Xin-Yi-Qing-Fei-Tang and its critical components reduce asthma symptoms by suppressing GM-CSF and COX-2 expression in RBL-2H3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine (TCM) XYQFT is composed of 10 herbs. According to the NHIRD, XYQFT is one of the top ten most commonly used TCM prescriptions for asthma treatment. AIM OF THE STUDY: The aim of this study was to explore whether XYQFT reduces asthma symptoms in a mouse model of chronic asthma and determine the immunomodulatory mechanism of mast cells. MATERIALS AND METHODS: BALB/c mice were intratracheally (it) stimulated with 40 µL (2.5 µg/µL) of Dermatophagoides pteronyssinus (Der p) once a week for 6 consecutive weeks and orally administered XYQFT at 1 g/kg 30 min before Der p stimulation. Airway hypersensitivity, inflammatory cells in the BALF and total IgE in the blood were assessed in mice. In addition, RBL-2H3 cells (mast cells) were stimulated with DNP-IgE, after which different concentrations of XYQFT were added for 30 min to evaluate the effect of XYQFT on the gene expression and degranulation of DNP-stimulated RBL-2H3 cells. After the compounds in XYQFT were identified using LC‒MS/MS, the PBD method was used to identify the chemical components that inhibited the expression of the GM-CSF and COX-2 genes in mast cells. RESULTS: The airway hypersensitivity assay demonstrated that XYQFT significantly alleviated Der p-induced airway hypersensitivity. Moreover, cell counting and typing of bronchoalveolar lavage fluid revealed a significant reduction in Der p-induced inflammatory cell infiltration with XYQFT treatment. ELISA examination further indicated a significant decrease in Der p-induced total IgE levels in serum following XYQFT administration. In addition, XYQFT inhibited the degranulation and expression of genes (IL-3, IL-4, ALOX-5, IL-13, GM-CSF, COX-2, TNF-α, and MCP-1) in RBL-2H3 cells after DNP stimulation. The compounds timosaponin AIII and genkwanin in XYQFT were found to be key factors in the inhibition of COX-2 and GM-CSF gene expression in mast cells. CONCLUSION: By regulating mast cells, XYQFT inhibited inflammatory cell infiltration, airway hypersensitivity and specific immunity in a mouse model of asthma. In addition, XYQFT synergistically inhibited the expression of the GM-CSF and COX-2 genes in mast cells through timosaponin AIII and genkwanin.

Anti-tumor effect and mechanisms of Timosaponin AIII across diverse cancer progression.

Timosaponin AIII (TAIII), a steroidal saponin derived from Anemarrhena asphodeloides Bunge, has gained attention for its versatile therapeutic properties. While well-established for its anti-inflammatory, antidepressant, and anticoagulant properties, emerging research highlights its potent anti-tumor capabilities. This review synthesizes recent findings on the intricate mechanisms and diverse functions of TAIII in cancer therapy, elucidating its impact on various tumor cells, encompassing the effects of TAIII on critical aspects of cancer progression, including metastasis, apoptosis, and autophagy. Additionally, the shared features of TAIII-induced anti-tumor activities, the factors contributing to the multifaceted anti-cancer activities of TAIII, and an exploration of the advantages and disadvantages associated with the regulation of multiple anti-tumor pathways by TAIII are discussed. Furthermore, the detailed regulation of signaling pathways is delineated and tailored to specific cancer types, providing a comprehensive overview of the potential development of TAIII as a promising anti-tumor agent.

Protective effect of Timosaponin AIII on Escherichia coli-induced endometritis in mice through inhibiting inflammatory response and regulating uterine microbiota structure.

