Acteoside Treatment Research Articles

Neuroprotective effects of acteoside in a glaucoma mouse model by targeting Serta domain-containing protein 4.

To explore the therapeutic effect and main molecular mechanisms of acteoside in a glaucoma model in DBA/2J mice. Proteomics was used to compare the differentially expressed proteins of C57 and DBA/2J mice. After acteoside administration in DBA/2J mice, anterior segment observation, intraocular pressure (IOP) monitoring, electrophysiology examination, and hematoxylin and eosin staining were used to analyze any potential effects. Immunohistochemistry (IHC) assays were used to verify the proteomics results. Furthermore, retinal ganglion cell 5 (RGC5) cell proliferation was assessed with cell counting kit-8 (CCK-8) assays. Serta domain-containing protein 4 (Sertad4) mRNA and protein expression levels were measured by qRT-PCR and Western blot analysis, respectively. Proteomics analysis suggested that Sertad4 was the most significantly differentially expressed protein. Compared with the saline group, the acteoside treatment group showed decreased IOP, improved N1-P1 wave amplitudes, thicker retina, and larger numbers of cells in the ganglion cell layer (GCL). The IHC results showed that Sertad4 expression levels in DBA/2J mice treated with acteoside were significantly lower than in the saline group. Acteoside treatment could improve RGC5 cell survival and reduce the Sertad4 mRNA and protein expression levels after glutamate injury. Sertad4 is differentially expressed in DBA/2J mice. Acteoside can protect RGCs from damage, possibly through the downregulation of Sertad4, and has a potential use in glaucoma treatment.

Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

Acteoside palliates d-galactose induced cognitive impairment by regulating intestinal homeostasis.

Acteoside, an important phenylethanol glycoside, is the main active component in Osmanthus fragrans flower. Our previous study found that acteoside showed high antiaging effect but its absorption rate was low. We speculated acteoside palliated aging-related cognitive impairment before being absorbed, that was intestinal homeostasis underlie the antiaging effect of acteoside. In this study, acteoside was confirmed to palliate cognitive impairment in d-galactose induced aging mice. Acteoside treatment dramatically reduced oxidative stress, alleviated intestinal inflammation, restored intestinal mucosal barrier, rebuilt gut microbiome structure and upregulated gut microbiome metabolites short-chain fatty acids (SCFAs) and amino acids (AAs). Furthermore, antibiotic treatment revealed that the antiaging ability of acteoside was abolished in microbiota depleted mice, which offered direct evidence for the essential role of gut microbiota in the attenuation of cognitive impairment of acteoside. Together, our study indicated that acteoside palliated cognitive impairment by regulating intestinal homeostasisand acteoside intake might be a promising nutritional intervention in prevention of neurodegenerative diseases.

Acteoside attenuates RSV-induced lung injury by suppressing necroptosis and regulating metabolism

Background: Necroptosis and inflammation are closely related to the pathogenesis of respiratory syncytial virus (RSV). Acteoside (AC), a natural phenylpropanoid glycoside from Kuding Tea, has significant anti-RSV effect. However, the roles of AC on RSV-induced lung necroptosis and inflammation are yet to be elucidated. Methods: The effects of AC were investigated in BALB/c mice and A549 cells. Lung histopathology was observed through H&E staining. The viral titer was assessed via plaque assay. The RSV-F expression was determined by RT-qPCR and immunohistochemistry assay. The levels of cytokines were detected by ELISA and RT-qPCR. The necroptosis rate and mitochondrial membrane potential were evaluated via flow cytometry. The expressions of HMGB1/NF-κB and RIP1/RIP3/MLKL/PGAM5/DRP1 were detected by western blot. Additionally, untargeted metabolomics was conducted to investigate the metabolic profiles and related metabolic pathways via Gas Chromatography-Mass Spectrometry. Results: The results showed that compared with the RSV-infected group, AC treatment significantly attenuated lung pathological damage, virus replication, and cytokines levels. AC also alleviated RSV-induced necroptosis and mitochondrial dysfunction in vitro and in vivo. Moreover, AC treatment down-regulated the expression of HMGB1, p-Iκbα/Iκbα, p-p65/p65, RIP1, RIP3, MLKL, PGAM5, and DRP1. Furthermore, metabolomic analyses suggested that the perturbations in major metabolites of AC therapy were related to variations in amino acid and energy metabolism. Conclusion: Our findings validated the beneficial effects of AC in suppressing necroptosis and regulating metabolism, suggesting AC may be a new drug candidate for RSV infection.

Acteoside promotes B cell-derived IL-10 production and ameliorates autoimmunity.

