KM Mice Research Articles

Effect and mechanism of citral against methicillin-resistant Staphylococcus aureus in vivo.

Citral is an active component of many plant extracts, and it is a safe additive used in food and cosmetics. A previous study showed that citral has a good antibacterial effect against methicillin-resistant Staphylococcus aureus (MRSA) in vitro, but its in vivo anti-infective activity has not been studied. Anti-MRSA activity and the preliminary mechanism of citral against MRSA were investigated in MRSA-infected KM mice. The ED50 was calculated using Karber's method. Groups were selected for inflammatory and oxidative stress level tests, and lung and liver tissues were counterstained with HE for detection of pathological changes. Cytokines and oxidative factors were evaluated using the ELISA method (one-way ANOVA computed using SPSS 19.0.). With the increase in the concentration of citral, the survival rate of MRSA-infected mice increased accordingly. The ED50 values of citral for intramuscular injection and intragastric administration were 0.09 and 0.26 g kg-1 respectively. Citral significantly reduced cytokines (IL-1β, IL-6, TNF-α) and oxidative factors (malondialdehyde and hydroxyl radicals) of MRSA-infected mice, whereas it increased gluthtione and superoxide dismutase levels. Citral can reduce the lung inflammatory infiltrates infected by MRSA. Citral exerted a dose-dependent anti-MRSA effect and ameliorated MRSA-induced abnormal changes in inflammation and oxidative stress. This indicates that citral has the potential for development as a new anti-MRSA drug. © 2019 Society of Chemical Industry.

Endoplasmic Reticulum Stress and Unfolded Protein Response Pathways Involved in the Health-Promoting Effects of Allicin on the Jejunum.

Intestinal endoplasmic reticulum stress (ERS) triggered by adverse factors disturbs the normal function of the intestine. Allicin has been reported to promote intestinal health and development. In the present study, we established in vivo (35-day-old weaned piglets, 4-week-old mice) and in vitro (IPEC-J2 cell line) ERS models to explore the possible mechanisms by which allicin may benefit intestinal health. This study revealed the following: (1) allicin supplementation improved intestinal morphological indices and ameliorated mild ERS in the jejunum of the weaned piglets; (2) allicin supplementation decreased cellular reactive oxygen species and upregulated the XBP-1s signaling pathways in IPEC-J2 cells; (3) allicin supplementation reduced the prolonged ERS-mediated apoptosis of IPEC-J2 cells and in the jejunal tissues of the KM mice; (4) allicin supplementation enhanced the intercellular junction protein levels of jejunal cells by alleviating the prolonged ERS. These novel findings suggest that eating garlic could alleviate some intestinal malfunctions associated with ERS.

High Density Lipoprotein from Egg Yolk (EYHDL) Improves Dyslipidemia by Mediating Fatty Acids Metabolism in High Fat Diet-induced Obese Mice.

We investigated the effect of high density lipoprotein from egg yolk (EYHDL) on serum, hepatic and fecal lipid and fatty acids (FAs) levels and on gene expression involved in FAs metabolism. Male KM mice were fed either normal diet (ND; n=20), high fat diet (HFD; n=20), or high fat diet containing EYHDL (EYHDL; 0.6 mg/g, every day by oral gavage, n=20) for 100 days. At the end of the experiment, the effects of treatments on biochemical parameters, FAs profiles and involved gene expression were analyzed. Our results revealed that EYHDL markedly suppressed the body weight gain, accumulation of abdominal fat tissues, serum concentrations of LDL-cholesterol (LDL-C) and triglycerides, hepatic triglycerides and cholesterol accumulation, while increased serum concentration of HDL-cholesterol (HDL-C). EYHDL intake also increased total cholesterol (TC) excretions compared with HFD group. Moreover, it alleviated the severity of fatty liver and improved glucose and insulin tolerance compared with HFD. More importantly, EYHDL partially normalized FAs profiles in serum, liver and fecaces and neutralized the HFD-induced upregulation of SREBP-1c, Acaca, Fasn, GPAT and Scd1. In conclusion, our findings indicate that EYHDL may have the potential to improve metabolic disturbances that occur in HFD mice and can be considered as an appropriate dietary recommendation for the treatment of metabolic syndrome (MetS).