Endometritis is a sort of general reproductive disease, which can lead to infertility in both humans and animals. Escherichia coli (E. coli) is recognised as the main bacterial etiology of endometritis among livestock and causes huge economic losses to dairy farming industry. Antibiotics are frequently used in the clinical treatment of endometritis; nevertheless, long-term use may result in adverse effects, including bacterial resistance and food safety concerns. TSAIII, one of the active pharmacological components of A. asphodeloides, has exhibited multiple biological activities, including anticancer, anti-angiogenesis, and anti-inflammatory properties. However, the protective effects of TSAIII in E. coli-challenged endometritis remain unclear. This study aimed to clarify the role of TSAIII in E. coli-induced endometritis in mice and elucidate its specific molecular mechanisms. In the present research, TSAIII treatment markedly alleviated the E. coli-induced uterine histopathological injury, and decreased myeloperoxidase (MPO) activity and pro-inflammatory cytokines levels in uterine tissue. Our results further demonstrated that TSAIII improved uterine epithelial barrier function by restoring the expressions of tight junction proteins. Furthermore, TSAIII administration noticeably suppressed the activation of the TLR4/NF-κB pathway and the NLRP3 inflammasome. Importantly, we found that TSAIII could regulate the uterine microbiota structure and composition in E. coli-induced mouse endometritis. In conclusion, these data demonstrate that treatment with TSAIII protects against E. coli-induced endometritis via modulating uterine microbiota composition, inhibiting TLR4/NF-κB pathway and NLRP3 inflammasome activation, in addition to improving uterine epithelial barrier function. Therefore, the results of this study provide a new therapeutic to potentially prevent endometritis.

The Potential Role of Timosaponin-AIII in Cancer Prevention and Treatment.

Cancer, as one of the leading causes of death worldwide, has challenged current chemotherapy drugs. Considering that treatments are expensive, alongside the resistance of tumor cells to anticancer drugs, the development of alternative medicines is necessary. Anemarrhena asphodeloides Bunge, a recognized and well-known medicinal plant for more than two thousand years, has demonstrated its effectiveness against cancer. Timosaponin-AIII (TSAIII), as a bioactive steroid saponin isolated from A. asphodeloides, has shown multiple pharmacological activities and has been developed as an anticancer agent. However, the molecular mechanisms of TSAIII in protecting against cancer development are still unclear. In this review article, we provide a comprehensive discussion on the anticancer effects of TSAIII, including proliferation inhibition, cell cycle arrest, apoptosis induction, autophagy mediation, migration and invasion suppression, anti-angiogenesis, anti-inflammation, and antioxidant effects. The pharmacokinetic profiles of TSAII are also discussed. TSAIII exhibits efficacy against cancer development. However, hydrophobicity and low bioavailability may limit the application of TSAIII. Effective delivery systems, particularly those with tissue/cell-targeted properties, can also significantly improve the anticancer effects of TSAIII.

Functional Axis of PDE5/cGMP Mediates Timosaponin-AIII-Elicited Growth Suppression of Glioblastoma U87MG Cells.

Glioblastoma (GBM) is the most aggressive brain tumor, with high mortality. Timosaponin AIII (TIA), a steroidal saponin isolated from the medicinal plant Anemarrhena asphodeloides Bge., has been shown to possess anticancer properties in various cancer types. However, the effect of TIA on GBM is unknown. In this study, we reveal that TIA not only inhibited U87MG in vitro cell growth but also in vivo tumor development. Moreover, we found that the cause of TIA-induced cell growth suppression was apoptosis. When seeking to uncover antitumor mechanisms of TIA, we found that TIA diminished the expression of cGMP-specific phosphodiesterase 5(PDE5) while elevating the levels of guanylate cyclases (sGCß), cellular cGMP, and phosphorylation of VASPser239. Following the knockdown of PDE5, PDE5 inhibitor tadalafil and cGMP analog 8-Bro-cGMP both inhibited cell growth and inactivated ß-catenin; we reason that TIA elicited an antitumor effect by suppressing PDE5, leading to the activation of the cGMP signaling pathway, which, in turn, impeded ß-catenin expression. As ß-catenin is key for cell growth and survival in GBM, this study suggests that TIA elicits its anti-tumorigenic effect by interfering with ß-catenin function through the activation of a PDE5/cGMP functional axis.

Timosaponin AIII promotes non-small-cell lung cancer ferroptosis through targeting and facilitating HSP90 mediated GPX4 ubiquitination and degradation.

