Timosaponin A III promotes cisplatin sensitivity in A549/DDP cells via JAK-2/STAT3/PD-L1 signaling pathway / 中国药理学通报

Aim To explore the mechanism of timosaponin A III increasing cisplatin sensitization of non-small cell lung cancer cisplatin-resistant A549/DDP cells. Methods The cell proliferation was detected by CCK8,the cell apoptosis was detected by Annexin V/ PI,and the cell cycle distribution was measured by PI. The effect of the combined drug on cell proliferation was detected by cell clone formation. The mRNA expression levels of C-myc, cysteinyl aspartate-specific protease-3 (caspase-3) were detected by reverse transcription polymerase chain reaction (RT-PCR). Western blot and Flow Cytometry were performed for the mechanism study. Results The results of CCK8 and clone formation showed that the timosaponin AM group significantly increased the sensitivity of cisplatin to A549/DDP cells. Compared with the cisplatin group a-lone,the combination of timosaponin AM and cisplatin significantly elevated the apoptosis rate of A549/DDP cells (P < 0.05) , induced cell cycle arrest in G

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.

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.

The enhancing immune response and anti-inflammatory effects of Anemarrhena asphodeloides extract in RAW 264.7 cells.

BACKGROUND: Anemarrhena asphodeloides has been widely used in traditional medicine for thousands of years; it has been reported to improve learning and memory, and to reduce inflammation. However, the role of A. asphodeloides in enhancing the immune response has remained unclear. PURPOSE: This study aimed to evaluate the effect of A. asphodeloides extract (AA-Ex) on enhancing the immune response in macrophages and to identify the active compounds causing these effects. STUDY DESIGN/METHODS: To determine the enhancing immune response of AA-Ex and its active compounds, cell proliferation and cell cycle of RAW 264.7 cells were analyzed by MTS assay and flow cytometry. The gene expression of p53, p27, cyclin D2, and cyclin E2 was measured by real-time PCR. To evaluate the anti-inflammatory effects of AA-Ex and its active compounds, the production of nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines was analyzed by Griess reagent, flow cytometry, and real-time PCR. The phosphorylation of p38, c-Jun N-terminal kinase, inhibitory kappa B alpha, and p65 was examined by western blot analysis. RESULTS: AA-Ex increased cell proliferation by extending the cell cycle S-phase; timosaponin B and timosaponin B-II affected cell proliferation and the cell cycle as active compounds of A. asphodeloides. Next, we determined that A. asphodeloides displayed anti-inflammatory effects, including the inhibition of the production of NO, ROS, and pro-inflammatory cytokines through the suppression of mitogen-activated protein kinase and nuclear factor kappa B phosphorylation downstream of the toll-like receptor 4 signaling pathway. Moreover, we identified that timosaponin B and timosaponin B-II were the active compounds for these effects. CONCLUSION: Our results suggest that A. asphodeloides promotes the immune response and has anti-inflammatory effects. Moreover, timosaponin B and B-II played important roles as the active compounds of A. asphodeloides in enhancing the immune and anti-inflammatory responses in this model.

Therapeutic Potential of the Rhizomes of Anemarrhena asphodeloides and Timosaponin A-III in an Animal Model of Lipopolysaccharide-Induced Lung Inflammation.

Investigations into the development of new therapeutic agents for lung inflammatory disorders have led to the discovery of plant-based alternatives. The rhizomes of Anemarrhena asphodeloides have a long history of use against lung inflammatory disorders in traditional herbal medicine. However, the therapeutic potential of this plant material in animal models of lung inflammation has yet to be evaluated. In the present study, we prepared the alcoholic extract and derived the saponin-enriched fraction from the rhizomes of A. asphodeloides and isolated timosaponin A-III, a major constituent. Lung inflammation was induced by intranasal administration of lipopolysaccharide (LPS) to mice, representing an animal model of acute lung injury (ALI). The alcoholic extract (50-200 mg/kg) inhibited the development of ALI. Especially, the oral administration of the saponin-enriched fraction (10-50 mg/kg) potently inhibited the lung inflammatory index. It reduced the total number of inflammatory cells in the bronchoalveolar lavage fluid (BALF). Histological changes in alveolar wall thickness and the number of infiltrated cells of the lung tissue also indicated that the saponin-enriched fraction strongly inhibited lung inflammation. Most importantly, the oral administration of timosaponin A-III at 25-50 mg/kg significantly inhibited the inflammatory markers observed in LPS-induced ALI mice. All these findings, for the first time, provide evidence supporting the effectiveness of A. asphodeloides and its major constituent, timosaponin A-III, in alleviating lung inflammation.

Timosaponin A-III inhibits oncogenic phenotype via regulation of PcG protein BMI1 in breast cancer cells.

