Ophiopogonin D alleviates acute lung injury by regulating inflammation via the STAT3/A20/ASK1 axis.

BACKGROUND: Acute lung injury (ALI) is characterized by acute pulmonary inflammatory infiltration. Alveolar epithelial cells (AECs) release numerous pro-inflammatory cytokines, which result in the pathological changes seen in ALI. Ophiopogonin D (OD), extracted from the roots of Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), reduces inflammation; however, the efficacy of OD in ALI has not been reported and the underlying molecular mechanisms remain unclear. PURPOSE: This study investigated the anti-inflammatory effects of OD, as well as the underlying mechanisms, in AECs and a mouse ALI model. METHODS: Lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) were used to stimulate macrophages and A549 cells, and a mouse ALI model was established by intratracheal LPS administration. The anti-inflammatory effects and mechanisms of OD in the TNF-α-induced in vitro inflammation model was evaluated using real-time quantitative polymerase chain reaction qPCR), enzyme-linked immunosorbent assay (ELISA), western blotting, nuclear and cytoplasmic protein extraction, and immunofluorescence. The in vivo anti-inflammatory activity of OD was evaluated using hematoxylin and eosin staining, qPCR, ELISA, and western blotting. RESULTS: The bronchoalveolar lavage fluid and lung tissue of LPS-induced ALI mice exhibited increased TNF-α expression. TNF-α induced a significantly greater pro-inflammatory effect in AECs than LPS. OD reduced inflammation and mitogen-activated protein kinase (MAPK) and transcription factor p65 phosphorylation in vivo and in vitro and promoted signal transducer and activator of transcription 3 (STAT3) phosphorylation and A20 expression, thereby inducing apoptosis signal-regulating kinase 1 (ASK1) proteasomal degradation. CONCLUSION: OD exerts an anti-inflammatory effect by promoting STAT3-dependent A20 expression and ASK1 degradation. OD may therefore have therapeutic value in treating ALI and other TNF-α-related inflammatory diseases.

Polyhydroxylated Spirostanol Saponins from the Rhizomes of Paris dulongensis.

Three new polyhydroxylated spirostanol steroidal saponins, dulongenosides B-D (2-4), along with 14 known compounds, dulongenoside A (1), padelaoside B (5), parisyunnanoside G (6), polyphyllin D (7), ophiopogonin C' (8), formosanin C (9), dioscin (10), paris saponin VII (11), paris H (12), parisyunnanoside I (13), protodioscin (14), proprotogracillin (15), crustecdysone (16), and stigmasterol-3-O-ß-d-glucopyranoside (17), were isolated from the rhizomes of Paris dulongensis (Melanthiaceae). Their chemical structures were elucidated based on extensive analyses of NMR and MS data and acidic hydrolyses. The isolates were evaluated for their cytotoxicity to five human cancer cell lines (HL-60, SW480, MDA-MB-231, A549, and A549/Taxol) and the normal human bronchial epithelial cell line BEAS-2B by the MTS test. Compounds 7-12 and 14 showed cytotoxic activity, with IC50 values ranging from 0.20 to 4.35 µM. Proprotogracillin selectively inhibited A549 (IC50=0.58 µM) and A549/Taxol (IC50=0.74 µM) cells, with no significant cytotoxic activity against HL-60, SW480, MDA-MB-231, or BEAS-2B cells, with IC50 values greater than 40 µM.

Two New Isospirostanol-Type Saponins from the Bulbs of Lilium Brownii and Their Anti-Hepatocarcinogenic Activity.

Bulbs of Lilium brownii, commonly known as "Bai-he" in China, serve both edible and medicinal purposes in clinical practice. In this study, two new isospirostanol-type saponins were isolated from L. brownii, and their structures were identified by spectroscopic method, and absolute configurations were elucidated by comprehensive analysis of spectral data obtained from combined acid hydrolysis. Two compounds were finally identified as 3-O-[α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside]-(22R,25R)-5α-spirosolane-3ß-ol (1) and 3-O-{α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→4)]-ß-D-glucopyranoside}-(22R,25R)-5α-spirosolane-3ß-ol (2), respectively. Further, we found that compound 2 significantly suppressed the proliferation of SMMC-7721 and HepG2 cells with IC50 values of 26.3±1.08 µM and 30.9±1.59 µM, whereas compound 1 didn't inhibit both of the two hepatocellular carcinoma. Subsequently, compound 2 effectively decreased the levels of interleukin-1ß and tumor necrosis factor-α and the expression of Bcl-2, and increased the expression of Bax and Caspase-3 proteins. Which indicated that the anti-hepatocellular carcinoma effect of compound 2 involves reducing the level of inflammation and inducing apoptosis.