Timosaponin AIII (Tim-AIII), a steroid saponin, exhibits strong anticancer activity in a variety of cancers, especially breast cancer and liver cancer. However, the underlying mechanism of the effects of Tim-AIII-mediated anti-lung cancer effects remain obscure. In this study, we showed that Tim-AIII suppressed cell proliferation and migration, induced G2/M phase arrest and ultimately triggered cell death of non-small cell lung cancer (NSCLC) cell lines accompanied by the release of reactive oxygen species (ROS) and iron accumulation, malondialdehyde (MDA) production, and glutathione (GSH) depletion. Interestingly, we found that Tim-AIII-mediated cell death was reversed by ferroptosis inhibitor ferrostatin-1 (Fer-1). Meanwhile, the heat shock protein 90 (HSP90) was predicted and verified as the direct binding target of Tim-AIII by SwissTargetPrediction (STP) and surface plasmon resonance (SPR) assay. Further study showed that Tim-AIII promoted HSP90 expression and Tim-AIII induced cell death was blocked by the HSP90 inhibitor tanespimycin, indicating that HSP90 was the main target of Tim-AIII to further trigger intracellular events. Mechanical analysis revealed that the Tim-AIII-HSP90 complex further targeted and degraded glutathione peroxidase 4 (GPX4), and promoted the ubiquitination of GPX4, as shown by an immunoprecipitation, degradation and in vitro ubiquitination assay. In addition, Tim-AIII inhibited cell proliferation, induced cell death, led to ROS and iron accumulation, MDA production, GSH depletion, as well as GPX4 ubiquitination and degradation, were markedly abrogated when HSP90 was knockdown by HSP90-shRNA transfection. Importantly, Tim-AIII also showed a strong capacity of preventing tumor growth by promoting ferroptosis in a subcutaneous xenograft tumor model, whether C57BL/6J or BALB/c-nu/nu nude mice. Together, HSP90 was identified as a new target of Tim-AIII. Tim-AIII, by binding and forming a complex with HSP90, further targeted and degraded GPX4, ultimately induced ferroptosis in NSCLC. These findings provided solid evidence that Tim-AIII can serve as a potential candidate for NSCLC treatment.

GPP-TSAIII nanocomposite hydrogel-based photothermal ablation facilitates melanoma therapy.

BACKGROUND: Photothermal therapy (PTT) is a promising cancer treatment, but its application is limited by low photoconversion efficiency. In this study, we aimed to develop a novel graphene oxide (GO)-based nanocomposite hydrogel to improve the bioavailability of timosaponin AIII (TSAIII) while maximizing PTT efficacy and enhancing the antitumor effect. METHODS: GO was modified via physical cross-linking with polyvinyl alcohol. The pore structure of the gel was adjusted by repeated freeze-thawing and the addition of polyethylene glycol 2000 to obtain a nanocomposite hydrogel (GPP). The GPP loaded with TSAIII constituted a GPP-TSAIII drug delivery system, and its efficacy was evaluated by in vitro cytotoxicity, apoptosis, migration, and uptake analyses, and in vivo antitumor studies. RESULTS: The encapsulation rate of GPP-TSAIII was 66.36 ± 3.97%, with slower in vitro release and higher tumor cell uptake (6.4-fold) compared to TSAIII. GPP-TSAIII in combination with PTT showed better bioavailability and antitumor effects in vivo than did TSAIII, with a 1.9-fold higher tumor suppression rate than the TSAIII group. CONCLUSIONS: GPP is a potential vehicle for delivery of TSAIII-like poor water-soluble anticancer drugs. The innovative PTT co-delivery system may serve as a safe and effective melanoma treatment platform for further anticancer translational purposes.

Blockage of Autophagy Increases Timosaponin AIII-Induced Apoptosis of Glioma Cells In Vitro and In Vivo.

Timosaponin AIII (TSAIII), a saponin isolated from Anemarrhena asphodeloides and used in traditional Chinese medicine, exerts antitumor, anti-inflammatory, anti-angiogenesis, and pro-apoptotic activity on a variety of tumor cells. This study investigated the antitumor effects of TSAIII and the underlying mechanisms in human glioma cells in vitro and in vivo. TSAIII significantly inhibited glioma cell viability in a dose- and time-dependent manner but did not affect the growth of normal astrocytes. We also observed that in both glioma cell lines, TSAIII induces cell death and mitochondrial dysfunction, consistent with observed increases in the protein expression of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, cytochrome c, and Mcl-1. TSAIII also activated autophagy, as indicated by increased accumulation of the autophagosome markers p62 and LC3-II and the autolysosome marker LAMP1. LC3 silencing, as well as TSAIII combined with the autophagy inhibitor 3-methyladenine (3MA), increased apoptosis in GBM8401 cells. TSAIII inhibited tumor growth in xenografts and in an orthotopic GBM8401 mice model in vivo. These results demonstrate that TSAIII exhibits antitumor effects and may hold potential as a therapy for glioma.

