Vehicle Control Group Research Articles

Four-Week GLP Immunotoxicity Assessment of Lactoferrin Alpha Produced by Komagataella phaffii in Sprague-Dawley Rats

Oral toxicity and toxicokinetic properties of human lactoferrin (LF) alpha produced in Komagataella phaffii (effera™) were investigated in adult Sprague-Dawley rats over a 28-day period under good laboratory practice conditions. Main study dosing used groups of 10 rats/sex/dose, and a secondary study evaluating toxicokinetic parameters used 6 rats/sex/dose. The vehicle control group received sodium citrate buffer, test groups received daily doses of 200, 600, and 2000 mg of effera™ per kg body weight, and the comparative control group received 2000 mg bovine LF (bLF)/kg body weight per day. T-cell-dependent antibody response against keyhole limpet hemocyanin and immunophenotyping of the spleen were performed as measures of immunotoxicity. Clinical observations, body weight, hematology, coagulation, clinical chemistry, urinalysis, immunotoxicity, gross necropsy, and histopathology were assessed. Toxicokinetic parameters were analyzed as an indication of LF bioavailability, and anti-LF antibody assays were conducted to detect antibodies produced against LF to measure immunogenicity. No treatment related toxicologically significant changes were observed. Based on the absence of toxicologically relevant changes, effera™ is well tolerated in rats at doses up to 2000 mg rhLF/kg/day, an amount ∼400 times that of the estimated daily intake at the 90th percentile proposed for human adult use.

ANTIDEPRESSANT-LIKE ACTIVITY OF AQUEOUS EXTRACT OF ROSA DAMASCENA IN MICE

Objective: Plant-based drugs have the potential to be very effective substitutes for prescription antidepressants. Rosa damascena has therapeutic potential as an analgesic, anticonvulsant, antitussive, bronchodilatory, antibacterial, anti-diabetic, anti-inflammatory, antioxidant, and laxative. Given this context, the goal of the current study was to assess Rosa damascena's potential antidepressant effects. Methods: Maceration was used to create an aqueous extract of Rosa damascena. The Tail Suspension Test (TST) on BALB/c mice and the Forced-Swimming test (FST) on C57BL/6 mice were used to quantify the antidepressant activity. Mice were divided into three groups: control (saline), standard (citalopram and desipramine), and Rosa damascena aqueous extract (n = 6 per group). Intraperitoneally (1 ml/100 g) injections of drugs were administered. Analysis of variance was used to examine the data, and then LSD post-hoc tests were performed. The data are expressed as mean±SEM. Results: Antidepressant-positive controls, citalopram and desipramine, significantly decreased the time of immobility in the FST and TST as compared to the vehicle control group (p<0.001). In FST, the immobility durations were significantly reduced by the Rosa damascena aqueous extract at a dose of 40 mg/kg compared to lesser doses of the same extract (10 and 20 mg/kg) (p<0.001). Similarly, the 40 mg/kg dose of Rosa damascena aqueous extract significantly reduced the length of immobility in TST (p<0.001). Conclusion: The present findings demonstrate Rosa damascena's antidepressant-like effects in mice. Further research is necessary to determine the underlying mechanism by which Rosa damascena generates effects akin to those of an antidepressant in light of this observation.

A Novel Lipid Nanoparticle NBF-006 Encapsulating Glutathione S-Transferase P siRNA for the Treatment of KRAS-driven Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers, and KRAS mutations occur in 25-30% of NSCLC. Our approach to developing a therapeutic with the potential to target KRAS mutant NSCLC was to identify a new target involved in modulating signaling proteins in the RAS pathway. Glutathione S-Transferase P (GSTP) known as a Phase II detoxification enzyme has more recently been identified as a modulator of MAP kinase-related cell-signaling pathways. Therefore, developing a GSTP siRNA may be an effective therapeutic approach to treat KRAS mutant NSCLC. The lead drug product candidate (NBF-006) is a proprietary siRNA-based lipid nanoparticle (LNP) comprising GSTP siRNA (NDT-05-1040). Here, studies using a panel of KRAS mutant NSCLC cell lines demonstrated that NDT-05-1040 is a very potent and selective GSTP siRNA inhibitor. Our Western blot analysis showed that NDT-05-1040 effectively decreased the phosphorylation of MAPK and PI3K pathway components while upregulating apoptotic signaling cascade. Our in vivo studies revealed statistically significant higher distribution of NBF-006 to the lungs and tumor as compared to liver. In the subcutaneous and orthotopic tumor models, NBF-006 led to a statistically significant and dose dependent anti-tumor growth inhibition. Further, quantitative image analysis of PCNA and PARP staining showed that NBF-006 decreased proliferation and induced apoptosis, respectively, in tumors. Additionally, in a surgically implanted orthotopic lung tumor model, the survival rate of the NBF-006 treatment group was significantly prolonged (P <0.005) as compared to the vehicle control group. Together, these preclinical studies supported advancement of NBF-006 into clinical studies.

