosis. In virus binding assay, kappa carrageenan was shown able to bind EV 71 firmly, forming carrageenan-viruses complexes, whereby the virus-receptor interaction is likely disrupted. Added together, kappa carrageenan may be an ideal candidate worthwhile to develop into anti-EV 71 agents.     

Enhanced potency and efficacy of 29-mer shRNAs in inhibition of Enterovirus 71

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It has been associated with severe neurological complications and has caused significant mortalities in large-scale outbreaks in Asia. In this study, we demonstrated an enhanced silencing of EV71 through the use of chemically synthesized 29-mer shRNAs. The 29-mer shRNAs were designed to target three highly conserved regions of EV71 genome. Transfection of rhabdomyosarcoma (RD) cells with the 29-mer shRNAs significantly inhibited EV71 replication in a dose-dependent manner as demonstrated by reduction of viral RNA, VP1 protein and plaque forming units. The inhibitory effects were more potent and were achieved at 10-fold lower concentrations when compared to 19-mer siRNAs reported previously [Sim, A.C.N., Luhur, A., Tan, T.M.C., Chow, V.T.K., Poh, C.L., 2005. RNA interference against Enterovirus 71 infection. Virology 341, 72-79]. The viral inhibitory effects lasted 72 h post-infection and there was no adverse off-target silencing effect. Gene silencing by 29-mer shRNAs targeted at the 3D(pol) region (sh-3D) was the most effective, achieving 91% viral inhibition. Further evaluation found that no enhanced inhibitory effects were observed when sh-3D was cotransfected with each of the other two candidates. This study showed an improvement in triggering RNAi using the more potent 29-mer shRNAs, indicating its therapeutic potential against EV71.     

Antiviral activity of lactoferrin against canine herpesvirus

Lactoferrin (LF) is an iron-binding protein that is found in milk and other mammalian secretions. We found that bovine lactoferrin (bLF) inhibited both the in vitro infection and replication of canine herpesvirus (CHV) in Madin-Darby canine kidney (MDCK) cells. Incubation of CHV with bLF prevented subsequent infection of MDCK cells. Furthermore, proteins from CHV-infected MDCK cells were resolved by SDS–PAGE, and then bLF CHV-binding proteins were identified by far Western blotting. We demonstrated that the anti-CHV activity of bLF was due to its interaction with CHV as well as with MDCK cells. Both the apo- and holo-forms of bLF inhibited virus multiplication independently of their iron-withholding properties. We also demonstrated that human LF had anti-CHV activity. Our findings suggest that LF could be effective in dogs to provide protection against CHV infection.     

Design, synthesis, and structure-activity relationship of pyridyl imidazolidinones: a novel class of potent and selective human enterovirus 71 inhibitors

When skeletons of Win compounds were used as templates, computer-assisted drug design led to the identification of a novel series of imidazolidinone derivatives with significant antiviral activity against enterovirus 71 (EV 71), the infection of which had resulted in about 80 fatalities during the 1998 epidemic outbreak in Taiwan. In addition to inhibiting all the genotypes (A, B, and C) of EV 71 in the submicromolar to low micromolar range, compounds 1 and 8 were extensively evaluated against a variety of viruses, showing potent activity against coxsackievirus A9 (IC(50) = 0.47-0.55 microM) and coxsackievirus A24 (IC(50) = 0.47-0.55 microM) as well as moderate activity against enterovirus 68 (IC(50) = 2.13 microM) and echovirus 9 (IC(50) = 2.6 microM). Our SAR studies revealed that imidazolidinone analogues with an aryl substituent at the para position of the phenoxyl ring, such as compounds 20, 21, 27, 57, 58, and 61, in general exhibited the highest activity against EV 71. Among them, compound 20 and its corresponding hydrochloride salt 57, in terms of potency and selectivity index, appear to be the most promising candidates in this series for further development of anti-EV-71 agents. Preliminary results of the study on the mode of action by a time-course experiment suggest that test compounds 1 and 8 can effectively inhibit the virus replication at the early stages, referring to virus attachment or uncoating. This indicates that the surface protein may be the target for this type of compounds.     