Timosaponin AIII Inhibits Migration and Invasion Abilities in Human Cervical Cancer Cells through Inactivation of p38 MAPK-Mediated uPA Expression In Vitro and In Vivo.

Cervical cancer is one of the most common gynecologic cancers globally that require novel approaches. Timosaponin AIII (TSAIII) is a steroidal saponin that displays beneficial effects in antitumor activities. However, the effect of TSAIII on human cervical cancer remains unknown. In this study, we found that TSAIII showed no influence on cell viability, cytotoxicity, cell cycle distribution and apoptosis induction in human cervical cancer cells. TSAIII was revealed to have a significant inhibitory effect on cell migration and invasion through the downregulation of invasion-related uPA expression and p38 MAPK activation in both human cervical cancer cells and cervical cancer stem cells (CCSCs), indicating that the p38 MAPK-uPA axis mediated the TSAIII-inhibited capacity of cellular migration and invasion. In a synergistic inhibition assay, a TSAIII plus p38 siRNA cotreatment revealed a greater inhibition of uPA expression, migration and invasion in human cervical cancer cells. In an immunodeficient mouse model, TSAIII significantly inhibited lung metastases from human cervical cancer SiHa cells without TSAIII-induced toxicity. These findings first revealed the inhibitory effects of TSAIII on the progression of human cervical cancer through its downregulation of p38 MAPK-uPA axis activation. Therefore, TSAIII might provide a potential strategy for auxiliary therapy in human cervical cancer.

Novel Timosaponin AIII-Based Multifunctional Liposomal Delivery System for Synergistic Therapy Against Hepatocellular Carcinoma Cancer.

INTRODUCTION: As high cholesterol level has been reported to be associated with cancer cell growth and cholesterol is vulnerable to oxidation, the conventional liposomes including cholesterol in the formulation seem to be challenged. Timosaponin AIII (TAIII), as a steroid saponin from Anemarrhena asphodeloides Bunge, possesses a similar structure with cholesterol and exhibits a wide range of antitumor activities, making it possible to develop a TAIII-based liposome where TAIII could potentially stabilize the phospholipid bilayer as a substitution of cholesterol and work as a chemotherapeutic drug as well. Meanwhile, TAIII could enhance the uptake of doxorubicin hydrochloride (DOX) in human hepatocellular carcinoma (HCC) cells and exhibit synergistic effect. Thus, we designed a novel thermally sensitive multifunctional liposomal system composed of TAIII and lipids to deliver DOX for enhanced HCC treatment. METHODS: The synergistic effects of DOX and TAIII were explored on HCC cells and the tumor inhibition rate of TAIII-based liposomes carrying DOX was evaluated on both subcutaneous and orthotopic transplantation tumor models. TAIII-based multifunctional liposomes were characterized. RESULTS: Synergistic HCC cytotoxicity was achieved at molar ratios of 1:1, 1:2 and 1:4 of DOX/TAIII. TAIII-based liposomes carrying a low DOX dose of 2 mg/kg exhibited significantly enhanced antitumor activity than 5 mg/kg of DOX without detected cardiotoxicity on both subcutaneous and orthotopic transplantation tumor models. TAIII-based liposomes were characterized with smaller size than cholesterol liposomes but exhibited favorable stability. Mild hyperthermia generated by laser irradiation accelerated the release of DOX and TAIII from liposomes at tumor site, and cell permeability of TAIII enhanced uptake of DOX in HCC cells. CONCLUSION: The innovative application of TAIII working as bilayer stabilizer and chemotherapeutic drug affords a stable multifunctional liposomal delivery system for synergistic therapy against HCC, which may be referred for the development of other types of saponins with similar property.

Biopharmaceutics and Pharmacokinetics of Timosaponin A-III by a Sensitive HPLC-MS/MS Method: Low Bioavailability Resulting from Poor Permeability and Solubility.

