20(S)-Protopanaxadiol Exerts Antidepressive Effects in Chronic Corticosterone-Induced Rodent Animal Models as an Activator of Brain-Type Creatine Kinase.

20(S)-Protopanaxadiol (PPD) is one of the bioactive ingredients in ginseng and possesses neuroprotective properties. Brain-type creatine kinase (CK-BB) is an enzyme involved in brain energy homeostasis via the phosphocreatine-creatine kinase system. We previously identified PPD as directly bound to CK-BB and activated its activity in vitro. In this study, we explored the antidepressive effects of PPD that target CK-BB. First, we conducted time course studies on brain CK-BB, behaviors, and hippocampal structural plasticity responses to corticosterone (CORT) administration. Five weeks of CORT injection reduced CK-BB activity and protein levels and induced depression-like behaviors and hippocampal structural plasticity impairment. Next, a CK inhibitor and an adeno-associated virus-targeting CKB were used to diminish CK-BB activity or its expression in the brain. The loss of CK-BB in the brain led to depressive behaviors and morphological damage to spines in the hippocampus. Then, a polyclonal antibody against PPD was used to determine the distribution of PPD in the brain tissues. PPD was detected in the hippocampus and cortex and observed in astrocytes, neurons, and vascular endotheliocytes. Finally, different PPD doses were used in the chronic CORT-induced depression model. Treatment with a high dose of PPD significantly increased the activity and expression of CK-BB after long-term CORT injection. In addition, PPD alleviated the damage to depressive-like behaviors and structural plasticity induced by repeated CORT injection. Overall, our study revealed the critical role of CK-BB in mediating structural plasticity in CORT-induced depression and identified CK-BB as a therapeutic target for PPD, allowing us to treat stress-related mood disorders.

Triphenylphosphonium-linked derivative of hecogenin with enhanced antiproliferative activity: Design, synthesis, and biological evaluation.

Hecogenin (HCG), a steroidal sapogenin, possesses good antitumor properties. However, the application of HCG for cancer treatment has been hindered primarily by its moderate potency. In this study, we incorporated triphenylphosphonium cation (TPP+) at the C-3 and C-12 positions through different lengths of alkyl chains to target mitochondria and enhance the efficacy and selectivity of the parent compound. Cytotoxicity screening revealed that most of the target compounds exhibited potent antiproliferative activity against five human cancer cell lines (MKN45, A549, HCT-116, MCF-7, and HepG2). Structure-activity relationship studies indicated that the TPP+ group significantly enhanced the antiproliferative potency of HCG. Among these compounds, 3c demonstrated remarkable potency against MKN45 cells with an IC50 value of 0.48 µM, significantly more effective than its parent compound HCG (IC50 > 100 µM). Further investigations into the mechanism of action revealed that 3c induced apoptosis of MKN45 cells through the mitochondrial pathway. In a zebrafish xenograft model, 3c inhibited the proliferation of MKN45 cells. Overall, these results suggest that 3c, with potent antiproliferative activity, may serve as a valuable scaffold for developing new antitumor agents.

Protopanaxatriol activates EGFR and HER2 to strengthen the molecules of skin protection in human keratinocytes.

