Safety and Toxicology Study of Hu7691, a Novel AKT Inhibitor, following Oral Administration in Rats.

Hu7691 represents a novel Pan-Akt kinase inhibitor, demonstrating excellent selectivity towards non-AGC kinase families and pronounced inhibitory effects on the proliferation of multiple tumor cell lines. However, there is currently a notable absence of in vivo toxicological research evidence concerning Hu7691. This study represents the first investigation into the 14-day repeated-dose toxicity of Hu7691 in male and female Sprague Dawley (SD) rats. Male rats were administered daily doses of 12.5, 50, 100, and 150 mg/kg/day, while female rats received doses of 12.5, 25, 50, and 75 mg/kg/day for 14 consecutive days. Hematological assessments, organ weights, and histopathological examinations revealed corresponding alterations, suggesting potential target organs for toxicity including the spleen, thymus, and gastrointestinal tract. It is worth noting that the test substance may also impact the liver, kidneys, heart, and ovaries. The No Observed Effect Level (NOAEL) was determined to be no greater than 12.5 mg/kg/day. Based on the observed gender-related toxicity differences in preliminary trials, it is recommended that the high dose reference dose for male animals in formal experiments should not be less than 100 mg/kg/day, while for female animals, it should be less than 50 mg/kg/day.

Ginseng Stem-Leaf Saponins in Combination with Selenium Promote the Immune Response in Neonatal Mice with Maternal Antibody.

Neonates acquire from their mothers maternal antibody (MatAb) which results in poor immune response to vaccination. We previously demonstrated that ginseng stem-leaf saponins in combination with selenium (GSe) had adjuvant effect on the immune response to an attenuated pseudorabies virus (aPrV) vaccine. The present study was to evaluate GSe for its effect on the immune response to aPrV vaccine in neonatal mice with MatAb. Results showed that GSe had adjuvant effect on the immune response to aPrV vaccine in neonates. When GSe was co-administered with aPrV vaccine (aP-GSe), specific gB antibody, Th1 cytokines (IL-2, IL-12 and IFN-γ) and Th2 cytokines (IL-4, IL-6 and IL-10) responses were significantly increased in association with enhanced protection of vaccinated neonates against the lethal PrV challenge even though MatAb existed when compared to the neonates immunized with aPrV vaccine alone. GSe-enhanced immune response depended on its use in the primary immunization. The mechanisms underlying the adjuvant effect of GSe may be due to more innate immune related pathways activated by GSe. Transcriptome analysis of splenocytes from neonates immunized with aP-GSe, aPrV or saline solution showed that there were 3976 differentially expressed genes (DEGs) in aP-GSe group while 5959 DEGs in aPrV group when compared to the control. Gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis showed that innate immune responses and cytokine productions related terms or pathways were predominantly enriched in aP-GSe group, such as "NOD-like receptor signaling pathway", "Natural killer cell mediated cytotoxicity", "NF-κB signaling pathway", "cytokine-cytokine receptor interaction", and "Th1 and Th2 cell differentiation". Considering the potent adjuvant effect of GSe on aPrV vaccine in neonatal mice with MatAb, it deserves further investigation in piglets.

Sunflower seed oil combined with ginseng stem-leaf saponins as an adjuvant to enhance the immune response elicited by Newcastle disease vaccine in chickens.

The present study was to evaluate the adjuvant effect of sunflower seed oil containing saponins extracted from the stem and leaf of Panax ginseng C.A. Meyer (E515-D) on the immune response induced by an inactivated Newcastle disease virus (NDV) in chickens. The results showed that E515-D promoted significantly higher serum NDV-specific HI and neutralizing antibody responses, IFN-γ and IL-4 levels, and lymphocyte proliferative responses to Con A, LPS, and NDV antigen than the conventional adjuvant Marcol 52. Different adjuvant effect between E515-D and Marcol 52 may be attributed to different genes expressed in two groups. Transcriptome analysis of splenocytes showed that there were 1198 differentially expressed genes (DEGs) with 539 up and 659 down regulated in E515-D group while 1395 DEGs with 697 up and 698 down regulated in Marcol 52 group in comparison with the control group. Analysis of gene ontology (GO) term and kyoto encyclopedia of Genes and Genomes (KEGG) pathways showed that the predominant immune related pathways included "Toll-like receptor signaling pathway", "NOD-like receptor signaling pathway", "C-type lectin receptor signaling pathway", and "Phosphatidylinositol signaling system" in E515-D group while Marcol 52 were "NOD-like receptor signaling pathway", "Phagosome", and "Lysosome", and the most relevant DEGs in E515-D group were STAT1, STAT2, PI3K, and IL-6. Considering the excellent adjuvant activity and vegetable origin, E515-D deserves further study as an adjuvant for vaccines used in food animals.

Immunomodulatory effect of ginseng stem-leaf saponins and selenium on Harderian gland in immunization of chickens to Newcastle disease vaccine.

