Exploring the effects of taurolidine on tumor weight and microvessel density in a murine model of osteosarcoma

Background:Osteosarcoma is the most common malignant primary bone tumor.The prognosis for patients with disseminated disease remains very poor despite recent advancements in chemotherapy.Moreover,current treatment regimens bear a significant risk of serious side effects.Thus,there is an unmet clinical need for effective therapies with improved safety profiles.Taurolidine is an antibacterial agent that has been shown to induce cell death in different types of cancer cell lines.Methods:In this study,we examined both the antineoplastic and antiangiogenic effects of taurolidine in animal models of osteosarcoma.K7M2 murine osteosarcoma cells were injected,both intramuscular and intraperitoneal,into 60 BALB/c mice on day zero.Animals were then randomized to receive treatment with taurolidine 2%(800 mg/kg),taurolidine 1%(400 mg/kg),or NaCl 0.9%control for seven days by intravenous or intraperitoneal administration.Results:After 35 days,mice were euthanized,and the tumors were harvested for analysis.Eighteen mice were excluded from the analysis due to complications.Body weight was significantly lower in the 2%taurolidine intraperitoneal treatment group from day 9 to 21,consistent with elevated mortality in this group.Intraperitoneal tumor weight was significantly lower in the 1%(p=0.003)and 2%(p=0.006)intraperitoneal taurolidine treatment groups compared to the control.No antineoplastic effects were observed on intramuscular tumors or for intravenous administration of taurolidine.There were no significant differences in microvessel density or mitotic rate between treatment groups.Reduced body weight and elevated mortality in the 2%taurolidine intraperitoneal group suggest that the lower 1%dose is preferable.Conclusions:In conclusion,there is no evidence of antiangiogenic activity,and the antitumor effects of taurolidine on osteosarcoma observed in this study are limited.Moreover,its toxic profile grants further evaluation.Given these observations,further research is necessary to refine the use of taurolidine in osteosarcoma treatment.

Chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor for neurotrophic keratopathy

Neurotrophic keratopathy is a persistent defect of the corneal epithelium,with or without stromal ulceration,due to corneal nerve deficiency caused by a variety of etiologies.The treatment options for neurotrophic keratopathy are limited.In this study,an ophthalmic solution was constructed from a chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor(CTH-mNGF).Its effectiveness was evaluated in corneal denervation(CD)mice and patients with neurotrophic keratopathy.In the preclinical setting,CTH-mNGF was assessed in a murine corneal denervation model.CTH-mNGF was transparent,thermosensitive,and ensured sustained release of mNGF for over 20 hours on the ocular surface,maintaining the local mNGF concentration around 1300 pg/mL in vivo.Corneal denervation mice treated with CTH-mNGF for 10 days showed a significant increase in corneal nerve area and total corneal nerve length compared with non-treated and CTH treated mice.A subsequent clinical trial of CTH-mNGF was conducted in patients with stage 2 or 3 neurotrophic keratopathy.Patients received topical CTH-mNGF twice daily for 8 weeks.Fluorescein sodium images,Schirmer’s test,intraocular pressure,Cochet-Bonnet corneal perception test,and best corrected visual acuity were evaluated.In total,six patients(total of seven eyes)diagnosed with neurotrophic keratopathy were enrolled.After 8 weeks of CTH-mNGF treatment,all participants showed a decreased area of corneal epithelial defect,as stained by fluorescence.Overall,six out of seven eyes had fluorescence staining scores<5.Moreover,best corrected visual acuity,intraocular pressure,Schirmer’s test and Cochet-Bonnet corneal perception test results showed no significant improvement.An increase in corneal nerve density was observed by in vivo confocal microscopy after 8 weeks of CTH-mNGF treatment in three out of seven eyes.This study demonstrates that CTH-mNGF is transparent,thermosensitive,and has sustained-release properties.Its effectiveness in healing corneal epithelial defects in all eyes with neurotrophic keratopathy suggests CTH-mNGF has promising application prospects in the treatment of neurotrophic keratopathy,being convenient and cost effective.