Efficacy and safety of a diffusion-based extended-release fluticasone propionate intra-articular injection (EP-104IAR) in knee osteoarthritis (SPRINGBOARD): a 24-week, multicentre, randomised, double-blind, vehicle-controlled, phase 2 trial.

Corticosteroids are among the few effective treatments for knee osteoarthritis, but short duration of action limits their utility. EP-104IAR, a long-acting formulation of fluticasone propionate for intra-articular injection, optimises the action of fluticasone propionate through novel diffusion-based extended-release technology. The SPRINGBOARD trial assessed the efficacy, safety, and pharmacokinetics of EP-104IAR in people with knee osteoarthritis. SPRINGBOARD was a randomised, vehicle-controlled, double-blind, phase 2 trial done at 12 research sites in Denmark, Poland, and Czech Republic. We recruited adults aged 40 years or older with primary knee osteoarthritis (Kellgren-Lawrence grade 2-3) who reported Western Ontario and McMaster Universities Osteoarthritis Arthritis Index (WOMAC) pain scores of at least 4 and no more than 9 out of 10. Participants were randomly assigned (1:1) to receive one intra-articular dose of 25 mg EP-104IAR or vehicle control. Randomisation was done via interactive web-based access to a central predefined computer-generated list with block size of six (allocated by clinical site). Participants and assessors were masked to treatment allocation. Participants were followed up for 24 weeks. The primary outcome was the difference between groups in change in WOMAC pain score from baseline to week 12, analysed in all participants who were randomly assigned and received treatment. Safety, including laboratory analyses, and pharmacokinetics from quantification of fluticasone propionate in peripheral blood were assessed in all participants who received a dose of randomly assigned treatment. A person with lived experience of knee osteoarthritis was involved in study interpretation and writing of the report. This trial is registered with ClinicalTrials.gov, NCT04120402, and the EU Clinical Trials Register, EudraCT 2021-000859-39, and is complete. Between Sept 10, 2021, and Nov 16, 2022, 1294 people were screened for eligibility, and 319 were randomly assigned to EP-104IAR (n=164) or vehicle control (n=155). One participant in the EP-104IAR group was excluded from all analyses because treatment was not administered due to an adverse event. 318 participants (135 [42%] male and 183 [58%] female, 315 [99%] White) received randomly assigned treatment and were included in the primary analysis and safety analysis (EP-104IAR, n=163; vehicle control, n=155). At week 12, least squares mean change in WOMAC pain score from baseline was -2·89 (95% CI -3·22 to -2·56) in the EP-104IAR group and -2·23 (-2·56 to -1·89) in the vehicle control group, with a between-group difference of -0·66 (-1·11 to -0·21; p=0·0044); a significant between-group difference persisted to week 14. 106 (65%) of 163 participants in the EP-104IAR group had one or more treatment-emergent adverse event compared with 89 (57%) of 155 participants in the vehicle control group. Effects on serum glucose and cortisol concentrations were minimal and transient. There were no treatment-emergent deaths or treatment-related serious adverse events. Plasma concentrations of fluticasone propionate showed a blunted initial peak with terminal half-life of approximately 18-20 weeks. These phase 2 results suggest that EP-104IAR has the potential to offer clinically meaningful pain relief in knee osteoarthritis for an extended period of up to 14 weeks, longer than published data for currently marketed corticosteroids. There were minimal effects on glucose and cortisol, and stable fluticasone propionate concentrations in plasma. The safety and efficacy of EP-104IAR will be further evaluated in phase 3 trials, including the possibility of bilateral and repeat dosing with EP-104IAR. Eupraxia Pharmaceuticals. For the Danish translation of the abstract see Supplementary Materials section.

Empagliflozin ameliorates olfactory bulbectomy-induced depression by mitigating oxidative stress and possible involvement of brain derived neurotrophic factor in diabetic rats

Empagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, has recently reported to prevent the depression as chronic animal model. However, its impact on neuroinflammation-mediated depression were remains unexplored. The present study aimed to explore the antidepressant potential of empagliflozin using a neuroinflammation-mediated depression involving the olfactory bulbectomy (OBX) model in rats. To establish this model, initially a low dose of streptozotocin was injected to induce diabetes in all group of animals. Following the confirmation of hyperglycemia, OBX surgery was performed. Post-surgery, the drug treatments were administered orally for 14 consecutive days. The study evaluated the effects of daily oral administration of empagliflozin at doses of 5 and 10 mg/kg, alongside metformin (200 mg/kg) and clomipramine (50 mg/kg), on OBX-induced behavioral depression in rats. Separate sham and vehicle control groups were also maintained. Behavioral parameters in open field, forced swim test, elevated plus maze and splash test were recorded on 28th day. Results showed that empagliflozin, particularly at the higher at the higher dose, significantly enhanced behavioral outcomes, evidenced by increased distance traveled, greater open arm entries, and reduced immobility, alongside a notable reduction in grooming time. Moreover, empagliflozin significantly restored the antioxidants level specifically Glutathione (GSH) and Catalase (CAT) in OBX insulted rat brain and reversed Lipid peroxidase (LPO). Notably, molecular docking study demonstrated a good binding affinity of empagliflozin for Brain-Derived Neurotrophic Factor (BDNF), suggesting that its antidepressant effects may be mediated through the modulation of the BDNF pathway. These findings support the potential therapeutic application of empagliflozin for depression, particularly in cases associated with neuroinflammation and oxidative stress.