Acute and sub-chronic 90-day oral toxicity study of Perilla seed oil in rodents and Beagle dogs

Perilla seeds are used as food and traditional medicine in China. This study aimed to investigate the toxicity profile of Perilla seed oil (PSO), which is the main constituent of Perilla seeds in rodents and Beagle dogs. No significant treatment-associated toxicity or mortality was observed at PSO dosages of up to 50 g/kg and 20 g/kg in KM mice and Wistar rats, respectively, suggesting that PSO was well tolerated by the experimental rodents. Sub-chronic oral toxicity of PSO was studied in dogs at doses of 3, 6 and 12 g/kg/d for 90 days followed by a 30 day recovery period. The results indicated that the body weight increased in all-dose groups more than control group, typical of animals on diets rich in fatty acids. Treatment-related side effects, including changes in hematology and serum biochemistry parameters, histopathology of liver and lymph glands, were observed in the high and moderate-dose dogs. However, these changes disappeared after the doses were withdrawn during the recovery period, except for alteration of liver in the high-dose group. In conclusion, the “no observed adverse effect level” (NOAEL) of oral administration of PSO for 90 days in Beagle dogs was considered to be 3 g/kg/d.

Hepatitis B virus X reduces hepatocyte apoptosis and promotes cell cycle progression through the Akt/mTOR pathway in vivo

Hepatitis B virus X (HBx), a viral onco-protein encoded by HBV, can promote oncogenesis of HCC. However, the mechanism of HBx in hepatocarcinogenesis is still unclear. In this study, we establish a new mouse model with normal immune system to investigate the role of HBx and its functional mechanisms under normal immune function. The animal model was established by injecting HBx-EGFP-14-19 cells into the hepatic portal vein of KM mice. To verify the mouse model, the expression of HBx in the liver tissue of mice was detected by qRT-PCR, western blotting and immunohistochemistry. The apoptosis index was calculated using the terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay, and the expression levels of apoptosis-related and cell cycle-related factors were measured. Moreover, expression of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway was detected in HBx-EGFP-14-19 mice with and without use of an Akt inhibitor. The results showed the HBx was successfully overexpressed in liver of KM mice. After overexpressing HBx, the apoptosis index was downregulated in HBx-EGFP-14-19 liver tissue, and the expression levels of caspase-9 and Bad were reduced, but Bcl-xl was increased in HBx-EGFP-14-19 liver tissue. Overexpression of HBx increased the expression of the cyclin-dependent kinase 2 (CDK2), cyclinD1 and cyclinE. Moreover, compared with the low-level HBx group, p-Akt and p-mTOR were increased in the livers of mice with high levels of HBx. However, inactivation of apoptosis by overexpression of HBx was abolished by the treatment with an Akt inhibitor. These results indicate that HBx can induce anti-apoptosis mechanisms in hepatocytes in vivo, which is mediated by the Akt/mTOR signaling pathway.

Disruption of the Golgi apparatus mediates zinc deficiency-induced impairment of cognitive function in mice.

Zinc deficiency is reported to be a global problem that affects cognitive function. The mechanism underlying zinc deficiency-induced impairment of cognitive function is still obscure. In this study, we treated KM mice (Kun Ming mice) with zinc chelator TPEN (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine) by i.p. injection. NOR (New Object Recognition) tests demonstrated that TPEN can impair the cognitive function of KM mice. Disruption of the GRASP55/Golgin45 complex, and even the Golgi apparatus, was also observed in hippocampus cells by TPEN treatment. Further investigation by IHF showed that enrichment of Aβ peptides occurs in neurons of the cerebral tissue of mice, suggesting that amyloidosis may mediate TPEN-induced impairment of cognitive function. This research not only clarifies that zinc plays an important role in Golgi organization in vivo, but also gives us a possible novel pathway underlying AD development.

Allyl methyl trisulfide protected against acetaminophen (paracetamol)-induced hepatotoxicity by suppressing CYP2E1 and activating Nrf2 in mouse liver.

