Objective This study aimed to investigate the tumorigenic properties of MMTV-PyMT breast cancer transgenic mice at different ages(in weeks)and the changes in the composition of immune cells in the tumor microenvironment.Methods Eight groups of 4,6,8,10,12,14,16 and 18 weeks of age MMTV-PyMT female mice(FVB mice as the background)and one group of 8 weeks of FVB female mice were prepared for routine blood testing,the pathological changes of the mammary gland and lung metastases were observed by histopathological sections,and the immune cells in blood,spleen,and tumor were analyzed by flow cytometry.Results MMTV-PyMT mice showed adenular ductal lesions at 4～6 weeks of age;the ductal portion expanded to the growth boundary at 8～9 weeks of age,and then gradually broke through the glandular boundary to form early breast cancer at 8～12 weeks of age,and advanced breast cancer at 10～14 weeks of age.At 12 weeks of age,metastases were visible in the lungs of some mice,and at 14 weeks of age,the number of metastases in the lungs increased significantly.As the age of the mice increased,the number of white blood cells,neutrophils,and platelets in their blood increased gradually,while the lymphocytes and erythrocytes showed a gradual downward trend.Flow cytometry showed that with the increase in age,the proportion of T cells in the spleen and tumor gradually decreased,the MDSCs in the blood,spleen,and tumor gradually increased,and the NK cells in the tumor also gradually increased.Conclusions This study analyzed routine blood tests,pathology,and immune cells in the tissues of MMTV-PyMT mouse models of different weeks of age,providing a novel perspective on the dynamic alterations of the tumor immune microenvironment during the malignant progression of breast cancer.

Objective This study aimed to investigate the tumorigenic properties of MMTV-PyMT breast cancer transgenic mice at different ages(in weeks)and the changes in the composition of immune cells in the tumor microenvironment.Methods Eight groups of 4,6,8,10,12,14,16 and 18 weeks of age MMTV-PyMT female mice(FVB mice as the background)and one group of 8 weeks of FVB female mice were prepared for routine blood testing,the pathological changes of the mammary gland and lung metastases were observed by histopathological sections,and the immune cells in blood,spleen,and tumor were analyzed by flow cytometry.Results MMTV-PyMT mice showed adenular ductal lesions at 4～6 weeks of age;the ductal portion expanded to the growth boundary at 8～9 weeks of age,and then gradually broke through the glandular boundary to form early breast cancer at 8～12 weeks of age,and advanced breast cancer at 10～14 weeks of age.At 12 weeks of age,metastases were visible in the lungs of some mice,and at 14 weeks of age,the number of metastases in the lungs increased significantly.As the age of the mice increased,the number of white blood cells,neutrophils,and platelets in their blood increased gradually,while the lymphocytes and erythrocytes showed a gradual downward trend.Flow cytometry showed that with the increase in age,the proportion of T cells in the spleen and tumor gradually decreased,the MDSCs in the blood,spleen,and tumor gradually increased,and the NK cells in the tumor also gradually increased.Conclusions This study analyzed routine blood tests,pathology,and immune cells in the tissues of MMTV-PyMT mouse models of different weeks of age,providing a novel perspective on the dynamic alterations of the tumor immune microenvironment during the malignant progression of breast cancer.

Objective To investigate whether a stable and reliable hyperuricemia model can be established in mice with an ICR background via a triple-modeling method(combined potassium oxazine,hypoxanthine,and 30％yeast paste),and to evaluate the effect of the positive drug febuxostat on the model.Methods A hyperuricemia model of ICR mice was established using a single drug or double-or triple-drug combinations.Serum uric acid and creatinine concentrations,xanthine oxidase(XOD)and urate oxidase(UOX)activity,and uric acid transporter(URAT)1,glucose transporter(Glut)9,anion transporter(OAT)1,and ATP-binding box subfamily G member(ABCG)2 mRNA levels were detected to evaluate whether the hyperuricemia model was formed successfully.Results The serum uric acid levels of ICR mice were not significantly changed by potassium oxazine alone,as they showed an increase but were not significantly different to those of the 30％yeast paste diet or hypoxanthine combined groups.Serum uric acid levels in the triple administration group were significantly increased at 7 days(P＜0.01),while XOD enzyme activity had increased(P＜0.01)and UOX enzyme activity decreased(P＜0.001)at the same timepoint.There were increased expression levels of URAT1 and Glut9(P＜0.05,P＜0.001),and decreased expression levels of OAT1 and ABCG2(P＜0.001).During dynamic monitoring,the blood uric acid levels of triple administration-induced ICR mice peaked at 7 days.In addition,triple administration-induced hyperuricemia in ICR mice was sensitive to the positive drug febuxostat,which caused a significant decrease in blood uric acid levels(P＜0.001).Conclusions A hyperuricemia model in ICR mice can be stably induced by triple administration for 7 days.

