Selective synergistic anticancer effects of cisplatin and oridonin against human p53-mutant esophageal squamous carcinoma cells.

Oridonin (ORI) is known to pose anticancer activity against cancer, which could induce the therapeutic impact of chemotherapy drugs. However, such simple combinations have numerous side effects such as higher toxicity to normal cells and tissues. To enhance the therapeutic effects with minimal side effects, here we used ORI in combination with cisplitin (CIS) against different esophageal squamous cell carcinoma (ESCC) cell lines in vitro, to investigate the synergistic anticancer effects of the two drugs against ESCC. Calcusyn Graphing Software was used to assess the synergistic effect. Apoptosis, wound healing and cell invasion assay were conducted to further confirm the synergistic effects of ORI and CIS. Intracellular glutathione (GSH) and reactive oxygen species assay, immunofluorescence staining and western blot were used to verify the mechanism of synergistic cytotoxicity. ORI and CIS pose selective synergistic effects on ESCC cells with p53 mutations. Moreover, we found that the synergistic effects of these drugs are mediated by GSH/ROS systems, such that intracellular GSH production was inhibited, whereas the ROS generation was induced following ORI and CIS application. In addition, we noted that DNA damage was induced as in response to ORI and CIS treatment. Overall, these results suggest that ORI can synergistically enhance the effect of CIS, and GSH deficiency and p53 mutation, might be biomarkers for the combinational usage of ORI and CIS.

Pharmacological Approaches to Attenuate Inflammation and Obesity with Natural Products Formulations by Regulating the Associated Promoting Molecular Signaling Pathways.

Obesity is a public health problem characterized by increased body weight due to abnormal adipose tissue expansion. Bioactive compound consumption from the diet or intake of dietary supplements is one of the possible ways to control obesity. Natural products with adipogenesis-regulating potential act as obesity treatments. We evaluated the synergistic antiangiogenesis, antiadipogenic and antilipogenic efficacy of standardized rebaudioside A, sativoside, and theasaponin E1 formulations (RASE1) in vitro in human umbilical vein endothelial cells (HUVECs), 3T3-L1 preadipocytes respectively, and in vivo using a high-fat and carbohydrate diet-induced obesity mouse model. Orlistat was used as a positive control, while untreated cells and animals were normal controls (NCs). Adipose tissue, liver, and blood were analyzed after dissection. Extracted stevia compounds and green tea seed saponin E1 exhibited pronounced antiobesity effects when combined. RASE1 inhibited HUVEC proliferation and tube formation by suppressing VEGFR2, NF-κB, PIK3, and-catenin beta-1 expression levels. RASE1 inhibited 3T3-L1 adipocyte differentiation and lipid accumulation by downregulating adipogenesis- and lipogenesis-promoting genes. RASE1 oral administration reduced mouse body and body fat pad weight and blood cholesterol, TG, ALT, AST, glucose, insulin, and adipokine levels. RASE1 suppressed adipogenic and lipid metabolism gene expression in mouse adipose and liver tissues and enhanced AMP-activated protein kinase levels in liver and adipose tissues and in serum adiponectin. RASE1 suppressed the NF-κB pathway and proinflammatory cytokines IL-10, IL-6, and TNF-α levels in mice which involve inflammation and progression of obesity. The overall results indicate RASE1 is a potential therapeutic formulation and functional food for treating or preventing obesity and inflammation.

Synergistic combination therapy of lung cancer using lipid-layered cisplatin and oridonin co-encapsulated nanoparticles.

Lung cancer treatment using cisplatin (DDP) in combination with other drugs are effective for the treatment of non-small cell lung cancer (NSCLC). The aim of this study was to prepare a layer-by-layer nanoparticles (NPs) for the co-loading of DDP and oridonin (ORI) and to evaluate the antitumor activity of the system in vitro and in vivo. Novel DDP and ORI co-loaded layer-by-layer NPs (D/O-NPs) were constructed. The mean diameter, surface change stability and drug release behavior of NPs were evaluated. In vitro cytotoxicity of D/O-NPs was investigated against DDP resistant human lung cancer cell line (A549/DDP cells), and in vivo anti-tumor efficiency of D/O-NPs was tested on mice bearing A549/DDP cells xenografts. D/O-NPs have a diameter of 139.6 ± 4.4 nm, a zeta potential value of +13.8 ± 1.6 mV. D/O-NPs could significantly enhance in vitro cell toxicity and in vivo antitumor effect against A549/DDP cells and lung cancer animal model compared to the single drug loaded NPs and free drugs. The results demonstrated that the D/O-NPs could be used as a promising lung cancer treatment system.