BACKGROUND: Protopanaxatriol (PPT) is an important ginsenoside produced by ginseng, a tonic plant used in many areas. PPT has beneficial effects against many disease states including inflammation, diabetes, and cancer. However, PPT's protective effects on skin integrity have been rarely studied. Previously, we reported that PPT can maintain skin moisture through activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) pathways. However, the cellular targets for enhancing skin moisturizing effects via PPT are still unknown. PURPOSE: We wanted to identify the upstream targets of PPT on upregulating moisturizing factor (HAS-2) expression. STUDY DESIGN: We investigated which upstream proteins can be directly stimulated by PPT to modulate NF-κB, MAPKs and other signaling cascades. Then, the targeted proteins were overexpressed to check the relationship with HAS-2. Next, the cellular thermal shift assay (CETSA) was conducted to check the relationship between targeted proteins and PPT. METHODS: A human keratinocyte HaCaT were employed to measure the levels of moisturizing factors and the signaling proteins activated by PPT. Transfection conditions were established with DNA constructs expressing epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and their mutants prepared by site-directed mutagenesis. Further investigation on molecular mechanisms was conducted by RT-PCR, luciferase reporter gene assay, CETSA, or Western blot. RESULTS: We found that PPT can activate the phosphorylation of EGFR and HER2. These stimulations caused Src phosphorylation, which resulted in the activation of phosphoinositide 3-kinases (PI3K)/pyruvate dehydrogenase kinase 1 (PDK1)/protein kinase B (AKT)/NF-κB and MAPKs signaling cascades. Additionally, EGFR and HER2 activation resulted in phosphorylation of signal transducer and activator of transcription 3 (STAT3) and calcium/calmodulin-dependent protein kinase II (CaMKII). This induced the AMP-activated protein kinase alpha (AMPKα) signaling pathway. Additionally, PPT blocked peroxisome proliferator activated receptor gamma (PPARγ), which also contributed to the phosphorylation of Src. CONCLUSION: Overall, we first found that PPT offers excellent protection of the skin barrier and hydrogen supply in keratinocytes. Moreover, growth factor receptors such as EGFR and HER2 were revealed to be central enzymes to be directly targeted by PPT. These results suggest a potentially valuable role as a cosmetic ingredient.

Comparison of interactions between alpha-lactalbumin and three protopanaxadiol ginsenosides: Impacts on the structure and antitumor properties.

In this research, interactions between α-lactalbumin (ALA) and three protopanaxadiol ginsenosides [20(S)-Rg3, 20(S)-Rh2, and 20(S)-PPD] were compared to explore the effects of similar ligand on structure and cytotoxicity of ALA. Multi-spectroscopy revealed the binding between ALA and ginsenoside changed the conformation of ALA, which related to different structures and solubility of ligands. Scanning electron microscope illustrated that all ALA-ginsenoside complexes exhibited denser structures via hydrophobic interactions. Additionally, the cytotoxic experiments confirmed that the cytotoxicity of ginsenoside was enhanced after binding with ALA. Molecular docking showed all three ginsenosides were bound to the sulcus depression region of ALA via hydrogen bonding and hydrophobic interaction. Furthermore, molecular dynamics simulation elucidated the precise binding sites and pertinent system properties. Among all three composite systems, 20(S)-Rh2 had optimal binding affinity. These findings enhanced understanding of the synergistic utilization of ALA and ginsenosides as functional ingredients in food, medicine, and cosmetics.

Synthesis, antioxidant, antiproliferative activity, molecular docking and DFT studies of novel isoxazole derivatives of diosgenin, a steroidal sapogenin from Dioscorea deltoidea.

Diosgenin [25R-spirost-5-en-3ß-ol], isolated from Dioscorea deltoidea was used as a starting material for synthesizing its various isoxazole derivatives. A library of fifteen isoxazole analogues (DG1-DG15) were synthesised via modification at the C-3 hydroxyl group. The resulting analogues were fully characterized by spectral techniques and evaluated for their antioxidant and anticancer activity against four breast cancer cell lines; MDA-MB-231, MDA-MB-468, MCF-7, and 4 T1, using MTT assay. Molecular docking studies were carried out for all analogues with EGFR protein (PDB id: 6LUD) to check their activity by inhibiting EGFR protein, which is an effective strategy for cancer cell death. Furthermore, DFT studies were carried out for four analogues. Among all analogues, compound DG6 and DG9 showed the highest scavenging activity and compound DG9 exhibited a maximum cytotoxic effect on the MDA-MB-468 and MCF-7 cell lines with an IC50 value of 6.25 µg/mL and 6.81 µg/mL, while compound DG5 was the least potent (IC50 25.89 µg/mL). Molecular docking results revealed that DG8 and DG9 afforded the highest binding energy of -14.33 and - 14.71 kcal/mol, respectively for the target EGFR protein. These results demonstrate the potential of diosgenin analogues as drug candidates for breast cancer therapy. Furthermore, DFT studies revealed that the molecules are more polarizable and have smaller energy gap between their HOMO and LUMO orbitals, the smallest being of DG9 (3.221 eV) and hence are more reactive.

Anti-inflammatory Activity: In silico and In vivo of Sapogenins Present in Agave brittoniana subsp. brachypus (Trel.).