Toxicology profile of a novel GLP-1 receptor biased agonist-SAL0112 in nonhuman primates

Oral small-molecule GLP-1 receptor biased agonists exhibit promising treatment efficacy of type 2 diabetes and obesity. SAL0112 is a novel compound that has demonstrated remarkable efficacy in preclinical animal models. Herein, both in vitro and in vivo preclinical toxicity investigations were conducted to explore the safety profile of SAL0112. The HTRF assay and TR-FRET assay were utilized for cAMP detection. Patch clamp assay was employed for hERG potassium ion channel determination. Cynomolgus monkeys were used in a cardiovascular safety pharmacology study and a 13-week repeated dose toxicity study. The telemetry system was employed to detect cardiovascular indicators such as ECG, HR, and BP. During the repeated dose toxicity study, body weight, food intake, hematology, coagulation function test, serum biochemistry tests, and urine analysis were measured. Macroscopic and microscopic observations were conducted at the end of the study. TK studies were conducted on Day 1 and Day 91. SAL0112 exhibited a high degree of potency in activating the monkey GLP-1 receptor whereas had no effect on the rodent GLP-1 receptor. In contrast to Danuglipron, which demonstrated high potency on hERG with an IC50 value of 6.9 μM, the IC50 of SAL0112 on hERG was greater than 100 μM. Compared to the Vehicle Control group, no significant changes in cardiovascular indicators were observed in the cardiovascular safety pharmacology study after a single dose of SAL0112 up to 250 mg/kg (P > 0.05). A repeated dose toxicity study revealed moderate anorexigenic effects and a reduction in body weight, effects that were found to be reversible and not associated with any pathological changes. The NOAEL of SAL0112 is 150 mg/kg, providing an approximate safety margin of threefold. SAL0112 demonstrated a favorable safety profile in cynomolgus monkeys, with a substantial therapeutic window that supports the progression of this compound into clinical studies.

Administration of Aripiprazole Alleviates Memory Impairment and Restores Damaged Glutamatergic System in 5xFAD Mice.

Many patients with Alzheimer's disease (AD) also have psychosis, and it has been reported that these patients have more severely impaired cognitive functions than patients without psychosis. The glutamatergic system in the brain is known to play an important role in memory and learning in the neural circuits. However, there has been limited research on how antipsychotic drugs affect the glutamatergic system of AD. Therefore, we aimed to investigate the effects of aripiprazole on the glutamatergic system in an animal model of AD using functional molecular imaging. In this study, 5xFAD mice were used as the animal model. At the age of 5months, the mice were divided into wild-type, vehicle control, and aripiprazole-treated groups (n = 6 per group). The aripiprazole-treated group was administered aripiprazole for 2months at a dose of 1mg·kg-1·day-1. At 7months of age, the animals underwent behavioral tests and glutamate positron emission tomography (PET) scans. The aripiprazole-treated group exhibited alleviated memory impairment in a novel object recognition test. Moreover, this group displayed 7-8% higher binding in the glutamate PET scan than the vehicle-treated 5xFAD group. Postmortem examination confirmed the recovery of glutamatergic damage. The administration of aripiprazole alleviated memory impairment and restored the damaged glutamatergic system in 5xFAD mice. Although the use of aripiprazole in AD patients may be a constraint in terms of safety, we confirmed the possibility that the administration of antipsychotic drugs can be effective in AD.

Clarification of a unique mucosal vaccination route for improved systemic and mucosal immune response in broiler