The Role of NLRP3 Inflammasome in Cerebrovascular Diseases Pathology and Possible Therapeutic Targets.

Cerebrovascular diseases are pathological conditions involving impaired blood flow in the brain, primarily including ischaemic stroke, intracranial haemorrhage, and subarachnoid haemorrhage. The nucleotide-binding and oligomerisation (NOD) domain-like receptor (NLR) family pyrin domain (PYD)-containing 3 (NLRP3) inflammasome is a protein complex and a vital component of the immune system. Emerging evidence has indicated that the NLRP3 inflammasome plays an important role in cerebrovascular diseases. The function of the NLRP3 inflammasome in the pathogenesis of cerebrovascular diseases remains an interesting field of research. In this review, we first summarised the pathological mechanism of cerebrovascular diseases and the pathological mechanism of the NLRP3 inflammasome in aggravating atherosclerosis and cerebrovascular diseases. Second, we outlined signalling pathways through which the NLRP3 inflammasome participates in aggravating or mitigating cerebrovascular diseases. Reactive oxygen species (ROS)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), ROS/thioredoxin-interacting protein (TXNIP) and purinergic receptor-7 (P2X7R) signalling pathways can activate the NLRP3 inflammasome; activation of the NLRP3 inflammasome can aggravate cerebrovascular diseases by mediating apoptosis and pyroptosis. Autophagy/mitochondrial autophagy, nuclear factor E2-related factor-2 (Nrf2), interferon (IFN)-ß, sirtuin (SIRT), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) reportedly alleviate cerebrovascular diseases by inhibiting NLRP3 inflammasome activation. Finally, we explored specific inhibitors of the NLRP3 inflammasome based on the two-step activation of the NLRP3 inflammasome, which can be developed as new drugs to treat cerebrovascular diseases.

Effects of the natural sweetener rebaudioside A on intake, nutrient digestion, rumen fermentation, and blood biochemical parameters in adult goats during summer.

The effects of dietary rebaudioside A inclusion on feed intake, digestion of nutrients, rumen fermentation, and blood biochemical parameters of goats were evaluated in a replicated 3 × 3 Latin square study. Nine adult goats during summer were fed a basal forage/concentrate-based diet and the forage was chopped rice straw. The three dietary treatments were 0, 350, and 700 mg rebaudioside A per kg chopped rice straw on a DM basis. No significant improvement was observed in dry matter intake (DMI) of forage and diet among treatments. Nutrient digestibility of DM and organic matter (OM) showed a significant trend (p < .10) across groups. Rebaudioside A inclusion significantly (p < .01) increased the concentration of total volatile fatty acids in the rumen, however, there were no differences in concentration of ruminal ammonia, and molar proportions of acetate, propionate, and butyrate. About blood metabolites, increasing rebaudioside A in the diet caused a quadratic response in glucose and total protein, and albumin concentrations. Under the conditions of this study, supplementation with rebaudioside A at 350 and 700 mg/kg forage did not improve consumption of rice straw-based diet in adult goats in summer. However, the responses in digestibility, rumen fermentation, and blood metabolites appear to indicate the potential of rebaudioside A as a bio-active substance in goats.

Promote anti-inflammatory and angiogenesis using a hyaluronic acid-based hydrogel with miRNA-laden nanoparticles for chronic diabetic wound treatment.

Chronic diabetic wound causes serious threat to human health due to its long inflammatory phase and the reduced vascularization. Herein, we develop a hydrogel system for the treatment of diabetic wound, which can short the inflammatory stage (through the use of ori) and promote the angiogenesis (through the addition of siRNA-29a gene). Based on the Schiff base bonds, the Gel/Alg@ori/HA-PEI@siRNA-29a hydrogel was prepared by mixing oxidized hydroxymethyl propyl cellulose (OHMPC), adipic dihydrazide-modified hyaluronic acid (HA-ADH), oridonin (ori) loaded alginate microspheres (Alg@ori) and siRNA-29a gene-loading hyaluronic acid-polyethyleneimine complex HA-PEI@siRNA-29a (HA-PEI@siRNA-29a) under physiological conditions, which had moderate mechanical strength, appropriate swelling property, impressive stability, and slow release ability of ori and siRNA-29a. Excellent biocompatibility of the prepared hydrogel was also confirmed by in vitro mouse fibroblasts L929 cells culture study. Moreover, in vivo experiments further demonstrated that the prepared Gel/Alg@ori/HA-PEI@siRNA-29a hydrogel not only significantly accelerated the diabetic wound healing, angiogenesis factors (α-SMA and CD31) production, but also inhibited pro-inflammatory factors (IL-6 and TNF-α). In summary, we believe that the prepared hydrogels exhibit great potential for the treatment of chronic diabetic wound.

