Abstract: Objective To establish tumor necrosis factor receptor superfamily member 4 (TNFRSF4) knockout and humanized mice for TNFRSF4-targeted cancer therapeutic antibodies development, and to study the function of TNFRSF4 in the immune response. Methods The TNFRSF4 knockout and humanized mice were established with the CRISPR/ Cas9 method. The mice were characterized by PCR, RT-PCR, HE staining and fluorescence-activated cell sorting. Results We successfully generated mice expressing the human TNFRSF4 gene, as well as TNFRSF4 gene knockout mice. TNFRSF4 humanized mice expressed human TNFRSF4, but not murine TNFRSF4. No TNFRSF4 expression was detected in TNFRSF4 knockout mice. TNFRSF4 humanized mice and knockout mice survived normally within 6 months after birth, and there were no obvious abnormalities in histopathology and immune system. Conclusions TNFRSF4 humanized mice and knockout mice produced using CRISPR/ Cas9 technology can be used as animal models for screening and evaluating of TNFRSF4-targeted cancer therapeutic antibodies or for studying the role of TNFRSF4 in immune processes.

Abstract: Objective Dysfunction of the cholinergic system is an important pathogenetic feature of Alzheimer’s disease (AD). However, the efficacy of cholinomimetic agents in treating AD is low, which may be related to the complexity of the affected pathways. In this study, we established an animal model of oxytocin receptor (OXTR) gene silencing in the nucleus basalis of Meynert (NBM) in the basal forebrain, and we investigated the effects of silencing on learning and memory behaviors and on the cholinergic pathway. Methods Recombinant adeno-associated virus ( rAAV) containing short hairpin RNA (shRNA) to the OXTR gene (rAAV9-EGFP-Oxtr-shRNA (OXTR-shRNA)) was injected at different dosages into the NBM region of the basal forebrain. Then, 2, 3, 4 and 5 weeks later, EGFP expression was measured to assess the rate of infection. An optimal dose of OXTR-shRNA and infection duration were selected for subsequent experiments. Rats were randomly divided into Sham, negative control shRNA (Con-shRNA) and OXTR-shRNA groups. Morris water maze and light-dark box were used to assess spatial learning and fear memory, respectively. To evaluate the effect of silencing on OXTR protein expression, immunofluorescence staining was used to detect the protein in the NBM region. Acetylcholine (Ach) level and the expression of choline acetyltransferase (ChAT) protein in the cortex were assessed by ELISA and immunofluorescence staining, respectively. Results The effects of OXTR-shRNA infection showed dose-dependency and time-dependency. In the water maze test, escape latency was longer in the OXTR-shRNA group than in the Sham and Con-shRNA groups. During the consolidation stage of the light-dark box test, the latency to enter the dark chamber was shorter in the OXTR-shRNA group than in the Sham and Con-shRNA groups. Immunofluorescence staining showed that OXTR expression was decreased in the NBM in the OXTR-shRNA group. Ach levels and ChAT expression in the cortex were reduced in the OXTR-shRNA group compared with the two other groups. Conclusions An animal model of OXTR gene silencing in the basal forebrain was successfully established in this study. This animal model has phenotypes of learning and memory impairment and cholinergic system damage, and therefore can be used to study diseases associated with cognitive dysfunction.

