Abstract:Objective　To establish and characterize GSK-3β knockout thymic epithelial cells in miceand evaluate their immunological characteristics. Methods　The Cre-LoxP system was used for the F0 generation of GSK-3β^{flox/ flox}(GSK-3β^{f/ f} ) mice with targeted gene knockout in embryonic stem cells. PCR was used to analyze GSK-3β^{f/ f} FoxN1-Cre^{+/ -}(GSK-3β^{-/ -} ) mice with specific knockout of the GSK-3β gene in thymus epithelial cells. The general biological characteristics, reproductive ability, and progeny survival rate of gene knockout mice were observed. HE staining, flow cytometry, and immunofluorescence were used to compare the immune organ structure, thymus, spleen, and proportion and proliferation of immune cells in peripheral blood between knockout and wildtype mice. Results　Mice with specific knockout of the GSK-3β gene in thymus epithelial cells were established successfully. Compared with wildtype mice, GSK-3β^{-/ -} mice showed no significant difference in general biological characteristics, and the survival rate of offspring was > 90%. In the aging process, the thymus of knockout mice was larger than that of wildtype mice with a higher thymus index and more thymic cells. The numbers of initial T cells and recently migrated cells in the spleen were significantly higher than those in wildtype mice. Conclusions　Mice with specific knockout of the GSK-3β gene in thymic epithelial cells was established successfully. These mice provide a good tool to study the function and mechanism of the GSK-3β gene in thymus epithelial cells.

Abstract:Objective 　By combining a high fat diet with balloon injury to the left carotid artery, a carotid atherosclerosis (CAS) model in Tibetan miniature pigs was established, and hematobiochemical, imaging, and pathological features of the CAS model were analyzed. Methods　Ten male Tibetan miniature pigs were randomly divided into two groups, one of which received a normal diet, whereas the other had an balloon injury to the left carotid artery after 2 weeks while being fed a high fat diet. The modeling period was 12 weeks. Body weight and blood biochemical indexes were measured before high fat feeding (-2 weeks) and balloon injury (0 weeks) and after 2, 6, and 10 weeks. Ultrasound, Doppler flow imaging, and angiography were performed at 10 weeks after the operation. To investigate morphology, inflammation, fibrosis, and lipid deposition, and lesion characteristics of Tibetan mini-pigs, the left carotid artery of the animals was stained with HE, Masson, and oil red O after euthanasia. Results　Numerous blood biochemical parameters in the CAS model were similar to the clinical parameters. Imaging findings revealed significant narrowing and increased resistance of the left carotid artery in the CAS model. Resultsing in reduced blood flow velocity. Pathological observations revealed significant inflammatory cell infiltration, collagen fiber proliferation, vessel wall thickening, and lipid deposition, which were similar to clinical manifestations. Conclusions　The CAS model in Tibet miniature pig is a an ideal model to investigate CAS because it is sensitive, stable, and resembles clinical aspects.

Abstract:Objective　A visual metastasis model of hepatoma cells and cancer-associated fibroblasts (CAFs) was established using zebrafish to provide a preclinical model for CAFs-mediated mechanism research and therapeutic efficacy evaluation of patients with tumor metastasis. Methods 　After establishing a co-culture system, the proliferation and migration of Huh7 cells were assessed by CCK8 and Transwell assays, respectively. Huh7 cells and CAFs labeled with different fluorescent dyes were injected into the perivitelline space of zebrafish to establish a visual metastasis model. The effects of CAFs on Huh7 cell migration and Huh7 cell binding to CAFs in blood vessels were observed. Results　The proliferation and migration of Huh7 cells co-cultured with CAFs were increased significantly. A visual metastasis model of hepatoma cells and CAFs in zebrafish was established successfully. The migration and binding rate of Huh7 cells co-injected with CAFs were increased significantly. Conclusions　CAFs promote migration of hepatoma cells, and most hepatoma cells are tightly bound to CAFs in distal metastases. Therefore, fibroblasts of solid tumors can be targeted to block their support of tumors and provide a new strategy for cancer therapy.