IL-10-producing regulatory B (Breg) cells are well recognized for maintaining immune tolerance. The impaired Breg cell function with decreased IL-10-producing capacity has been found in autoimmune diseases, such as rheumatoid arthritis, lupus, and primary Sjogren's syndrome (pSS). However, seldom therapeutic agents targeting Breg cells are available to treat those autoimmune diseases. Here, we showed that acteoside (AC), a caffeoyl phenylethanoid glycoside from a medicinal herb Radix Rehmanniae, could promote IL-10 production from both human and murine B cells via critically regulating the TLR4/PI3K axis. Moreover, TLR4 was found increased in Breg cells from mice with experimental SS (ESS), a mouse model that recapitulates human pSS. Thus, B cells from the ESS mice were susceptible to AC treatment, showing higher IL-10-producing capacity than those from naïve controls. In addition, AC treatment also promoted the production of IL-10 from TLR4+ CXCR4+ plasma cells of ESS mice. Notably, we found that AC was able to enter lymphoid organs upon oral administration. AC treatment effectively increased IL-10+ B cells in ESS mice and ameliorated disease pathology accompanied by reduced T effector cells, including Th17 and T follicular helper cells in the ESS mice. In conclusion, AC could promote Breg cell function and attenuate ESS pathology in vivo, which may be a promising drug candidate for treating pSS and other autoimmune diseases.

Anti-infammatory and antioxidant activities of acteoside isolated from Acanthus ilicifolius var. xiamenensis

Acanthus ilicifolius var. xiamenensis (Acanthaceae), a mangrove found in southeastern China, is an herb with strong antiinflammatory property. Phytochemical study of the mangrove showed that the plant has a high content of phenylethanol glycoside acteoside (AC). In this research, the anti-inflammatory activity of acteoside on dextran sodium sulphate (DSS)-mediated mouse ulcerative colitis model were studied. For DSS- mediated colitis in mice, AC can reduce weight loss and DAI score in UC mice, suppress colon shortening and alleviate colon pathological injury. Moreover, AC treatment notably up-regulates IL-10, down-regulates the levels of IL-1β and TNF-α, and inhibits the protein expression of JAK2/STAT3, NF-κB p65, IKKα/β and IKB of colon. In addition, after AC treatment, the level of MDA and NO in colonic tissue were remarkably decreased, while the levels of GSH, SOD, and Nrf2 and HO-1 protein expression levels were significantly increased. These results indicate that AC can activate the Nrf2 signaling pathway by inhibiting the JAK/STAT, iNOS/eNOS and NF-κB signaling cascades, enhance the intestinal barrier function, and effectively reduce DSS-induced UC in mice.

Acteoside protects retinal ganglion cells from experimental glaucoma by activating the PI3K/AKT signaling pathway via caveolin 1 upregulation.

BackgroundGlaucoma is the second leading cause of blindness in the world and is characterized by optic neuropathy and degeneration of retinal ganglion cells (RGCs). Our preliminary research found that acteoside can inhibit autophagy-induced apoptosis of RGCs via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. However, it is unclear how acteoside activates the PI3K/AKT signaling pathway to prevents RGCs autophagic apoptosis.MethodsAnimal and cell models were used in this study. Hematoxylin-eosin staining revealed pathological histology of retinas. The number of RGCs in retinas was counted using immunofluorescence. Malondialdehyde and superoxide dismutase were determined using enzyme-linked immunosorbent assay kits. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were used to detect cell apoptosis. The reactive oxygen species was determined by the Flow cytometry. The proteins were determined by Western blot.ResultsThe results showed that acteoside treatment significantly reduced RGC loss, oxidative stress, and autophagy, thereby preventing glaucoma exacerbation. Acteoside reversed caveolin 1 (Cav1) expression and PI3K/AKT signaling activation, according to Western blot results. Cav1 knockdown also reversed acteoside’s effects on RGC loss, PI3K/AKT signaling pathway activation, autophagy and oxidative stress. Notably, 3-methyladenine, a PI3K inhibitor, reversed the effects of acteoside and Cav1 overexpression on RGC loss, oxidative stress, and autophagy.ConclusionsThese finding imply that acteoside alleviates RGC loss and oxidative stress by activating of the PI3K/AKT signaling pathway by upregulating Cav1.