A novel pulmonary fibrosis murine model with immune-related liver injury

Idiopathic pulmonary fibrosis(IPF),characterized by aggravated alveolar destruc-tion and fibrotic matrix deposition,tendentiously experiences the stage called acute exacerbation IPF(AE-IPF)and progresses to multiple organ damage,especially liver injury.Recent studies have found a variety of immune microenvironment disorders associated with elevated IPF risk and secondary organ injury,whereas current animal models induced with bleomycin(BLM)could not completely reflect the pathologi-cal manifestations of AE-IPF patients in clinic,and the exact underlying mechanisms are not yet fully explored.In the current study,we established an AE-IPF model by tracheal administration of a single dose of BLM and then repeated administrations of lipopolysaccharide in mice.This mouse model successfully recapitulated the clinical features of AE-IPF,including excessive intrapulmonary inflammation and fibrosis and extrapulmonary manifestations,as indicated by significant upregulation of Il6,Tnfa,Il1b,Tgfb,fibronectin,and Col1a1 in both lungs and liver and elevated serum aspartate transaminase and alanine transaminase levels.These effects might be attributed to the regulation of Th17 cells.By sharing this novel murine model,we expect to pro-vide an appropriate experimental platform to investigate the pathogenesis of AE-IPF coupled with liver injury and contribute to the discovery and development of targeted interventions.

Evaluation of antiviral activities of Houttuynia cordata Thunb.extract,quercetin,quercetrin and cinanserin on murine coronavirus and dengue virus infection

Objective:To evaluate the in vitro activities of the ethyl acetate(EA) fraction of Houttuynia cordata(H.cordata) Thunb.(Saururaceae) and three of its constituent flavonoids(quercetin.quercitrin and rutin) against murine coronavirus and dengue virus(DENV).Methods:The antiviral activities of various concentrations of the EA fraction of H.cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus(MHV) and DENV type 2(DENV-2).Cinanserin hydrochloride was also tested against MHV.The EA fraction of H.cordata was tested for acute oral toxicity in C57BL/6 mice.Results:The EA fraction of H.cordata inhibited viral infectivity up to 6 d.Cinanserin hydrochloride was able to inhibit MHV for only 2 d.The 50%inhibitory concentrations(IC_(50)) of the EA fraction of H.cordata added before the viral adsorption stage were 0.98 μg/mL for MHV and 7.50 μg/mL for DENV-2with absence of cytotoxicity.The mice fed with the EA fraction up to 2 000 mg/kg did not induce any signs of acute toxicity,with normal histological features of major organs.Certain flavonoids exhibited comparatively weaker antiviral activity,notably quercetin which could inhibit both MHV and DENV-2.This was followed by quercitrin which could inhibit DENV-2but not MHV,whereas rutin did not exert any inhibitory effect on either virus.When quercetin was combined with quercitrin,enhancement of anti-DENV-2 activity and reduced cytotoxicity were observed.However,the synergistic efficacy of the flavonoid combination was still less than that of the EA fraction.Conclusions:The compounds in H.cordata contribute to the superior antiviral efficacy of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vim.H.cordata has much potential for the development of antiviral agents against coronavirus and dengue infections.

Gonadotrophin-releasing hormone-Ⅰ and -Ⅱ stimulate steroidogenesis in prepubertal murine Leydig cells in vitro

Aim： To study the effect and mechanism of gonadotrophin-releasing hormone （GnRH） on murine Leydig cell steroidogenesis. Methods： Purified murine Leydig cells were treated with GnRH-Ⅰ and -Ⅱ agonists, and testosterone production and steroidogenic enzyme expressions were determined. Results： GnRH-Ⅰ and -Ⅱ agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner （P 〈 0.05）. The mRNA expressions of steroidogenic acute regulatory （STAR） protein, P450scc, 3β-hydroxysteroid dehydrogenase （HSD）, but not 17β-hydroxylase or 17β-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase （P 〈 0.05）. However, only 3β-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase （P 〈 0.05）. Conclusion： GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3β-HSD enzyme expression.

Two stomach-originated lactobacillus strains improve Helicobacter pylori infected murine gastritis

AIM:To investigate the potential anti-Helicobacter pylori(H.pylori ) and anti-inflammation in vivo effects of two lactobacillus strains from human stomach.METHODS:Forty H.pylori infected Balb/c mice were randomly divided into 4 groups:proton pump inhibitor and antibiotics triple treated group,Lactobacillus fermenti(L.fermenti ) treated group,Lactobacillus acidophilus treated group and normal saline control group.Ten uninfected mice were also included as blank control group.The infection of H.pylori was detected by rapid urease tests,Giemsa staining and bacterial culture.The colonization of H.pylori was assessed in bacterial density score and gastric inflammation was assessed in histological score.The colonization of L.fermenti was performed by fluorescent probe.RESULTS:Histopathologic evaluation showed significant release of mucosal inflammation in gastric antrum and gastric body in lactobacillus treated groups and triple treated group.H.pylori eradication rate in both lactobacillus treated groups and triple treated group were higher than normal saline control group.Lactobacillus treated groups and triple treated group showed significant decrease of H.pylori bacterial density.CONCLUSION:Both lactobacillus strains have a significant anti-H.pylori activity;L.fermenti displays more efficient antagonistic activity in vivo against H.pylori infection.