Objective To investigate the differences in susceptibility to Lewis lung carcinoma and T lymphocyte subsets in the immune microenvironment between young and elderly mice.Methods Six C57/B6 mice at two months(young)and six mice at twelve months(aged)were injected with Lewis lung carcinoma cells at the dose of 1 × 106 in the left armpit to establish a murine model of lung carcinoma.The weight and tumor growth were monitored.Blood samples for routine blood tests were collected after 24 days.The proportions of CD4+ and CD8+T cells in the spleen were detected by flow cytometry,and the infiltration of CD4+,CD8+ T cells and related effector T cells in the tumor microenvironment were determined in the same way.Results The body weight of tumor bearing mice in the aged group was significantly higher than that in the young group(P ＜0.001);The tumor weight in the aged group(5.084±0.528)g was significantly higher than that in the young group(2.963 ±0.378)g(t =3,349,P =0.012);Routine blood tests showed that the numbers of leukocytes and subsets(except mononuclear)in the aged group were significantly lower than in the young group(P ＜0.05);Flow cytometry found that the effector and memory/effector CD4+T cell ratios in the spleen were significantly higher in the aged group than in the young group(P ＜0.001)and the expression of effector and memory/effector CD8+T cells in the tumor microenvironment was also significantly higher than in the young group(P ＜0.05);Quantitative expression values of IL-6 and IL-10 in the tumor microenvironment were 25090±3820 and 10670± 1793 in the aged group and 6252±864 and 3061±451 in the young group,respectively.Moreover,the expression levels of IL-6 and IL-10(t =3.925,P =0.01;t =3.552,P =0.02)in the tumor microenvironment in the aged group were significantly lower than those in the young group.Conclusions Young mice are more susceptible to Lewis lung carcinoma,probably as a result of differences in inflammation and immunity.

Objective To study the application of implantable telemetry and whole-body plethysmography to observe the changes of circadian rhythm in conscious rats and evaluate the pharmacological safety of doxofylline, and to provide a basis for the future application of this technological system for drug safety evaluation.Methods Eight healthy SPF Sprague-Dawley rats were used in this study, 4 males and 4 females.The rats were implanted with telemetry transmitters by surgery to establish a telemetry system combined with plethysmography to observe the changes of 24 h physiological parameters and circadian rhythm in conscious rats at 14 d after operation, including heart rate (HR), blood pressure, the time interval from the Q wave to point A in the ECG of the aortic pressure wave (QA interval), respiration, activity, body temperature and pulmonary function parameters.The rats were divided into 3 groups: normal control group, doxofylline 40 mg/kg and 80 mg/kg groups, and the performance was validated by aerosolizing saline, doxofylline 40 mg/kg and 80 mg/kg inhalation, respectively, to observe the changes in physiological parameters after the drug administration.Results The physiological parameters of rats showed obvious changes in circadian rhythms at 14 d after operation.Compared with the normal control group, the doxofylline 40 mg/kg-treated group showed significantly increased changes of HR, tidal volume (TV), minute ventilation (MV), 50% expiratory flow (EF50), peak inspiratory flow (PIF) and peak expiratory flow (PEF) (P<0.01), significantly decreased respiratory frequency, QA interval and enhance pause (Penh) (P<0.05, P<0.01), but no significant differences in the blood pressure, activity and body temperature (P>0.05).Compared with the normal control group, the group treated with doxofylline 80 mg/kg had significantly increased HR, blood pressure, TV, MV, EF50, PIF and PEF (P<0.01), significantly decreased respiratory frequency, QA interval and Penh not (P<0.01), but not significantly changed activity and body temperature (P>0.05).Conclusions The application of implantable telemetry and whole-body plethysmography in this study does not obviously affect the circadian rhythm, and can sensitively monitor the relevant cardiovascular and respiratory parameters in conscious rats.It can be used in drug safety pharmacological research of cardiovascular and respiratory systems in conscious rats.