There are often outbreaks of infectious diseases on farms, which not only disrupt production but also cause significant economic losses. Vaccines are given to prevent the spread of these infectious diseases, but they produce only systemic antibodies or antibodies in the mucosa of a particular system. So, a new mucosal vaccination route is needed where the vaccine will produce antibodies in the systemic circulation as well as in the mucosa of many systems. In our study, the cloaca was targeted because it is associated with the mucosa of many systems. Whole-mount and routine histological staining show both lymphatic nodules and diffuse lymphatic tissues in the lamina propria of cloaca. These lymphatic tissues are made up of Bu-1+ B-cells, CD3+ T-cells, and KUL01+ macrophages and monocytes. So, this is a new mucosa-associated lymphoid tissue, named cloaca-associated lymphoid tissue (CALT). The CALT contains antigen-presenting cells (dendritic cells, macrophages, B cells, MHC II molecules, and T cells) and is equipped with blood vessels and high endothelial venules, which indicate its functional status. More antibodies were produced in the treatment group compared to the vehicle control group after administration of the infectious bursal disease (IBD) and the Newcastle disease (ND) vaccine through cloaca. In addition, the cloaca-associated route produces a higher number of antibodies than the other traditional routes, which reveals the uniqueness of this route. Cloacal-vaccinated chickens showed less damage to the follicle and epithelium of the bursa of Fabricius compared to other groups, indicating its lower cytotoxic effect. Therefore, the cloaca-associated mucosal vaccination route produces more antibodies than other mucosal vaccination routes, which will protect the chickens on the farm to a greater extent.

Modulation of regenerative responses by retinoic and ascorbic acids in human apical papilla cells

ObjectiveThis study investigated the bioactive effects of retinoic acid and ascorbic acid on hSCAPs in vitro. DesignCells were obtained from human third molars (n=4) and characterized for mesenchymal stem cell markers by flow cytometry. The experimental groups: control (α-MEM); vehicle control group (α-MEM + 0.17 % DMSO); retinoic acid 0.1, 1, and 10 µM; and ascorbic acid 3, 30, and 300 µM (n=8) were tested for cell viability (alamarBlue; 1, 3, and 7 days), total collagen synthesis (Sirius Red; 1 and 7 days), mineralized matrix formation (Alizarin red; 14 days), and the regulation of gene expression related to mineralization (ALPL and DSPP), cell migration (ITGAV and CXCL12) angiogenesis (VEGFA) and collagen synthesis (COL1A1 and COL3A1; RT-qPCR) on 1 and 7 days. ACTB and GAPDH were used as reference genes. Data were analyzed by ANOVA and complementary tests at a 5 % significance level. ResultsAscorbic acid 300 µM increased viability, and retinoic acid reduced it dose-dependently. Retinoic acid 0.1 µM and ascorbic acid 30 and 300 µM increased mineralized matrix formation and total collagen synthesis, and retinoic acid 10 µM decreased. On day 1, 0.1 µM retinoic acid upregulated the gene expression of COL1A1, COL3A1, VEGFA, CXCL12, ALPL, DSPP e ITGAV, and 300 µM ascorbic acid upregulated COL1A1, COL3A1 and DSPP. However, on day 7, retinoic acid downregulated ALPL, COL3A1, CXCL12, and VEGFA and downregulated ITGAV and VEGFA. ConclusionRetinoic acid 0.1 µM and ascorbic acid 300 µM biostimulated hSCAPs to differentiate into pro-regenerative phenotypes with potential application for REPs.

99 The Effects of β-caryophyllene on butyrate utilization and metabolism in Caco 2 cells

Abstract Butyrate is important for regulating energy status and cellular metabolism. Beta-caryophyllene (BCP) is a plant compound which may exert potentially beneficial effects on intestinal epithelial cells, such as barrier integrity and modulation of nutrient utilization. The goal of this preliminary study was to investigate the effects of BCP on butyrate utilization and metabolism in intestinal epithelial cells. Caco 2 cells were used as a model for the intestinal barrier. Cells were seeded in multi-well plates with hanging inserts (n = 4) and grown for 18 d. On d 19, 1 of 4 treatments was added to triplicate wells per plate. Treatments included vehicle control (VC), 40 µM BCP, 2 mM butyrate (BTR), and butyrate plus BCP (BB). Treatments were added to the apical side and incubated for 24 and 48 h. Transepithelial electrical resistance (TEER; ohms/cm2) readings and supernatant samples from both the apical and basolateral sides of the cell layer were taken at each time point, and butyrate and β- hydroxybutyrate (BHB) concentrations measured. Butyrate utilization was calculated as absolute nmol lost. Statistical analyses were performed using lm and emmeans packages of R, with treatment, time and side (of cell layer) as fixed effects. All wells started with the same statistical TEER values on h 0. After 24 and 48 h BCP showed no changes in TEER (P &amp;gt; 0.05). Compared with VC, the mean TEER for BTR and BB was greater at 24 h (579 vs 878 and 1,008 ± 35, respectively; P &amp;lt; 0.001). After 48 h, all groups returned to baseline, except BB which had a greater TEER (883 ± 35; P &amp;lt; 0.001) compared with other treatments. After 24 and 48 h BCP showed no changes for both sides of the cell layer (P &amp;gt; 0.05). Overall, absolute BHB concentrations increased from 24 to 48 h in all treatments (P &amp;lt; 0.05). Production of BHB in each of the VC and BC groups was less than 15 nmol at each time point. For BB and BTR groups, more nmol BHB was detected on the basolateral side (31.9 and 22.2 ± 1.3) vs apical (14.0 and 10.4 ± 1.3, respectively; P &amp;lt; 0.001) after 24 h. After 48 h, BHB was also greater on the basolateral side (58.2 and 45.2 ± 1.3 nmol for BB and BUT, respectively) vs apical side (30.2 and 24.0 ± 1.3 nmol; P &amp;lt; 0.001). When looking at BTR utilization, the BB treatment had greater butyrate disappearance than BTR in 24 h (254 vs 169 ± 12.6 nmol; P = 0.0005) and 48h (707 vs 573 ± 12.6 nmol; P &amp;lt; 0.0001). BCP may promote the metabolism of butyrate by intestinal epithelial cells. More research is needed to understand the mechanism of how BCP increases cellular butyrate utilization.