Antiviral activities of extracts and selected pure constituents of Ocimum basilicum

1. Ocimum basilicum (OB), also known as sweet basil, is a well known medicinal herb in traditional Chinese medicine preparations. In the present study, extracts and purified components of OB were used to identify possible antiviral activities against DNA viruses (herpes viruses (HSV), adenoviruses (ADV) and hepatitis B virus) and RNA viruses (coxsackievirus B1 (CVB1) and enterovirus 71 (EV71)). 2. The results show that crude aqueous and ethanolic extracts of OB and selected purified components, namely apigenin, linalool and ursolic acid, exhibit a broad spectrum of antiviral activity. Of these compounds, ursolic acid showed the strongest activity against HSV-1 (EC50 = 6.6 mg/L; selectivity index (SI) = 15.2), ADV-8 (EC50 = 4.2 mg/L; SI = 23.8), CVB1 (EC50 = 0.4 mg/L; SI = 251.3) and EV71 (EC50 = 0.5 mg/L; SI = 201), whereas apigenin showed the highest activity against HSV-2 (EC50 = 9.7 mg/L; SI = 6.2), ADV-3 (EC50 = 11.1 mg/L; SI = 5.4), hepatitis B surface antigen (EC50 = 7.1 mg/L; SI = 2.3) and hepatitis B e antigen (EC50 = 12.8 mg/L; SI = 1.3) and linalool showed strongest activity against AVD-II (EC50 = 16.9 mg/L; SI = 10.5). 3. No activity was noted for carvone, cineole, beta-caryophyllene, farnesol, fenchone, geraniol, beta-myrcene and alpha-thujone. 4. The action of ursolic acid against CVB1 and EV71 was found to occur during the infection process and the replication phase. 5. With SI values greater than 200, the potential use of ursolic acid for treating infection with CVB1 and EV71 merits further investigation.     

BNIP3 deletion ameliorated enterovirus 71 infection-induced hand,foot and mouth disease via inhibiting apoptosis,autophagy, and inflammation in mice

Bcl2/adenovirus E1B protein-interacting protein 3 (BNIP3) plays a key role in cellular response to stress by regulating apoptosis and selective autophagy. The present study aimed to determine the effects of BNIP3 on enterovirus (EV) 71 infection-induced hand, foot and mouth disease (HFMD), and the apoptosis, autophagy and inflammatory in mice and SH-SY5Y human neuroblastoma cell line. Neonatal BALB/c mice were injected with EV 71 strain to induce the HFMD. Western blotting and ELISA were used to measure the protein expression and cytokine levels. The BNIP3 mRNA and protein levels in the brain were increased in EV 71-infected mice. By contrast, the BNIP3-knockout (KO) mice with EV 71 infection had higher health score and survival rate. BNIP3 deletion reversed the increase of cleaved-caspase 3, cleaved-caspase 8, Bax, LC3 II and LC3 II/LC3 I levels, and the decrease of Bcl2 and Bcl2/Bax and LC3 I levels in the brain of mice with EV 71 infection. The EV 71 infection-induced increase of tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1β, IL-6, interferon (IFN)-α and IFN-γ levels were inhibited in BNIP3-KO mice. BNIP3 knockdown with small interfering RNA (siRNA) inhibited the EV 71 infection-induced the increases of apoptosis, autophagy and inflammatory factors in SH-SY5Y cells. BNIP3 overexpression further facilitated the EV 71 infection-induced increase of these inflammatory factors in SH-SY5Y cells. These results demonstrated that BNIP3 deletion ameliorated EV 71 infection-induced HFMD via inhibiting apoptosis, autophagy and inflammation in mice. BNIP3 may be a therapeutic target for HFMD.     

Antiviral Activity of Hederasaponin B from Hedera helix against Enterovirus 71 Subgenotypes C3 and C4a

Enterovirus 71 (EV71) is the predominant cause of hand, foot and mouth disease (HFMD). The antiviral activity of hederasaponin B from Hedera helix against EV71 subgenotypes C3 and C4a was evaluated in vero cells. In the current study, the antiviral activity of hederasaponin B against EV71 C3 and C4a was determined by cytopathic effect (CPE) reduction method and western blot assay. Our results demonstrated that hederasaponin B and 30% ethanol extract of Hedera helix containing hederasaponin B showed significant antiviral activity against EV71 subgenotypes C3 and C4a by reducing the formation of a visible CPE. Hederasaponin B also inhibited the viral VP2 protein expression, suggesting the inhibition of viral capsid protein synthesis.These results suggest that hederasaponin B and Hedera helix extract containing hederasaponin B can be novel drug candidates with broad-spectrum antiviral activity against various subgenotypes of EV71.     