Objective To investigate the relatio nship between the DNA methylation status of gluco-corticoid receptor (GR) gene promoter and mRNA expression level of GRα gene of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Methods Fifteen new onset SLE patients and fifteen healthy controls were enrolled in this study. The DNA methylation status of GR gene promoter 1 of PBMCs was detected through bisulfite sequencing polymerase chain reaction (PCR). The mRNA expression of GRα, DNA methyltransferases, growth arrest and DNA damage-induced 45α (GADD45α) of PBMCs was detected using the quantitative real-time polymerase chain reaction method. T-test and χ2-test were used to detect the differences between the two groups, Pearson's correlation coefficient was used to analyze the linear correlation between two variables. Results Compared with healthy controls, the mRNA expression of GRα was signi-ficantly declined in SLE patients (10±5, 17±7, t=2.69, P<0.05), and the mRNA expression of DNMT1 and GADD45α was significantly elevated in SLE patients (t=3.11, P<0.05 and t=2.98, P<0.05). The overall mean methylation status of the 142 CpG islands of the four promoters was significantly elevated in SLE patients [(16±8)%vs (11±6)%, t=2.75, P<0.05]. The global methylation status of PBMCs in SLE patients was obviously lower than healthy controls (t=4.39, P<0.05). Conclusion Hypermethylation of GRα promoter may result in GRαgene low expression in PBMCs of patients with SLE.

Objective To observe the changes of mRNA expression related to kidney yang deficiency and the ul-trastructure of hypothalamus in Lewis rats, and further study the constitution of kidney yang deficiency in Lewis rats.Meth-ods Ten 7-8-week old SPF male Lewis rats and twenty 7-8-week old SPF male Wistar rats were used in this study.The rats received subcutaneous injection of hydrocortisone to establish kidney yang deficiency model.The expressions of TNF-α, IFN-γ, IL-10, CRH, MR and GR mRNA in the hypothalamus of the two groups were detected and the ultrastructural changes of hypothalamus and adrenal gland were observed to compare the differences between the two groups, and to ex-plore the mechanism of kidney yang deficiency constitution in the Lewis rats.Results Compared with the normal Wistar rats, the expressions of GR and IL-2 mRNA in the hypothalamus were significantly increased ( P<0.01 ) , while the ex-pressions of CRH, TNF-α, IFN-γand IL-10 mRNA were significantly decreased in the Lewis rats (P<0.01).The zona fasciculata of the adrenal cortex was slightly thinner, the mitochondria were slightly swollen and the amount of mitochondria was decreased.The nuclei of hypothalamic neurons were larger, and the mitochondria, neuron synapses and secretory vesi-cles in the presynaptic neurons were decreased.Conclusions There are abnormal expression of immune-related cytokines and CRH mRNA, and ultrastructural changes in the hypothalamus and adrenal gland of Lewis rats with kidney yang defi-ciency.Such changes may be related to the functional imbalance of the hypothalamus-pituitury-adrenal axis.

OBJECTIVETo observe the effects ofGynura segetum in rats with hepatic veno-occlusive disease (HVOD).

METHODSSixty Sprague-Dawley rats were assigned to a blank control group, one of three Gynura segetum treatment groups (low-dose group, 5.0 g/kg; mid-dose group, 10 g/kg; high-dose group, 20 g/kg), or a pseudo-drug group (10 g/kg of pseudo-ginseng). After 28 days of treatment, effects on white blood cell count, coagulation, secreted factors from vascular endothelium, and histopathology of the spleen were observed and inter-group differences were statistically assessed.