Abstract:Objective　To explore and evaluate a mouse model of dry eye with liver meridian stagnation heat. Methods　Thirty C57 mice were randomly divided into blank, model, and Danzhi groups (n = 10 in each group). The blank group did not receive any treatment, and the other groups were treated with a benzalkonium chloride solution as eye drops in a drying oven for 28 days combined with chronic pain stimulation (tail clamping) to establish a model of liver meridian stagnation heat syndrome in dry eyes. After modeling was complete, the Danzhi group were administered a modified formula of Danzhi Xiaoyao powder (12. 48 g/ (kg·d)) by gavage, and blank and model groups were administered deionized water (1 mL/100 (g·d)) by gavage for 14 days. Body weight, anal temperature, tear secretion, tear film rupture time, and corneal fluorescein staining were evaluated. The elevated cross maze test and open field test were used to assess the behavioral changes. Enzyme-linked immunosorbent assays and immunohistochemical staining were used to assess tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β). Results　Compared with the blank group, the model group exhibited high tension, anxiety, decreased body weight, and increased body temperature (P< 0. 01). Horizontal and vertical scores in the open field test were increased (P< 0. 01). OE% and OT% values in the elevated cross maze test were decreased (P< 0. 01). Tear film rupture time and tear secretion were decreased (P< 0. 01). The cornea was seriously damaged, and proinflammatory factors was increased (P< 0. 01). After treatment with modified Danzhi Xiaoyao powder, all indicators were significantly improved (P< 0. 05 or P< 0. 01). Conclusions　On the basis of measuring syndrome by prescription, a mouse model of dry eye with liver meridian stagnation heat syndrome was successfully established, which may facilitate systematic and in-depth study of treating dry eye with a prescription.

Abstract:Objective　To investigate the effects of fenofibrate and simvastatin on liver lipid accumulation and mitochondrial function-related signaling pathways in non-alcoholic fatty liver disease (NAFLD) induced by a high fat diet. Methods　Forty male 6-week-old C57BL/6J mice were randomly and equally assigned into four groups: Control group (normal diet), High fat diet group (14 weeks of a high fat diet), Fenofibrate group (14 weeks of a high fat diet and administered 0. 04 g/ (kg·d) fenofibrate by gavage at 11 weeks for 4 weeks), and Simvastatin group (14 weeks of a high fat diet and administered 0. 004 g/ (kg·d) simvastatin by gavage at 11 weeks for 4 weeks). This study verified the role of mitochondrial functions in the pathogenesis of NAFLD by changes in body weight, liver weight, liver index, serum biochemical indexes, pathological changes of the liver, oxidative stress indicators, mitochondrial function indexes, and related signaling pathways. It also explored the effects of fenofibrate and simvastatin on mitochondrial functions in NAFLD model mice. Results 　There were significant differences between NAFLD model and control groups in terms of body weight, liver weight, liver index, serum biochemical indexes, oxidative stress indicators, pathological changes of the liver, and mitochondrial function indexes ( P< 0. 05). Fenofibrate and simvastatin improved these objective indicators. Conclusions　Fenofibrate and simvastatin upregulated the PPARα/ PGC-1α signaling pathway related to mitochondrial functions, promoted mitochondrial β oxidation, and reduced oxidative stress damage and lipid accumulation in the liver.