Murine models based on acute myeloid leukemia-initiating stem cells xenografting

Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based on somatic mutations, AML is still characterized by poor outcomes, with a 5-year survival rate of only 30%-40%, the majority of the patients dying due to AML relapse. Leukemia stem cells(LSC) are considered to be at the root of chemotherapeutic resistance and AML relapse. Although numerous studies have tried to better characterize LSCs in terms of surface and molecular markers, a specific marker of LSC has not been found, and still the most universally accepted phenotypic signature remains the surface antigens CD34+CD38- that is shared with normal hematopoietic stem cells. Animal models provides the means to investigate the factors responsible for leukemic transformation, the intrinsic differences between secondary post-myeloproliferative neoplasm AML and de novo AML, especially the signaling pathways involved in inflammation and hematopoiesis. However, AML proved to be one of the hematological malignancies that is difficult to engraft even in the most immunodeficient mice strains, and numerous ongoing attempts are focused to develop "humanized mice" that can support the engraftment of LSC. This present review is aiming to in-troduce the field of AML pathogenesis and the concept of LSC, to present the current knowledge on leukemic blasts surface markers and recent attempts to develop best AML animal models.

A murine model for human immune thrombocytopenic purpura and comparative analysis of multiple gene expression in bone marrow and spleen

Homeostasis of platelet number in human and other mammals is well maintained for prevention of minor bleeding and for other im- munological functions, but the exact molecular mechanism responsible for immune thrombocytopenic purpura （ITP） has not been fully understood. In an effort to identify genetic factors involved in initiation of platelet production in response to bleeding injury or platelet destruction, we have successfully generated an animal model of human ITP via intraperitoneal injection of anti-platelet antibody into the Balb/c mouse. Platelet counts were dropped dramatically in animals that received antibody injection within 4 h, maintained at the mini- mum level for a period of 44 h, started to rebound after 48 h, and reached to the maximum at 144 h （6 days）. Final homeostasis reached at approximately 408 h （17 days）, following a minor cycle of platelet number fluctuation. Using semi-quantitative RT-PCR, we assessed and compared mRNA level of CD41, c-myb, c-mpl, caspase-3, caspase-9, GATA-1, and Bcl-xl in bone marrow and spleen. Alteration of mRNA expression was correlated with the change of platelet level, and an inverse relationship was found for expression of the genes be- tween bone marrow and spleen. No transcription was detectable for any of the seven genes in bone marrow at the time when platelet number reached the maximum （144 h）. In contrast, mRNA transcripts of the seven genes were found to be at the highest level in spleen tissue. This is the first study of simultaneous detection of multiple platelet related genes in a highly reproducible ITP animal model. Our results provided the supportive evidence that expression of the above seven genes are more related to negative regulation of platelet number in spleen tissue, at least in the model animals.

Maternal Murine Cytomegalovirus Infection during Pregnancy Up-regulates the Gene Expression of Toll-like Receptor 2 and 4 in Placenta

Increasing evidence has revealed that maternal cytomegalovirus （CMV） infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 （TLR2） and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL- 10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV （MCMV） infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide （LPS）-treated and uninfected mice were used as controls. At E13.5, E 14.5 and E 18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 ktg/kg could decrease the IL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more pro- inflammatory cytokine IL-6. High dose of LPS stimulation （50 gg/kg） during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.

Local Expression of Vaginal Th1 and Th2 Cytokines in Murine Vaginal Candidiasis under Different Immunity Conditions

To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 （IL-2）/Th2 （IL-4, IL-10, TGF-β1） cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-β1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-β1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans （C. albicans）, and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.

Cytokine profile in murine toxoplasmosis

Objective:To investigate which cytokines are produced after acute infection of mice with Toxoplasma gondii(T.Gondii) RH strain.Methods:Mus domesticus domesticus mice in infected group were inoculated with with highly virulent T.Gondii RH strain by intraperitoneally.Serum samples were obtained from infected and non-infected mice for cytokine levels for ELISA assay.Results:The concentrations of tumor necrosis factor-α,interferon-γ,interleukin(IL)- 10 and IL-12 in the cardiac blood sample of the infected mice were significantly higher than those in uninfected controls(P【0.05).The levels of transforming growth factor-1βdecreased in mice infected with T.gondii compared to those of the controls,the decrease was statistically significant(P【0.05).No significant difference was observed in levels of IL-4 between infected and healty control groups(P】0.05).Conclusions:According to our findings,immune response into T helper type 1 was predominant during acute T.gondii infection.Further characterization and purification of Toxoplasma molecule(s) implicated in the regulation of cytokines could lead to the development of new drug prospects to control Toxoplasma infection.