RESULTSAfter the 4-week administration, all rats in the Gynura segetum treatment groups showed decrease in body weight, increases in numbers of leukocytes, neutrophils, lymphocytes, monocytes and eosinophils.decreases in platelets and platelet hematocrit, and increases in mean platelet volume and platelet distribution.In addition, the Gynura segetum treatments increased the prothrombin time, activated partial thromboplastin time, thrombin time, prothrombin ratio and international normalized ratio, but decreased the PT%, fibrinogen level and platelet aggregation.Serum levels of endothelin and nitric oxide were also elevated by the Gynura segetum treatments.All measured parameters showed significant differences from the control group (P less than 0.01 or less than 0.05).Finally, the splenic follicles were significantly reduced and the spleens showed an absence of germinal centers along with a large number of diffuse lymphocytes and reduced red pulp sinusoids.

CONCLUSIONThe Gynura segetum treatment has some toxic effects; it can reduce platelet count and platelet hematocrit, inhibit blood clotting time and platelet aggregation, increase the secretion of factors from the vascular endothelium and disrupt spleen histology.

Animals ; Asteraceae ; Blood Coagulation ; Blood Platelets ; Disease Models, Animal ; Endothelium, Vascular ; Hepatic Veno-Occlusive Disease ; Platelet Count ; Prothrombin Time ; Rats ; Rats, Sprague-Dawley

Objective To observation dynamic changes and gender difference of heart rate variability and blood pressure variability in unbonded spontaneously hypertensive rats using Telemetry technology .Method Taking sixteen spontaneously hypertensive rats of 3-month-old in SPF grade, there were implanted TL11M2-C50-PXT device intraperitoneally.Seven days later, respectively monitoring chainless blood pressure and electrocardiogram for 24 h continuously time using telemetry system when 3, 5, 7 months of age in spontaneously hypertensive rats .Analysing 24 h systolic pressure , mean blood pressure , diastolic blood pressure , heart rate variability and blood pressure variability . Result Systolic pressure , diastolic pressure and mean pressure , blood pressure variability increased with age of the moon increased in spontaneously hypertensive rats , moreover female SHR rats of blood pressure variability was significantly lower than that of the male rats .Correlated indexes of heart rate variability had no obvious change Between three to seven month -old in male spontaneously hypertensive rats .But RR interval , SDNN, TP, VLF and HF increased significantly with age of the moon increased in female spontaneously hypertensive rats , moreover the female SHR rats of TP , VLF and HF index of heart rate variability were lower than the male rats .Conclusion With the SHR rats age and blood pressure , blood pressure variability increased , and the female SHR rat variability in blood pressure and heart rate was significantly lower than that of the male .

ObjectiveToinvestigatethekidneyyangdeficiencyconstitutionandhypothalamus-pituitary-adrenal ( HPA) axis dysfunction in Lewis rats .Method Two kidney yang deficiency models were established by subcutaneous injection of hydrocortisone and by adrenalectomy to induce hypothalamic -pituitary-adrenal ( HPA) axis dysfunction in Lewis rats.Wistar rats were used as control and compared with the two types of Lewis rat models of kidney yang deficiency .The degree of kidney yang deficiency of the Lewis rats was evaluated by examination and detection of the general signs , behavior , and neuroendocrine function , and so on .Result Compared with the normal Wistar rats , the body weight of Lewis rats was significantly higher than that of Wistar rats at the same age .The body temperature , urine volume and grip strength were significantly lower than those of Wistar rats ( P<0.01 for all ) .The memory ability of the Lewis rats was slightly decreased.The liver, kidney and adrenal indexes were significantly decreased in the Lewis rats (P<0.01).The levels of serum ACTH, CRH, cGMP, Cort and urine 17OHCS were significantly decreased (P<0.01).Conclusion A very slight deficiency of adrenal cortex function of Lewis rats is caused by genetic inheritance , and Lewis rats have the constitution of congenital kidney yang deficiency ( criticality or prophase of kidney yang deficiency ) .