In order to investigate the protective effects of allyl methyl trisulfide (AMTS) on acetaminophen (APAP)-induced hepatotoxicity, 75 KM mice were randomized into 5 groups, i.e. a control group, an APAP group, and three AMTS/APAP groups. The mice in the AMTS/APAP groups and APAP group were gavaged with 25-100 mg kg-1 AMTS or corn oil for 7 d followed by intraperitoneal injection of 300 mg kg-1 APAP, while mice in the control group were treated with a vehicle. We found that AMTS significantly attenuated APAP-induced hepatotoxicity shown by reduced mortality, decreased serum aminotransferase activities, and improved liver histological morphology. APAP overdose resulted in a significant increase of hepatic malondialdehyde (MDA) level and a decrease of the protein levels of NQO-1, γ-GCS, HO-1, and SOD, which was suppressed by AMTS pretreatment. Furthermore, AMTS inhibited the APAP-induced elevation of hepatic p62 and LC3II protein levels. Interestingly, AMTS attenuated the APAP-induced decline of hepatic CYP2E1 protein levels, but AMTS alone led to the decrease of CYP2E1 protein expression in mouse liver. Collectively, these data suggest that AMTS could attenuate APAP-induced hepatotoxicity by suppressing CYP2E1 and activating Nrf2.

Polymeric micelles loading with ursolic acid enhancing anti-tumor effect on hepatocellular carcinoma.

Ursolic acid (UA) is widely found in many dietary plants, which has been proved to be effective in cancer therapy. But unfortunately its hydrophobic property limits its clinical application. Polymer micelles (PMs) are constructed from amphiphilic block copolymers that tend to self-assemble and form the unique core-shell structure consisting of a hydrophilic corona outside and a hydrophobic inner core. PMs could entrap the hydrophobic substance into its hydrophobic inner core for solubilizing these poorly water-soluble drugs and it is widely applied as a novel nano-sized drug delivery system. This study aimed to develop the drug delivery system of UA-loaded polymer micelles (UA-PMs) to overcome the disadvantages of UA in clinical application thus enhancing antitumor effect on hepatocellular carcinoma. UA-PMs was prepared and characterized for the physicochemical properties. It was investigated the cell-growth inhibition effect of UA-PMs against the human hepatocellular carcinoma cell line HepG2 and human normal liver cell line L-02. UA-PMs was evaluated about the in vivo toxicity and the antitumor activity. We took a diblock copolymer of methoxy poly (ethylene glycol)-poly(L-lactic acid) (mPEG-PLA) as carrier material to prepare UA-PMs by the thin-film dispersion method. MTT assay and wound-healing assay were investigated to assess the inhibition effect of UA-PMs against HepG2 cells on cell-growth and cell-migration. Further, we chose KM mice for the acute toxicity experiment and assessed the antitumor effect of UA-PMs on the H22 tumor xenograft. UA-PMs could markedly inhibit the proliferation and migration of HepG2 cells. In vivo study showed that UA-PMs could significantly inhibit the growth of H22 xenograft and prolong the survival time of tumor-bearing mice. It demonstrated that UA-PMs possess great potential in liver cancer therapy and may enlarge the application of UA in clinical therapy.

The impact of Bacillus subtilis 18 isolated from Tibetan yaks on growth performance and gut microbial community in mice

The purpose of this study was to evaluate the effects of Bacillus subtilis 18 (BS-18) isolated from free-ranging Tibetan yaks in high altitude regions of Tibet (3600 m) on growth performance and gut microbial community in mice. In this study, mice (15-day-old) were used as an animal model and raised under standard conditions. A total of 20 KM mice were divided equally into two groups: control group (feed and drink freely), experimental group (feed and drink freely + 1 × 109 CFU/day BS-18). The intestines (duodenum, jejunum, ileum, cecum) and organs (liver, spleen, kidney) were collected from all the mice at day 18 for high throughput sequencing and HE staining. During the whole experiment, the mice treated by BS-18 displayed no abnormal behavior or macroscopic lesions on dissection. Meanwhile, there were no pathological changes observed using HE staining compared with the control group. The results show that BS-18 isolated from Tibetan yaks was safe and could increase average daily gain (ADG) and reduce feed conversion ratio (FCR). Furthermore, supplementation with BS-18 could improve the mucosal morphology and the ratio of villi to crypt cells (P < 0.05 or P < 0.01). The high-throughput sequencing results showed that the abundance and diversity of duodenum and jejunum in the experimental group were higher than control group. Lactobacillus in experimental group had higher abundance than control group. In addition, the quantity of Candidatus arthromitu was increased after BS-18 intake, which is associated with immune system activity. Acinetobacter induced brain abscess and bronchopneumonia were reduced in the experimental group. In conclusion, this study demonstrated that BS-18 isolated from Tibetan yaks was safe, beneficial and had the potential to serve as a probiotic.