Great efficacy of sulfachloropyrazine-sodium against acute murine toxoplasmosis

Objective:To identify more effective and less toxic drugs to treat animal toxoplasmosis.Methods:Efficucy of seven kinds of sulfonamides against Toxoplasma gondii(T.gondii)in an acute murine model was evaluated.The mice used throughout the study were randomly assigned to many groups(10 mice each),which either remained uninfected or were infected intraperitoneally with lachyzoites of T.gondii(strains RH and CN).All groups were then treated with different sulfonamides and the optimal treatment protocol was determined candidates.Sulfadiazine-sodium(SD)was used for comparison.Results:The oplimal therapy involved gavaging mice twice per day with 250 mg/kg bw of sulfachloropyrazine-sodium(SPZ)for five days.Using this protocol,the average survival time and the time-point of 50%fatalities were prolonged significantly compared with SD treatment.Treatment with SFZ protected 40%of mice from death,and the heart and kidney tissue of these animals was parasite-free,as determined by nestedPCK.SPZ showed excellent therapeutic effects in the treatment of T.gondii in an acute murine model and is therefore a promising drug candidate for the treatment and prevention of T.gondii in animals.Conclusions:It can be concluded that the effective drug sulfachloropyrazine may be the new therapeutic options against animal toxoplasmosis.

Comparison of the Effects of Three Different Anti-fungus Drugs on Candida Albicans of Murine Vaginal Mucosa

To compare the therapeutic effects of three different anti-fungal drugs （i.e., terbinafine, fluconazole and intraconazole） in the treatment of experimental vaginitis caused by Candida albicans （C. albicans） in mice, the fungal vaginitis model was established in female ICR mice by intravaginal inoculation of suspension of C. albicans after the animal had been pretreated with estradiol. Mice were divided at random into different groups and then respectively treated with terbinafine, fluconazole and intraconazole given by gastrogavage. The burden of the fungus in the vaginal lavage fluids in the mice of the different groups was measured dynamically at different time points after the beginning of the drug treatment. The fungal burdens in the vaginal lavage fluids taken at different time points from the mice treated with terbinafine were significantly higher than those taken at corresponding time points from mice treated with fluconazole or itraconazole （P〈0.01）. The fungal burdens in the vaginal lavage fluids taken from mice 1 week after the beginning of the treatment with terbinafine remained at a relatively high level. A dramatic drop in the fungal burden was noted in the vaginal lavage fluids taken on the 2nd day of the treatment from mice treated with itraconazole or fluconazole group and the fungal burden on the 3rd day of the treatment in these mice were at a very low level, suggesting that fluconazole or itraconazole were highly effective for the treatment. However, the difference in the therapeutic effect between the two drugs was not significant （P〉0.05）. Itraconazole or fluconazole, but not terbinafine, is very effective for the treatment of fungal vaginitis caused by C. albicans in mice.

A ModelSystem of Prim ary Murine Hepatocytes Infected byMurine Cytom egalovirus

In order to establish a model system of the murine hepatocyte infection by murine cytomegalovirus (MCMV), the primary cultured murine hepatocytes were obtained in a modified low serum medium system by a non perfusion method, and then infected by Smith strain MCMV. Infected hepatocytes showed characteristic cytopathic effect (CPE) at 30 h after infection, in which a large number of viral particles was found and ultrastructures were destroyed (as revealed by disappearance of bile canalicula and organelles) under the electron microscope and MCMV immediate early genes were detected by in situ hybridization. Meanwhile, infected cells produced albumin significantly less than corresponding uninfected controls. On the contrary, uninfected controls simultaneously cultured under the same conditions showed normal function and ultratructure (glycogen rosettes, bile canalicula, wheel like mitochondria and well developed rough and smooth endoplasmic reticula). These results demonstrated that a model system of primary cultured murine hepatocytes infected by MCMV was successfully set up.

Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B

AIM: To evaluate the expression of fibrinogenlike protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinaseactivity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury.RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice.In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2)was detected in 21 of 23 patients (91.30%)with severe AOC hepatitis B, while only 1 of 13 patients(7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1of 10 with mild chronic hepatitis B had detectable hfgl2expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B.CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2protein play a pivotal role in initiating severe hepatitis.The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.