15ß-Senecioyl-oxy-ent-kaur-16-en-19-oic Acid, a Diterpene Isolated from Baccharis lateralis, as Promising Oral Compound for the Treatment of Schistosomiasis.

Praziquantel is the only available drug to treat schistosomiasis, and therefore, urgent studies must be performed to identify new anthelmintic agents. This study reports the anthelmintic evaluation of two related ent-kaurane diterpenes isolated from aerial parts of Baccharis lateralis (Asteraceae), ent-kaur-16-en-19-oic acid (1) and 15ß-senecioyl-oxy-ent-kaur-16-en-19-oic acid (2) against Schistosoma mansoni in vitro and in a murine model of schistosomiasis. Both compounds exhibited in vitro activity with lethal concentration 50% (LC50) values of 26.1 µM (1) and 11.6 µM (2) as well as reduced toxicity against human cell lines, revealing a good selectivity profile, mainly with compound 2 (selectivity index > 10). Compound 2 also decreased egg production and caused morphological alterations in the parasite reproductive system. In mice infected with S. mansoni, oral treatment with compound 2 at 400 mg/kg, the standard dose used in this model of schistosomiasis, caused a significant reduction in a total worm burden of 61.9% (P < 0.01). S. mansoni egg production, a key mechanism for both transmission and pathogenesis, was also markedly reduced. In addition, compound 2 achieved a significant reduction in hepatosplenomegaly. Therefore, the diterpene 15ß-senecioyl-oxy-ent-kaur-16-en-19-oic acid (2) has an acceptable cytotoxicity profile and is orally active in a murine schistosomiasis model.

Oridonin promotes osteogenesis through Wnt/ß-catenin pathway and inhibits RANKL-induced osteoclastogenesis in vitro.

AIMS: To study the molecular mechanism of oridonin (ORI) on osteoblast differentiation and osteoclast formation in vitro. MAIN METHODS: Rat bone marrow mesenchymal stem cells (BMSCs) were treated with different concentrations of ORI in osteogenic medium (OM). CCK-8 assay and were used to detect the effect on BMSCs viability. Alizarin red staining and ALP activity were used to illuminate the effect of ORI on osteogenic differentiation. Expressions of osteogenic differentiation related genes were detected by real-time quantitative PCR (qRT-PCR), and expressions of osteogenic related proteins were detected by Western blot (WB) and immunofluorescence. Similarly, bone marrow mononuclear cells (BMMs) were treated with different concentrations of ORI. CCK-8 assay and Live/Dead staining were used to detect the effect of ORI on BMMs activity. TRAP staining was used to detect its effect on osteoclast differentiation. Expressions of osteoclast-related genes were detected by qRT-PCR, and expressions of osteoclast-related proteins were detected by WB and immunofluorescence. KEY FINDINGS: (1) ORI (2 µM) promoted the ALP activity of BMSCs differentiation into osteoblasts and increased the number of calcium nodules. (2) ORI stimulated the expressions of wnt1, ß-catenin and Runx2, but with no significantly effect on p-GSK-3ß and GSK-3ß. (3) ORI promoted the expression of OPG and inhibited the expression of RANKL. (4) ORI directly/indirectly inhibited the osteoclast formation and expressions of osteoclast-related genes TRAP, NFATc1 and c-Fos. SIGNIFICANCE: ORI may promote BMSCs differentiate into osteoblasts through the Wnt/ß-catenin signaling pathway. At the same time, it may also inhibit the formation of osteoclasts mediated by RANKL.

Oridonin is an antidepressant molecule working through the PPAR-γ/AMPA receptor signaling pathway.