Polycomb group (PcG) protein BMI1 is an important regulator of oncogenic phenotype and is often overexpressed in several human malignancies including breast cancer. Aberrant expression of BMI1 is associated with metastasis and poor prognosis in cancer patients. At present, therapy reagents that can efficiently inhibit the expression of BMI1 are not very well known. Here, we report that Timosaponin A-III (TA-III), a steroidal saponin obtained from the rhizomes of an herb, Anemarrhena asphodeloides, strongly inhibits expression of BMI1 in breast cancer cells. Treatment of breast cancer cells with TA-III resulted in inhibition of oncogenic phenotypes such as proliferation, migration and invasion, and induction of cellular senescence. Inhibition of these oncogenic phenotypes was accompanied by downregulation of BMI1 expression and histone posttranslational modification activity of PRC1. The mechanistic analysis of TA-III-induced inhibition of oncogenic activity and BMI1 expression suggests that downregulation of c-Myc mediates TA-III effect on BMI1. We further show that exogenous BMI1 overexpression can overcome TA-III-induced inhibition of oncogenic phenotypes. We also show that TA-III induces expression of tumor suppressive miR-200c and miR-141, which are negatively regulated by BMI1. In summary, our data suggest that TA-III is a potent inhibitor of BMI1 and that it can be successfully used to inhibit the growth of tumors where PcG protein BMI1 and PcG activities are upregulated.

Therapeutic Potential of the Rhizomes of Anemarrhena asphodeloides and Timosaponin A-III in an Animal Model of Lipopolysaccharide-Induced Lung Inflammation

Investigations into the development of new therapeutic agents for lung inflammatory disorders have led to the discovery of plant-based alternatives. The rhizomes of Anemarrhena asphodeloides have a long history of use against lung inflammatory disorders in traditional herbal medicine. However, the therapeutic potential of this plant material in animal models of lung inflammation has yet to be evaluated. In the present study, we prepared the alcoholic extract and derived the saponin-enriched fraction from the rhizomes of A. asphodeloides and isolated timosaponin A-III, a major constituent. Lung inflammation was induced by intranasal administration of lipopolysaccharide (LPS) to mice, representing an animal model of acute lung injury (ALI). The alcoholic extract (50–200 mg/kg) inhibited the development of ALI. Especially, the oral administration of the saponin-enriched fraction (10–50 mg/kg) potently inhibited the lung inflammatory index. It reduced the total number of inflammatory cells in the bronchoalveolar lavage fluid (BALF). Histological changes in alveolar wall thickness and the number of infiltrated cells of the lung tissue also indicated that the saponin-enriched fraction strongly inhibited lung inflammation. Most importantly, the oral administration of timosaponin A-III at 25–50 mg/kg significantly inhibited the inflammatory markers observed in LPS-induced ALI mice. All these findings, for the first time, provide evidence supporting the effectiveness of A. asphodeloides and its major constituent, timosaponin A-III, in alleviating lung inflammation.

Induction of mitochondria-dependent apoptosis in HepG2 human hepatocellular carcinoma cells by timosaponin A-III.

Timosaponin A-III (TSA-III), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. However, the molecular mechanism underlying the anticancer activity of TSA-III has not been fully elucidated. In this study, the apoptotic effects of TSA-III were investigated in HepG2 cells. Treatment with TSA-III significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis in HepG2 cells. This induction was associated with increased fluorescence intensity of Annexin V-FITC, activation of caspases, and altered expression of inhibitor of apoptosis protein (IAP) family members. In addition, TSA-III mediated mitochondrial dysfunction with the release of HtrA2/Omi, Smac/Diablo, and cytochrome c. These findings suggest that TSA-III induces mitochondria-mediated and caspase-dependent apoptosis in HepG2 cells by altering expression of the IAP family. Thus, TSA-III could possibly be used to treat other types of cancer with similar pathologic mechanisms.

Simultaneous determination of timosaponin B-II and A-III in rat plasma by LC-MS/MS and its application to pharmacokinetic study.

A rapid, specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of timosaponin B-II (TB-II) and A-III (TA-III) in rat plasma. Plasma samples were pretreated via simple protein precipitation with acetonitrile and ginsenoside Rg2 was used as internal standard. Chromatographic separation was carried out on an Agilent XDB-C8 (150 mm × 2.1mm i.d., 5 µm) column by isocratic elution with acetonitrile-2 mmol/L ammonium acetate (55:45, v/v). The detection was performed on a Sciex API 4000(+) triple-quadrupole tandem mass spectrometer with TurboIonSpray ionization (ESI) inlet via the negative ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 3-3,000 ng/mL for TB-II and 0.3-3,000 ng/mL for TA-III, respectively. The intra- and inter-day precisions were less than 13.25%, and the accuracy ranged from 100.88% to 104.07% at three QC levels for both. The pharmacokinetic profiles of TB-II and TA-III in timosaponins (total timosaponin) at three dose levels (TB-II 150, 300, 600 mg/kg and TA-III 0.59, 1.17, 2.34 mg/kg, respectively) and in timosaponins-Huangbai alkaloids mixtures (1:1, 1:3, w/w, TB-II 300 mg/kg and TA-III 1.17 mg/kg) were studied for the first time in rats by this LC-MS/MS method. After single oral administration of timosaponins, mean Cmax and AUC0-t of TB-II and TA-III increased but non-proportional to the oral doses. When timosaponins-Huangbai alkaloids (1:1, 1:3, w/w) mixtures were administered, Cmax and AUC0-t of TB-II in the mixtures were obviously higher than the corresponding values in timosaponins at the same dose level.