BACKGROUND: Timosaponin A-III is one of the most promising active saponins from Anemarrhena asphodeloides Bge. As an oral chemotherapeutic agent, there is an urgent need to clarify its biopharmaceutics and pharmacokinetics to improve its development potential. OBJECTIVE: This research explores the bioavailability of timosaponin A-III and clarifies its absorption and metabolism mechanisms by a sensitive and specific HPLC-MS/MS method. METHODS: Pharmacokinetics and bioavailability studies of timosaponin A-III were performed in Sprague- Dawley rats by oral (20 mg/kg) and intravenous administration (2 mg/kg). Control group was given the same volume of normal saline. The absorption of timosaponin A-III was investigated in a rat intestinal perfusion model in situ and a Caco-2 cell transport model in vitro. The metabolic rate of timosaponin A-III was determined in a rat liver microsome incubation system. RESULTS: After the oral administration, timosaponin A-III reached Cmax of 120.90 ± 24.97 ng/mL at 8 h, and the t1/2 was 9.94 h. The absolute oral bioavailability of timosaponin A-III was 9.18%. The permeability coefficients of timosaponin A-III in four intestinal segments ranged from 4.98 to 5.42 × 10-7 cm/s, indicating a difficult absorption. A strikingly high efflux transport of timosaponin A-III was found, PappBA 3.27 ± 0.64 × 10-6 cm/s, which was abolished by a P-gp inhibitor. Rat liver microsome incubation studies showed that timosaponin A-III could hardly be metabolized, with a t1/2 of over 12 h. In addition, the solubility test showed a low solubility in PBS solution, i.e. 30.58 µg/mL. CONCLUSION: Timosaponin A-III exhibited low oral bioavailability by oral and intravenous administration, which was probably caused by its low permeability and solubility. This study may provide a reference for its rational clinical use and further study on the pharmacology or toxicology of timosaponin A-III.

Pharmacological Activity, Pharmacokinetics, and Toxicity of Timosaponin AIII, a Natural Product Isolated From Anemarrhena asphodeloides Bunge: A Review.

Anemarrhena asphodeloides Bunge is a famous Chinese Materia Medica and has been used in traditional Chinese medicine for more than two thousand years. Steroidal saponins are important active components isolated from A. asphodeloides Bunge. Among which, the accumulation of numerous experimental studies involved in Timosaponin AIII (Timo AIII) draws our attention in the recent decades. In this review, we searched all the scientific literatures using the key word "timosaponin AIII" in the PubMed database update to March 2020. We comprehensively summarized the pharmacological activity, pharmacokinetics, and toxicity of Timo AIII. We found that Timo AIII presents multiple-pharmacological activities, such as anti-cancer, anti-neuronal disorders, anti-inflammation, anti-coagulant, and so on. And the anti-cancer effect of Timo AIII in various cancers, especially hepatocellular cancer and breast cancer, is supposed as its most potential activity. The anti-inflammatory activity of Timo AIII is also beneficial to many diseases. Moreover, VEGFR, X-linked inhibitor of apoptosis protein (XIAP), B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1), thromboxane (Tx) A2 receptor, mTOR, NF-κB, COX-2, MMPs, acetylcholinesterase (AChE), and so on are identified as the crucial pharmacological targets of Timo AIII. Furthermore, the hepatotoxicity of Timo AIII was most concerned, and the pharmacokinetics and toxicity of Timo AIII need further studies in diverse animal models. In conclusion, Timo AIII is potent as a compound or leading compound for further drug development while still needs in-depth studies.

Timosaponin AIII attenuates inflammatory injury in AGEs-induced osteoblast and alloxan-induced diabetic osteoporosis zebrafish by modulating the RAGE/MAPK signaling pathways.