Oridonin is a diterpene compound that regulates the activity of PPAR-γ (peroxisome proliferator-activated receptor gamma) transcription factor. Cumulative evidence indicates that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors (AMPARs) play an important role in the treatment of depression. In the article, we found that after treatment with oridonin, the immobility time of mice was significantly reduced in the tail suspension test (TST) and the forced-swim test (FST). After five consecutive days of treatment in mice, oridonin significantly enhanced the expression of PPAR-γ, GluA1 (Ser845) phosphorylation, and GluA1 in the total protein extract of the prefrontal cortex (PFC). Blocking PPAR-γ was able to block antidepressant effects of oridonin. In synaptosome fractions of the PFC, oridonin treatment also significantly increased the GluA1 (Ser845) phosphorylation and GluA1 levels. Moreover, antidepressant actions of oridonin were blocked by AMPA receptor-specific antagonist GYKI 52466. This study demonstrates that oridonin regulates PPAR-γ/AMPA receptor signaling in the prefrontal cortex, and that oridonin can be identified as a novel antidepressant with clinical potential.

Structural dependence of antidiabetic effect of steviol glycosides and their metabolites on streptozotocin-induced diabetic mice.

BACKGROUND: Stevia has been proposed as a potential antidiabetic sweetener, mainly based on inconsistent results from stevioside or the plant extract, yet lacking relative experimental evidence from individual steviol glycosides (SGs) and their metabolites. RESULTS: The results systematically revealed that the typical SGs and their final metabolite (steviol) presented an antidiabetic effect on streptozotocin (STZ) diabetic mice in all assayed antidiabetic aspects. In general, the performance strength of the samples followed the sequence steviol > steviol glucosyl ester > steviolbioside > rubusoside > stevioside > rebaudioside A, which is opposite to their sweetness strength order, and generally in accordance with the glucosyl group numbers in their molecules. This may imply that the antidiabetic effect of the SGs might be achieved through steviol, which presented antidiabetic performance similar to that of metformin with a dose of 1/20 that of metformin. Moreover, the 18 F-fluorodeoxyglucose traced micro-PET experiment revealed that stevioside and steviol could increase the uptake of glucose in the myocardium and brain of the diabetic mice within 60 min, and decrease the accumulation of glucose in the liver and kidney. CONCLUSIONS: The SGs and steviol presented an antidiabetic effect on STZ diabetic mice in all assayed aspects, with an induction time to start the effect of the SGs. Stevioside and steviol could increase uptake of glucose in the myocardium and brain of the diabetic mice, and decrease accumulation of glucose in the liver and kidney. The performance strength of the SGs is generally in accordance with glucosyl group numbers in their molecules.

Novel ultrasmall nanomicelles based on rebaudioside A: A potential nanoplatform for the ocular delivery of pterostilbene.

Rebaudioside A (RA) self-assembled into ultrasmall nanomicelles can be utilized as ocular drug delivery system; nevertheless, the therapeutic efficacy of RA micelles has not evaluated thus far. In this manuscript, the RA micelles are thought to strengthen the therapeutic effects of pterostilbene (Pt). Results showed that Pt can be highly encapsulated into RA micelles with ultrasmall particle sizes (3.99 ± 0.03 nm) with a uniform distribution (polydispersity index, PDI = 0.184 ± 0.008). RA-Pt exhibited pronounced improvement for the in vitro antioxidant activity and the in vitro membrane permeation of Pt. RA-Pt exhibited good ocular tolerance. The use of RA-Pt led to the significant improvement in the in vivo intraocular permeation of Pt as well as the improvement in the in vivo anti-inflammatory efficacy. The in vivo evaluation of antioxidant effects revealed that RA-Pt exhibits improvement in suppressing the generation of oxidative stress and inducing the enzymes of reactive oxygen species (ROS) degradation. Furthermore, RA-Pt was confirmed to exhibit a strong treatment efficiency for corneal alkali burns. These findings indicate that RA-based self-assembled ultrasmall nanomicelles demonstrate tremendous potential toward the improvement in the ocular bioavailability as well as a therapeutic effect of poor aqueous soluble drugs such as Pt.

Oridonin suppresses autophagy and survival in rheumatoid arthritis fibroblast-like synoviocytes.