Effect of total glucosides of paeony on the changes of IL-4 and ICAM-1 levels in eczema mouse model serum.

Effect of total glucosides of paeony on the changes of IL-4 and ICAM-1 levels in eczema mouse model serum was investigated. A total of 38 KM mice of SPF grade were divided into 3 groups: the control group (n=10), the model group (n=15) and the treatment group (n=13). The pathological model of chronic eczema in mouse right ear was induced using dinitrochlorobenzene acetone solution. Two ears of mice in the control group and the left ear of mice in the model and treatment groups were smeared with acetone as control. The mice in the treatment group were treated by administration with total glucoside of paeony. The changes of IL-4 and ICAM-1 levels were measured using caudal vein blood collection. The mouse ear weight was measured and the relationship among IL-4 and ICAM-1 levels, ear thickness and treatment time was analyzed. Mouse ear thickness in the model group was higher than that in the treatment and control groups (P<0.05). The weight of the mouse right ear in the model and treatment groups was significantly higher than that of the left ear (P<0.05). Furthermore, The IL-4 and ICAM-1 levels of mice in the model group were higher than that in the treatment and control groups (P<0.05). The IL-4 and ICAM-1 levels of mice in the model and treatment groups increased compared to that before modeling (P<0.05). The IL-4 and ICAM-1 levels of mice were positively correlated with ear thickness in the model group (r=0.865, P=0.002; r=0.833, P=0.009). In addition, the IL-4 level of mice was positively correlated with the ICAM-1 level in the model group (r=0.812, P=0.014). Finally, IL-4 and ICAM-1 may be involved in the pathologic process of chronic eczema. Therefore, the study showed that the total glucosides of paeony may play a role in the treatment of chronic eczema by regulating the IL-4 and ICAM-1 levels.

The low protein diet affects the nonspecific inflammatory response of middle-aged and old mice through mTOR.

To explore the effect of low protein diet on nonspecific inflammatory changes in mice during aging and related mechanisms. Thirty-two 14-month-old female KM mice were randomly divided into 4 groups: control group, low protein group, high protein group, high protein + rapamycin group. Hematoxylin-eosin (HE) staining was performed to observe the pathological changes of the liver. Immunohistochemistry of liver sections was performed to detect the expression of CD68 protein. HE staining of colon sections was performed to observe intestinal lymphocyte infiltration. The percentage of spleen CD4+ T and CD8+ T cells was detected by flow cytometry. The mTOR expression in the liver was detected by Western blot and immunohistochemistry. Compared with the control group, HE staining of liver tissue sections in high-protein group showed the cytoplasm of hepatocytes was loose and disordered, and the hepatic sinus was significantly expanded. Immunohistochemistry of the liver showed a significant increase in CD68 protein expression. Colorectal HE staining showed extensive lymphocyte infiltration. The number of CD4+ T and CD8+ T cells in spleen flow cytometry was significantly decreased (*p < 0.05). Western blot and immunohistochemistry detected a significant increase in mTOR expression in the liver (*p < 0.05, *p < 0.05). In the High Protein+Rapamycin group and Low-protein group, the time-dependent changes were reduced, the numbers of CD4+ T cell and CD8+ T cell in the spleen were significantly increased (*p < 0.05) and the expression of mTOR was significantly reduced (*p < 0.05). Low-protein diet is beneficial for delaying the non-specific inflammatory changes of liver and intestines in middle-aged and aged mice, and this effect may be achieved through down-regulation of mTOR.