Abstract: Objective To investigate the protective effect and the underlying mechanisms of action of sheep bone collagen peptide chelated calcium (CPCC) on the kidney of estrogen-deficient rats. Methods Rats were subjected to bilateral ovariectomy. The CPCC dosages for the high-dose (CPCC-H) and low-dose (CPCC-L) groups were 5 g / (kg·d) and 1 g / ( kg·d), respectively. In the model and sham groups, the same volume of distilled water was intragastrically administered every day. After 8 weeks, the kidney antioxidant indexes were measured, and the morphological changes in kidney tissues were observed. qRT-PCR was used to measure the mRNA expression of JAK/ STAT signaling pathway-related genes. Results Compared with the sham group, the kidney T-SOD, GSH-Px and T-AOC activities in the model group were decreased significantly, and the MDA content was increased significantly ( P< 0. 01). Furthermore, normal renal tissue structure was perturbed and lymphocyte infiltration was observed. Levels of the serum inflammatory factors IL-2, TNF-α and IFN-γ were increased significantly (P< 0. 01). qRT-PCR analysis showed that the mRNA expression levels of the JAK2, STAT1 and STAT3 genes were increased significantly (P< 0.01). After CPCC treatment, compared with the model group, T-SOD, GSH-Px and T-AOC levels increased significantly ( P< 0. 01), and MDA content decreased significantly (P< 0.01). Renal tissue lesions were significantly reduced, and the number of lymphocytes was decreased. Furthermore, serum levels of various inflammatory factors were decreased significantly (P< 0.01), and mRNA expression levels of JAK2, STAT1 and STAT3 genes were decreased significantly (P< 0.01). Conclusions CPCC inhibits oxidative stress in ovariectomized rats and may ameliorate renal tissue pathology through the JAK/ STAT signaling pathway.

Abstract: Objective The purpose of this study was to investigate the effect of royal jelly ( RJ) on serum hormone levels and mammary gland tissues of female non-pregnant rats (late puberty to early sexual maturity). Methods Fifty Wistar rats were randomly divided into 5 groups with 10 rats in each group. Rats in the treatment group were given RJ at different doses (100, 200, 400, 800 mg / (kg·d)), while rats in the control group (CK group) were given sterile water ( 2 mL). After 35 d, the growth index and nipple diameter were measured. The serum levels of estrogen ( E2), progesterone ( P) and prolactin ( PRL) were measured by ELISA. After hematoxylin-eosin staining of breast tissue, histological morphological changes were observed. Results The result showed that serum E2 and PRL levels in the RJ200 group and RJ400 group were significantly higher than those in the CK group (P< 0. 05), while serum P levels in the RJ200, RJ400 and RJ800 groups were significantly lower than those in the CK group (P< 0. 05). Serum E2, P and PRL levels in the RJ100 and CK groups were not significantly different (P< 0. 05). Breast duct diameter measurements showed that compared with the CK group, duct diameter in each of the treatment groups was significantly increased (P< 0. 05). The histological changes in the breast were consistent with duct diameter in the RJ100 group being lower than that in the RJ200, RJ400 and RJ800 groups. In addition, the diameter of the nipple in the RJ200 and RJ800 groups was slightly increased compared with the CK group (P< 0. 05). Conclusions Daily intake of low-dose (100 mg / kg) RJ did not affect serum hormone levels or breast development in female non-pregnant rats. However, at higher doses of 200 and 400 mg / (kg ·d), it may adversely affect the development of breast tissue and the stability of serum hormone levels in female non- pregnant rats.

Abstract: Objective To study the proteomic changes in the liver of aging female mice, to find the core age- related differential proteins, and to explore the aging mechanism based on proteomics, with the aim of providing molecular targets for the study of the mechanisms of aging. Methods In this study, differential proteins between 12- and 3-month-old female mice were analyzed using relative and absolute quantitative isotopic labeling (iTRAQ), LC-MS and bioinformatics. Results A total of 369 different proteins were identified by comparing the two groups, among which 182 were upregulated and 187 were downregulated. After screening, 13 age-related differential proteins in the core DEP were obtained. Conclusions The exploration and in-depth study of differential proteins will help elucidate the mechanisms of aging and identify potential markers, providing new molecular targets for the prevention of aging and the diagnosis and treatment of aging-related diseases.