Total synthesis and cytotoxicity evaluation of the spirostanol saponin gitonin.

The spirostanol saponin gitonin was efficiently synthesized in 12 steps (longest linear sequence) in 18.5% overall yield from the commercially available isopropyl ß-D-1-thiogalactopyranoside (IPTG) and tigogenin. A cascade two-step glycosylation and Schmidt's inverse procedure significantly facilitated the synthesis of gitonin and its derivatives. The cytotoxic activities of gitonin and its structural analogues were evaluated against A549, HepG2, and MCF-7, and most of them exhibited moderate to excellent inhibitory activity. Our study demonstrates that the removal of the ß-D-galactopyranosyl residue (attached at C-2 of the glucose unit) from gitonin would not decrease the inhibition activities; however, further cleavage of sugar units could seriously reduce the activities. A bioassay on these cancer cell lines also suggested that the presence of 2α-hydroxy on the aglycone weakened the cytotoxicity of the designed saponin.

Anticancer Effect of Ruscogenin in B(a)P-Induced Lung Cancer in Mice via Modulation of Proinflammatory Cytokines and Mitochondrial Enzymes.

Lung cancer, one of the most often diagnosed malignancies, is the top cause of death in both men and women globally. In both developed and emerging countries, high incidences of cancer are becoming a huge health burden. Natural resources, including plants, have always been a possible source of lead compounds in the identification of optimal medications for cancer treatment, with natural resources accounting for around half of all anticancer drugs. Ruscogenin, a natural saponin, is a major component of Radix Ophiopogon japonicus with a well-established anticancer activity. In this study, the anticancer potential of ruscogenin against a B(a)P-challenged lung cancer model in mice was assessed. The mice were categorized into four groups: group I was as the control group, group II mice were challenged with B(a)P, group III rodents were treated with ruscogenin prior to challenge with B(a)P, and group IV rodents were treated with ruscogenin after B(a)P administration. Tumor incidence was calculated, and the following parameters were analyzed: body weight, lung weight, immunoglobulin (Ig) levels (IgG, IgA, and IgM), key marker enzymes, and proinflammatory cytokines in both treated and control mice. Lung tissues were analyzed via histopathological analysis. According to our results, all the markers that favor the growth of cancer were increased in the lung cancer group. After administration of ruscogenin, all the markers returned to their original levels, revealing the anticancer potential of ruscogenin.

Chemistry, Biosynthesis and Pharmacology of Sarsasapogenin: A Potential Natural Steroid Molecule for New Drug Design, Development and Therapy.

Sarsasapogenin is a natural steroidal sapogenin molecule obtained mainly from Anemarrhena asphodeloides Bunge. Among the various phytosteroids present, sarsasapogenin has emerged as a promising molecule due to the fact of its diverse pharmacological activities. In this review, the chemistry, biosynthesis and pharmacological potentials of sarsasapogenin are summarised. Between 1996 and the present, the relevant literature regarding sarsasapogenin was obtained from scientific databases including PubMed, ScienceDirect, Scopus, and Google Scholar. Overall, sarsasapogenin is a potent molecule with anti-inflammatory, anticancer, antidiabetic, anti-osteoclastogenic and neuroprotective activities. It is also a potential molecule in the treatment for precocious puberty. This review also discusses the metabolism, pharmacokinetics and possible structural modifications as well as obstacles and opportunities for sarsasapogenin to become a drug molecule in the near future. More comprehensive preclinical studies, clinical trials, drug delivery, formulations of effective doses in pharmacokinetics studies, evaluation of adverse effects and potential synergistic effects with other drugs need to be thoroughly investigated to make sarsasapogenin a potential molecule for future drug development.

Ophiopogonin­B targets PTP1B to inhibit the malignant progression of hepatocellular carcinoma by regulating the PI3K/AKT and AMPK signaling pathways.