PO-106 Anisochronous aerobic exercise improves glucose and lipid metabolism of obstructive jaundice by activating Akt signaling pathway

Objective To observe the effect of moderate intensity and different interval aerobic exercise on hepatic injury and AKT signaling pathway induced by severe obstructive jaundice in mice.&#x0D; Methods 40 male KM mice were randomly divided into 4 groups: sham operation (OJ) group, model (OM) group, 3-week exercise (ST) group and 6-week exercise (SU) group. Mice in OM group, ST group and SU group all adopted the orthotopic hanging choledochotomy method modified by this study group to construct the animal model of obstructive jaundice. The slope of group ST and group SU were 0% and the speed was 10m/min. After the above 6 weeks of intervention, HE staining was used to observe the morphological changes of hepatocytes. Methods of automatic biochemical analyzer test serum total bilirubin (TBIL), alanine aminotransferase (ALT), aspertate aminotransferase (AST), liver function and fasting plasma glucose (GLU), glycosylated hemoglobin (HbA1c), glycosylated serum protein (GSP), total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL - C) of sugar, lipid metabolism and biochemical indexes such as detection; Immunohistochemical staining and qrt-pcr technology were used to observe the expression changes of AKT related molecules such as SREBP-1c, LDL-C, gsk-3β, GCK and G6Pase in liver tissues.&#x0D; Results HE staining showed that the liver cell cords of the normal group were orderly. In the model group, the hepatocytes of the rats were fibrosed in large amounts, which showed degeneration, necrosis and even disordered structure of hepatocytes. In the 3-week exercise group, a small number of hepatic cells were found with patchy necrosis, hepatic lobule structure was changed, and hepatic cords were not well arranged. There was no obvious tissue necrosis in the 6-week exercise group, the hepatic lobule structure was basically normal, and the liver cord was arranged in order. Serological results showed that the levels of TG, TC, LDL-C, HDL - C and GLU in the 6-week exercise group were significantly lower than those in the model group (P &lt; 0.01), and the levels of TG and TC in the 3-week group were also significantly decreased (P &lt; 0.05). In liver tissues, the mRNA and protein expression of related molecules of AKT pathway such as SREBP-1c, LDL-C, gsk-3β, GCK and G6Pase were significantly decreased. &#x0D; Conclusions Moderate intensity aerobic exercise can regulate glucose and lipid metabolism in mice with severe liver injury caused by obstructive jaundice. The underlying mechanism may be related to regulating the AKT pathway.

PO-105 Exercise regulates HMGB1 / TLR4 / NF- κ B pathway by H2S to improve OJ intestinal injury

Objective To study the effect of aerobic exercise on the damage of intestinal mucosal barrier function caused by obstructive jaundice（OJ）and to explore its mechanism of action.&#x0D; Methods 50 male KM mice were randomly divided into 5 groups: sham operation group (S), model group (M), exercise group (TM), DL-Propargylglycine + exercise (PT) group and sodium hydrosulfide + exercise (NT) group.In addition to the S group which are in the common bile duct to the abdominal wall hanging 48 hours to build mouse obstructive jaundice model. In the PT group, PAG (40 mg/kg) was intraperitoneally injected 7 days after surgery; NaHS (50 μmol/kg) was intraperitoneally injected in the NT group 7 days after surgery; TM group, NT group and PT group were graded at 0%, and the speed was 10m/min no weight training (30min/day).After 6 weeks, HE staining was used to observe the morphological changes of the intestinal mucosa.Biochemical analysis was used to detect the concentration of hydrogen sulfide (H2S) in blood and ileum, and total bilirubin (TBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) Liver function, diamine oxidase (DAO), D-lactic acid intestinal barrier function biochemical index; qRT-PCR and immunohistochemical staining were used to observe the expression changes of H2S-mediated related channel mRNA and protein（HMGB1, TLR4 and NF-Kbp6）in intestinal tissues.&#x0D; Results HE staining showed that the intestinal mucosa of group M was atrophied and the villus was broken.Compared with M group, the intestinal mucosa arrangement in TM group was relatively regular. Compared with TM group, intestinal mucosa atrophy in PT group, fluff hair loss, sparseness and disorder, partial mucosa The layer was separated from the lamina propria and the gland was severely damaged. The intestinal mucosa of the NT group was relatively regular, and the changes of intestinal mucosa atrophy were restored. Serum test results showed that H2S levels were higher in the TM group than in the M group; compared with the TM group, the PT group decreased and the NT group increased. DAO level: The TM group was lower than the M group; compared with the TM group, the PT group was elevated and the NT group was decreased. Changes in serum D-lactic acid levels were similar to DAO. The results of qRT-PCR and immunohistochemical staining showed that the expressions of HMGB1, TLR4 and NF-Kbp6 mRNA and protein in the intestinal tissues of mice in TM group were significantly lower than those in M group and PT group, and the mRNA and protein expression levels in NT group were the lowest.&#x0D; Conclusions Aerobic exercise inhibits the HMGB1 / TLR4 / NF-κB signaling pathway through the H2S / CSE system, thereby exerting a protective effect on the intestinal mucosal barrier.