Local Th1/Th2 Cytokine Expression in Experimental Murine Vaginal Candidiasis

In order to investigate the expression of Th1 and Th2 cytokines in the vaginal candidiasis caused by Candida, the fungal vaginitis model was established in female ICR mice by intravaginal inoculation of suspension of C. albicans after the animals were pretreated with estradiol. Reverse transcriptase-polymerase chain reaction （RT-PCR） was used to detect the expression of IL-2, IL-4, IL-10 and TGF-β1 in the vagina in the mice of different groups at different time points after the beginning of the experiment. The average expression level of IL-2 mRNA in group D （estrogen-treated mice） was significantly higher than that in groups H （estrogen-untreated mice） and I （control group） on the day 2. The average expression level of IL-4 mRNA in group D was significantly higher than that in groups I and H on the day 5. The average expression level of IL-10 mRNA in group D was significantly higher than that in groups H and I from day 7 to 11. The average expression level of TGF-β1 mRNA in group D was significantly higher than that in groups H and I at all time points. It was concludes that the high-level expression of IL-2 mRNA during early infection was associated with clearance of mucosal C. albicans, and the high-level expression of IL-10 mRNA during late stage of the infection was related to susceptibility to infection. TGF-β1 may play a predominant role when the virtual absence of changes in other Th-type cytokines during infection.

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.

N-acetylcysteine and glycyrrhizin combination:Benefit outcome in a murine model of acetaminophen-induced liver failure

BACKGROUND Acetaminophen overdose is the most frequent cause of drug-induced liver failure in developed countries.Substantial progress has been made in understanding the mechanism of hepatocellular injury,but N-acetylcysteine remains the only effective treatment despite its short therapeutic window.Thus,other hepatoprotective drugs are needed for the delayed treatment of acetaminopheninduced hepatotoxicity.Our interest focused on glycyrrhizin for its role as an inhibitor of high mobility group box 1(HMGB1)protein,a member of the family of damage-associated molecular pattern,known to play an important pathological role in various diseases.AIM To investigate the efficacy of the N-acetylcysteine/glycyrrhizin combination compared to N-acetylcysteine alone in the prevention of liver toxicity.METHODS Eight-week-old C57BL/6J wild-type female mice were used for all our experiments.Mice fasted for 15 h were treated with acetaminophen(500 mg/kg)or vehicle(phosphate-buffered saline)by intraperitoneal injection and separated into the following groups:Glycyrrhizin(200 mg/kg);N-acetylcysteine(150 mg/kg);and N-acetylcysteine/glycyrrhizin.In all groups,mice were sacrificed 12 h following acetaminophen administration.The assessment of hepatotoxicity was performed by measuring plasma levels of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase.Hepatotoxicity was also evaluated by histological examination of hematoxylin and eosin-stained tissues sections.Survival rates were compared between various groups using Kaplan-Meier curves.RESULTS Consistent with data published in the literature,we confirmed that intraperitoneal administration of acetaminophen(500 mg/kg)in mice induced severe liver injury as evidenced by increases in alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase but also by liver necrosis score.Glycyrrhizin administration was shown to reduce the release of HMGB1 and significantly decreased the severity of liver injury.Thus,the co-administration of glycyrrhizin and N-acetylcysteine was investigated.Administered concomitantly with acetaminophen,the combination significantly reduced the severity of liver injury.Delayed administration of the combination of drugs,2 h or 6 h after acetaminophen,also induced a significant decrease in hepatocyte necrosis compared to mice treated with N-acetylcysteine alone.In addition,administration of N-acetylcysteine/glycyrrhizin combination was associated with an improved survival rate compared to mice treated with only N-acetylcysteine.CONCLUSION We demonstrate that,compared to N-acetylcysteine alone,co-administration of glycyrrhizin decreases the liver necrosis score and improves survival in a murine model of acetaminophen-induced liver injury.Our study opens a potential new therapeutic pathway in the prevention of acetaminophen hepatotoxicity.

Therapeutic effect of intravesical or intratumoral injection of oncolysate transfected with recombinant vaccinia virus encoding human IL-2 gene on murine melanoma model

Oncolysate, a debris of tumor cells, has been provento be effective in tumor active immunotherapy, it wasreported that the vaccinia virus, especially recombinantvaccinia viruses encoding human IL-2 (rVV-IL-2 ),enhanced the immunogenicity of transfected tumor cells.In this experiment, the murine melanoma cell B16-F10oncolysates trans fected by rVV-IL-2 (IL-2VBO) wereused as vaccine. The IL-2VBO or TK-VBO was preparedby incubating B16-F10 cells with rVV-IL-2 or rVV-TK