基于转录组测序的EV71感染RD细胞的基因表达变化研究

目的　基于转录组测序技术探讨肠道病毒71型（EV71）感染对人横纹肌肉瘤（RD）细胞自噬和免疫的影响及其作用机制。方法　用不同剂量EV71感染RD细胞确定感染复数（MOI）。在MOI=1时选择不同时间点（0、0.5、1、1.5、3、6、9和12 h）共8组进行后续实验。倒置显微镜观察细胞形态和数量变化;蛋白免疫印迹（Western blot）检测蛋白表达情况;运用转录组测序技术分析细胞功能及基因表达的变化;高内涵细胞成像技术观察细胞内蛋白表达情况。结果　倒置显微镜可见细胞在病毒感染3 h由梭形贴壁逐渐变为圆形悬浮,细胞数量相比0 h明显减少（P＜0.05）。Western blot结果显示细胞在感染后6 h、9 h、12 h,病毒在RD细胞内已经表达病毒衣壳蛋白1（VP-1）,感染后9 h自噬相关蛋白LC-3Ⅱ/LC-3Ⅰ与0 h组相比明显升高（P＜0.01）。转录组测序结果显示EV71感染RD细胞后3 h细胞内基因表达变化达到峰值,上调基因1 199个,下调基因126个。KEGG分析提示改变的信号通路分子大多与自噬和免疫相关。高内涵细胞成像显示细胞在感染后数量减少,细胞内自噬蛋白平均荧光强度增加（P＜0.05）。结论　EV71感染RD细胞通过自噬相关信号通路促进病毒在细胞内完成复制和组装。     

RNA干扰技术抗肠道病毒71型复制

目的肠道病毒71型(Enterovirus 71,EV 71)是手足口病(hand,foot and mouth disease,HFMD)主要的病原体之一。研究RNA干扰技术抑制肠道病毒71型复制。方法以RNA干扰技术(RNAi)为干预手段,抑制病毒EV 71在人横纹肌肉瘤(rhabdomyosarcoma,RD)细胞中的复制。通过Western blot、Real-time PCR和病毒滴度3种方法验证,其抑制效果体现在感染细胞内部病毒RNA,病毒蛋白质的表达水平和培养基上清中子代病毒颗粒的数量上。结果靶向病毒基因组5'UTR和VP2的siRNAs具有明显的病毒抑制效应。结论此研究证实RNAi方法具有特异性抑制病毒复制的潜能和可行性。     

复方一枝蒿颗粒对防治手足口病的体内外药效学研究

目的 通过观察复方一枝蒿颗粒在体内外对肠道病毒的抑制作用,评价复方一枝蒿颗粒防治手足口病的药效作用。方法 采用复方一枝蒿颗粒的3岁儿童临床2倍、等倍和1/2倍剂量分别对肠道病毒EV71 H株感染的Babl/c乳鼠手足口病动物模型进行预防性给药和治疗性给药,通过观察乳鼠感染的严重程度、死亡数,来计算动物死亡率、死亡保护率和生命延长率;采用细胞病变的CPE法观察复方一枝蒿颗粒对肠道病毒EV71 H株,BrCr株,柯萨奇病毒B3、B4、B5株的抑制作用;采用real time PCR法观察复方一枝蒿颗粒对肠道病毒EV71 BrCr株在RD细胞中增殖的抑制作用。结果 复方一枝蒿颗粒对肠道病毒EV71 H株感染的乳鼠模型具有明显的治疗作用,其可降低感染程度和死亡率,具有明显的死亡保护和延长生命的作用;复方一枝蒿颗粒在无明显毒性的浓度下,对肠道病毒EV71 H株、BrCr株具有明显的抑制作用,其能明显抑制EV71 BrCr株在细胞内的增殖。结论 复方一枝蒿颗粒在体内外对手足口病均具有较好的治疗作用,其作用机制可能与抑制肠道病毒在细胞内增殖有关。     

微量细胞培养法在手足口病毒研究中的应用

目的建立应用于手足口病毒分离和对手足口EV71病毒抑制药物筛选的微量细胞培养方法。方法比较微量细胞培养结合real-timePCR和直接应用real-timePCR两种方法检测手足口病毒的差别,并应用微量细胞培养采用细胞病变效应法和MTT分析法,观察利巴韦林对EV71病毒的抑制作用。结果经微量细胞培养后EV71、CA16阳性检出率为90%和80%,但PE阴性样本20%检测为EV71阳性;利巴韦林浓度为0.4、0.2、0.1mg/mL时对EV71病毒有抑制作用,抑制率分别为30.28%、28.09%和29.16%。结论微量细胞培养在手足口病毒分离检测和对其抑制作用药物筛选研究中具有可行性,且其具有操作简便、省时省力等优点。     

