Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.

Two birds with one stone: YQSSF regulates both proliferation and apoptosis of bone marrow cells to relieve chemotherapy-induced myelosuppression.

ETHNOPHARMACOLOGICAL RELEVANCE: Yiqi Shengsui formula (YQSSF) is a commonly used formula to treat chemotherapy-induced myelosuppression, but little is known about its therapeutic mechanisms. AIM OF THIS STUDY: This study aims to examine the effect of YQSSF in treating myelosuppression and explore its mechanism. MATERIALS AND METHODS: A myelosuppression BALB/c mouse model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CTX). The efficacy of YQSSF in alleviating chemotherapy-induced myelosuppression was evaluated by blood cell count, immune organ (thymus, spleen, liver) index, bone marrow nucleated cell (BMNC) count and histopathological analysis of bone marrow and spleen. Then, ultra-performance liquid chromatograph quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to analyze the ingredients of YQSSF extract. Key effects and potential mechanism of YQSSF extract in alleviating myelosuppression were predicted by network pharmacology method. Finally, cell cycle and TUNEL staining of bone marrow cells was detected to verify the key effects, and RT-qPCR or Western blotting were performed to measure the gene and protein expressions of the effect targets respectively to confirm the predicted mechanism of YQSSF for myelosuppression. RESULTS: YQSSF up-regulated the number of peripheral blood leukocytes and BMNC, reduced spleen index and liver index, improved the pathological morphology of bone marrow and spleen. A total of 40 ingredients were isolated from YQSSF extract using UPLC-Q/TOF-MS analysis. Network pharmacology revealed that YQSSF regulated both proliferation and apoptosis to alleviate myelosuppression. Finally, YQSSF decreased G0/G1 ratio, increased the proportion of bone marrow cells in S phase and proliferation index (PI), and reduced apoptotic cells in femur bone marrow. RT-qPCR and Western blotting showed that YQSSF up-regulated the expression levels of CDK4, CDK6, CyclinB1, c-Myc and Bcl-2, as well as down-regulated the expression levels of Cyt-c, Fas, Caspase-8/3 and p53. CONCLUSIONS: YQSSF promotes the proliferation and inhibits the apoptosis of bone marrow cells to relieve chemotherapy-induced myelosuppression.

Cyclodextrin metal-organic framework as vaccine adjuvants enhances immune responses.

It is urgently needed to develop novel adjuvants for improving the safety and efficacy of vaccines. Metal-organic frameworks (MOFs), with high surface area, play an important role in drug delivery. With perfect biocompatibility and green preparation process, the γ-cyclodextrin metal-organic framework (γ-CD-MOF) fabricated with cyclodextrin and potassium suitable for antigen delivery. In this study, we modified γ-CD-MOF with span-85 to fabricate the SP-γ-CD-MOF as animal vaccine adjuvants. The ovalbumin (OVA) as the model antigen was encapsulated into particles to investigate the immune response. SP-γ-CD-MOF displayed excellent biocompatibility in vitro and in vivo. After immunization, SP-γ-CD-MOF loaded with OVA could induce high antigen-specific IgG titers and cytokine secretion. Meanwhile, SP-γ-CD-MOF also significantly improved the proliferation of spleen cells and activated and matured the bone marrow dendritic cells (BMDCs). The study showed the potential of SP-γ-CD-MOF in vaccine adjuvants and provided a novel idea for the development of vaccine adjuvants.

Calycosin-7-O-ß-Glucoside Isolated from Astragalus membranaceus Promotes Osteogenesis and Mineralization in Human Mesenchymal Stem Cells.

Stem cells have received attention in various diseases, such as inflammatory, cancer, and bone diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that are critical for forming and repairing bone tissues. Herein, we isolated calycosin-7-O-ß-glucoside (Caly) from the roots of Astragalus membranaceus, which is one of the most famous medicinal herbs, and investigated the osteogenic activities of Caly in MSCs. Caly did not affect cytotoxicity against MSCs, whereas Caly enhanced cell migration during the osteogenesis of MSCs. Caly increased the expression and enzymatic activities of ALP and the formation of mineralized nodules during the osteogenesis of MSCs. The osteogenesis and bone-forming activities of Caly are mediated by bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, Wnt3a, phospho-GSK3ß, and phospho-AKT, inducing the expression of runt-related transcription factor 2 (RUNX2). In addition, Caly-mediated osteogenesis and RUNX2 expression were attenuated by noggin and wortmannin. Moreover, the effects were validated in pre-osteoblasts committed to the osteoblast lineages from MSCs. Overall, our results provide novel evidence that Caly stimulates osteoblast lineage commitment of MSCs by triggering RUNX2 expression, suggesting Caly as a potential anabolic drug to prevent bone diseases.