Ophiopogonin­B (OP­B) is a bioactive component from the root of Ophiopogon japonicus, which can exert anticancer effects on multiple malignant tumors. The present study aimed to uncover the effects of OP­B on hepatocellular carcinoma (HCC) and the underlying mechanisms. An HCC­xenografted mouse model was established and subsequently treated with OP­B (15 and 75 mg/kg) to observe the effects of OP­B on HCC progression and protein tyrosine phosphatase 1B (PTP1B) expression in vivo. The HCC cell line MHCC97­H was transfected with either PTP1B overexpression (Ov)­PTP1B or empty vector control, and then exposed to different concentrations of OP­B. Subsequently, PTP1B expression, cell viability, proliferation, apoptosis, migration, invasion and angiogenesis were evaluated by western blotting, reverse transcription­quantitative PCR, Cell Counting Kit­8, colony formation, TUNEL staining, wound healing, Transwell and tube formation assays. The expression of phosphatidylinositol 3 kinase (PI3K)/AKT and adenosine 5'­monophosphate­activated protein kinase (AMPK) was also assessed by western blot assay. The results showed that OP­B inhibited tumor growth and the expression of Ki67, CD31, VEGFA and PTP1B in HCC xenograft model. The expression of PTP1B in HCC cells was also inhibited by OP­B in a concentration­dependent manner. Results from the in vitro studies revealed that OP­B suppressed cell proliferation, migration, invasion and angiogenesis, and promoted apoptosis of HCC cells. However, PTP1B overexpression reversed the effect of OP­B on HCC cells. PI3K/AKT was inactivated and AMPK was activated by OP­B exposure in HCC cells, and PTP1B overexpression blocked these effects. In conclusion, OP­B effectively inhibited the progression of HCC both in vivo and in vitro. These effects may depend on downregulating PTP1B expression, thereby inactivating the PI3K/AKT pathway and activating the AMPK pathway.

Spirostane saponins with a rearranged A/B ring system isolated from the rhizomes of Ophiopogon japonicus.

In this study, the popular food and medicinal herb Ophiopogon japonicus was investigated alongside a 70% ethanol extract of its rhizomes, revealing twenty-three steroidal glycosides with four undescribed steroidal saponins, named ophiopogonols A-D. Among them, ophiopogonols A-B are two unusual spirostanols with a rearranged A/B ring system (5/7/6/5/5/6 ring system) that have not previously been identified in plants. The chemical structures of all isolated steroidal glycosides were elucidated by comprehensive analysis through chemical methods, HRESIMS, and NMR spectroscopy. Further, putative biosynthetic pathways for ophiopogonols A-B were proposed. In addition, based on traditional applications of O. japonicus, cytotoxic effects of the isolates were evaluated using human large cell lung carcinoma cells (NCI-H460 cells). Sprengerinin C displayed a remarkable cytotoxic effect with IC50 values of 2.1 ± 0.8 µM by inducing apoptosis and G2/M phase cycle arrest in the NCI-H460 cell line.

The anti-tumor effect of OP-B on ovarian cancer in vitro and in vivo, and its mechanism: An investigation using network pharmacology-based analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Maidong (Liliaceae) is used as a yin-nourishing medication for the treatment of cardiovascular disease, inflammation, and assistant cancer chemotherapy in the clinic. Ophiopogonin B (OP-B), a major saponin extracted from Maidong, is reported to have potential antitumor activities against various human cancers. However, the effects of OP-B on human ovarian cancer (OC) and the potential mechanisms of action are yet elusive. AIM OF THE STUDY: In this study, we aimed to explore the potential molecular mechanisms of OP-B in the treatment of OC using network pharmacology. In vivo and in vitro experiments were conducted to further verify the therapeutic effects of OP-B on OC. MATERIALS AND METHODS: To investigate the functions of OP-B against OC holistically, the related targets of OP-B and OC were each predicted based on four public databases. Subsequently, the identified PPI network was constructed to detect the hub potential targets. In addition, GO and KEGG enrichment analysis were applied by Metascape database. Furthermore, we simultaneously investigated the anticancer effects of OP-B on SKOV3 and A2780 human ovarian cancer cells using a cell viability assay, transwell assay, and an image-based cytometric assay. The quantitative real-time PCR and western-blot assay were used to validate the RNA and protein levels of target genes in OP-B treated OC cells. At last, SKOV3-bearing BALB/c nude mice were applied to observe the effectiveness and toxicity of OP-B. RESULTS: Through network pharmacological analysis, OP-B was found to play a critical role in OC via multiple targets and pathways, especially the STAT3 signaling pathways. In addition, in vitro experiments found OP-B suppressed SKOV3 and A2780 cells proliferation in a time and concentration dependent manner, and markedly impaired cancer cell migration. Flow cytometry analysis revealed that OP-B significantly increased early and late apoptosis, induced G2/M phase cell cycle arrest in SKOV3 cells and G0/G1 phase cell cycle arrest in A2780 cells. Moreover, OP-B administration down-regulated the expression of p-STAT3 protein, whereas the RNA expression and total protein levels of STAT3 were not altered. Finally, in vivo experiments confirmed the therapeutic effects of OP-B on OC in nude mice with low toxicity in heart, liver, lung, and kidney. CONCLUSION: OP-B could efficiently suppress OC cellular proliferation, migration and induce apoptosis, cell cycle arrest mainly via the regulation of STAT3 signaling pathway. This study provides a promising potential application for an alternative to chemotherapy in ovarian cancer.