Abstract:Objective　To investigate the effect of aerobic and resistance exercise on immune functions in SAMP8 mice. Methods　Twenty-eight-week-old male SAMP8 mice were divided into a model group, aerobic exercise group and resistance exercise group, with eight mice in each group. Another eight SAMR1 mice of the same age were used as the control group. The aerobic exercise group received 8 weeks of uniform horizontal exercise running platform training, and the resistance exercise group received 8 weeks of increasing weight climbing exercise training. The relative grip strength and rotarod test were performed every 2 weeks. The index of immune organs was measured. The histomorphology of immune organs was observed, and the serum IL2 concentration was measured by an ELISA. Immunohistochemistry was used to detect expression of IL2Rα protein in the spleen. RT-qPCR was used to detect gene expression of IL2, IL2Rα, IL2Rβ, and IL2Rγ in the spleen. Results　Compared with the control group, the relative grip strength and rotarod test time of model group were decreased significantly (P< 0. 0001). Thymus and spleen indexes were decreased significantly (P< 0. 001). Aging of immune organs was observed. The serum IL2 concentration was decreased significantly (P< 0. 0001). The positive rate of IL2Rα protein in the spleen was decreased significantly (P< 0. 001). Gene expression of IL2, IL2Rα, IL2Rβ and IL2Rγ in the spleen was decreased significantly (P< 0. 01). After 8 weeks of aerobic and resistance exercise, compared with model group, the relative grasping strength and the rotarod test time of aerobic exercise and resistance exercise groups were increased significantly (P< 0. 05). Compared with aerobic exercise group, the relative grasping strength of resistance exercise group was increased significantly (P< 0. 05). Thymus and spleen indexes of aerobic exercise and resistance exercise groups were increased significantly (P< 0. 05). Delays in aging of immune organs were observed. The serum IL2 concentration in aerobic exercise and resistance exercise groups was increased significantly (P<0. 01). The positive rate IL2Rα protein in the spleen in aerobic exercise and resistance exercise groups was increased significantly (P< 0. 05). Gene expression of IL2, IL2Rα, IL2Rβ, and IL2Rγ the in spleen in aerobic exercise and resistance exercise groups was increased significantly (P< 0. 05). Conclusions　Aerobic and resistance exercise delays age-related decline of immune functions in SAMP8 mice, and the expression of IL2/ IL2R upregulated by exercise may be its target.

Abstract:Objective　To investigate modeling drugs in animal models of chronic bronchitis (CB) and provide a reference for experimental method and model refinement of this animal model. Methods　Using chronic bronchitis and animal models as subject terms, literature related to animal models of chronic bronchitis from January 2000 to January 2022 was collected from the China Knowledge Network, Wanfang database, and PubMed database. The model animal species, modeling method , test indexes, positive control drugs, administration time, and evidence of modelling in the literature were collated and summarized to establish a database for statistical analysis. Results　We screened out 243 articles that met the criteria. SD rats (126, 51. 85%) were the most common animal species, followed by Wistar rats (70, 28. 81%) and Kunming mice (30, 12. 35%). The gender of the animals was mostly male (120, 49. 38%). The modeling method was mostly smoking (111, 46. 91%), followed by intratracheal injection of lipopolysaccharide (LPS) combined with smoking (44, 18. 11%). LPS was mostly injected into the trachea (43, 17. 70%), and the modeling cycle was mostly 20 ~ 30 days (146, 60. 08%). The commonly used test indicators were bronchopulmonary histopathology (143, 20. 82%), serum (99, 14. 41%), bronchoalveolar lavage fluid (82, 11. 93%), and general condition (66, 9. 61%). Among the 131 articles with positive control drugs, the most used was Guilong Cough and Panning Capsules (30, 21. 58%), followed by dexamethasone acetate (28, 20. 14%). Animal models of Chinese medicine syndrome had 10 pulmonary qi deficiency syndrome and one pulmonary yin deficiency syndrome. Conclusions　The current CB animal models are mostly established by a single-factor fumigation method and LPS intratracheal injection combined with fumigation. The model animals are SD/Wistar rats or Kunming mice, and the modeling period is mostly within 20 ~ 30 days, which effectively improves the experimental efficiency. The evaluation criteria of the model are mostly combined with bronchopulmonary histopathology, general condition, serum, alveolar lavage fluid, and other indicators for comprehensive judgment. We should focus on establishment of animal models for CB by developing unified and standardized modeling criteria and relevant model evaluation indicators to establish more reasonable animal models of CB.