Inhibitory Effects of Cucurbitane-Type Triterpenoids from Momordica charantia Fruit on Lipopolysaccharide-Stimulated Pro-Inflammatory Cytokine Production in Bone Marrow-Derived Dendritic Cells.

The bitter melon, Momordica charantia L., was once an important food and medicinal herb. Various studies have focused on the potential treatment of stomach disease with M. charantia and on its anti-diabetic properties. However, very little is known about the specific compounds responsible for its anti-inflammatory activities. In addition, the in vitro inhibitory effect of M. charantia on pro-inflammatory cytokine production by lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs) has not been reported. Phytochemical investigation of M. charantia fruit led to the isolation of 15 compounds (1-15). Their chemical structures were elucidated spectroscopically (one- and two-dimensional nuclear magnetic resonance) and with electrospray ionization mass spectrometry. The anti-inflammatory effects of the isolated compounds were evaluated by measuring the production of the pro-inflammatory cytokines interleukin IL-6, IL-12 p40, and tumor necrosis factor α (TNF-α) in LPS-stimulated BMDCs. The cucurbitanes were potent inhibitors of the cytokines TNF-α, IL-6, and IL-12 p40, indicating promising anti-inflammatory effects. Based on these studies and in silico simulations, we determined that the ligand likely docked in the receptors. These results suggest that cucurbitanes from M. charantia are potential candidates for treating inflammatory diseases.

Nicotinamide riboside attenuates age-associated metabolic and functional changes in hematopoietic stem cells.

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

Assessment of reproductive toxicity and genotoxicity of Aconiti Lateralis Radix Praeparata and its processed products in male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconiti Lateralis Radix Praeparata (Chinese name: Fuzi), the root of Aconitum carmichaelii Debx., is a representative medicine for restoring yang and rescuing patient from collapse. However, less studies had been reported on the reproductive toxicity and genotoxicity of Fuzi. According to the principle of reducing toxicity and preserving efficiency, only processed products of Fuzi are commonly applied in clinic, including Baifupian, Heishunpian and Danfupian. However, whether processing could alleviate the reproductive toxicity and genotoxicity of Fuzi had not been revealed. AIM OF THE STUDY: To assess the effect and possible mechanism of Fuzi and its processed products on reproductive toxicity and genotoxicity in male mice. MATERIALS AND METHODS: Aqueous extracts of Fuzi and its processed products (Baifupian, Heishunpian and Danfupian, 5.85 g/kg) were administrated by gavage once daily for fourteen consecutive days. The reproductive toxicity was evaluated by testis weight, testis ratio, testis histopathology, sperm count, sperm viability rate and sperm deformity rate. The genotoxicity was evaluated by comet assay and micronucleus test in sperm, peripheral blood cell and bone marrow cell. Possible mechanisms of attenuating toxicity by processing were analyzed by detecting the level of testosterone, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and catalase (CAT). RESULTS: Fuzi significantly caused different degrees of reproductive toxicity and genotoxicity, specifically reducing the weight and testicular coefficient of testis, causing obvious pathological changes in testicular tissue, reducing sperm count and sperm viability rate, increasing sperm deformity rate and DNA damage in sperm/peripheral blood cells/bone marrow cells. Moreover, Fuzi decreased the level of testosterone, SOD, GSH and CAT, while increased the level of MDA in serum. Notably, the reproductive toxicity and genotoxicity induced by the processed products, especially Heishunpian and Danfupian, were significantly lowered compared to Fuzi. Processing could increase the level of testosterone, SOD, GSH, CAT and decrease the level of MDA compared to Fuzi. CONCLUSION: Fuzi and its processed products had reproductive toxicity and genotoxicity, but the toxicity of processed products was significantly weakened compared to Fuzi. The protective mechanism of processing to reduce the toxicity of Fuzi might be related to increasing the level of testosterone and decreasing oxidative stress.