Acteoside exerts immunomodulatory effects on dendritic cells via aryl hydrocarbon receptor activation and ameliorates Th2-mediated allergic asthma by inducing Foxp3+ regulatory T cells

Dendritic cells (DCs) are professional antigen-presenting cells that play a key role in directing T-cell responses and are involved in the pathogenesis of allergic asthma. Acteoside, an active phenylethanoid glycoside, is widely distributed in many medicinal plants. Herein, we explored the immunomodulatory effects of acteoside on bone marrow-derived DCs in vitro, and further investigated the immunosuppressive ability of acteoside to manipulate T helper type 2 (Th2)-mediated allergic asthma in mice. Following lipopolysaccharide activation, 50 μM of acteoside significantly reduced the production of proinflammatory mediators, including interleukin (IL)-12 and tumor necrosis factor (TNF)-α, whereas it enhanced secretion of the anti-inflammatory cytokine, IL-10, by DCs. However, these effects of acteoside on DCs were reversed by pretreatment with CH223191, an aryl hydrocarbon receptor (AhR) antagonist. Additionally, coculture of acteoside-treated DCs with CD4+ T cells promoted the generation of forkhead box P3-positive (Foxp3+) regulatory T cells (Tregs) via AhR activation. Using a murine asthma model, our results demonstrated that oral administration of 50 mg/kg of acteoside decreased levels of Th2-type cytokines, such as IL-4, IL-5, and IL-13, whereas the level of IL-10 and the frequency of CD4+Foxp3+ Tregs were augmented. Moreover, acteoside treatment markedly inhibited the elevated serum level of ovalbumin-specific immunoglobulin E, attenuated the development of airway hyperresponsiveness, and reduced inflammatory cell counts in bronchoalveolar lavage fluid. Additionally, histological results reveled that acteoside ameliorated pulmonary inflammation in asthmatic mice. Taken together, these results indicated that acteoside exhibits immunomodulatory effects on DCs and plays an anti-inflammatory role in the treatment of allergic asthma.

Effects of acteoside on the expressions of MCP-1 and TGF-β1 in the diabetic nephropathy mice

Objective: Immune inflammatory cells and cytokines play an important role in the occurrence and development of diabetic nephropathy (DN). Acteoside has been reported to regulate the inflammation and immune response. The study aims to investigate the effects of acteoside on the expressions of MCP-1 and TGF-β1 on nephropathy in diabetic mice. Methods: C57BL/6J mice in the model group were given a single intraperitoneal injection of STZ (150 mg/kg). Model mice were divided randomly into two groups: 5 without treatment, 5 treated with acteoside. After continuous administration for 8 weeks, serum, urine, and kidney tissue were collected, then, ralated biochemical parameters, pathological characteristics and MCP-1 and TGF-β1 mRNA or protein were detected. The NRK-52E cells were divided into three groups as follows: the normal control group (NC group), the high glucose group (HS group), the high glucose+acteoside group (HS+ACT group). The expressions of MCP-1 and TGF-β1 in the mRNA and protein levels were assessed with RT-PCR, western blot and ELISA. Results: The expressions of MCP-1 and TGF-β1 were significantly enhanced in DN group and dramatically reduced after acteoside treatment. Compared with those in NC group, the expressions of MCP-1 and TGF-β1 in NRK-52E cell of HS group were significantly enhanced, while both were significantly decreased in HS+ACT group compared with HS group. Conclusion: Our findings indicate that Acteoside has protective effects on DN via inhibiting the expressions of MCP-1 and TGF-β1.

Acteoside ameliorates experimental autoimmune encephalomyelitis through inhibiting peroxynitrite-mediated mitophagy activation

Multiple sclerosis (MS) is an inflammatory disease in central nervous system (CNS) with limited therapeutic drugs. In the present study, we explored the anti-inflammatory/neuroprotective properties of Acteoside (AC), an active compound from medicinal herb Radix Rehmanniae (RR), and neuroprotective effects of AC on MS pathology by using an experimental autoimmune encephalomyelitis (EAE) model. We tested the hypothesis that AC could alleviate EAE pathogenesis through inhibiting inflammation and ONOO−-mediated mitophagy activation in vivo and in vitro. The results showed that AC treatment effectively ameliorated neurological deficit score and postponed disease onset in the EAE mice. AC treatment inhibited inflammation/demyelination, alleviated peripheral activation and CNS infiltration of encephalitogenic CD4+ T cells and CD11b+ activated microglia/macrophages in the spinal cord of EAE mice. Meanwhile, AC treatment reduced ONOO− production, down-regulated the expression of iNOS and NADPH oxidases, and inhibited neuronal apoptotic cell death and mitochondrial damage in the spinal cords of the EAE mice. Furthermore, AC treatment decreased the ratio of LC3-II to LC3-I in mitochondrial fraction, and inhibited the translocation of Drp1 to the mitochondria. In vitro studies further proved that AC possessed strong ONOO− scavenging capability and protected the neuronal cells from nitrative cytotoxicity via suppressing ONOO−-mediated excessive mitophagy. Taken together, Acteoside could be a potential therapeutic agent for multiple sclerosis treatment. The suppression of ONOO−-induced excessive mitophagy activation could be one of the critical mechanisms contributing to its anti-inflammatory and anti-demyelinating properties.