Abstract:Objective　Establish immortalized brain microvascular endothelial cells from tree shrews, clarify the effect of D-galactose on senescence of brain microvascular endothelial cells, and explore the potential mechanisms. Methods BMECs were transfected with a lentivirus carrying the SV40T gene, and immortalized cells were characterized by morphological observation, growth curve determination, immunofluorescence, and karyotype after expansion for > 50 passages. Next, BMECs were treated with various concentrations of D-gal, and the optimal concentration was determined by cell proliferation assays. Senescence was analyzed by staining with senescence-associated β-galactosidase. Expression of senescence-associated p53 was measured by Western Blot. Intracellular oxidative stress levels were measured by superoxide dismutase and lipid oxidation assays. Results　Immortalized cells grew with consistent morphology throughout passages. Single cells were shaped as short spindles or polygonal with a typical paving stone shape. The cell growth curve showed cells were in the logarithmic growth phase on days 2 ~ 5. Cell growth peaked at day 6 and then the cells grew slowly to a plateau. High levels of tree shrew vWF, CD31, and SV40T were detected by immunofluorescence. Karyotyping showed that the number of chromosomes in immortalized cells matched the number of chromosomes in tree shrew (Tupaia belangeri chinensises). D-galactose reduced BMEC proliferation in time- and dose-dependent manners. In the experimental group, i.e. BMECs treated by D-gal at a concentration of 10 g/ L for 48 h, positive β-galactosidase staining was significantly increased, p53 was significantly increased, intracellular SOD activity was reduced, and MDA content was increased. Conclusions　A tree shrew immortalized brain microvascular endothelial cell line was established successfully. The optimal concentration to establish an immortalized BMEC senescence model was 10 g/ L D-gal. The mechanism by which D-gal promotes cellular senescence in vitro may be mediated through promotion of oxidative stress and inhibition of cell proliferation.

Abstract:Objective　To establish a real-time monitoring mouse model for primary liver cancer (HCC) research. Methods　We applied an approach for stable gene expression in mouse hepatocytes by hydrodynamic injection in combination with PiggyBac transposon system-mediated somatic gene integration. We fused an oncogene with a luciferase reporter gene for live imaging of mouse hepatocytes. Results 　Mouse hepatocytes integrated with a YAP1 mutant gene (YAP5SA) were used to form liver tumors with characteristics similar to the Hippo signaling pathway-inactivated HCC subclass. Live imaging of the liver showed strong a fluorescence signal from hepatocytes integrated with the oncogene and luciferase. The peak fluorescence signal decreased rapidly in the early stage after hydrodynamic injection because of hepatocyte apoptosis. The fluorescence signal was significantly increased after tumor initiation and progression. We applied this real-time monitoring model to confirm that PTEN was a HCC tumor suppressor gene by an additionally fused oncogene with shRNA expression elements. We also validated small molecular targeted drug sorafenib by oral administration. Conclusions　A real-time monitoring mouse HCC model was successfully established. This method can be applied to molecular characterization of HCC subtypes and targeted drug screening.