Safety evaluation of fermented Platycodon grandiflorus (Jacq.) A.DC. extract: Genotoxicity, acute toxicity, and 13-week subchronic toxicity study in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Platycodon grandiflorus (Jacq.) A.DC. is a well-known traditional herbal medicine administered for bronchitis and inflammatory diseases. Especially, anti-inflammatory effect of fermented P. grandiflorus (Jacq.) A.DC. extract (FPGE) was higher than that of P. grandiflorus (Jacq.) A.DC. extract. However, toxicological information for FPGE is lacking. AIM OF THE STUDY: In this study, we establish a toxicological profile for FPGE by testing genotoxicity, acute and 13-week subchronic toxicity. MATERIALS AND METHODS: FPGE was evaluated with bacterial reverse mutation, chromosome aberration, and micronucleus test. For the acute- and 13-week subchronic toxicity tests, FPGE was administered orally at doses of 0, 750, 1500, and 3000 mg/kg in SD rats. RESULTS: The results of the genotoxic assays indicated that FPGE induced neither mutagenicity nor clastogenicity. The acute toxicity test showed that FPGE did not affect animal mortality, clinical signs, body weight changes, or microscopic findings at ≤ 3000 mg/kg. The approximate lethal dose (ALD) of FPGE in SD rats was >3000 mg/kg. For the 13-week subchronic toxicity assay, no FPGE dose induced any significant change in mortality, clinical signs, body or organ weight, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings and histopathologic examination in either SD rat sex. The rat no observed adverse effects level (NOAEL) for FPGE was set to 3000 mg/kg. CONCLUSIONS: The present study empirically demonstrated that FPGE has a safe preclinical profile and indicated that it could be safely integrated into health products for atopic dermatitis treatment.

Mitigation of X-ray induced DNA damages and expression of DNA-repair genes by antioxidative Potentilla fulgens root extract and its ethyl-acetate fraction in mammalian cells.

Potentilla fulgens is a medicinal plant in North-East India whose root is reported to have anti-diabetic, anticarcinogenic and antioxidant properties. The potential of hydro-alcoholic extract of P. fulgens root (PRE) for providing protection to mammalian cells exposed to ionising radiation was investigated in this study. The methanolic extract of PRE shows an enhanced radical scavenging ability in a concentration dependent manner. PRE-pre-treatment to stimulated human blood lymphocytes (HBLs) reduced the frequency of deletion and exchange aberrations induced by X-irradiation. Similar protection of chromosome aberrations was also observed in mouse bone marrow cells (BMCs) where mice were given PRE extract (1 mg extract/day/mice) ad libitum in the drinking water for 45 days before whole-body X-irradiation. Of the various extracts prepared by partitioning of the methanol extract, the ethyl-acetate (EA) fraction was found to possess better antioxidant, radical scavenging and DNA-damage reduction activities. PRE-pre-treatment also reduced the radiation-induced cell-cycle delay effectively in HBL. In HEK-293 cells, PRE reduced radiation-induced G2-block in cell kinetics. Interestingly, PRE-treatment alone increased the concentration of endogenous glutathione (GSH) in mouse BMC and in stimulated HBL along with the elevated expression of γ-glutamyl-cysteine synthetase heavy/catalytic subunit, a key determinant of GSH synthesis. Studies on expression of two DNA-repair genes revealed that there was a marked increase in the expression of GADD45 and H2AX genes after X-irradiation in stimulated HBL, and such expression was reduced significantly if PRE-treatment was given prior to radiation. The present findings show the ability of PRE to reduce radiation-induced DNA damages probably by free radical scavenging whereas modulation of expression of DNA-repair genes' and endogenous GSH-increment emerge as effective strategies. The present study is the first report on the selected medicinal plant species that suggests it to be a potential natural radioprotector when used as root extract or its EA fraction for mitigating radiation toxicity.

Structural characterization and antioxidant activity of an acetylated Cyclocarya paliurus polysaccharide (Ac-CPP0.1).