BACKGROUND: Agave brittoniana subsp. brachypus is an endemic plant of Cuba, which contains different steroidal sapogenins with anti-inflammatory effects. This work aims to develop computational models which allow the identification of new chemical compounds with potential anti-inflammatory activity. METHODS: The in vivo anti-inflammatory activity was evaluated in two rat models: carrageenaninduced paw edema and cotton pellet-induced granuloma. In each study, we used 30 Sprague Dawley male rats divided into five groups containing six animals. The products isolated and administrated were fraction rich in yuccagenin and sapogenins crude. RESULTS: The obtained model, based on a classification tree, showed an accuracy value of 86.97% for the training set. Seven compounds (saponins and sapogenins) were identified as potential antiinflammatory agents in the virtual screening. According to in vivo studies, the yuccagenin-rich fraction was the greater inhibitor of the evaluated product from Agave. CONCLUSION: The evaluated metabolites of the Agave brittoniana subsp. Brachypus showed an interesting anti-inflammatory effect.

Diosgenin, a Natural Steroidal Sapogenin, Alleviates the Progression of Diabetic Retinopathy in Diabetic Mice.

BACKGROUND/AIM: Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and a major cause of blindness in working-age adults. Diosgenin (DG), a natural steroidal sapogenin extracted from fenugreek seeds and wild yam roots, has hypolipidemic, hypoglycemic, anticancer, and anti-inflammatory properties. Given its pharmacological effects, we speculated that DG may be a promising treatment for DR. Therefore, this study was aimed at evaluating the effectiveness of DG in preventing or slowing DR progression in a mouse model (+Leprdb/+Leprdb strain) of type 2 diabetes (T2D). MATERIALS AND METHODS: DG (5.0 mg/kg body weight) or phosphate-buffered saline (PBS) was administered to 8-week-old T2D mice via oral gavage daily for 24 weeks. Paraffin-embedded eye tissues from the mice were collected and stained with hematoxylin and eosin to evaluate retinal histopathology. Apoptosis-related proteins BCL2-associated X (Bax), B-cell lymphoma 2 (Bcl-2), and cleaved caspase-3 were evaluated by western blotting of mouse retinas. RESULTS: Body weight was slightly reduced in the DG-treated group; however, glucose levels were not markedly different between the DG- and PBS-treated groups. Total retinal thickness, thickness of the photoreceptor and outer nuclear layers, and loss of ganglion cells significantly improved in the retina of the DG-treated T2D mice compared with those in the PBS-treated T2D mice. Cleaved caspase-3 level significantly decreased in the retina of the DG-treated T2D mice. Conclusion: DG alleviates DR pathology and exerts a protective effect on the T2D mouse retina. The inhibitory effects of DG on DR may involve mechanisms of the anti-apoptotic pathway.

Hecogenin and its derivates: A pharmacology review.

Despite the several uses of drugs from natural compounds in the pharmaceutical industry, new molecules have been discovered and associated with pharmacological activities over the years. Hecogenin, a steroidal saponin, has been the subject of several studies due to reports of pharmacological activities. This study combines the articles published to date that show the pharmacological activity and the mechanism of action of hecogenin, its acetate, and its derivates. This compilation shows that the compounds can act in different pathologies that affect many systems of the human body. They showed pharmacological properties in inflammation, mediating cytokines, cells, and environment. Also, it participated in tumoral processes by pathways like PPGARγ, ERK½, and MMP-2 and showed antimicrobial effects against organisms like Candida and Aedes aegypti's larvae. This review indicates that continuing studies with these molecules are essential once they have the potential to be a future drug.

Molecular docking and dynamic simulation approach to decipher steroidal sapogenins (genus Trillium) derived agonists for glucocorticoid receptor.