Investigation of The Effect of Metal-based Medicine Arsenicum album on Humoral Immune Response in SRBC-immunized Mice.

In complementary and alternative medicinal systems, the Arsenicum album in ultra-high dilution was used in various therapeutic conditions, considering its effects on the body's immune system, including the COVID-19 pandemic. However, scientific evidence regarding its immunomodulatory effects is insufficient. The current study aimed to investigate the immunomodulatory effects of Arsenicum album in an experimental mouse model. Immunomodulatory activity of potentized dilutions of Arsenicum album i.e., 6C, 30C, 200C in BALB/c mice was evaluated by humoral antibody titer and delayedtype hypersensitivity assays wherein a fixed concentration (0.5 ml of 1× 109 cells/ml) of freshly prepared sheep RBC was administered as a foreign antigen to generate primary and secondary antibodies. Arsenicum album showed significant immunomodulatory activity by increasing primary antibody titer evaluated on day 21 of the treatment in all the dilutions as compared to SRBC and vehicle control group in humoral immune response assay without showing any effect on delayed-type hypersensitivity. The results of this preliminary study indicate that oral administration of Arsenicum album has the potential to augment primary humoral response at all dilutions. Hence, the possibility of using the Arsenicum album could be explored to treat immunological conditions, infections, etc., as an alternative therapy alongwith modern medicines.

27-Hydroxycholesterol contributes to hypercholesterolemia-associated aggravation of apical periodontitis in ovariectomized rats and raloxifene counteracts its action.

The influence of hypercholesterolemia on the development of apical periodontitis (AP) is inconclusive. Recent studies revealed that cholesterol metabolite 27-hydoxycholesterol (27HC) can affect cellular responses to bacterial infections and oestrogen status and raloxifene may influence its action. Herein, we aimed to examine the impact of 27HC on production of inflammatory mediators by macrophages and the regulatory function of raloxifene. The contribution of 27HC to AP development and the therapeutic effect of raloxifene were evaluated in a rat model. Murine macrophages J774 cells were used. The expression of inducible nitric oxide synthase (iNOS) was examined by Western blot. The concentrations of C-C motif chemokine ligand (CCL) 2 and 27HC were assessed by enzyme-linked immunosorbent assay. Colorimetric assay was used to evaluate cholesterol levels. Experimental AP was induced in ovariectomized (OVX) or un-operated rats receiving high-fat/high-cholesterol diet (HFHCD) or normal diet (ND). Micro-computed tomography and immunohistochemistry were employed to evaluate disease severity and the therapeutic effect of raloxifene. Cholesterol enhanced 27HC production in macrophages. 27HC induced iNOS and CCL2 synthesis by macrophages and estradiol suppressed the responses. In our animal model of AP, HFHCD plus OVX significantly augmented serum and lesion tissue levels of 27HC (p < .05 versus the ND group). Lesion size, infiltration of CD68+ cells, and iNOS+ monocytes were increased in parallel with 27HC accumulation. Raloxifene inhibited pro-inflammatory effects of 27HC on macrophages and suppressed AP progression in HFHCD/OVX rats (p < .05 versus the vehicle control group). Our results suggested that 27HC contributes to AP aggravation associated with hypercholesterolemia. Oestrogen deficiency may both enhance 27HC production and exacerbate its downstream action.

Acute and sub-chronic toxicity studies of methanol extract of Trema orientalis (Linn) blume in albino wistar rats