Abstract:Objective　The advantages and disadvantages of 12 and 24 h intervals of observation to evaluate the degree of estrous cycle disturbance were compared, and the significance of interphase sampling to evaluate a Tripterygium wilfordii-induced premature ovarian failure model in rat was explored. Methods　Sixty female SD rats with a regular estrous cycle were randomly divided into blank groups 1 and 2, and Tripterygium wilfordii groups 1 and 2 (n= 15). The blank groups were gavaged with normal saline, and the TG groups were gavaged with a Tripterygium wilfordii polyside tablet (TG) suspension for 15 days. Blank group 1 and TG group 1 were observed for vaginal exfoliation cytology at 8:00 every day, and blank group 2 and TG group 2 were observed for vaginal exfoliation cytology at an interval of 12 h. Rats were weighed weekly. Rats with a regular estrous cycle were sacrificed on days 15 ~ 17 after treatment. Rats with an irregular estrous cycle were sacrificed on day 15 after treatment. Serum sex hormones were measured, ovarian and uterine indexes were calculated, and pathological changes of the ovary were observed. Results　Compared with the black group, the degree of estrous cycle disorder in the TG group was increased. The number of normal estrous cycles was significantly decreased, the estrous period was significantly shortened, and the diestrus period was significantly prolonged when observed at an interval of 24 h. The difference between the two groups was better observed at 8:00 than at 20:00, and there was a significant difference in the level of estrous cycle disturbance (I and IV) between the two groups when observed at an interval of 12 h. Rats sacrificed in diestrus showed significantly decreased serum INHB and E2 in the TG group, while FSH was significantly increased. Rats sacrificed in any subcycle stage showed no significant differences between the groups. Because the serum AMH level fluctuated independently of the estrous cycle, the serum AMH level of rats in the TG group sacrificed in diestrus or any subcycle stage was significantly decreased. Conclusions　Tripterygium wilfordii causes significant ovarian function decline, follicle development disorder, and ovarian reserve decline in rats. Cytological analysis of vaginal exudation at an interval of 12 h facilitates capturing cytological changes of the subcycle stage and reflects real changes of the estrous cycle. Disturbance of the estrous cycle in the model group observed at an interval of 12 h was better reflected than that observed at an interval of 24 h, but its feasibility needs to be further explored. Rats were sacrificed in diestrus, which reflected the basic sex hormone level and was beneficial to evaluate the success rate of model establishment of premature ovarian failure induced by Tripterygium wilfordii. Comparison of the ovarian pathological changes between the two groups in the subcycle stage objective ly and faithfully reflected the adverse effects of Tripterygium wilfordii on follicle development and luteal function.

Abstract:Objective　To explore the effect of colchicine on atherosclerotic unstable plaque formation in high fat diet-fed New Zealand rabbits and the underlying mechanism. Methods　A total of 32 male New Zealand rabbits at 3 months of age were randomly assigned into a control group (n= 8), model group (n= 8), colchicine group (n= 8), and pitavastatin group (n= 8). Serum blood lipids, high sensitivity C-reactive protein (hsCRP), and interleukin-6 (IL-6) were measured at 8 and 12 weeks postoperatively. Rabbits were sacrificed at 12 weeks postoperatively, and blood vessels were collected to prepare paraffin-embedded sections, followed by observation of blood vessel morphology by HE staining. AMP-activated protein kinase ( AMPK), sirtuin 1 ( SIRT1), and peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) mRNA levels in unstable plaques were measured by qRT-PCR. p-AMPK, SIRT1, and PGC-1α protein levels were measured by Western Blot and immunohistochemistry. Malondialdehyde ( MDA) and superoxide dismutase (SOD) activities in unstable plaques were determined by chromogenic reaction. Intravenous ultrasound was performed to visualize plaque formation. Results　Relative levels of blood lipids and inflammatory indicators in model, colchicine, and pitavastatin groups at 12 weeks postoperatively were significantly higher than those in the control group (P< 0. 05). Triglycerides, low-density lipoprotein-cholesterol, hsCRP, and IL-6 levels in the colchicine group were significantly lower than those in the model group (P< 0. 05). Neointimal hyperplasia was observed in model, colchicine, and pitavastatin groups, and typical atherosclerotic plaque formation was seen in the model group. Upregulation of p-AMPK, SIRT1, and PGC-1α was detected in rabbit plaques and hyperplastic intima, which was most pronounced in the colchicine group (P< 0. 05). Intravenous ultrasound scans showed moderate-severe lumen stenosis, plaque rupture, and subintimal hemorrhage in the model group. Moderate lumen stenosis was seen in colchicine and pitavastatin groups without plaque rupture. Decreased MDA content and increased SOD activity were found in the colchicine group compared with model and pitavastatin groups. Conclusions　Colchicine delays atherosclerotic plaque formation in high fat diet-fed New Zealand rabbits by reducing lipid accumulation in plaques, AMPK-induced activation of deacetylase, upregulation of SIRT1 and PGC-1α, and inhibition of oxidative stress and inflammatory responses by enhancing the release threshold of proinflammatory factors in plaques.