Steroidal sapogenins (SS) are structural analogues of steroidal drugs, which are frequently used for the treatment of several diseases including reproductive, malignancies, neurological, and inflammation-related diseases. The glucocorticoid receptor (GR) is a nuclear receptor that regulates development, metabolism, and inflammation, in response to steroidal ligands. Therefore, GR is considered as a potential therapeutic target for steroidal agents to the treatment of inflammation-related diseases. We hypothesized that SS may act as an agonist for GR due to structural similarity with corticosteroids. In this study, we carried out in silico screening of various SS from the genus Trillium to check their potential as an agonist for GR. Our data suggest that out of 42 SS, only 7 molecules have interacted with GR. However, molecular mechanics with generalized Born and surface area (MM-GBSA) analysis revealed that only two SS (SS 38 and SS 39) molecules bind favorably to GR. Among these, SS 38 (docking score: -9.722 Kcal/mol and MM-GBSA ΔGbind: -50.192 Kcal/mol) and SS 39 (docking score: -11.20 Kcal/mol and MM-GBSA ΔGbind: -58.937 Kcal/mol) have best docking and MM-GBSA scores. Molecular dynamics (MD) simulation studies of SS 38, SS 39, and dexamethasone-GR complex revealed that both SS shows hydrogen bonding and hydrophobic interaction with GR over the 120 ns simulation with mild fluctuations. The current study suggests that SS 38 and SS 39 may be further explored as a potential agonist to treat several disease conditions mediated by GR.

20(S)-Protopanaxadiol from Panax ginseng Induces Apoptosis and Autophagy in Gastric Cancer Cells by Inhibiting Src.

20(S)-protopanaxadiol (PPD), a metabolite of Panax ginseng, has multiple pharmacological properties. However, the effects of PPD against human gastric cancer have not been elucidated. Our purpose in this study was to investigate if PPD has anticancer effects against human gastric cancer in vitro. Cell viability, migration, clone formation, and invasion were assessed to explore the effects of PPD on cancer cells. PI and annexin V staining as well as immunoblotting were employed to determine if PPD-induced apoptosis and autophagy of MKN1 and MKN45 cells. The target of PPD was identified using immunoblotting, overexpression analysis, and flow cytometric analysis. PPD exhibited significantly suppressed cell viability, migration, colony formation, and invasion. Phosphorylation of Src and its down-stream effectors were inhibited by PPD. PPD-enhanced apoptosis and autophagy in a dose- and time-dependent manner by inhibiting Src. Collectively, our results demonstrate that PPD induces apoptosis and autophagy in gastric cancer cells in vitro by inhibiting Src.

Transgenic Rice Seed Extracts Exert Immunomodulatory Effects by Modulating Immune-Related Biomarkers in RAW264.7 Macrophage Cells.

Protopanaxadiol (PPD), a native active triterpenoid present in Panax ginseng, has been reported to exert immune-related effects. We previously created PPD-producing transgenic rice by introducing the P. ginseng protopanaxadiol synthase and dammarenediol-II synthase genes into Dongjin rice. In the present study, the seeds of the T4 generation of this transgenic rice were tested for their immunomodulatory effects in RAW264.7 macrophage cells. Treatment with transgenic rice seed extract in RAW264.7 cells (i) significantly enhanced nitric oxide (NO) production in a dose-dependent manner without any cytotoxicity (up to 100 µg/mL), (ii) upregulated the expression of immune-related genes and increased production of the inflammation mediator prostaglandin E2 (PGE2), and (iii) activated nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) by promoting the phosphorylation of NF-κB p65, p38 MAPK, and c-Jun N-terminal protein kinase (JNK). In lipopolysaccharide (LPS)-treated RAW264.7 cells used to mimic the inflammation condition, treatment with transgenic rice seed extract significantly reduced NO production, proinflammatory cytokine expression, and PGE2 production, all of which are LPS-induced inflammation biomarkers, by inhibiting the phosphorylation of NF-κB p65, p38 MAPK, and JNK. Collectively, these results indicate that PPD-producing transgenic rice has immunomodulatory effects.

Production of Prosaikogenin F, Prosaikogenin G, Saikogenin F and Saikogenin G by the Recombinant Enzymatic Hydrolysis of Saikosaponin and their Anti-Cancer Effect.