BackgroundThe safety potential of the methanol extract of Trema orientalis (TOM) leaf was evaluated in albino Wistar rats using biochemical, haematological, and histological indices in both acute and sub-chronic toxicity studies. MethodsThe animals were managed following the National Institute of Health (NIH) stipulated protocols for handling laboratory animals. The weight of each animal was recorded upon arrival and monitored throughout the study. The animals were allowed to acclimatize for 14 days, after which they were reweighed and randomly distributed into four groups of three female rats (n=12) for acute toxicity studies. Group A was given distilled water, and Groups B, C, and D were given a single dose of 2000, 4000, and 5000 mg/kg bw TOM extract, respectively. On day 15, each animal was anaesthetized and then euthanized. For the sub-chronic toxicity study, animals were randomly distributed into five groups of eight female rats (n=40). They were dosed daily for 28 days. Group A (Negative control group) was given distilled water. Groups B, C, and D had 200, 400, and 800 mg/kg bw TOM extract and Group E (Vehicle control group) were given 0.25 % of sodium carboxyl methyl cellulose (CMC). Five animals were anaesthetized and then euthanized on day 29, while three animals were kept for recovery evaluation for another two weeks without further administration of the extract. Ten organs were excised from each animal and weighed. The liver and kidney were processed for histopathological studies, while the blood samples were collected for biochemical and haematological assays. ResultsFrom acute toxicity studies, the LD50 value of TOM extract was estimated to exceed 5000 mg/kg bw via oral passage. From Sub-chronic toxicity studies, biochemical results showed a significant (p < 0.05) reduction in total protein, albumin, globulin, AST, ALP, and ALT in a dose-dependent manner. Histology of the liver and kidney tissues of all the animals except the kidney of the 800 mg/kg group had no visible lesions; this sets the safety dose for TOM at above 400 mg/kg but below 800 mg/kg. Recovery animals had significantly (p < 0.05) increased total protein, total bilirubin, and ALP and decreased albumin and direct bilirubin levels. ConclusionThis study reports the safety dose of TOM, a reputable medicinal plant extract. This is the first study reporting that the LD50 value of TOM extract exceeds 5000 mg/kg bw via oral passage.

Potassium nitrate suppresses hyperactivities of Vc neurons of the model with dentin hypersensitivity

ObjectivePotassium nitrate (KNO3) suppresses nociception induced by dental hypersensitivity (HYS). We aimed to examine the effects of KNO3 on the neural activity of the trigeminal spinal subnucleus caudalis (Vc) in HYS model rats. MethodsKNO3 or vehicle was applied to the exposed dentin of HYS rats for 3 days. c-Fos expression and neuronal activity in the Vc after acetone treatment for cold stimulation were examined to evaluate the effects of KNO3 application on dentin. ResultsThe number of c-Fos-immunoreactive cells in the Vc was lower in the group that received KNO3 (KNO3 group) than in the group that received vehicle (control group). Spike firing of Vc neurons in response to cold stimulation of the dentin was recorded before and after KNO3 application to the cavity, and the increased neural activity was effectively suppressed by KNO3 application. Scanning electron microscopy revealed that the dentin tubules were not occluded by deposits in any of the groups. ConclusionsKNO3-induced suppression of Vc neuronal activity does not involve physical occlusion of the dentin tubules but likely involves suppression of Aδ or C-fiber activities in the tooth pulp, resulting in the suppression of Vc neuronal activities.

The effect of pregnancy-related hormones on hepatic transporters: studies with premenopausal human hepatocytes.

Pregnancy results in significant changes in drug pharmacokinetics (PK). While previous studies have elucidated the impact of pregnancy-related hormones (PRH) on mRNA or protein expression and activity of major hepatic metabolizing enzymes, their effect on hepatic drug transporters remains largely unexplored. Therefore, we investigated the effect of a cocktail of PRH on the mRNA expression and activity of hepatic transporters. Plated human hepatocytes (PHH) from 3 premenopausal donors were incubated, in triplicate, for 72h, with vehicle (DMSO < 0.01%), rifampin (10μM; positive control) or a cocktail of PRH consisting of estrone, estradiol, estriol, estetrol, progesterone, cortisol, testosterone, oxytocin, and placental growth hormone. The PRH concentrations replicated 0.1×, 1×, or 10× of the plasma concentrations of these hormones observed during each of the three trimesters of pregnancy. After treatment, mRNA expression (quantified by qPCR) of hepatic influx and efflux transporters as well as the activity of influx transporters was quantified (uptake of a selective substrate ± corresponding transporter inhibitor). The data were expressed relative to that in the control (vehicle) group. Significance was evaluated by ANOVA (followed by Dunn's multiple comparisons) or unpaired t-test when the within-lot data were analyzed, or repeated measures ANOVA (followed by Dunn's multiple comparisons) or paired t-test when data from all 3 lots were analyzed (p < 0.05). In general, a) PRH cocktails significantly induced transporter mRNA expression in the following order OAT2 ≈ NTCP ≈ OCT1 > OATP2B1 and repressed mRNA expression in the following order OATP1B3 > OATP1B1; b) these changes translated into significant induction of OAT2 (T1-T3) and NTCP (T2-T3, in only two lots) activity at the 1× PRH concentration. Compared with the influx transporters, the induction of mRNA expression of efflux transporters was modest, with mRNA expression of MRP2 and BSEP being induced the most. Once these data are verified through in vivo probe drug PK studies in pregnancy, they can be populated into physiologically based pharmacokinetic (PBPK) models to predict, for all trimesters of pregnancy, transporter-mediated clearance of any drug that is a substrate of the affected transporters.