Our previous study demonstrated that ginseng stem-leaf saponins (GSLS) in combination with selenium (GSLS-Se) have adjuvant effect on the live vaccine of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) in intraocular-and-intranasal immunization in chickens. The present study was to investigate the potential molecular mechanisms involved in the immunomodulation of GSLS-Se on the Harderian gland (HG). It was found that the window allowing animals susceptible to infections due to low antibody titers became smaller or even completely closed because of increased NDV-specific HI titers when NDV vaccine and GSLS-Se were coadministered for immunization at early life in chickens. In addition, NDV-specific sIgA and the numbers of IgG+, IgA+, IgM+ plasma cells were significantly more in GSLS-Se group than the control in the HGs. Transcriptome analysis of HGs identified 1184 differentially expressed genes (DEGs) between GSLS-Se treated and non-treated groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified 42 significantly enriched GO terms and 13 canonical immune pathways. These findings indicated that GSLS-Se might exert immunomodulatory effects through influencing the antioxidant regulation and modulating the activity of immune related enzymes. Besides, Toll-like receptor (TLR) signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway might be involved primarily in the immunomodulation. Therefore, enhanced antibody responses in GSLS-Se group may be attributed to the immunomodulatory effects of GSLS-Se on the immune-related gene profile expressed in the immunocompetent cells of the HGs.

Receptor and signaling pathway involved in bovine lymphocyte activation by Atractylodis macrocephalae polysaccharides.

The present study was to investigate the molecular mechanism underlying lymphocyte activation by total polysaccharides from Atractylodis macrocephalae (RAMPtp). The results showed that RAMPtp significantly promoted the secretions of cytokines (IFN-γ, IL-1α, IL-21, IFN-α, CCL4, CXCL9 and CXCL10), increased the proportions of CD4+ and CD8+ subpopulations, and enhanced the expressions of c-JUN, NFAT4, STAT1 and STAT3. microRNA sequencing identified 67 differentially expressed miRNAs (DEMs) in RAMPtp-stimulated SMLN lymphocytes, including 55 up-regulated and 12 down-regulated. GO and KEGG enrichment analyses of the predicted DEMs-targeted genes indicated that they were associated with immune system pathways, including PI3K-Akt, MAPKs, Jak-STAT and Calcium signaling pathways, which were confirmed by western blot and pathway inhibition assays. RAMPtp was further observed to favor immunostimulatory effect on both T and B lymphocytes via binding to TCR and membrane Ig individually. These findings might explain the immunomodulatory mechanism of RAMPtp in ameliorating the bovine intramammary infection.

Protective Effect of Epigallocatechin-3-Gallate in Hydrogen Peroxide-Induced Oxidative Damage in Chicken Lymphocytes.

Epigallocatechin-3-gallate (EGCG) is one of the fundamental compounds in green tea. The present study was to evaluate the protective effect of EGCG in oxidative damage and apoptosis induced by hydrogen peroxide (H2O2) in chicken lymphocytes. Results showed that preincubation of lymphocytes with EGCG significantly decreased H2O2-reduced cell viability and apoptotic cells with DNA damage, restored the H2O2-dependent reduction in total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), glutathione (GSH), and glutathione disulfide (GSSG), and suppressed the increase in intracellular reactive oxygen species (ROS), nitric oxide (NO), nitric oxide synthesis (NOS), malondialdehyde (MDA), lipid peroxide (LPO), and protein carbonyl (Carbonyl). In addition, preincubation of the cells with EGCG increased mitochondrial membrane potential (MMP) and reduced calcium ion ([Ca2+]i) load. The protective effect of EGCG in oxidative damage in lymphocytes was accompanied by mRNA expression of SOD, Heme oxygenase-1 (HO-1), Catalase (CAT), GSH-PX, nuclear factor erythroid 2-related factor 2 (Nrf2), and thioredoxin-1 (Trx-1). As EGCG had been removed before lymphocytes were challenged with H2O2, the activation of genes such as Nrf2 and Trx-1 by preincubation with EGCG could be the main reason for EGCG to protect the cells from oxidative damage by H2O2. Since oxidative stress is an important mechanism of biological damage and is regarded as the reasons of several pathologies, the present findings may be helpful for the use of tea products to prevent oxidative stress and maintain healthy in both humans and animals.

Protective Effect of Ginsenoside Rg1 on Oxidative Damage Induced by Hydrogen Peroxide in Chicken Splenic Lymphocytes.

Previous investigation showed that ginsenoside Rg1 (Rg1) extracted from Panax ginseng C.A. Mey has antioxidative effect on oxidative stress in chickens. The present study was designed to investigate the protective effects of Rg1 on chicken lymphocytes against hydrogen peroxide-induced oxidative stress and the potential mechanisms. Cell viability, apoptotic cells, malondialdehyde, activity of superoxide dismutase, mitochondrial membrane potential, and [Ca2+]i concentration were measured, and transcriptome analysis and quantitative real-time polymerase chain reaction were used to investigate the effect of Rg1 on gene expression of the cells. The results showed that treatment of lymphocytes with H2O2 induced oxidative stress and apoptosis. However, pretreatment of the cells with Rg1 dramatically enhanced cell viability, reduced apoptotic cells, and decreased oxidative stress induced by H2O2. In addition, Rg1 reduced these H2O2-dependent decreases in mitochondrial membrane potential and reversed [Ca2+]i overload. Transcriptome analysis showed that 323 genes were downregulated and 105 genes were upregulated in Rg1-treated cells. The differentially expressed genes were involved in Toll-like receptors, peroxisome proliferator-activated receptor signaling pathway, and cytokine-cytokine receptor interaction. The present study indicated that Rg1 may act as an antioxidative agent to protect cell damage caused by oxidative stress via regulating expression of genes such as RELT, EDA2R, and TLR4.