The saponins of Bupleurum falcatum L., saikosaponins, are the major components responsible for its pharmacological and biological activities. However, the anti-cancer effects of prosaikogenin and saikogenin, which are glycoside hydrolyzed saikosaponins, are still unknown due to its rarity in plants. In this study, we applied two recombinant glycoside hydrolases that exhibit glycoside cleavage activity with saikosaponins. The two enzymes, BglPm and BglLk, were cloned from Paenibacillus mucilaginosus and Lactobacillus koreensis, and exhibited good activity between 30-37 °C and pH 6.5-7.0. Saikosaponin A and D were purified and obtained from the crude B. falcatum L. extract using preparative high performance liquid chromatography technique. Saikosaponin A and D were converted into saikogenin F via prosaikogenin F, and saikogenin G via prosaikogenin G using enzyme transformation with high ß-glycosidase activity. The two saikogenin and two prosaikogenin compounds were purified using a silica column to obtain 78.1, 62.4, 8.3, and 7.5 mg of prosaikogenin F, prosaikogenin G, saikogenin F, and saikogenin G, respectively, each with 98% purity. The anti-cancer effect of the six highly purified saikosaponins was investigated in the human colon cancer cell line HCT 116. The results suggested that saikosaponins and prosaikogenins markedly inhibit the growth of the cancer cell line. Thus, this enzymatic technology could significantly improve the production of saponin metabolites of B. falcatum L.

Photoaffinity Labeling-Based Chemoproteomic Strategy Reveals RBBP4 as a Cellular Target of Protopanaxadiol against Colorectal Cancer Cells.

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.

Pseudo-sapogenin DQ 3-Maleate Derivative Induces Ovarian Carcinoma Cell Apoptosis via Mitochondrial Pathway.

In the present study, four novel ginsenosides fatty acid and aromatic acid derivatives were designed and synthesized, and their cytotoxic effects on human ovarian carcinoma cells (SKOV3) were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated that all derivatives inhibited SKOV3 cell growth, and Compound 3 showed the most outstanding anti-proliferative effect on SKOV3 cells. The IC50 value of Compound 3 was 33.8 ± 2.21 µM, less than half of that of cis-platinum (70.1 ± 7.64 µM). Subsequent analysis revealed that Compound 3 could promote SKOV3 cell apoptosis, and the percentage of apoptotic cell population increased with increasing Compound 3 concentrations. In addition, the expression ratios of Bax/Bcl-2, cleaved-Caspase-3/Caspase-3 and cleaved-Caspase-9/Caspase-9 were gradually elevated in Compound 3-treated SKOV3 cells compared with control cells. Furthermore, translocation of Bax to mitochondria was associated with the release of Cytochrome C. Molecular docking analysis revealed three hydrogen-bonds existed in Compound 3 with poly(ADP-ribose)polymerase (PARP) receptor (PDB code: 5DSY), which may be the target of the anti-ovarian cancer effect of Compound 3. Altogether, our study indicates that Compound 3 induces SKOV3 cell apoptosis via reactive oxygen species (ROS)-dependent mitochondrial pathway, and can serve as an anti-cancer agent for treating ovarian carcinoma.

Molecular profiling of ginsenoside metabolites to identify estrogen receptor alpha activity.

20(S)-Protopanaxadiol (PPD) and 20(S)-Protopanaxatriol (PPT) are major metabolites of ginseng in humans and are considered to have estrogenic activity in cellular bioassays. In this study, we conducted in silico analyses to determine whether PPD and PPT interact with estrogen receptor alpha (ERα) and compared them with ERα agonists, partial agonists, and antagonists to identify their ERα activity. The transcriptome profile of 17ß-estradiol (E2), PPD, and PPT in MCF-7 cells expressing ERα was further compared to understand the ERα activity of ginsenoside metabolites. The results showed that PPD and PPT interacted with the 1ERE, 1GWR, and 3UUD ERα proteins in the E2 interaction model, the 3ERD protein in the diethylstilbestrol (DES) interaction model, and the 1X7R protein in the genistein (GEN) interaction model. Conversely, neither the 4PP6 protein of the interaction model with the antagonist resveratrol (RES) nor the 1ERR protein of the interaction model with the antagonist raloxifene (RAL) showed the conformation of amino acid residues. When E2, PPD, and PPT were exposed to MCF-7 cells, cell proliferation and gene expression were observed. The transcriptomic profiles of E2, PPD, and PPT were compared using a knowledge-based pathway. PPD-induced transcription profiling was similar to that of E2, and the neural transmission pathway was detected in both compounds. In contrast, PPT-induced transcription profiling displayed characteristics of gene expression associated with systemic lupus erythematosus. These results suggest that ginsenoside metabolites have ERα agonist activity and exhibit neuroprotective effects and anti-inflammatory actions. However, a meta-analysis using public microarray data showed that the mother compounds GRb1 and GRg1 of PPD and PPT showed metabolic functions in insulin signaling pathways, condensed DNA repair and cell cycle pathways, and immune response and synaptogenesis. These results suggest that the ginsenoside metabolites have potent ERα agonist activity; however, their gene expression profiles may differ from those of E2.