Context: Oridonin exhibits various pharmacological and physiological activities, including antioxidant, antibacterial, anti-inflammatory, pro-apoptotic, anticancer and neurological effects. However, its role in rheumatoid arthritis (RA) is yet to be revealed.Objective: We evaluated the effects of oridonin on the survival and autophagy of RA-fibroblast-like synoviocytes (FLSs).Materials and methods: RA-FLSs were treated with oridonin at serial concentrations of 0, 2, 4, 6, 8 and 10 µg/mL for 24, 48 and 72 h. Then, cell proliferation and apoptosis were measured. A GFP-LC3 plasmid was transfected into the cells to determine autophagy.Results: Oridonin suppressed RA-FLS proliferation in a dose-dependent manner. The half maximal inhibitory concentrations (IC50) of oridonin at 24, 48 and 72 h were 8.28, 7.88 and 8.35 µg/mL, respectively. Treatment with oridonin for 24 h increased apoptosis by 4.1%, and increased the protein levels of Bax and cleaved caspase-3 but significantly decreased the levels of IL-1ß in the culture supernatant (p < 0.05). In addition, 6 h of oridonin treatment significantly decreased the number of GFP-LC3 punctate dots and inhibited the protein levels of ATG5 and Beclin1 by 80.01% and 42.12%, respectively. Chloroquine (CQ) significantly reinforced the effects of oridonin on inhibition of autophagy, suppression of proliferation, and induction of apoptosis in RA-FLSs (p < 0.05).Conclusions: Our results indicate that treatment with oridonin in combination with CQ inhibits autophagy and cell proliferation and induces apoptosis in RA-FLSs more effectively than treatment oridonin alone. This finding indicates that oridonin is a potential therapeutic agent for RA.

Ceramide-Rubusoside Nanomicelles, a Potential Therapeutic Approach to Target Cancers Carrying p53 Missense Mutations.

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer in vivo bioavailability and restore p53-dependent tumor suppression in cancer cells carrying a p53 missense mutation. Natural RUB encapsulated short-chain C6-Cer so as to form Cer-RUB nanomicelles (∼32 nm in diameter) that substantially enhanced Cer solubility and its levels in tissues and tumors of mice dosed intraperitoneally. Intriguingly, Cer-RUB nanomicelle treatments restored p53-dependent tumor suppression and sensitivity to cisplatin in OVCAR-3 ovarian cancer cells and xenograft tumors carrying p53 R248Q mutation. Moreover, Cer-RUB nanomicelles showed no signs of significant nonspecific toxicity to noncancerous cells or normal tissues, including bone marrow. Furthermore, Cer-RUB nanomicelles restored p53 phosphorylated protein and downstream function to wild-type levels in p53 R172H/+ transgenic mice. Altogether, this study, for the first time, indicates that natural Cer-RUB nanomicelles offer a feasible approach for efficaciously and safely targeting cancers carrying p53 missense mutations.

Validation of an UHPLC-MS/MS Method for the Determination of Glaucocalyxin A, a Novel Potent Negative Akt Regulator in Rat Plasma, Lung and Brain Tissues: Application to a Pharmacokinetic Study.

Glaucocalyxin A, a novel potent negative Akt regulator, is a major active constituent of Rabdosia japonica. A simple, specific and sensitive ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantification of glaucocalyxin A in rat plasma, lung and brain tissues after intravenous administration. Sample preparation was carried out through a simple liqiud-liquid extraction with ethyl acetate using bullatine A as internal standard. The chromatographic separation was achieved by using an Agilent ZORBAX Eclipse Plus C18 column (3.0 mm × 100 mm, 1.8 µm) with a mobile phase of acetonitrile and water containing 0.1% formic acid in a gradient elution. Mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring transitions at m/z 333.2 â†’ 157.1 for glaucocalyxin A and m/z 344.2 â†’ 128.1 for IS. Calibration curves were linear over the ranges of 20.0-4000 ng/mL for both plasma and tissue samples (r2 > 0.99). The Lower limit of quantification (LLOQ) was 0.284 ng/mL. The intra-day and inter-day precision relative standard deviation (RSD%) were <14.9%, while the accuracy ranged from -12.5 to 17.0% for LLOQ and quality control samples. This UHPLC-MS/MS method was successfully applied in the pharmacokinetics, lung and brain tissue distributions of glaucocalyxin A after intravenous administration.

Angiotensin-converting enzyme inhibiting ability of ethanol extracts, steviol glycosides and protein hydrolysates from stevia leaves.