The aim of this study was to investigate the primary structure of an acetylated Cyclocarya paliurus polysaccharide (Ac-CPP0.1) and its protective effect on H2O2-treated dendritic cells. The backbone of Ac-CPP0.1 was →3)-ß-D-Galp-(1→, with some branches α-L-Araf-(1→ residues at O-6 and O-5, ß-D-Galp-(1→ and 3,5,6)-ß-D-Galf-(1 residues at O-4 and acetyl groups were substituted at the O-2 and O-6 positions of 3)-ß-D-Galp-(1 residues. The CPP0.1 and Ac-CPP0.1 significantly increased the levels of superoxide dismutase, glutathione peroxidase and catalase on H2O2-treated dendritic cells. Meanwhile, both CPP0.1 and Ac-CPP0.1 up-regulated the expression of Nrf2 (NF-E2-related factor 2) and down-regulated the Keap1 (Kelch-like ECH-associated protein-1), but Ac-CPP0.1 had a better effect on antioxidant capacity. These results indicated that potential application of Ac-CPP0.1 as an antioxidant agent.

Network pharmacology reveals pharmacological effect and mechanism of Panax notoginseng (Burk.) F. H. Chen on reproductive and genetic toxicity in male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cisplatin (CP), one of the most commonly used antitumor drugs in clinic, could induce reproductive and genetic toxicity. Traditional Chinese medicine believed that this side effect might be caused by the deficiency of both qi and blood. Panax notoginseng (Burk.) F. H. Chen (PN) is a traditional precious Chinese medicine for nourishing blood and hemostasis, which had the synergistic antitumor and reducing toxicity effects. However, the protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity were still unknown. AIM OF THE STUDY: This study was designed to illuminate the possible protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity. MATERIALS AND METHODS: Network pharmacology was first applied to analyze the potential components and targets of PN against CP-induced reproductive and genetic toxicity. Then, the results of network pharmacology were validated in a mouse model of reproductive and genotoxicity induced by CP. Body weight, testis weight, epididymis weight, sperm count, sperm viability and sperm morphology were used to assess protective effects of PN on CP-induced reproductive toxicity. Tail moment in peripheral blood cells and micronucleus in bone marrow cells were used to assess protective effects of PN on CP-induced genetic toxicity. Finally, possible protective targets obtained from network pharmacology, including 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), were experimentally validated by ELISA. RESULTS: One hundred and nineteen components of PN and sixty-eight targets of reproductive/genetic toxicity were acquired and constituted as the component-target network. Network pharmacology analysis showed alleviating oxidative stress might play important role in therapeutic mechanism of PN. In verified experiments, PN significantly improved the decline of body weight, testis weight and epididymis weight, increased sperm count and viability, decreased abnormal sperm morphology rate induced by CP in mice. Moreover, PN also significantly decreased the tail moment in peripheral blood cells and micronucleus formation rate in bone marrow cells in CP-induced mice. Finally, not only the decrease of T-SOD and GSH-Px level but also the increase of 8-OHdG and MDA level in serum were restored under PN treatment. CONCLUSION: Current study found that PN could improve CP-induced reproductive and genetic toxicity, which were probably attributed to alleviating oxidative stress. This finding provided the new perspective for understanding the therapeutic effect of PN on CP-induced reproductive and genetic toxicity and facilitating the clinical use of PN.

Flavonoids from the peels of Citrus unshiu Markov. and their inhibitory effects on RANKL-induced osteoclastogenesis through the downregulation of c-Fos signaling in vitro.

Phytochemical investigation of Citrus unshiu peels led to the isolation of eight new flavonols (7-9, 11-15) and sixteen known compounds (1-6, 10, 16-24). Their structures were elucidated using spectroscopic analysis (1D, 2D NMR, and HR-MS). Besides, all isolated compounds (1-24) were evaluated for their inhibitory effects on receptor activator of RANKL-induced osteoclastogenesis in BMMs. Among them, dimethylmikanin (1), quercetogetin (2), 3,3',4',5,7,8-hexamethoxyflavone (3), 3-methoxynobiletin (4) showed a significant inhibitory effect on RANKL-induced osteoclast differentiation at a concentration of 10 µM. Moreover, 3-methoxynobiletin (4) suppressed RANKL-induced osteoclastogenesis by decreasing the number of osteoclasts and osteoclast actin-ring formation in a dose-dependent manner without causing any cytotoxic effects on BMMs. At the molecular level, 3-methoxynobiletin (4) inhibited RANKL-induced c-Fos expression and subsequently NFATc1 activation, as well as the expression of osteoclastogenesis-related marker genes c-Src and CtsK. These findings suggested that 3-methoxynobiletin (4) attenuated osteoclast differentiation by inhibiting RANKL-mediated c-Fos signaling and that it may have therapeutic potential for treating or preventing bone resorption-related diseases, such as osteoporosis.