Cycloastragenol alleviates airway inflammation in asthmatic mice by inhibiting autophagy.

Cycloastragenol (CAG), a secondary metabolite from the roots of Astragalus zahlbruckneri, has been reported to exert anti­inflammatory effects in heart, skin and liver diseases. However, its role in asthma remains unclear. The present study aimed to investigate the effect of CAG on airway inflammation in an ovalbumin (OVA)­induced mouse asthma model. The current study evaluated the lung function and levels of inflammation and autophagy via measurement of airway hyperresponsiveness (AHR), lung histology examination, inflammatory cytokine measurement and western blotting, amongst other techniques. The results demonstrated that CAG attenuated OVA­induced AHR in vivo. In addition, the total number of leukocytes and eosinophils, as well as the secretion of inflammatory cytokines, including interleukin (IL)­5, IL­13 and immunoglobulin E were diminished in bronchoalveolar lavage fluid of the OVA­induced murine asthma model. Histological analysis revealed that CAG suppressed inflammatory cell infiltration and goblet cell secretion. Notably, based on molecular docking simulation, CAG was demonstrated to bind to the active site of autophagy­related gene 4­microtubule­associated proteins light chain 3 complex, which explains the reduced autophagic flux in asthma caused by CAG. The expression levels of proteins associated with autophagy pathways were inhibited following treatment with CAG. Taken together, the results of the present study suggest that CAG exerts an anti­inflammatory effect in asthma, and its role may be associated with the inhibition of autophagy in lung cells.

Protopanaxadiol and Protopanaxatriol Ginsenosides Can Protect Against Aconitine-induced Injury in H9c2 Cells by Maintaining Calcium Homeostasis and Activating the AKT Pathway.

ABSTRACT: This study aimed to investigate the effects of protopanaxadiol and protopanaxatriol ginsenosides on aconitine-induced cardiomyocyte injury and their regulatory mechanisms. The effects of ginsenosides on aconitine-induced cardiomyocyte damage were initially evaluated using H9c2 cells, and the molecular mechanisms were elucidated using molecular docking and western blotting. The changes in enzyme content, reactive oxygen species (ROS), calcium (Ca2+) concentration, and apoptosis were determined. Furthermore, an aconitine-induced cardiac injury rat model was established, the cardiac injury and serum physiological and biochemical indexes were measured, and the effects of ginsenoside were observed. The results showed that ginsenoside Rb1 significantly increased aconitine-induced cell viability, and its binding conformation with protein kinase B (AKT) protein was the most significant. In vitro and in vivo, Rb1 protects cardiomyocytes from aconitine-induced injury by regulating oxidative stress levels and maintaining Ca2+ concentration homeostasis. Moreover, Rb1 activated the PI3K/AKT pathway, downregulated Cleaved caspase-3 and Bax, and upregulated Bcl-2 expression. In conclusion, Rb1 protected H9c2 cells from aconitine-induced injury by maintaining Ca2+ homeostasis and activating the PI3K/AKT pathway to induce a cascade response of downstream proteins, thereby protecting cardiomyocytes from damage. These results suggested that ginsenoside Rb1 may be a potential cardiac protective drug.

Inhibitory activities of 20(R, S)-protopanaxatriol against epidermal growth factor receptor tyrosine kinase.