Protective effects of Panax notoginseng saponins on PME-Induced nephrotoxicity in mice.

Polymyxin E (PME) plays an important role in fighting against Gram-negative bacterial infections; however, it causes nephrotoxicity, which limits its clinical use. The aim of this study was to investigate the protective effects of a plant extract Panax notoginseng saponins (PNS) on PME-induced nephrotoxicity in mice. In vivo studies showed that PNS significantly reduced blood urea nitrogen (BUN), serum creatinine (CRE) and number of apoptotic cells in kidney, as well as renal histopathological damage which increased in the presence of PME, and suppressed PME-induced oxidative stress in kidney, as shown by the up-regulation of superoxide dismutase (SOD) and the down-regulation of malondialdehyde (MDA) levels. Furthermore, PNS inhibited the expression of Bax, while increased the expression of Bcl-2 compared to the PME-treated group. In vitro studies showed that PNS decreased intracellular reactive oxygen species (ROS) and MDA levels, increased glutathione (GSH) levels, and enhanced the activity of SOD and glutathione peroxidase (GSH-Px) in murine renal tubular epithelial cells (TCMK-1 cells). In addition, PNS enhanced cell viability and the expression of Bcl-2, restored the mitochondrial membrane potential, inhibited the expression of Bax, inhibited the activity of caspase-3 and caspase-9, and reduce apoptotic rate in PME-treated TCMK-1 cells. PNS could reduce PME-induced nephrotoxicity. The protective effects could result from inhibition of oxidative stress, and prevention of cell apoptosis via the mitochondrial pathway. These findings highlight the potential of PNS as a safe adjunct for ameliorating the nephrotoxicity.

Anti-inflammatory mechanism of ginsenoside Rg1: Proteomic analysis of milk from goats with mastitis induced with lipopolysaccharide.

Previous investigation showed that intravenous injection of ginsenoside Rg1 had a therapeutic effect on Escherichia coli lipopolysaccharide-induced mastitis in lactating goats and it protected animals from lipopolysaccharide challenge via toll-like receptor 4 signaling pathway. The present study was to use proteomic approach to explore the anti-inflammatory mechanisms of Rg1. Nine dairy goats were randomly divided into three groups with 3 animals in each: groups 1 and 2 received intra-mammary infusion of lipopolysaccharide and then intravenously injected with saline or Rg1 solution; animals in group 3 were first intramammarily and then intravenously administered saline solution, and served as a control group. Milk whey at 6 h post lipopolysaccharide challenge was prepared for tandem mass tags based quantitative proteomic analysis. The results showed that 791 proteins were totally identified from the whey. Of them, 98 proteins between groups 1 (lipopolysaccharide + Saline) and 3 (Saline + Saline), and 34 proteins between groups 2 (lipopolysaccharide + Rg1) and 1 were significantly different. Group 1 than group 3 had significantly more inflammatory factors such as interleukin 6, acute phase proteins, blood coagulation factors, complement proteins, and oxidative stress markers while these factors were reduced in group 2 treated with Rg1. In addition, proteins in group 2 associated with peroxisome-proliferator-activated receptor γ activation and recovery of milk fat and production were upregulated compared to group 1. Therefore, Rg1 may exert its anti-inflammatory effect on lipopolysaccharide-induced mastitis in goats via modulating expression of proteins relating to peroxisome-proliferator-activated receptor γ and toll-like receptor 4 signaling pathway.

Enhanced immune response to foot-and-mouth disease vaccine by oral administration of ginseng stem-leaf saponins.

Vaccination is an important approach to the control of foot-and-mouth disease (FMD). This study evaluated the effect of oral administration of ginseng stem-leaf saponins (GSLS) on the immune response to FMD vaccine and the gut mucosal immunity in mice. In experiment 1, mice were orally administered GSLS or not treated as a control. The animals were then immunized twice with FMD vaccine. Blood was sampled weekly within five weeks after the boost immunization for measurement of serum IgG and the isotypes. In experiment 2, mice were orally administrated GSLS or not treated as a control. After that, splenocytes were prepared from sacrificed mice for lymphocyte proliferation assay and intestinal tissues were sampled for immunohistochemistry and histological examination. The results showed that oral administration of GSLS significantly enhanced serum IgG and the isotype responses to FMD vaccine as well as the number of intestinal intraepithelial lymphocytes (IELs) and immunoglobulin A (IgA)+ cells. Therefore, GSLS may be a potent oral adjuvant and deserve further study to improve vaccination in susceptible animals.