Evaluation of in vitro and in vivo genotoxic and antigenotoxic effects of Rhus coriaria.

Rhus coriaria has been important in the treatment of many diseases in traditional use. In this content, the genotoxic, antigenotoxic, and oxidative stress effects of methanol extract of R. coriaria (RCE) were investigated in this study. Two hundred fifty, 500, or 750 µg/mL concentrations of RCE were not found to have DNA damaging effect on pET22-b(+) plasmid and were unable to induce micronuclei in human lymphocytes (24 or 48 h treatment period). However, it did not inhibit the genotoxic effect of mitomycin-c (0.25 µg/mL). Cytotoxic effects of RCE were investigated using mitotic index (MI) and nuclear division index (NDI). Five hundred, 1000, and 2000 mg/kg concentrations of RCE did not induce chromosome aberrations in rat bone marrow cells for 12 or 24 h treatment period. In addition, 2000 mg/kg concentration of RCE showed an antigenotoxic effect by decreasing to genotoxic effect of 400 mg/kg urethane at 12 and 24 h treatment periods. RCE showed cytotoxic effects by significantly decreasing NDI. Moreover, RCE increased cytotoxic effect of Mitomycin C (MMC). However, RCE did not induce cytotoxicity in rat bone marrow cells. The highest concentration of RCE reduced total oxidant level in 12 h treatment. Interestingly, the lowest total oxidant level was found in rats blood treated with the lowest concentration RCE and urethane together. Thousand and 2000 mg/kg concentrations of RCE decreased total antioxidant levels of rat blood at 24 h treatment period. Our results showed that RCE possess cytotoxic effect in short-term treatments in vitro. However, it does not demonstrate genotoxic or cytotoxic effects in vivo.

Jabuticaba peel extract modulates adipocyte and osteoblast differentiation of MSCs from healthy and osteoporotic rats.

INTRODUCTION: The jabuticaba peel extract (JPE) contains bioactive compounds that regulate fat metabolism. Because the negative correlation between fat accumulation and bone formation in bone marrow, we hypothesized that JPE inhibits adipocyte as well as favors osteoblast differentiation of mesenchymal stromal cells (MSCs) under healthy and osteoporotic conditions, a disease that display an imbalance between adipocyte and osteoblast differentiation resulting in reduced bone mass. MATERIAL AND METHODS: To test these hypotheses, bone marrow MSCs were harvested from healthy and osteoporotic rats and cultured in adipogenic and osteogenic media with three concentrations of JPE, 0.25, 5 and 10 µg/ml, and vehicle (control). After selecting the most efficient concentrations of JPE, we used them to evaluate adipocyte and osteoblast differentiation of MSCs from both sources. RESULTS: We observed that, in general, JPE inhibited adipocyte differentiation of MSCs with more pronounced effects in cells from healthy than osteoporotic rats. In addition, JPE increased osteoblast differentiation, exhibiting a slightly higher osteogenic potential on MSCs from osteoporotic compared to healthy condition. CONCLUSION: Our results demonstrated that JPE drives MSCs to inhibit adipocyte differentiation and toward osteoblast differentiation under healthy and osteoporotic conditions. These findings pave the way for further translational studies to investigate the therapeutic possibilities of JPE in both prevention and treatment of osteoporosis.