Behavioral and electrophysiological study in Colossoma macropomum treated with different concentrations of Nepeta cataria oil in an immersion bath revealed a therapeutic window for anesthesia.

The purpose of this study was to characterize the activity of essential oils from Nepeta Cataria (EON) at concentrations of 125 L-1, 150 L-1, 175 L-1, and 200 L-1 on the behavior of loss of the posture reflex and recovery of the posture reflex and electrocardiographic activity and recording of the opercular beat of Colossoma macropomum during immersion bathing for a period of 5min, in order to obtain a window for safe use during anesthesia. The fish (23.38 ± 3.5g) were assigned to the following experiments: experiment 1 (latency to loss and recovery of the posture reflex): (a) 125 L-1, (b) 150 L-1, (c) 175 L-1, and (d) 200 L-1 (n = 9) per group. Experiment 2 (electrocardiographic and heartbeat recordings): (a) control group; (b) vehicle control group (2ml of alcohol per liter of water), (c) 125 L-1, (d) 150 L-1, (e) 175 L-1, and (f) 200 L-1 (n = 9), per group. All the concentrations used showed efficacy in inducing loss of the posture reflex and reversibility with recovery of the posture reflex, but the electrocardiographic recordings indicated morphographic changes such as bradycardia during induction and p wave apiculation during recovery at the highest concentrations tested. In this way, we suggest a safe use window for short-term anesthesia with EON in the concentration range of 125 to 150 L-1 for juvenile Colossoma macropomum.

The role of alternative autophagy in cell viability and response to paclitaxel treatment in v-Ha-ras-transformed NIH 3T3 cells

In confluent v-Ha-ras-transformed NIH 3T3 fibroblasts (Ras-NIH 3T3), LC3 downregulation may precede a decrease in canonical autophagy, thus contributing to cell survival. Herein, we aimed to investigate the role of alternative autophagy in the viability of long-term cultures of Ras-NIH 3T3 cells and their parental NIH 3T3 cells. As cell confluence increased with the culture period, the level of alternative autophagy, as assessed through Lamp2-Rab9 co-localization, gradually decreased in both cell lines. However, Ras-NIH 3T3 cells maintained higher levels of alternative autophagy than the parental cells did. Rab9 knockdown minimally affected NIH 3T3 cells while drastically reducing the viability of Ras-NIH 3T3 cells, which suggested that alternative autophagy plays a critical role in Ras-NIH 3T3 cells. In contrast, reactive oxygen species (ROS) production in Ras-NIH 3T3 cells was higher than that in NIH 3T3 cells during long-term culture. Moreover, NIH 3T3 cells exhibited a continual decrease in mitochondrial mass, whereas Ras-NIH 3T3 cells maintained high mitochondrial mass. Immunofluorescence analysis of mitochondrial membrane marker proteins and mitochondrial membrane potential (MMP) also demonstrated a temporal pattern of changes similar to those of mitochondrial mass. This finding could be attributed to the relatively higher level of alternative autophagy in Ras-NIH 3T3 cells facilitating the removal of damaged mitochondria. Paclitaxel treatment in Ras-NIH 3T3 cells induced an increase in canonical autophagy rates along with suppression of alternative autophagy. Ras-NIH 3T3 cells showed high sensitivity to paclitaxel at the early stage of culture, but as cell confluence increased, resistance to paclitaxel increased, showing a similar level of cell viability to the vehicle control group. The study findings suggest that alternative autophagy is more important than canonical autophagy for maintaining cell survival in response to an unfavorable environment, such as during high cell confluence and exposure to anticancer agents.

Human induced pluripotent stem cell/embryonic stem cell-derived pyramidal neuronal precursors show safety and efficacy in a rat spinal cord injury model

Nerve regeneration and circuit reconstruction remain a challenge following spinal cord injury (SCI). Corticospinal pyramidal neurons possess strong axon projection ability. In this study, human induced pluripotent stem cells (iPSCs) were differentiated into pyramidal neuronal precursors (PNPs) by addition of small molecule dorsomorphin into the culture. iPSC-derived PNPs were transplanted acutely into a rat contusion SCI model on the same day of injury. Following engraftment, the SCI rats showed significantly improved motor functions compared with vehicle control group as revealed by behavioral tests. Eight weeks following engraftment, the PNPs matured into corticospinal pyramidal neurons and extended axons into distant host spinal cord tissues, mostly in a caudal direction. Host neurons rostral to the lesion site also grew axons into the graft. Possible synaptic connections as a bridging relay may have been formed between host and graft-derived neurons, as indicated by pre- and post-synaptic marker staining and the regulation of chemogenetic regulatory systems. PNP graft showed an anti-inflammatory effect at the injury site and could bias microglia/macrophages towards a M2 phenotype. In addition, PNP graft was safe and no tumor formation was detected after transplantation into immunodeficient mice and SCI rats. The potential to reconstruct a neuronal relay circuitry across the lesion site and to modulate the microenvironment in SCI makes PNPs a promising cellular candidate for treatment of SCI.