Curcumin inhibits the replication of enterovirus 71 in vitro

Human enterovirus 71 (EV71) is the main causative pathogen of hand, foot, and mouth disease (HFMD) in children. The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades, and no vaccine and effective antiviral medicine are available. Curcumin has been used as a traditional medicine for centuries to treat a diversity of disorders including viral infections. In this study, we demonstrated that curcumin showed potent antiviral effect again EV71. In Vero cells infected with EV71, the addition of curcumin significantly suppressed the synthesis of viral RNA, the expression of viral protein, and the overall production of viral progeny. Similar with the previous reports, curcumin reduced the production of ROS induced by viral infection. However, the antioxidant property of curcumin did not contribute to its antiviral activity, since N-acetyl-l-cysteine, the potent antioxidant failed to suppress viral replication. This study also showed that extracellular signal-regulated kinase (ERK) was activated by either viral infection or curcumin treatment, but the activated ERK did not interfere with the antiviral effect of curcumin, indicating ERK is not involved in the antiviral mechanism of curcumin. Unlike the previous reports that curcumin inhibited protein degradation through ubiquitin–proteasome system (UPS), we found that curcumin had no impact on UPS in control cells. However, curcumin did reduce the activity of proteasomes which was increased by viral infection. In addition, the accumulation of the short-lived proteins, p53 and p21, was increased by the treatment of curcumin in EV71-infected cells. We further probed the antiviral mechanism of curcumin by examining the expression of GBF1 and PI4KB, both of which are required for the formation of viral replication complex. We found that curcumin significantly reduced the level of both proteins. Moreover, the decreased expression of either GBF1 or PI4KB by the application of siRNAs was sufficient to suppress viral replication. We also demonstrated that curcumin showed anti-apoptotic activity at the early stage of viral infection. The results of this study provide solid evidence that curcumin has potent anti-EV71 activity. Whether or not the down-regulated GBF1 and PI4KB by curcumin contribute to its antiviral effect needs further studies.KEY WORDS: Curcumin, Enterovirus 71, Viral replication, GBF1, PI4KB, Ubiquitin–proteasome system, ApoptosisAbbreviations: CVB, coxsackieviurs B; DCFH-DA, dichloro-dihydro-fluorescein diacetate; ERK, extracellular signal-regulated kinase; EV71, enterovirus 71; GBF1, Golgi brefeldin A resistant guanine nucleotide exchange factor 1; GEF, guanine nucleotide exchange factor; HBV, hepatitis B virus; HCV, hepatitis C virus; HFMD, hand, foot, and mouth disease; HIV, human immunodeficiency virus; HPV, human papillomavirus; NAC, N-acetyl-l-cysteine; PARP-1, poly(ADP-ribose) polymerase; PGC-1α, peroxisome proliferator-activated receptor-gamma co-activator 1 alpha; p.i., post-infection; PI4KB, phosphatidylinositol 4-kinase class III catalytic subunit β; PI4P, phosphatidylinositol 4-phosphate; ROS, reactive oxygen species; siRNA, small interfering RNA; SLLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; UPS, ubiquitin–proteasome system     

IGF-I receptor signaling pathway is involved in the neuroprotective effect of genistein in the neuroblastoma SK-N-SH cells

Genistein, an isoflavone naturally found in soy products, displays estrogenic properties. Our previous study clearly demonstrated that genistein can activate the insulin-like growth factor-I receptor (IGF-IR) signaling pathway in human breast cancer MCF-7 cells. The present study aims to test the hypothesis that the IGF-I receptor signaling pathway is involved in the neuroprotective effects of genistein in neuroblastoma SK-N-SH cells. Our results revealed that pretreatment with genistein resulted in an enhancement in the survival of human neuroblastoma SK-N-SH cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. 6-OHDA arrested the cells at G(0)G(1) phase and prevented S phase entry. Genistein pretreatment could reverse the cytostatic effect of 6-OHDA on cell cycle. The decreased mitochondrial membrane potential induced by 6-OHDA could be also reversed by genistein pretreatment. These effects could be completely blocked by co-treatment with JB-1, which is the specific antagonist of the IGF-I receptor. Furthermore, genistein pretreatment restored the 6-OHDA-induced up-regulation of Bax and down-regulation of Bcl-2 mRNA and protein expression. Genistein treatment alone could significantly increase the phosphorylation level of MEK and induce ERE luciferase activity. Co-treatment with IGF-I could enhance the effect of genistein on cell proliferation and MEK phosphorylation. This study provides the first evidence that genistein has neuroprotective effects against 6-OHDA-induced neurotoxicity in SK-N-SH cells and activation of the IGF-I receptor signaling pathway might be involved in actions of genistein.