Protective effect of sarsasapogenin in TNBS induced ulcerative colitis in rats associated with downregulation of pro-inflammatory mediators and oxidative stress.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel condition considered by oxido-nitrosative stress and the release of pro-inflammatory cytokines that affects the mucosal lining of the colon. Sarsasapogenin (SG), as an active component, has been found in many plants, and it exhibits potential protective effects, such as anti-inflammatory, antioxidant, anti-psoriasis, anti-arthritis, anti-asthma, anti-depressant and anti-cancer. However, the effects of SG on UC remain unknown. OBJECTIVE: The purpose of this study was to investigate the effects of SG on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced UC in rats. METHOD: Thirty Wistar rats were randomized into five groups: (i) Normal control, (ii) Disease control (TNBS), (iii) Sarsasapogenin (SG) (50 µg/rat), (iv) Fluticasone (FC) (50 µg/rat), (v) Sarsasapogenin + Fluticasone (SG + FC) (25 µg/rat). UC was induced in rats by trans-rectal instillation of TNBS (10 mg/kg). SG, FC and SG + FC were administered for 11 days and on the 8th day colitis was induced. Several molecular, biochemical and histological alterations were evaluated in the colon tissue. All treatment group results were compared to the TNBS group results. RESULT: The study results revealed that treatment of rats with SG and SG + FC combination significantly decreased the colon weight/length ratio, macroscopic inflammation score, lesions score, diarrhea score and adhesion score. Combination treatment in rats significantly reduced the production of biochemical parameters, proinflammatory cytokines, haematological parameters, serum IgE levels and restored the oxidative stress markers. SG and SG + FC treatment also considerably restored the histopathological changes induced by TNBS. CONCLUSION: Thus, SG and SG + FC combination could alter the disease progression and could be a hopeful therapeutic target for the management of UC by reducing its dose in combination with FC to elude the long term adverse effects of FC.

Ophiopogonin B induces reactive oxygen species­dependent apoptosis through the Hippo pathway in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in South China and is characterized by a high death rate. Ophiopogonin B (OP­B) is a bioactive component of Radix Ophiopogon japonicus, which is frequently used in traditional Chinese medicine to treat cancer. The present study aimed to examine the anti­cancer properties of OP­B on NPC cells. Cell viability and cell proliferation were measured using MTT and EdU assays. Flow cytometry was used to measure cell apoptosis, reactive oxygen species and mitochondrial membrane potential. Western blotting was used to investigate the expression of apoptosis and Hippo signaling pathway proteins. OP­B inhibited the proliferation of NPC cells by inducing apoptosis and disturbing the mitochondrial integrity. OP­B enhanced ROS accumulation. In addition, OP­B promoted the expression of mammalian STE20­like kinase 1, large tumor suppressor 1 and phosphorylated yes­associated protein (YAP) and suppressed the expression of YAP and transcriptional enhanced associate domain in NPC cells. OP­B increased the expression of forkhead box transcription factor O1 in the nuclear fraction. In conclusion, OP­B has therapeutic potential and feasibility in the development of novel YAP inhibitors for NPC.