Protective Effect of Acteoside on Ovariectomy-Induced Bone Loss in Mice.

Acteoside, an active phenylethanoid glycoside compound isolated from herbs of Cistanche, was chosen for the investigation of anti-osteoporotic effect on postmenopausal osteoporosis by using an ovariectomized (OVX) mice model. The results from in vivo experiments showed that after daily oral administration of acteoside (20, 40, and 80 mg/kg body weight/day) for 12 weeks, bone mineral density and bone biomechanical properties of OVX mice were greatly enhanced, with significant improvement in bone microarchitecture. Furthermore, biochemical parameters of bone resorption markers as well as bone formation index, including tartrate-resistant acid phosphatase, cathepsin K, deoxypyridinoline, alkaline phosphatase, and bone gla-protein, were ameliorated by acteoside treatment, whereas the body, uterus, and vagina wet weights were seemingly not impacted by acteoside administration. Acteoside significantly affected osteoclastogenesis by attenuating nuclear factor kappa B (NF-κB) and stimulating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signal pathways through down-regulated levels of tumor-necrosis factor receptor-associated factor 6 (TRAF6), receptor activator of nuclear factor kappa B ligand (RANKL), RANK, NFKBIA, IκB kinase β, nuclear factor of activated T-cells c2 (NFAT2), and up-regulated expressions of PI3K, AKT, and c-Fos. Accordingly, the current research validated our hypothesis that acteoside possesses potent anti-osteoporotic properties and may be a promising agent for the prevention of osteoporosis in the future.

Acteoside inhibits autophagic apoptosis of retinal ganglion cells to rescue glaucoma-induced optic atrophy.

BackgroundGlaucoma is the world's second biggest cause of blindness, and patients progressively lose their eyesight. The current clinical treatment for glaucoma involves controlling intraocular pressure with drugs or surgery; however, some patients still progressively lose their eyesight. This treatment is also similar to the treatment of traumatic optic neuropathy. Thus, saving retinal ganglion cells (RGCs) from apoptosis is essential.MethodsThe role of Acteoside on autophagy modulation in the 661 W cell line.ResultsIn this study, we first find that Acteoside inhibits autophagy, Rapamycin alleviates this inhibition and the PI3K inhibitor, 3‐MA or LY294002, synergistically promotes it. In a mechanistic study, we find that Optineurin (OPTN) mediates Acteoside regulation of autophagy. OPTN overexpression or knockdown activates or inhibits autophagy, respectively. OPTN is inhibited by autophagy inhibitors, such as Acteoside and 3‐MA and is promoted by the autophagy activator, Rapamycin. Meanwhile, PI3K and AKT are elevated by Acteoside and 3‐MA and inhibited by Rapamycin. Finally, we find that Acteoside inhibits apoptosis in parallel to autophagy and that this inhibition is also mediated by OPTN.ConclusionIn summary, we conclude that Acteoside inhibits autophagy‐induced apoptosis in RGCs through the OPTN and PI3K/AKT/mTOR pathway, and glaucoma patients may benefit from Acteoside treatment alone or in combination with other autophagy inhibitors.

Effect of Acteoside as a Cell Protector to Produce a Cloned Dog.

Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 μM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects.

Acteoside inhibits irradiation-mediated decreases in the viability and DNA synthesis of MC3T3-E1 cells

Therapeutic irradiation can cause bone loss, whereas antioxidant supplementation is considered to attenuate irradiation-mediated damages. This study examined whether or not acteoside inhibits irradiation-mediated changes in viability and proliferation of MC3T3-E1 cells. X-ray radiation at >4 Gy not only decreased cell viability and DNA synthesis in the cells, but also increased intracellular levels of reactive oxygen species (ROS) and phosphorylated p66Shc protein. Irradiation at 8Gy also decreased intracellular levels of reduced glutathione (GSH) and induced G1 phase arrest of cell cycle progression with the attendant increase of p21 induction. Pretreatment with acteoside inhibited the irradiation-mediated decreases in viability and DNA synthesis by restoring the radiation-mediated changes in the levels of ROS, GSH, p21, and p-p66Shc to the untreated control levels. These inhibitory activities of acteoside were greater than that of a synthetic antioxidant compound or N-acetyl cysteine did. Collectively, acteoside treatment may prevent irradiation-induced oxidative damages to osteoblasts.