Treatment of Hyperoxia-Induced Lung Injury with Lung Mesenchymal Stem Cells in Mice.

Objective Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in preterm neonates and has no effective treatment. This study aimed to investigate the therapeutic effects of neonatal mouse lung resident mesenchymal stem cells (L-MSCs) on the hyperoxia-induced lung injury. Methods L-MSCs were separated and identified according to the MSC criterions. Hyperoxia-Induced Lung Injury (HILI) of neonatal KM mice was induced with hyperoxia (FiO2 = 60%) and investigated with pathological methods. Neonatal KM mice were divided into 3 groups (hyperoxia + L-MSC group, hyperoxia + PBS group, and air control group). Mice in the hyperoxia + L-MSC group were treated with L-MSCs at 3, 7, and 14 days after birth. After hyperoxia exposure for 21 days, the lung pathology, Radial Alveolar Count (RAC), CD31 expression, and vascular endothelial growth factor (VEGF) expression were investigated. Results After hyperoxia exposure, the body weight, RAC, CD31 expression, and VEGF expression in the hyperoxia + L-MSC group were significantly better than those in the hyperoxia + PBS group but inferior to those in the air control group significantly. These indicate L-MSCs are partially protective on the lung injury of mice with hyperoxia-induced BPD. Conclusion L-MSCs are helpful for the prevention and treatment of BPD, and endogenous L-MSCs may play a role in the postinjury repair of the lung.

Novel thioredoxin reductase inhibitor butaselen inhibits tumorigenesis by down-regulating programmed death-ligand 1 expression.

The thioredoxin system plays a role in a variety of physiological functions, including cell growth, differentiation, apoptosis, tumorigenesis, and immunity. We previously confirmed that butaselen (BS), a novel thioredoxin reductase inhibitor, can inhibit the growth of various human cancer cell lines, yet the underlying mechanism remains elusive. In this study, we investigated the anti-tumor effect of BS in vivo through regulating the immune system of KM mice. We found that BS inhibits tumor proliferation by promoting the activation of splenic lymphocytes in mice. BS can elevate the percentage of CD4-CD8+ T lymphocytes and the secretion of downstream cytokines in mice via down-regulating the expression of programmed death-ligand 1 (PD-L1) on the tumor cells' surface in vivo. Further study in HepG2 and BEL-7402 cells showed that decrease of PD-L1 level after BS treatment was achieved by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation. Taken together, our results suggest that BS has a role in promoting the immune response by reducing PD-L1 expression via the STAT3 pathway, and subsequently suppresses tumorigenesis.

Tumor suppression by Tegafur combined with Barbadian in S-180 tumor-bearing mice.