Gracillin Isolated from Reineckia carnea Induces Apoptosis of A549 Cells via the Mitochondrial Pathway.

BACKGROUND: Reineckia carnea is commonly used to treat cough, pneumonia and other diseases in China. In our previous study, it was found that the ethanol extracts of Reineckia carnea have a strong inhibitory effect on the proliferation of human lung cancer A549 cells. Here, we isolated gracillin from ethanol extracts for the first time. PURPOSE: Clarify the antiproliferation effect of gracillin on A549 cells and further explore its mechanisms via the mitochondrial pathway. METHODS: Gracillin was isolated and purified by silica gel, D-101 macroporous resin and preparative RP-HPLC, then identified by NMR and HR-MS. The inhibitory effects of gracillin on the proliferation of A549 cells were detected by the MTS method. Its mechanisms were further explored by flow cytometry and Western blot. RESULTS: A steroid saponin, gracillin, was isolated and identified from Reineckia carnea for the first time. In a concentration-dependent and time-dependent manner, gracillin significantly inhibited the proliferation of A549 cells with an IC50 value at 2.54 µmol/L and induced morphological changes. The results of flow cytometry analysis showed that the apoptosis rate of A549 cells was significantly increased (p < 0.05), and the cells proportion was obviously arrested in S phase. The concentration of intracellular calcium was raised (p < 0.01), and the mitochondrial membrane potential was greatly decreased (p < 0.01). In addition, the expression levels of Bax, caspase-3, cleaved caspase-3, and cytochrome C were dramatically up-regulated while Bcl-2 was down-regulated (p < 0.05) in A549 cells. CONCLUSION: This study confirmed that gracillin has a significant antiproliferative effect on A549 cells. Gracillin could induce the apoptosis of A549 cells through the mitochondrial pathway, which might be associated with regulation of the concentration of intracellular calcium, the mitochondrial membrane potential and the expression levels of Bax, Bcl-2, caspase-3, cleaved caspase-3, and cytochrome C.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. AIM OF THE STUDY: In this study, we aimed to observe the effects of OP-D on the diabetic myocardium. MATERIALS AND METHODS: Leptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. RESULTS: Blood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. CONCLUSIONS: Our findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.

Sarsasapogenin improves adipose tissue inflammation and ameliorates insulin resistance in high-fat diet-fed C57BL/6J mice.

Insulin resistance is a major cause of type 2 diabetes and metabolic syndrome. Macrophage infiltration into obese adipose tissue promotes inflammatory responses that contribute to the pathogenesis of insulin resistance. Suppression of adipose tissue inflammatory responses is postulated to increase insulin sensitivity in obese patients and animals. Sarsasapogenin (ZGY) is one of the metabolites of timosaponin AIII in the gut, which has been shown to exert anti-inflammatory action. In this study, we investigated the effects of ZGY treatment on obesity-induced insulin resistance in mice. We showed that pretreatment with ZGY (80 mg·kg-1·d-1, ig, for 18 days) significantly inhibited acute adipose tissue inflammatory responses in LPS-treated mice. In high-fat diet (HFD)-fed obese mice, oral administration of ZGY (80 mg·kg-1·d-1, for 6 weeks) ameliorated insulin resistance and alleviated inflammation in adipose tissues by reducing the infiltration of macrophages. Furthermore, we demonstrated that ZGY not only directly inhibited inflammatory responses in macrophages and adipocytes, but also interrupts the crosstalk between macrophages and adipocytes in vitro, improving adipocyte insulin resistance. The insulin-sensitizing and anti-inflammatory effects of ZGY may result from inactivation of the IKK /NF-κB and JNK inflammatory signaling pathways in adipocytes. Collectively, our findings suggest that ZGY ameliorates insulin resistance and alleviates the adipose inflammatory state in HFD mice, suggesting that ZGY may be a potential agent for the treatment of insulin resistance and obesity-related metabolic diseases.

Gracillin shows potent efficacy against colorectal cancer through inhibiting the STAT3 pathway.