Abstract: Objective To investigate the effect of sheep bone collagen peptide ( SBCP) on the immune function of macrophages with or without stimulation with lipopolysaccharide (LPS). Methods Rat peritoneal macrophages were treated with different doses of SBCP, with or without stimulation with LPS. The metabolic activity and phagocytic ability of macrophages were measured by MTT assay and the neutral red method , respectively. The production of NO was measured with the Griess reaction, and TNF-α and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of key genes in the Toll-like receptor pathway and the negative regulatory factor Single Ig IL-1-related receptor were measured by qRT-PCR. Results SBCP treatment of normal macrophages increased their metabolic activity, phagocytosis, NO, TNF-α and IL-6 production, and upregulated mRNA expression of key genes in the TLR pathway and downregulated SIGIRR mRNA expression in a dose-dependent manner. At a concentration of 10⁵ μg / mL, there was no significant difference between the SBCP and LPS group. SBCP treatment of LPS-stimulated macrophages inhibited their metabolic activity, with 10² μg / mL having the greatest effect, while 10³ μg / mL most strongly inhibited phagocytosis and the secretion of NO, TNF-α and IL-6. This concentration also downregulated the mRNA expression of TLR-related genes and upregulated the mRNA expression of SIGIRR. Conclusions SBCP has a dual regulatory effect on the inflammatory response through the TLR signaling pathway. At a concentration of 10 ~ 10⁴ μg / mL, it enhances the immune activity of macrophages, while at 10⁵ μg / mL, it induces macrophages to release various inflammatory mediators. However, SBCP inhibits the inflammatory reaction induced by LPS.

Abstract: Objective The maturation of mammalian epididymal sperm and the acquisition and maintenance of motility are prerequisites for normal sperm function and complete fertilization; however, the mechanism that regulates this process is still unclear. SRC kinase is involved in the regulation of mouse sperm capacitation, and the serine / threonine phosphatase PP1γ2 / PP2A is the key enzyme that regulates epididymal sperm maturation and motility. However, it is unclear whether these molecules interact with each other and whether this interaction regulates sperm motility ( including motility acquisition). In this study, we investigated the role of SRC kinase and phosphatase PP1γ2 / PP2A in Kunming mouse sperm and their regulation of sperm functions. Methods Using western blot, enzyme activity assay and co- immunoprecipitation assay, we examined threonine phosphorylation level as well as enzyme activity of SRC kinase and phosphatase PP1γ2 / PP2A in Kunming mouse sperm from caput and cauda epididymis. In addition, we investigated the interaction of SRC kinase and phosphatase PP1γ2 / PP2A.We also investigated the effects of a SRC inhibitor (SU6656) and an activator (sc-3052) on phosphatase activity and motility of sperm from cauda and caput epididymis. The phosphorylation level of threonine in sperm of cauda epididymis was higher than that in caput epididymis. SRC kinase activity in sperm from caput epididymis was lower than that in epididymal cauda sperm. The phosphatase activity of sperm from caput epididymis was significantly higher than that from cauda epididymis (P< 0. 05). SRC kinase in mouse epididymal sperm modulates phosphatase PP1γ2 or PP2A, which in turn influences sperm motility. Results In epididymal cauda sperm, where SRC is more active, when SRC activity is inhibited by SU6656, the activity of phosphatase PP1γ2 / PP2A is increased, while sperm motility is decreased. In epididymal caput sperm where SRC is less active, when SRC activity is enhanced by sc- 3052, the PP1γ2 / PP2A phosphatase activity is reduced, while sperm motility is increased. Conclusions The activity of SRC and phosphatase PP1γ2 / PP2A in caput sperm from mouse epididymis is significantly different from that in cauda sperm. SRC kinase interacts with phosphatase PP1γ2 or PP2A in mouse sperm. SRC kinase modulates sperm motility (including motility acquisition) by inhibiting the activity of phosphatase PP1γ2 / PP2A.