BACKGROUND: Advanced glycation end products (AGEs) deposition causes inflammatory injury in osteoblasts and contributes to diabetic osteoporosis. The receptor for advanced glycation end product/mitogen-activated protein kinase pathway (RAGE/MAPK) signaling pathway is closely linked to the pathogenesis of diabetic osteoporosis. Timosaponin AIII, a steroidal saponin isolated from Anemarrhena asphodeloides Bunge (Asparagaceae), shows anti-inflammatory and anti-osteoporosis effects. PURPOSE: The present study was aimed to investigate the therapeutic effects of timosaponin AIII on diabetic osteoporosis and whether its effect is dependent on protecting osteoblasts against AGEs-induced injury via RAGE/MAPK signaling suppression. METHODS: An alloxan-induced diabetic osteoporosis zebrafish model was applied to investigate the effects of timosaponin AIII in vivo, and alendronate was used as a positive control. Moreover, related mechanisms were explored in primary rat osteoblasts. Molecular docking was applied to investigate the interactions between timosaponin AIII and RAGE. RESULTS: Timosaponin AIII treatment reversed alloxan-induced reduction in the mineralized area of the larvae head skeleton, accompanied by a decreased level of triglyceride and total cholesterol in the zebrafish. Additionally, AGEs significantly influenced RAGE expression, alkaline phosphatase activity, interleukin 1ß expression, interleukin 6 expression, and tumor necrosis factor-α expression, and increased cell apoptosis. Timosaponin AIII significantly downregulated AGEs-induced interleukin 1ß, interleukin 6, and tumor necrosis factor-α levels, and upregulated alkaline phosphatase and osteocalcin levels. Timosaponin AIII also significantly reduced the expression of RAGE and had additive effects on downstream P38, extracellular signal-regulated kinase and c-Jun N-terminal kinase in AGEs-induced osteoblast. Molecular docking predicted that hydrogen and hydrophobic interactions occurred between timosaponin AIII and RAGE. CONCLUSION: These data clarified that timosaponin AIII attenuates diabetic osteoporosis via a novel mechanism involved suppressing the RAGE/MAPK signaling pathway. Our finding highlights the potential value of timosaponin AIII as an anti-diabetic osteoporosis agent.

Identification of a novel BACE1 inhibitor, timosaponin A-III, for treatment of Alzheimer's disease by a cell extraction and chemogenomics target knowledgebase-guided method.

BACKGROUND: Rhizoma Anemarrhenae (RA) has been conventionally used for treatment of Alzheimer's disease (AD) in Traditional Chinese Medicine, and thus, the active components from RA can be screened. PURPOSE: This research aimed to identify the active components of RA and their targets and further clarify the molecular mechanisms underlying its anti-AD activity. METHODS: First, the potential active compounds from RA were screened by neurocyte extraction and micro-dialysis methods. Second, the potential targets were predicted by a chemogenomics target knowledgebase and further explored by surface plasmon resonance and enzyme activity assays. Third, the pharmacological effects were evaluated by employing APP/PS1 transgenic mice and SH-SY5Y-APP cells. ELISAs and Western blot analyses were used to evaluate the expression of key molecules in the amyloidogenic and NMDAR/ERK pathways. RESULTS: Timosaponin A-III (TA-III) was screened and identified as a potential active component for the anti-AD activity, and BACE1 was proven to be a potential high-affinity target. Enzyme kinetic analysis showed that TA-III had strong noncompetitive inhibitory activity against BACE1. The in vitro and in vivo assays indicated that TA-III had pharmacological effects through improving memory impairment, reducing Aß aggregation via the amyloidogenic pathway and preventing neuronal impairment through downregulating the NMDAR/ERK signaling pathway. CONCLUSION: TA-III targets BACE1 to reduce Aß aggregation through down-regulating the NMDAR/ERK pathway for treating AD.

Antiangiogenesis effect of timosaponin AIII on HUVECs in vitro and zebrafish embryos in vivo.

Timosaponin AIII (Timo AIII) is a natural steroidal saponin isolated from the traditional Chinese herb Anemarrhena asphodeloides Bge with proved effectiveness in the treatment of numerous cancers. However, whether Timo AIII suppresses tumor angiogenesis remains unclear. In the present study, we investigated the antiangiogenesis effects of Timo AIII and the underlying mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish embryos in vivo. We showed that treatment with Timo AIII (0.5-2 µM) partially disrupted the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) growth in transgenic zebrafish Tg(fli-1a: EGFP)y1. Timo AIII (0.5-4 µM) dose-dependently inhibited VEGF-induced proliferation, migration, invasion, and tube formation of HUVECs, but these inhibitory effects were not due to its cytotoxicity. We further demonstrated that Timo AIII treatment significantly suppressed the expression of VEGF receptor (VEGFR) and the phosphorylation of Akt, MEK1/2, and ERK1/2 in HUVECs. Timo AIII treatment also significantly inhibited VEGF-triggered phosphorylation of VEGFR2, Akt, and ERK1/2 in HUVECs. Moreover, we conducted RNA-Seq and analyzed the transcriptome changes in both HUVECs and zebrafish embryos following Timo AIII treatment. The coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc., which was consistent with the antiangiogenesis effects of Timo AIII in HUVECs and zebrafish embryos. We conclude that the antiangiogenesis effect of Timo AIII is mediated through VEGF/PI3K/Akt/MAPK signaling cascade; Timo AIII potentially exerts antiangiogenesis effect in cancer treatment.