Antidyslipidaemic evaluation of coagulin C and withacoagulin isolated from Withania coagulans (Stocks) dunal berries in hypertension induced secondary dyslipidemia in albino mice

Vascular diseases like hypertension has often been associated with dyslipidemia. Sustained elevated blood pressure for not less than four weeks develops gross irregularities inside arteriolar lumen. The pathological changes might be due to intimal arteriosclerotic thickening causing tapering of the structural narrowing along the arterioles. It might also be due to occurrence of irregular damage in the vascular structure from focal arteriolar constriction and lead to secondary dyslipidemia. The pharmacological intervention may lead to rectification and therapeutic benefit against dyslipidemia.The withanolides coagulin C and withacoagulin were isolated by flash chromatography from the extracts of Withania coagulans (Stocks) Dunal berries. The withanolides were characterized using 1H NMR and 13C NMR, UV and mass spectrometry and confirmed as coagulin C and withacoagulin. The oral toxicity of coagulin C and withacoagulin were investigated in albino mice followed by their antidyslipidemic activity with two dose levels i.e. 25 mg/kg and 50 mg/Kg p.o. This dyslipidemia was secondary to hypertension which was developed by DOCA salt model (25 mg/kg; s.c.+ 2% NaCl). Mice were administered dose of 10, 50 and 500 mg/kg coagulin C and withacoagulin orally for 28 days daily for sub-acute toxicity effect.Coagulin C and withacoagulin were found to be tolerated upto 2000 mg/kg p.o. during acute toxicity study in mice. Coagulin C and withacoagulin were claimed to possess No-Observed Adverse Effect Level (NOEL) at the dose of 500 mg/kg orally. The high dose (50 mg/Kg) withacoagulin has highly significant difference in total cholesterol, triglyceride, HDL, LDL and VLDL levels in comparison to disease control group along with highly significant difference in triglyceride and VLDL level compared to animals of vehicle control group. Coagulin C at both the low (25 mg/Kg) and high dose (50 mg/Kg) have highly significant difference in comparison to vehicle control, disease control and positive control in controlling total cholesterol, triglyceride, LDL and VLDL levels in the experimental animals indicating that coagulin C is not much as effective as withacoagulin in the normalization of altered lipid profile of the experimental animals.On the basis of spectroscopic analysis the isolated compounds from Withania coagulans (Stocks) Dunal berries were confirmed to be coagulin C and withacoagulin. Acute oral toxicity test in mice was found to have safe profile even at high dose for both the compounds. On development of secondary dyslipidemia due to DOCA salt induced hypertension in albino mice, both the isolated compounds showed different degree of correction in the altered lipid profile of the animals. Whereas withacoagulin has been found to have more significant and successful in controlling the altered lipid profile, coagulin C has only marginal effect. The docking studies also confirm the result and hence it can be suggested that withacoagulin is better than coagulin C in managing the alteration of lipid profile of the experimental mice.

The Toxic Effect of Bisphenol A on Male Rat Testicles and The Possible Protective Value of Propolis and Vitamin E

This study aimed to examine the impact of propolis (Pro) on various physiological parameters in male rats experiencing induced testicular dysfunction by Bisphenol A. Five groups of ten male rats apiece were created by random selection from the rats. Group 1 the rats, which performed as the group under negative control, received normal food and water. In Group 2, rats were subjected to intraperitoneal administration of 0.2 mL of corn oil, utilized as the vehicle for bisphenol A (BPA), thereby serving as the vehicle control group. For Group 3, rats were administered in corn oil, BPA dissolved at a dosage of 50 mg/kg body weight administered three times a week for three weeks using an intraperitoneal injection. In Group 4rats received a dose of 250 mg/kg body weight of propolis (protected) orally via a gavage needle. Subsequently, intraperitoneal injections are administered to them from BPA delivered three times weekly for three weeks, the dosage is 50 mg per kilogram of body weight dissolved in corn oil. In Group 5, rats received protection with vitamin E orally, at a dose of 100 mg/kg body weight, using a gavage needle. The intraperitoneal injection of BPA, dissolved in corn oil at a dose of 50 mg/kg body weight, was then given three times a week for three weeks. After three weeks, animals were sacrificed, and testes and blood were gathered for additional analysis. Superoxide Dismutase (SOD), Sperm Viability, General motility, Progressive motility, and Morphology, were all significantly reduced in the BPA group compared to the control groups, according to the results. While malondialdehyde (MDA) increased in the BPA group compared to the control groups. In conclusion, propolis enhanced physiological parameters adversely affected by BPA, suggesting its potential as a protective agent against BPA-induced testicular dysfunction.