Abstract:Establishing an appropriate xenograft model is critical for cancer research. To date, the most commonly used xenograft model is the cancer cell line-based xenograft (CDX) model, in which tumor cells subcultured in vitro are transplanted into immunodeficient mice to form xenografts. Although this model is simple to establish and has a short modeling period, it cannot fully represent clinical cancer patients. Therefore, the patient-derived tumor xenograft (PDX) model that stably retains tumor heterogeneity has started to replace the CDX model in many applications. The PDX model develops tumors by directly implanting patient tissue or primary cells into immunodeficient mice in a manner that preserves the histopathology, molecular features, and drug responsiveness of the parental tumor. It can be used as a preclinical model and has significant advantages in drug screening, biomarker development, and combined clinical trials. In this review, we introduce the establishment method and application of the PDX model in detail, and summarize the problems that may be encountered in the process of model establishment and preclinical research.

Abstract:Alcohol use disorder (AUD) is a neuropsychiatric disorder associated with behavior, psychosociology, and neurobiology. AUD is mainly diagnosed by judgment of behavioral criteria. The animal model of AUD is the basis of research. However, because of the complexity of AUD diagnosis, it is too difficult to establish an animal model that includes all characteristics of AUD. This review summarizes the relevant literature and divides the AUD model into forced administration and self-administration models. The advantages and disadvantages of alcohol-only drinking, alcohol gavage, an alcohol liquid diet, chronic intermittent ethanol exposure, two-bottle choice, and alcohol self-administration included in both models are described and compared. Additionally, the modeling method were explored by face, predictive, and construct validities to provide a theoretical basis and reference for researchers to choose AUD models.

Abstract:Dengue virus (DENV) infection is the most widespread viral disease transmitted by insect vectors worldwide. Mild and severe clinical dengue disease processes can occur after human infection. DENV escapes recognition and attack by the immune system, mainly by resisting transduction of the human interferon (IFN) signaling. Moreover, mice are naturally resistant to DENV infection. Therefore, researchers have been attempting to induce DENV infection by establishing a mouse model devoid of IFN signaling pathway-related molecules. However, it remains difficult to accurately replicate the clinical characteristics of DENV infection. This review summarizes the immune escape mechanism of dengue virus by IFN signaling pathways. This article also analyzes the applications of the mouse model devoid of such pathways to uncover the pathogenic mechanisms of dengue virus and develop effective vaccines and drugs. In conclusion , we in propose that a better mouse model that closely simulates the processes of different diseases after clinical DENV infection may be established by modifying the present models in humans.

Abstract:Medical functional experiments are important in the medical experimental curriculum system, and experimental animals are essential research objects. This article discusses the phenomenon that animal experiments are partially inconsistent with experimental animal ethics on the basis of the connotation and meaning of experimental animal ethics education. This review explores the necessity, path, and measures of integrating medical functional experiments into animal ethics education to organically combine the legal and standardized management of experimental animals with knowledge and moral education, guide students to abide by laws and technical standards, treat patients with respect to life, and care for them.

Abstract:Chemotherapy-induced peripheral neuropathy pain ( CIPNP) seriously affects quality of life and treatment of cancer patients, but its mechanism is unknown and there is no effective therapy. Animal models are important to study CIPNP and play an extremely important role in the study of pathogenesis and treatments. This article reviews the research status of CIPNP animal models in the previous 5 years, analyzes the advantages and disadvantages of several common modeling method , and introduces its pathogenesis. This review aims to provide a reference for researchers to choose more effective animal experimental models and new ideas for treatment.

Abstract:The incidence and mortality of lung cancer are high. It is very important to establish an appropriate animal model of lung cancer to conduct reductive studies on its pathogenesis, mechanism, and drug effects. Animal models of transplanted lung cancer are widely used because of the various transplantation method and simple operation. This article reviews orthotopic and heterotopic transplantation in animal models of transplanted lung cancer from three aspects, the transplantation host, source of the graft, and method of establishment, to provide ideas to establish and select lung cancer models.