Efficient treatment of hypertension is vital. The inhibition of angiotensin-converting enzyme activity has been one of the major strategies for treating hypertension. The ethanol extract of stevia leaves, steviol glycosides (with 95% purity; natural sweeteners widely used in the food industry) isolated from the ethanol extract and stevia leaf protein hydrolysates inhibited 26.60%, 59.56% and 74.38% of angiotensin-converting enzyme activities, respectively. Their effect was dose-dependent, which can be beneficial for avoiding hypertension or hypotension just by the proper control of the amount of their intake, and it was found to be superior to that of pharmaceutical drugs. A sensory test indicated that the application of the mixtures of steviol glycosides and stevia protein hydrolysates to decaffeinated coffee or tea as well as a formulated peanut protein drink was found to be well accepted, and an animal test showed that they had a significantly antihypertensive effect in spontaneously hypertensive rats. Steviol glycosides and stevia leaf protein hydrolysates can be good ingredients for making functional or healthy food products or beverages targeted for the prevention or treatment of hypertension.

The antibacterial mechanism of oridonin against methicillin-resistant Staphylococcus aureus (MRSA).

Context: Methicillin-resistant Staphylococcus aureus (MRSA) is a very harmful bacterium. Oridonin, a component in Rabdosia rubescens (Hemsl.) Hara (Lamiaceae), is widely used against bacterial infections in China. Objective: We evaluated oridonin effects on MRSA cell membrane and wall, protein metabolism, lactate dehydrogenase (LDH), DNA and microscopic structure. Materials and methods: Broth microdilution and flat colony counting methods were used to measure oridonin minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against USA300 strain. Electrical conductivity and DNA exosmosis were analysed to study oridonin effects (128 µg/mL) on cell membrane and wall for 0, 1, 2, 4 and 6 h. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to detect effects on soluble protein synthesis after 6, 10 and 16 h. LDH activity was examined with an enzyme-linked immunosorbent assay. Effects of oridonin on USA300 DNA were investigated with DAPI staining. Morphological changes in MRSA following oridonin treatment were determined with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results: Oridonin MIC and MBC values against USA300 were 64 and 512 µg/mL, respectively. The conductivity and DNA exosmosis level of oridonin-treated USA300 improved by 3.20±0.84% and increased by 58.63 ± 1.78 µg/mL, respectively. LDH and soluble protein levels decreased by 30.85±7.69% and 27.51 ± 1.39%, respectively. A decrease in fluorescence intensity was reported with time. Oridonin affected the morphology of USA300. Conclusions: Oridonin antibacterial mechanism was related to changes in cell membrane and cell wall permeability, disturbance in protein and DNA metabolism, and influence on bacterial morphology. Thus, oridonin may help in treating MRSA infection.

Preliminary safety assessment of oridonin in zebrafish.

Context: Oridonin, isolated from the leaves of Isodon rubescens (Hemsl.) H.Hara (Lamiaceae), has good antitumor activity. However, its safety in vivo is still unclear. Objective: To investigate the preliminary safety of oridonin in zebrafish. Materials and methods: Embryo, larvae and adult zebrafish (n = 40) were used. Low, medium and high oridonin concentrations (100, 200 and 400 mg/L for embryo; 150, 300 and 600 mg/L for larvae; 200, 400 and 800 mg/L for adult zebrafish) and blank samples were administered. At specific stages of zebrafish development, spontaneous movement, heartbeat, hatching rate, etc., were recorded to assess the developmental effects of oridonin. VEGFA, VEGFR2 and VEGFR3 gene expression were also examined. Results: Low-dose oridonin increased spontaneous movement and hatching rate with median effective doses (ED50) of 115.17 mg/L at 24 h post-fertilization (hpf) and 188.59 mg/L at 54 hpf, but these values decreased at high doses with half maximal inhibitory concentrations (IC50) of 209.11 and 607.84 mg/L. Oridonin decreased heartbeat with IC50 of 285.76 mg/L at 48 hpf, and induced malformation at 120 hpf with half maximal effective concentration (EC50) of 411.94 mg/L. Oridonin also decreased body length with IC50 of 324.78 mg/L at 144 hpf, and increased swimming speed with ED50 of 190.98 mg/L at 120 hpf. The effects of oridonin on zebrafish embryo development may be attributed to the downregulation of VEGFR3 gene expression. Discussions and conclusions: Oridonin showed adverse effects at early stages of zebrafish development. We will perform additional studies on mechanism of oridonin based on VEGFR3.

Investigation of Metal-Organic Framework-5 (MOF-5) as an Antitumor Drug Oridonin Sustained Release Carrier.