Shuangshen granules attenuate lung metastasis by modulating bone marrow differentiation through mTOR signalling inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Shuangshen granules (SSG) have been used to treat lung cancer patients with Qi deficiency and blood stasis for decades. According to clinical experience, SSG indeed improve the quality of life and prolong the survival time of patients with lung cancer after surgery. Each of the components herbs was proved to be effective in anti-cancer therapy. Both the American ginseng and notoginseng belong to genus Panax of the family Araliaceae. Preclinical and clinical studies demonstrated that ginsenosides of them have anti- or preventive activities to various tumors, including cancers of gastric, breast, liver, lung, ovarian, colon, melanoma and leukemia. PDS, such as ginsenoside Rb1, and PTS, such as ginsenoside Rg1 are the main anticancer compositions. Cordyceps sinensis had also been found effective in inhibiting tumour growth and metastasis, especially on tumour associated immune cells, such as macrophages. However, limited information is available regarding potential mechanisms of SSG. Myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, which is closely associated with poor clinical outcomes in cancer patients, may be the target of SSG, which regulate immune function. AIM OF THE STUDY: The present study aimed to explore whether SSG attenuate the differentiation of bone marrow cells (BMCs) into MDSCs by blocking the mTOR signalling, leading to the suppression of lung metastasis. MATERIALS AND METHODS: First, we observed the differentiation of BMCs into MDSCs in vitro and in vivo. BMCs were cultured alone or co-cultured with Lewis lung carcinoma (LLC) cell supernatant in vitro. The effects of different concentrations of SSG, or LLC cell supernatant as a control, on BMC differentiation were detected by flow cytometry and western blotting. Male C57BL/6J mice were subcutaneously implanted with LLC cells, and SSG were administered by gavage twice daily before and after implantation for 7 or 14 days, respectively. The tumour weight, proportion of MDSCs, presence of CD11b+Ly6C+Ly6G- and CD11b+Ly6C+Ly6G+ cells in the bone marrow, blood, and lungs, as well as the expression levels of differentiation-related proteins in the bone marrow and lungs were evaluated. RESULTS: SSG attenuated the differentiation of BMCs into MDSCs, and reduced the fraction of CD11b+Ly6C+Ly6G+ cells by inhibiting the mTOR/S6K1/Myc signalling pathway. In vivo, SSG attenuated differentiation-associated protein markers and reduced the fractions of MDSCs and CD11b+Ly6C+Ly6G+ cells in the bone marrow, blood, and lungs. In addition, SSG administration reduced the tumour weight and inhibited lung metastasis. CONCLUSIONS: SSG may reduce lung metastasis by attenuating BMC differentiation into CD11b+Ly6C+Ly6G+ cells by inhibiting mTOR signalling in vitro and in vivo.

Clinical plasma concentration of vinpocetine does not affect osteogenic differentiation of mesenchymal stem cells.

AIM: Vinpocetine (Vin) has long been used as a medicine to treat cerebrovascular disorders and as a dietary supplement to improve cognitive functions. Previous studies have revealed that the transcription factor nuclear factor kappa B (NF-κB) activity plays an important role in osteogenic differentiation of mesenchymal stem cells (MSC). Vin inhibits NF-κB-dependent inflammatory responses; however, the effect of Vin on the osteogenic differentiation of MSCs has not been reported. In this study, we aimed to the investigate effect of Vin on the osteogenic differentiation of rat bone marrow-derived MSCs (BMSCs). METHODS: We treated BMSCs with clinical plasma (0.17 µM) or higher concentrations (5 and 20 µM) of Vin with no significant effect on the cell viability. Alizarin Red S and alkaline phosphatase (ALP) stainings were used to evaluate mineralizations on days 14 and 21. Moreover, expressions of target genes were detected using qRT-PCR analysis. RESULTS: Osteogenic differentiation of BMSCs did not significantly change with Vin's clinical plasma concentration, but significantly decreased with higher concentrations. Calcium mineralization, ALP staining and mRNA gene expressions of Runx2 and ALP were decreased significantly with high concentrations of Vin, paticularly on day 21. CONCLUSION: Our in vitro findings suggest that clinically relevant concentration of Vin seems safe to use in elderly patients with respect to osteoporosis. On the other hand, Vin at high concentrations appears to be harmful to bone homeostasis.