Colorectal cancer (CRC) accounts for about 10% of all annually diagnosed cancers and cancer-related deaths worldwide. STAT3 plays a vital role in the occurrence and development of tumours. Gracillin has shown a significant antitumour activity in tumours, but its mechanism remains unknown. The human CRC cell lines HCT116, RKO, and SW480 and immunodeficient mice were used as models to study the effects of gracillin on cell proliferation, migration and apoptosis. These were evaluated by cell viability, colony formation, wound-healing migration and cell apoptosis assays. Luciferase reporter assay, and immunostaining and western blot analyses were used to explore the specific mechanism through which gracillin exerts its effects. Gracillin significantly reduces viability and migration and stimulates apoptosis in human CRC cells. It also significantly inhibits tumour growth with no apparent physiological toxicity in animal model experiments. Moreover, gracillin is found to inhibit STAT3 phosphorylation and STAT3 target gene products. In addition, gracillin inhibits IL6-induced nuclear translocation of P-STAT3. Gracillin shows potent efficacy against CRC by inhibiting the STAT3 pathway. It should be further explored as a unique STAT3 inhibitor for the treatment of CRC.

Sarsasapogenin Suppresses RANKL-Induced Osteoclastogenesis in vitro and Prevents Lipopolysaccharide-Induced Bone Loss in vivo.

INTRODUCTION: Osteoclasts are giant polynuclear cells; their main function is bone resorption. An increased number of osteoclasts and enhanced bone resorption exert significant effects on osteoclast-related bone-lytic diseases, including osteoporosis. Given the limitations of current therapies for osteolytic diseases, it is urgently required to develop safer and more effective alternatives. Sarsasapogenin, a major sapogenin from Anemarrhena asphodeloides Bunge, possesses potent antitumor effects and inhibits NF-κB and MAPK signaling. However, the manner in which it affects osteoclasts is unclear. METHODS: We investigated the effects of anti-osteoclastogenic and anti-resorptive of sarsasapogenin on bone marrow-derived osteoclasts. RESULTS: Sarsasapogenin inhibited multiple RANKL-induced signaling cascades, thereby inhibiting the induction of key osteoclast transcription factor NFATc1. The in vivo and in vitro results were consistent: sarsasapogenin treatment protected against bone loss in a mouse osteolysis model induced by lipopolysaccharide. CONCLUSION: Our research confirms that sarsasapogenin can be used as a new treatment for osteoclast-related osteolytic diseases.

Ophiopogonin D suppresses TGF-ß1-mediated metastatic behavior of MDA-MB-231 breast carcinoma cells via regulating ITGB1/FAK/Src/AKT/ß-catenin/MMP-9 signaling axis.

Ophiopogonin D, a steroidal glycoside extracted from the Traditional Chinese Medicine Ophiopogon japonicus, shows anti-tumor property in several lines of cancers; however, its effect on triple-negative breast cancer (TNBC) has not been investigated. In this study, the anti-metastatic effect of Ophiopogonin D in TNBC cells as well as the underlying mechanism in such process was explored. Ophiopogonin D dose-dependently decreased cell proliferation of MDA-MB-231 cells. Meanwhile, Ophiopogonin D significantly inhibited TGF-ß1-induced metastatic behavior of MDA-MB-231 cells, including EMT, anoikis resistance as well as migration and invasion, via suppressing MMP-9 activity. Mechanically, Ophiopogonin D achieved its effect through efficiently abolishing ITGB1 expression, thus reducing the phosphorylation of FAK, Src and AKT, as well as upregulating nuclear ß-catenin. ITGB1 overexpression partly recovered Ophiopogonin D's inhibitory effect on metastatic behavior via activating MMP-9. These results demonstrated that Ophiopogonin D could suppress TGF-ß1-mediated metastatic behavior of MDA-MB-231 cells by regulating ITGB1/FAK/Src/AKT/ß-catenin/MMP-9 signaling axis, which might provide new insight for the control of TNBC metastasis.

Cytotoxicity of a naturally occuring spirostanol saponin, progenin III, towards a broad range of cancer cell lines by induction of apoptosis, autophagy and necroptosis.