The aim of this study was to discuss the antitumor effect of Tegafur combined with Barbadian on S-180 tumor-bearing mice. A murine tumor model was prepared by subcutaneous injection of S-180 sarcoma cells to the armpit of the right limb of healthy female SPF KM mice. The 24 tumor-bearing mice were randomly divided into 4 groups: Combination therapy group of Tegafur and Barbadian, Barbadian group, Tegafur group and normal saline control group. Corresponding test substances were given to each group by intragastric administration, respectively, 0.2 ml/mouse, once/day, continuous 5 days, interval for 2 days, recorded as 1 period, 3 periods were continuously performed. Antitumor rate, immune cells, blood biochemistry and inflammatory mediators and other indexes were then respectively measured. Result showed that the antitumor rate for the Combination group was 78%; Barbadian group, 72%; and Tegafur group, −89%. White blood cells (WBC) in Barbadian group was significantly higher than that in the control group (P<0.01); lymphocytes (LYMPH), Barbadian group was significantly higher than that in the control group (P<0.01), Tegafur group was significantly lower than the control group (P<0.01); monocytes (MONO), all drug groups were significantly higher than the control group (P<0.01); neutrophils (NEUT), combination group (P<0.01) and Barbadian group (P<0.05) were significantly higher than the control group; blood sugar for the combination (P<0.05) and Barbadian (P<0.01) groups were significantly higher than the control group, while the Tegafur group was significantly lower than the control group (P<0.01). Cholesterol and BUN in the Tegafur group were significantly higher than that in control group (P<0.05). For IL-1, the combination and Barbadian groups were significantly higher than the control group (P<0.05), while for IL-6, all the drug groups were significantly higher than the control group (P<0.05). TNF-α in the Tegafur group was significantly lower than that in the control group (P<0.05). In conclusion, the combination of Tegafur and Barbadian has the significant effect of inhibiting mice S-180 sarcoma. The single use of chemotherapeutic drug Tegafur has no significant inhibitory effect on mice S-180 sarcoma. The single use of Barbadian has good antitumor effect and can resist the significant decrease of lymphocytes caused by the chemotherapeutic drug Tegafur. Thus, Barbadian has a good antitumor effect and can protect the immune system of the body, making it a viable treatment option.

Arylamino containing hydroxamic acids as potent urease inhibitors for the treatment of Helicobacter pylori infection

A novel series of aniline-containing hydroxamic acids were designed, synthesized and evaluated as anti-virulence agents for the treatment of gastritis and gastric ulcer caused by Helicobacter pylori. In vitro enzyme-based screen together with in vivo assays and structure−activity relationship (SAR) studies led to the discovery of three potent urease inhibitors 3-(3,5-dichlorophenylamino)N-hydroxypropanamide (3a), 3-(2-chlorophenylamino)N-hydroxypropanamide (3d) and 3-(2,4-dichlorophenylamino)N-hydroxypropanamide (3n). Compounds 3a, 3d and 3n showed excellent urease inhibition with IC50 values 0.043 ± 0.005, 0.055 ± 0.008 and 0.018 ± 0.002 μM, and significantly depressed gastritis developing at the dose of 32 mg/kg b. i.d with eradication rates of H. pylori reaching 92.3, 84.6 and 100%, respectively. Preliminary safety studies (acute toxicity in mice) disclosed that 3a, 3d and 3n was well-tolerated in KM mice with LD50s of 2982.8, 3349.4 and 3126.9 mg/kg, respectively. Collectively, the data obtained in this study indicate that 3a, 3d and 3n, in particular 3n, could considered as promising candidates for the potential treatment of H. pylori caused gastritis and gastric ulcer, and hence merit further studies.

Genetic and neuroattenuation phenotypic characteristics and their stabilities of SA14-14-2 vaccine seed virus

Japanese encephalitis (JE) live attenuated vaccine SA14-14-2 is the most widely used JE vaccine in the world. Large-scale clinical trials have demonstrated satisfactory safety and efficacy profiles. The establishment of genetic and attenuated neurovirulence characteristics and their stabilities of SA14-14-2 virus are important in relation to vaccine safety in humans. Therefore, several researchers have studied and analyzed the full-length gene sequences of the SA14-14-2 virus strain. However, sequencing results have shown a significant difference. Here, we further studied the full-length sequence of three class seed virus banks of the vaccine as well as two vaccine viruses with different passages in primary hamster kidney cells, and compared them with our original stored SA14 parent virus (low passage in mouse brain). The full-length gene sequence determined in this study indicates there were 57 nucleotide and 25 amino acid substitutions of the SA14-14-2 strain compared to its parental SA14 virus strain. The full-length sequences of the three class seed bank viruses and the vaccine virus PHKC8 were completely identical among them, but the working seed virus passaged in primary hamster kidney cells for 17 generations (PHKC17) had a single nucleotide change at the 5′ NCR. Both KM and ICR mice tested by intracerebral (i.c.) or subcutaneous (s.c.) routes with the three class seed viruses and vaccine viruses with ≥5.7 lgpfu/mL remained healthy, but all the mice inoculated with the SA14 parental virus strain died as early as day 5 post-inoculation. The present study provided new information on the full-length gene sequence and attenuated neurovirulence of SA14-14-2. They can be used as a reference sequence for vaccine quality control and surveillance of neurovirulence reversion following vaccination. Moreover, the present results further demonstrated the high genetic and phenotypic stabilities of the SA14-14-2 virus, suggesting the neurovirulence reversion of the vaccine strain will be highly unlikely.