Clinical evaluation of the safety and efficacy of a timosaponin A-III-based antiwrinkle agent against skin aging.

BACKGROUND: Timosaponin A-III (TA-III) is known to exist in the medicinal herb of Anemarrhena asphodeloides as one of major chemical components. AIMS: The photoprotective properties of TA-III on UVB-exposed HaCaT cells were evaluated on the antiwrinkle effects and skin safety in terms of clinical trial. METHODS: The level of matrix metalloproteinase (MMP)-1, tissue inhibitor of metalloproteinases (TIMPs), and pro-inflammatory cytokines were measured in HaCaT cells following UVB irradiation. To evaluate the clinical safety of an agent containing 0.25% of TA-III for use on human skin. Female subjects (n = 21) between the ages of 43 and 55 who met the criteria for subject selection were selected. They were beginning to form or had already formed wrinkles. RESULTS: UVB irradiation increased MMP-1 expression and pro-inflammatory cytokines. These increases were attenuated by TA-III pretreatment of UVB-exposed HaCaT cells. We found that the agent containing 0.25% of TA-III ameliorated skin wrinkling. A comparison between groups showed that wrinkle parameters were significantly reduced after 12 weeks of product use (P < 0.05). According to skin safety result, TA-III showed no dermatological toxicity was found in participants. CONCLUSIONS: In conclusion, TA-III could provide protection against photoaging and daily application of TA-III for 12 weeks significantly reduced signs of facial aging by limiting wrinkle formation.

Timosaponin AIII Induces G2/M Arrest and Apoptosis in Breast Cancer by Activating the ATM/Chk2 and p38 MAPK Signaling Pathways.

Timosaponin AIII (TAIII), a steroidal saponin, exerts potent anti-tumor activity in various cancers, especially breast cancer. However, the concrete molecular mechanisms of TAIII against breast cancer are still unclear. Here, we find that TAIII triggers DNA damage, leads to G2/M arrest, and ultimately induces apoptosis in breast cancer both in vitro and in vivo. TAIII induced G2/M phase arrest and apoptosis in MDA-MB-231 and MCF7 cells accompanied with down-regulation of CyclinB1, Cdc2 and Cdc25C. Further data showed that the ATM/Chk2 and p38 pathways were activated representing by up-regulated levels of p-H2A.X and p-p38, which indicated an induction of DNA damage by TAIII, leading to cell cycle arrest and apoptosis. The effects of TAIII were further confirmed by employing inhibitors of ATM and p38 pathways. In vivo, TAIII suppressed the growth of subcutaneous xenograft tumor without obvious toxicity, which indicated by Ki67 and TUNEL analysis. Data also showed that TAIII stimulated the ATM/Chk2 and p38 MAPK pathways in vivo, which in consistent with the effects in vitro. Hence, our data demonstrate that TAIII triggers DNA damage and activates ATM/Chk2 and p38 MAPK pathways, and then induces G2/M phase arrest and apoptosis in breast cancer, which provide theoretical evidence for TAIII utilized as drug against breast cancer.

Synergistic anticancer effects of timosaponin AIII and ginsenosides in MG63 human osteosarcoma cells.

BACKGROUND: Timosaponin AIII (TA3) is a steroidal saponin extracted from Anemarrhena asphodeloides. Here, we investigated the anticancer effects of TA3 in MG63 human osteosarcoma cells. TA3 attenuates migration and invasion of MG63 cells via regulations of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, which are involved with cancer metastasis in various cancer cells. TA3 reduced enzymatic activities and transcriptional expressions of MMP-2 and MMP-9 in MG63 cells. TA3 also inhibited Src, focal adhesion kinase, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38, ß-catenin, and cAMP response element binding signaling, which regulate migration and invasion of cells. TA3 induced apoptosis of MG63 cells via regulations of caspase-3, caspase-7, and poly(ADP-ribose) polymerase (PARP). Then, we tested several ginsenosides to be used in combination with TA3 for the synergistic anticancer effects. We found that ginsenosides Rb1 and Rc have synergistic effects on TA3-induced apoptosis in MG63 cells. METHODS: We investigated the anticancer effects of TA3 and synergistic effects of various ginseng saponins on TA3-induced apoptosis in MG63 cells. To test antimetastatic effects, we performed wound healing migration assay, Boyden chamber invasion assays, gelatin zymography assay, and Western blot analysis. Annexin V/PI staining apoptosis assay was performed to determine the apoptotic effect of TA3 and ginsenosides. RESULTS: TA3 attenuated migration and invasion of MG63 cells and induced apoptosis of MG63 cells. Ginsenosides Rb1 and Rc showed the synergistic effects on TA3-induced apoptosis in MG63 cells. CONCLUSIONS: The results strongly suggest that the combination of TA3 and the two ginsenosides Rb1 and Rc may be a strong candidate for the effective antiosteosarcoma agent.