Abstract: Objective To establish a mouse model of liver cancer by injecting mutant p53 gene CRISPR/ Cas9 plasmid and H22 cells into the tail vein. Methods Healthy male BALB/ c mice were randomly divided into blank group (normal saline), plasmid group ( Plasmid), control group ( H22 cells + saline) and experimental group ( H22 cells + Plasmid), each with 25 mice. Tail vein injection was used to establish the liver cancer model. Samples were taken at 2, 3, 4 and 5 weeks after the injection, and blood was collected from the orbit to detect serum aminotransferase (ALT/ AST). Liver and lung surface changes and the number of nodules were observed, and successful modeling rate, mortality and organ index in each group were assessed. HE staining was used to observe the pathological and morphological changes in the liver. Immunohistochemistry, western blot and other method were used to evaluate the expression of p53 and PCNA protein in the liver. Results Both the experimental group and the control group showed successful generation of a mouse model of metastatic liver cancer. The rates of successful modeling in the experimental and control groups were 60. 87% and 45. 83%, respectively, and the mortality rate was 4. 35% and 29. 17%, respectively. The serum ALT and AST levels in the two groups of mice increased significantly, and cysts and white granular nodules of varying sizes appeared on the liver surface over time. The HE result showed that the liver lobules in the experimental and control groups were damaged to varying degrees after 5 weeks, showing obvious tumor-related pathological changes. The expression of p53 protein in the liver of the plasmid and experimental groups was significantly reduced (P< 0. 05). The expression of PCNA protein in the liver tissue of the experimental and control groups was increased significantly, and expression was significantly higher in the experimental group compared with the control group (P< 0. 05). Conclusions The method of injecting mutant p53 gene CRISPR/ Cas9 plasmid and H22 cells through the tail vein can successfully and rapidly construct a mouse model of metastatic liver cancer.

Abstract: Objective To investigate the antitumor activity of Fomes Officinalis polysaccharide (FOPS) and the underlying mechanisms. Methods The mice tumor-bearing model was established by subcutaneous injection of S180 ascites tumor cells into the axilla. Mice were randomly divided into control group, model group, CTX group, FOPS-L group, FOPS-M group and FOPS-H group, with 10 mice in each group. The mice were treated with NS, cyclophosphamide or FOPS for 15 d. Tumor inhibition rate and organ index were assessed, and white blood cell ( WBC) and lymphocyte (LYM) were measured. Serum TNF-α, IFN-γ, IL-2, IL-1β and IL-6 were detected by ELISA. The mRNA expression levels of p38MAPK and p-c-jun in tumor tissues were detected by qRT-PCR. The protein expression levels of p38MAPK, p-c-jun, NF-κB and p-NF-κB were detected by Western Blot. Results (1) The mean tumor weights in the CTX and three FOPS groups were significantly decreased compared with the model group (P< 0. 05). The tumor inhibition rate in the CTX and three FOPS groups (-L, -M, -H) were 84. 87%, 54. 29%, 40. 57% and 30. 84%, respectively. (2) Compared with the model group, the spleen index, thymus index, WBC and LYM were increased significantly in the FOPS-L groups (P< 0. 05). (3) Compared with the model group, the levels of TNF-α, IFN-γ, IL-2 in the FOPS-L group were significantly increased (P< 0. 05), while the levels of IL-1β and IL-6 in the CTX and three FOPS groups were significantly decreased (P< 0. 01). (4) The mRNA expression levels of p38MAPK and p-c-jun, and the protein expression levels of p38MAPK, p-c-jun and p-NF-κB in tumor tissue were significantly increased compared with the model group (P< 0.05). Conclusions FOPS has significant anti-tumor activities, and the mechanism of action is related to activation of the MAPK/ NF-κB signaling pathway.

Abstract: Objective Verruca is a dermatological disease caused by human papillomavirus (HPV), and includes conditions such as verruca vulgaris and condyloma acuminatum. Although treatments are available to remove skin warts, effective therapies are still lacking. Because of species specificity, the development of animal models of HPV infection has been limited. In this study, mouse papillomavirus (MmuPV1) was used to infect immunodeficient mice to establish a murine model of skin papillomatous hyperplasia. Methods After anesthesia, the tail skin was gently scraped with a blade to produce a slight wound, which was then inoculated with 1. 5 × 10⁸ copies of viral DNA. Changes in the tail skin were observed, viral DNA and load were examined, and pathological analysis was performed. Results Sixteen weeks post-infection with MmuPV1, papillomatous hyperplasia with hyperkeratosis had developed at the infection sites. HE staining showed hyperplasia of squamous epithelium and vacuolar degeneration in the spinous and granular layers. MmuPV1 E4 transcript was detected by RNAscope in situ hybridization. MmuPV1 DNA in the samples was detected by PCR amplification of the E2 gene. Viral DNA was tested in all MmuPV1-infected NU/ NU mice samples by qRT-PCR. Conclusions Our result suggest that MmuPV1 skin infection in mice mimics HPV disease, and can therefore serve as a useful model for studying the pathogenesis and natural history of HPV infection.