Immune Tolerance Induced by Decitabine Combined With Multiple Allogeneic Bone Marrow Mononuclear Cell Infusion

ObjectiveThe aim of our study was to investigate the strategy of immune tolerance induced by the demethylating drug decitabine (DAC) combined with multiple allogeneic bone marrow mononuclear cell (BMMNC) infusions. MethodsAn animal model with C57BL/6 mice as recipients and a BALB/c mouse as the donor was established. C57BL/6 mice were randomly divided into the following groups: DAC, BMMNC, DAC+BMMNC, and the control groups. Mice in the DAC and DAC+BMMNC groups were given DAC (5 μg/kg) on days 1, 2, 7, 8, 13, and 14. On experimental days 3, 9, and 15, BMMNCs (5 × 106/mouse/time) from BALB/c mice were infused into mice of the BMMNC and DAC+BMMNC groups. One-way mixed lymphocyte reaction (MLR), interleukin-2 reverse test, and in vitro adoptive transfer experiments were performed. The ratio of regulatory T cells in splenocytes, chimera detection, and median survival time of skin grafts were recorded. ResultsMLR showed that the stimulation index of the DAC+BMMNC group was significantly lower compared with the other 3 groups (P < .01), but still showed a strong proliferative response to the third-party unrelated donor KM mice, with no significant difference compared with the other 3 groups. The MLR low reactivity of the DAC+BMMNC group could be reversed by exogenous interleukin-2. After adding an equal amount of splenocytes from a C57BL/6 mouse in the DAC+BMMNC group, MLR of control group significantly decreased (P < .05). Flow cytometry showed the ratio of regulatory T cells in splenocytes from DAC and DAC+BMMNC groups was significantly higher than that from the BMMNC and control groups (P < .05). Chimeric rate of the DAC+BMMNC (60%) group was significantly higher than that of the BMMNC group (30%). Moreover, survival time of skin grafts in the DAC+BMMNC group was significantly longer than that in the other 3 groups (P < .001). No death or graft-vs-host disease was observed. ConclusionDAC combined with multiple allogeneic BMMNC infusion could successfully induce specific immune tolerance in mice, which may provide some new strategies to improve immune tolerance after organ transplant.

Anti-Aging Effect of Chitosan Oligosaccharide on d-Galactose-Induced Subacute Aging in Mice.

Chitosan oligosaccharide (COS), a natural polysaccharide with good antioxidant and anti-inflammatory properties, is the depolymerized product of chitosan possessing various biological activities. The present study was designed to investigate the possible anti-aging effect of COS on the aging model mouse induced by d-galactose (d-gal) and explore the underlying mechanism. In the experiment, 48 male Kunming mice (KM mice) were randomly divided into the normal group, model group, positive group, and low-medium-high dose polysaccharide groups (300, 600, 1200 mg/kg/day). The results showed that COS, by intragastric gavage after subcutaneous injection of d-gal (250 mg/kg/day) into the neck of mice consecutively for eight weeks, gradually recovered the body weight, the activity of daily living, and organ indices of mice, as well as effectively ameliorated the histological deterioration of the liver and kidney in mice triggered by d-gal. To be specific, COS obviously improved the activities of antioxidant enzymes in liver and kidney of KM mice, including catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD), as well as decreased malondialdehyde (MDA) levels when compared with those in model group mice. Furthermore, COS not only elevated the diminished levels of serum immunoglobulin G (IgG) and IgM induced by d-gal, but also significantly inhibited the d-gal-caused upregulation of serum alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), uric acid (UA) and creatinine (CREA) levels as compared with those of mice in the model group. These results demonstrate that COS has an obvious anti-aging activity in d-gal-induced subacute aging mice, the mechanism of which, to some extent, is associated with enhancing the antioxidant defenses, reducing oxidative stress, and improving the immune function of aging model mice.