Oridonin (ORI) is a natural active ingredient with strong anticancer activity. But its clinical use is restricted due to its poor water solubility, short half-life, and low bioavailability. The aim of this study is to utilize the metal organic framework material MOF-5 to load ORI in order to improve its release characteristics and bioavailability. Herein, MOF-5 was synthesized by the solvothermal method and direct addition method, and characterized by Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectrometer (FTIR), Thermogravimetric Analysis (TG), Brunauer-Emmett-Teller (BET), and Dynamic Light Scattering (DLS), respectively. MOF-5 prepared by the optimal synthesis method was selected for drug-loading and in vitro release experiments. HepG2 cells were model cells. MTT assay, 4',6-diamidino-2-phenylindole (DAPI) staining and Annexin V/PI assay were used to detect the biological safety of blank carriers and the anticancer activity of drug-loaded materials. The results showed that nano-MOF-5 prepared by the direct addition method had complete structure, uniform size and good biocompatibility, and was suitable as an ORI carrier. The drug loading of ORI@MOF-5 was 52.86% ± 0.59%. The sustained release effect was reliable, and the cumulative release rate was about 87% in 60 h. ORI@MOF-5 had significant cytotoxicity (IC50:22.99 µg/mL) and apoptosis effect on HepG2 cells. ORI@MOF-5 is hopeful to become a new anticancer sustained release preparation. MOF-5 has significant potential as a drug carrier material.

Sweet taste receptor agonists alter ovarian functions and ovarian cycles in aged mice.

Saccharine sodium and rebaudioside A are low-calorie sweeteners, and the biologic effects of these sweeteners in rat ovaries are related to the activity of sweet taste receptors. Data on the impact and regulatory mechanisms underlying such sweeteners on the reproduction of aged animals are currently lacking. In the present study we assessed how the consumption of sweeteners affects the ovarian cycle, ovulation, biochemical indices, and other biologic functions. Thirty-six 1-year-old mice were randomly divided into 3 groups: a control (C) group receiving regular water, a saccharin sodium group receiving a 7.5 mM solution, and the rebaudioside A group receiving a 2.5 mM solution for 30 days. We observed no significant changes in body weights in any group. However, uterine weight in the rebaudioside A group significantly increased in diestrus, and we recorded a significant increase in the percentage of abnormal estrous cycles and the number of corpora lutea in the treatment groups. TUNEL staining and Immunoreactivity for the apoptosis-inducing factor (AIF) confirmed apoptosis in granulosa cells, oocyte, and corpus luteum. Serum glucose increased significantly in both treatment groups and there was a significant increase in cholesterol in the rebaudioside A group. Furthermore, the saccharin sodium-treated group exhibited elevated serum progesterone levels compared with the other groups. In conclusion, sweeteners manifested deleterious effects on reproductive indices in aged mice.

Low-Dose Stevia (Rebaudioside A) Consumption Perturbs Gut Microbiota and the Mesolimbic Dopamine Reward System.

Stevia is a natural low-calorie sweetener that is growing in popularity in food and beverage products. Despite its widespread use, little is understood of its impact on the gut microbiota, an important environmental factor that can mediate metabolism and subsequent obesity and disease risk. Furthermore, given previous reports of dysbiosis with some artificial low-calorie sweeteners, we wanted to understand whether prebiotic consumption could rescue potential stevia-mediated changes in gut microbiota. Three-week old male Sprague-Dawley rats were randomized to consume: (1) Water (CTR); (2) Rebaudioside A (STV); (3) prebiotic (PRE); (4) Rebaudioside A + prebiotic (SP) (n = 8/group) for 9 weeks. Rebaudioside was added to drinking water and prebiotic oligofructose-enriched inulin added to control diet (10%). Body weight and feces were collected weekly and food and fluid intake biweekly. Oral glucose and insulin tolerance tests, gut permeability tests, dual X-ray absorptiometry, and tissue harvest were performed at age 12 weeks. Rebaudioside A consumption alone did not alter weight gain or glucose tolerance compared to CTR. Rebaudioside A did, however, alter gut microbiota composition and reduce nucleus accumbens tyrosine hydroxylase and dopamine transporter mRNA levels compared to CTR. Prebiotic animals, alone or with Rebaudioside A, had reduced fat mass, food intake, and gut permeability and cecal SCFA concentration. Adding Rebaudioside A did not interfere with the benefits of the prebiotic except for a significant reduction in cecal weight. Long-term low-dose Rebaudioside A consumption had little effect on glucose metabolism and weight gain; however, its impact on gut microbial taxa should be further examined in populations exhibiting dysbiosis such as obesity.