Upregulation of Heme Oxygenase-1 by Acteoside Through ERK and PI3 K/Akt Pathway Confer Neuroprotection Against Beta-Amyloid-Induced Neurotoxicity

Our previous study has shown that acteoside, an antioxidative phenylethanoid glycoside, protect against beta-amyloid (Aβ)-induced cytotoxicity in vitro. However, the precise protective mechanisms remains unclear. Heme oxygenase-1 (HO-1) is a crucial factor in the response to oxidative injury, protecting neurons against Aβ-induced injury. In the present study we examined to determine whether acteoside upregulates HO-1 expression, and thereby protects PC12 cells against Aβ-induced cell death. It was revealed that acteoside is an activator of Nrf2 and inducer of HO-1 expression. We showed that acteoside increased HO-1 expression in vitro and in vivo. Acteoside treatment resulted in nuclear translocation of the transcription factor NF-E2-related factor 2 (Nrf2). Acteoside activated both ERK and PI3 K/Akt, and treatments with the specific ERK inhibitor PD98059, the PI3 K inhibitor LY294002, and the specific Nrf2 siRNA suppressed the acteoside-induced HO-1 expression. The HO-1 inhibitor ZnPP, PD98059, and LY294002 markedly abolished the neuroprotective effect of acteoside against Aβ-induced neurotoxicity. Taken together, these results demonstrate that acteoside is an activator of Nrf2 and inducer of HO-1 expression. We also showed that acteoside increased HO-1 expression through activation of ERK and PI3 K/Akt signal pathways in vitro. Upregulation of HO-1 by acteoside may involve in the neuroprotection against Aβ-induced neurotoxicity.

Effect of acteoside on mesangial proliferation in rat anti-Thy 1 nephritis.

We investigated whether acteoside can inhibit mesangial matrix expansion or mesangial cell proliferation in mesangioproliferative anti-Thy 1 nephritis. Untreated control rats were compared to the rats treated with acteoside either during early (day 1 to 8) or late (day 4 to 12) period after the induction of anti-Thy 1 nephritis. The result showed that acteoside and prednisolone treatments (in either early or late period) significantly reduced proteinuria, mesangial matrix expansion (the index of matrix expansion) and mesangial proliferation as determined by the number of proliferating nuclear cell antigen (PCNA)-positive cells. Acteoside also reduced glomerular macrophage infiltration and ICAM-1 expression in glomeruli of anti-Thy 1 nephritic rats. Furthermore, acteoside treatment markedly increased the activities of matrix metaloproteinases (MMP) in glomeruli. These results suggest that acteoside can inhibit mesangial cell proliferation and extracellular matrix overproduction by either inhibiting ICAM-1 expression or increasing activities of MMP.

Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G0/G1 phase and differentiation into monocyte

We investigated the in vitro effects of acteoside on the proliferation, cell cycle regulation and differentiation of HL-60 human promyelocytic leukemia cells. Acteoside inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner with an IC50, approximately 30 microM. DNA flow cytometric analysis indicated that acteoside blocked cell cycle progression at the G1 phase in HL-60 human promyelocytic leukemia cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent protein kinase (CDK)2, CDK6, cyclin D1, cyclin D2, cyclin D3 and cyclin E were reduced by acteoside, whereas the steady-state level of CDK4 was unaffected. The protein and mRNA levels of CDK inhibitors (cyclin-dependent kinase inhibitors), such as p21(CIP1/WAF1) and p27(KIP1), were gradually increased after acteoside treatment in a time-dependent manner. In addition, acteoside markedly enhanced the binding of p21(CIP1/WAF1) and p27(KIP1) to CDK4 and CDK6, resulting in the reduction of CDK2, CDK4 and CDK6 activities. Moreover, the hypophosphorylated form of retinoblastoma increased, leading to the enhanced binding of protein retinoblastoma (pRb) and E2F1. Our results further suggest that acteoside is a potent inducer of differentiation of HL-60 cells based on biochemical activities and the expression level of CD14 cell surface antigen. In conclusion, the onset of acteoside-induced G1 arrest of HL-60 cells prior to the differentiation appears to be tightly linked to up-regulation of the p21(CIP1/WAF1) and p27(KIP1) levels and decreases in the CDK2, CDK4 and CDK6 activities. These findings, for the first time, reveal the mechanism underlying the anti-proliferative effect of acteoside on human promyelocytic HL-60 cells.