This study was aimed to investigate the cytotoxic potential of a natural compound, progenin III on a broad range of cancer cell lines, including various sensitive and drug-resistant phenotypes. The cytotoxicity, progenin III-induced autophagic, ferroptotic and necroptotic cell death were evaluated by the resazurin reduction assay (RRA). Spectrophotometric analysis of caspases activity was performed using caspase-Glo assay. Flow cytometry was applied for cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H2DCFH-DA). Progenin III and the reference molecule, doxorubicin exerted cytotoxic effects towards the 18 cancer cell lines tested including animal and human cell lines. The IC50 values obtained ranged from 1.59 µM (towards CCRF-CEM leukemia cells) to 31.61 µM (against the BRAF-V600E homozygous mutant SKMel-28 melanoma cells) for progenin III. Normal sensitivity was achieved with CEM/ADR5000 cells and HCT116p53-/- adenocarcinoma cells respectively compared to their sensitive congeners CCRF-CEM cells and HCT116 p53+/+ cells. Progenin III induced apoptosis in CCRF-CEM cells mediated by caspases 3/7 activation, MMP alteration and increase ROS production, and otherwise autophagy and necroptosis. Progenin III is a potential anticancer molecule that deserves further investigations to develop a novel drug to combat malignant diseases including refractory cancers.

Synthesis and anti-tumour, immunomodulating activity of diosgenin and tigogenin conjugates.

A series of novel diosgenin (DSG) and tigogenin (TGG) derivatives with diosgenin or tigogenin steroid aglycons linked to levulinic and 3,4-dihydroxycinnamic acids, dipeptides and various amino acids by an ester bond at the C3-oxygen atom of the steroid skeleton has been synthesized. Diosgenyl esters have been prepared by an esterification reaction (DCC/DMAP) of diosgenin with the corresponding acids. All analogues have been evaluated in vitro for their antiproliferative profile against cancer cell lines (MCF-7, MDA-MB-231, PC-3) and human umbilical vein endothelial cells (HUVEC). Analogue2c (l-serine derivative of TGG), the best representative of the series showed IC50 of 1.5 µM (MCF-7), and induced apoptosis in MCF-7 by activating caspase-3/7. The immunomodulatory properties of six synthesized analogues have been determined by examining their effects on the expression of cytokine genes essential for the functioning of the human immune system (IL-1, IL-4, IL-10, IL-12 and TNF-α). Biological evaluation has revealed that new compounds 4c and 16a do not induce the expression of pro-inflammatory cytokines in THP-1 cells after the lipopolysaccharide (LPS) stimulation. They also stimulate the expression of anti-inflammatory IL-10 that acts stronger than diosgenin itself. An in silico ADME properties(absorption, distribution, metabolism, excretion) study was also performed to predict the pharmacokinetic profile of the synthesized compounds. To shed light on the molecular interactions between the synthesized compounds and the glucocorticoid receptor and the estrogen receptor, 2c, 4c and 16a compounds were docked into the active binding sites of these receptors. The in silico and in vitro data suggested that this new group of compounds might be considered as a promising scaffold for further modification of more potent and selective anticancer and immunomodulatory agents.

Ophiopogonin D promotes bone regeneration by stimulating CD31hi EMCNhi vessel formation.

OBJECTIVES: CD31hi EMCNhi vessels (CD31, also known as PECAM1 [platelet and endothelial cell adhesion molecule 1]; EMCN, endomucin), which are strongly positive for CD31 and endomucin, couple angiogenesis and osteogenesis. However, the role of CD31hi EMCNhi vessels in bone regeneration remains unknown. In the present study, we investigated the role of CD31hi EMCNhi vessels in the process of bone regeneration. MATERIALS AND METHODS: We used endothelial-specific Krüppel like factor 3 (Klf3) knockout mice and ophiopogonin D treatment to interfere with CD31hi EMCNhi vessel formation. We constructed a bone regeneration model by surgical ablation of the trabecular bone. Immunofluorescence and micro-computed tomography (CT) were used to detect CD31hi EMCNhi vessels and bone formation. RESULTS: CD31hi EMCNhi vessels participate in the process of bone regeneration, such that endothelial-specific Klf3 knockout mice showed increased CD31hi EMCNhi vessels and osteoprogenitors in the bone regeneration area, and further accelerated bone formation. We also demonstrated that the natural compound, ophiopogonin D, acts as a KLF3 inhibitor to promote vessels formation both in vitro and in vivo. Administration of ophiopogonin D increased the abundance of CD31hi Emcnhi vessels and accelerated bone healing. CONCLUSIONS: Our findings confirmed the important role of CD31hi Emcnhi vessels in bone regeneration and provided a new target to treat bone fracture or promote bone regeneration.