Toxicological safety evaluation of the cultivated Chinese cordyceps.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese cordyceps, a parasitic Thitarodes insect-Ophiocordyceps sinensis fungus complex in the Qinghai-Tibet Plateau, is one of the most valuable traditional Chinese medicines and health food for ameliorating conditions associated with aging and for treating fatigue, night sweats, hyperglycemia, hyperlipidemia, respiratory, renal and liver diseases, and hyposexuality. The natural Chinese cordyceps resource is rare due to its harsh growing environment, limited geographical distribution and global climate warming. Artificial cultivation of Chinese cordyceps has been successfully established to meet its high demand in market. AIMS OF THE STUDY: The present study aims to evaluate the toxicological safety of the cultivated Chinese cordyceps and provide scientific data for subsequent development and utilization of this valuable biological resource. MATERIALS AND METHODS: The Chinese cordyceps was cultivated by mimicking the habitat environment in low-altitude areas and identified by morphological and microscopic characteristics. Its phytochemical profile was determined by the HPLC. Toxicological studies based on the cultivated Chinese cordyceps were conducted, including chromosomal aberration test of Chinese hamster lung (CHL) cells, Ames test, acute toxicity test and micronucleus (MN) test of bone marrow cells. RESULTS: The Chinese cordyceps successfully cultivated in low-altitude areas exhibited the same morphological and microscopic characteristics as natural Chinese cordyceps. The adenosine content was in accordance with the Chinese Pharmacopoeia (2015 Edition). The HPLC fingerprint was determined and five main chromatographic peaks representing uracil, uridine, inosine, guanosine and adenosine were identified. No dose-dependent increase in the rates of chromosomal aberration was detected in the presence or absence of metabolic activation system. Ames test also demonstrated no dose-dependent increase in the number of reversion mutation for five bacterial strains, with or without rat liver microsomal enzyme mixture (S9) metabolic activation, at a quantity range of 128-5000 µg cultivated Chinese cordyceps per plate. The acute toxicity test with mice showed that after 20 g/kg oral administration of cultivated Chinese cordyceps, neither animal death nor any abnormal change in general dissection of various tissues and organs of the animals were found within 14 days. The median lethal dose (LD50) was greater than 5 g/kg, which is regarded as a non-toxic level, and maximum tolerable dose (MTD) of cultivated Chinese cordyceps in ICR mice was more than 20 g/kg. MN test of mouse bone marrow cells indicated no significant differences among each sample dose and the negative control. CONCLUSION: Based on the results from four toxicological tests, it was concluded that the cultivated Chinese cordyceps was classified as non-toxic in one single administration at high doses by intragastric route in mice. This study provides scientific experimental basis for its safety.

Eucalyptus oil reduces allergic reactions and suppresses mast cell degranulation by downregulating IgE-FcεRI signalling.

Eucalyptus oil has been used since ancient times for its bactericidal, anti-inflammatory, analgesic and sedative effects. In recent years, the action of Eucalyptus oil has been scientifically proven, and there have been reports that Eucalyptus oil suppresses the production of chemokines, cytokines and lipid mediators in basophils, alveolar macrophages and monocytes. Based on this information, we aimed to verify whether Eucalyptus oil can be used for allergic dermatitis, the incidence of which has been increasing among human skin diseases. This effect was verified using a mouse IgE-mediated local allergic model. In conclusion, topical application of Eucalyptus oil suppressed oedema and vascular permeability enhancement due to IgE-mediated allergic on the skin. In addition, we also verified the degranuration of mast cells, which is a part of its action, and examined whether 1,8-cineole, which is the main component of Eucalyptus oil, suppresses the phosphorylation of PLCγ and p38 directly or indirectly. 1,8-cineole was found to suppress degranulation of mast cells.

Effect of Tussilago farfara L. Polysaccharides on the Content of Hematopoietic Stem Cells (CD117+34+) in the Bone Marrow of Mice Treated with Cyclophosphamide.

Myelotoxicity is a serious side effect of anticancer drugs. The search for drugs that can reduce the hematological complications of chemotherapy through modulation of hematopoietic stem cells is an urgent task of oncopharmacology. In the present study we showed that administration of Tussilago farfara L. polysaccharides to C57BL/6 mice treated with cyclophosphamide can increase the number of hematopoietic stem cells (CD117+34+) in the bone marrow.

Expression of RUNX2 and Osterix in Rat Mesenchymal Stem Cells during Culturing in Osteogenic-Conditioned Medium.

We studied the expression of transcription factors RUNX2 and Osterix after addition of a concentrate of osteogenic-conditioned medium to the culture medium for osteogenic differentiation of mesenchymal stem cells (MSC). The obtained concentrate of osteogenic-conditioned medium containing a complex of bioactive substances with a molecular weight >10 kDa provided MSC differentiation into osteoblasts, which was confirmed by high level of expression of transcription factors RUNX2 and Osterix in comparison with the negative control. The highest expression of transcription factor Osterix was revealed on day 14 of MSC culturing in the presence of osteogenic supplement StemPro (positive control) and the studied concentrate of osteogenic-conditioned medium.