As major metabolites of protopanaxatriol-type ginsenosides, 20(R, S)-protopanaxatriol [20(R, S)-PPT] display multiple bioactivities. This work aimed to investigate the inhibitory activities of 20(R, S)-PPT against epidermal growth factor receptor tyrosine kinase and the potential mechanism. 20(R, S)-PPT inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked cell cycle progression at G1/G0 phase. Then 20(R, S)-PPT were found to influence the protein expressions involved in epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) signaling pathway. Molecular docking suggested that 20(R, S)-PPT could bind to the active sites of all target proteins in EGFR-MAPK pathway. It is worth noting that 20(R, S)-PPT showed stronger binding capacities with EGFR, compared with other proteins. Hence, this work further investigated the binding interactions and binding stabilities between 20(R, S)-PPT and EGFR. Both hydrophobic interactions and hydrogen bonds contributed to the 20(R, S)-PPT-EGFR binding. In addition, the in vitro inhibitory activities of 20(R, S)-PPT against EGFR tyrosine kinase were observed in a homogeneous time-resolved fluorescence assay, with the IC50 values of 24.10 ± 0.17 and 33.19 ± 0.19 µM respectively. Taken together with the above results, both of 20(R)-PPT and 20(S)-PPT might serve as potential EGFR tyrosine kinase inhibitors.

Protopanaxadiol improves endometriosis associated infertility and miscarriage in sex hormones receptors-dependent and independent manners.

Background: Patients with endometriosis (EMs) have high risks of infertility and spontaneous abortion. How to remodel the fertility of patients with EMs has always been the hot spot and difficulty in the field of reproductive medicine. As an aglycone of ginsenosides, protopanaxadiol (PPD) possesses pleiotropic biological functions and has high medicinal values. We aimed to investigate the effect and potential mechanism of PPD in the treatment of EMs-associated infertility and spontaneous abortion. Methods: The EMs mice models were constructed by allotransplantation. The pregnancy rates, embryo implantation numbers and embryo resorption rates of control and EMs were counted. RNA sequencing, qRT-PCR, enzyme linked immunosorbent assay (ELISA) and FCM analysis were performed to screen and confirm the expression of endometrial receptivity/decidualization-related molecules, inflammation cytokines and NK cell function-related molecules in vitro and/or in vivo. The SWISS Target Prediction, STRING and Cytoscape were carried out to predict the potential cellular sensory proteins, the protein-protein interaction (PPI) network between sensory proteins and fertility-related molecules, respectively. Micro-CT detection, liver and kidney function tests were used to evaluate the safety. Results: Here, we observe that PPD significantly up-regulates endometrial receptivity-related molecules (e.g., Lif, Igfbp1, Mmps, collagens) and restricts pelvic inflammatory response (low levels of IL-12 and IFN-γ) of macrophage, and further remodel and improve the fertility of EMs mice. Additionally, PPD increases the expression of decidualization-related genes and Collagens, and promotes the proliferation, residence, immune tolerance and anagogic functions of decidual NK cells (low levels of CD16 and NKp30, high levels of Ki67, VEGF, TGF-ß) in pregnant EMs mice, and further triggers decidualization, decidual NK cell-mediated maternal-fetal immune tolerance and angiogenesis, preventing pregnant EMs mice from miscarriage. Mechanically, these effects should be dependent on ESRs, PGR and other sensory proteins (e.g., AR). Compared with GnRHa (the clinic first-line drug for EMs), PPD does not lead to the decline of serum estrogen and bone loss. Conclusion: These data suggest that PPD prevents EMs-associated infertility and miscarriage in sex hormones receptors-dependent and independent manners possibly, and provides a potential therapeutic strategy with high efficiency and low side effects to remodels the fertility of patients with EMs.

Synthesis and evaluation of novel thiosemicarbazone and semicarbazone analogs with both anti-proliferative and anti-metastatic activities against triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive cancer with high mortality and recurrence rates. Hecogenin, a steroidal sapogenin, is reported as a potential anti-tumor agent against breast cancer. However, the moderate activity limits its further application in clinical. With the aim to identify novel analogues that are especially efficacious in therapy of TNBC, a series of novel hecogenin thiosemicarbazone and semicarbazone derivatives were designed, synthesized and biologically evaluated. Screening of cytotoxicity revealed that 4c could potently inhibit the proliferation of breast cancer cells (MCF-7 and MDA-MB-231 cells), lung cancer cells (A549) and colon cancer cells (HT-29) at low µM level. Importantly, further mechanism studies indicated the ability of 4c in inducing apoptosis of MDA-MB-231 cells by arresting the cell cycle. Moreover, 4c notably suppressed the migration and invasion of MDA-MB-231 cells compared to its parent hecogenin at the equal concentration.