Protective Effects of Timosaponin AIII against UVB-Radiation Induced Inflammation and DNA Injury in Human Epidermal Keratinocytes.

UVB radiation changes several photoaging pathway in the body, thereby prompting skin injury. Besides, chronic UVB radiation leads to photoaging, sustained immunosuppression, and photocarcinogenesis. We investigated the protective effect of Timosaponin AIII (TA-III), a naturally occurring steroidal saponin separated from Anemarrhena asphodeloides, against UVB-induced invasive properties of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDF). No cytotoxicity was observed up to 50 nM concentration of TA-III. Similarly, TA-III inhibited UVB-induced cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9) transcription level and protein expression in a dose-dependent manner at non-cytotoxic dose. Further, TA-III decreased UVB-induced invasion in primary skin cells. Additionally, TA-III suppressed UVB-stimulates mitogen-activated protein kinase (MAPK) signaling, activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) activation, thereby preventing the overexpression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and COX-2 in human epidermal keratinocytes cells. Furthermore, TA-III prevented UVB-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and activation of DNA repair enzymes and, cell cycle arrest genes like as proliferating cell nuclear antigen (PCNA), structural maintenance of chromosomes protein 1 (SMC1). This results support that understanding into the molecular action of TA-III, which can be useful for developing photoprotective agents.

Integrated pathological cell fishing and network pharmacology approach to investigate main active components of Er-Xian decotion for treating osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative damage to osteoblasts was a key factor in the development of osteoporosis. Er-Xian Decotion (EXD) is widely used in China for the treatment of osteoporosis, which has a variety of antioxidant active ingredients. EXD may be an important source of protection against oxidative damage in osteoblasts, but the anti-osteoporotic active components of EXD is currently unclear. AIM OF THE STUDY: This work established an effective and reliable drug screening method to find main active ingredients in EXD (M-EXD) that can protect osteoblasts against oxidative stress and achieve anti-osteoporosis effects. MATERIALS AND METHODS: H2O2-induced osteoblast cell fishing with UHPLC-QTOF/MS was firstly used to discover the potential active components from EXD. Afterword, the EXD compound-osteoporosis target network was constructed using network pharmacology, thus potentially anti-osteoporosis ingredients were founded, and their combination were defined as the M-EXD. Finally, pharmacology effects of M-EXD was evaluated by ovariectomized rats, prednisolone induced-zebrafish and H2O2-induced osteoblasts. RESULTS: 40 candidate active ingredients in EXD were initially screened out via pathological cell fishing. According to network pharmacology result, M-EXD consisted of 13 ingredients since they had a close relationship with 65 osteoporosis-related targets. Pharmacological evaluation showed that M-EXD significantly ameliorated oxidative stress in H2O2-induced osteoblast model, evidently reversed the activity of ALP, ROS, GSH-px, NO and MDA compared with the model group. M-EXD showed better anti-oxidative activities than individual ingredients, presenting obvious synergetic effects. In osteoporosis rat and zebrafish models, M-EXD also demonstrated good anti-osteoporotic properties by mitigating the osteoporosis bone loss and increasing serum bone morphogenetic protein 2, and reversing osteocalcin expression in bone tissue. It significantly ameliorated oxidative stress in the in-vivo models. Moreover, M-EXD and EXD showed similar anti-osteoporotic and anti-oxidative properties, while the rest components of EXD had no satisfactory anti-osteoporotic efficacy. CONCLUSIONS: Our work successfully identified the main active components in EXD, which could represent the efficacy of EXD on treating osteoporosis, and meanwhile, it also provided an effective strategy to investigate active ingredients from natural medicines, which might be helpful for drug development and application.