Abstract: Objective Hand, foot and mouth disease ( HFMD) mainly affects children under the age of 5. Because HFMD is caused by a group of enteroviruses, a vaccine targeting only one pathogen would be greatly limited, and a multivalent vaccine would be more effective. In this study, we prepared a trivalent VP1 protein vaccine against EV71, CA16 and CA10 viruses. This vaccine was then injected into mice to examine immune activities and to evaluate safety. Methods After immunization with the trivalent VP1 protein vaccine, virus-specific antibodies and their neutralizing activities were analyzed, and virus-specific T cell immune responses and changes in cytokines were assessed. Furthermore, passive immunoprotective effects passed from immunized mothers to their neonatal offspring were evaluated with in vivo infection experiments. Results The trivalent VP1 protein vaccine continuously induced virus-specific IgGs and IgMs, which had the ability to neutralize the viruses. In addition to inducing virus-specific humoral immunity, the trivalent VP1 protein vaccine activated the virus-specific T cell response as well. The vaccine also provided passive immunity to neonatal mice against EV71 virus infection. Additionally, the stable levels of inflammatory cytokines during the immune response indicated that the vaccine was safe. Conclusions The trivalent VP1 protein vaccine was effective against EV71, CA16 and CA10 viral challenge, and could therefore be useful for the development of multivalent HFMD vaccines.

Abstract: Objective To establish an animal model of type 2 diabetes with deficiency of Qi and Yin, and its evaluation index. Methods Forty-eight Sprague Dawley rats were randomly divided into blank, disease and syndrome model, and high sugar and fat groups. The blank group was fed an ordinary diet, whereas the other two groups were fed a high fat diet. After 4 weeks of feeding, rats in the disease model group were fasted for 12 h and then injected with 40 mg / kg streptozotocin intraperitoneally. After 1 week, the fasting blood glucose level of the rats was measured, and a fasting blood glucose level of >11. 1 mmol / L was considered to be successful modeling of diabetes. The rats were continued to be fed until the end of the experiment at 14 weeks. Changes in rat physical signs and various laboratory indexes were monitored. Results Compared with those of rats in the blank group, the mental and motor scores of rats in the disease and syndrome model group were increased significantly at weeks 8 ~ 14 (P< 0. 01). The weight of rats was decreased significantly from week 4 (P< 0. 05 or P< 0. 01), food intake and water intake were increased significantly from week 8 (P< 0. 05 or P< 0. 01), and grip strength was significantly decreased from week 12 (P< 0. 01). At weeks 6~ 14, both random and fasting blood glucose were increased significantly, and urine sugar was increased significantly (P< 0. 01). The respiratory rate, pulse amplitude, and tongue values, PT, APTT, thymus index, and CD4 and CD4 / CD8 values were reduced significantly (P< 0. 05 or P< 0. 01). TC, TG, LDL-C, cAMP, and cAMP / cGMP values were increased significantly (P< 0. 05 or P< 0. 01). Pathological result showed that islet tissue was seriously damaged, and its function was reduced significantly. Conclusions Rats fed with high glucose and fat for 4 weeks were intraperitoneally injected with 40 mg / kg streptozotocin and further fed with high glucose and fat for 14 weeks to prepare an animal model of type 2 diabetes combined with Qi and Yin deficiency. Food intake, water intake, movement score, mental state score, grasping ability, respiration, pulse, tongue image and other information were collected. PT, APTT, TC, TG, LDL-C, cAMP, cAMP / cGMP, CD4, CD4 / CD8 and thymus index were detected. Combined with pathological changes of pancreatic tissue, the syndrome characteristics of this model could be well reflected.

Abstract:Castration-resistant prostate cancer (CRPC) has high morbidity and mortality, and the prognosis for patients is poor. Establishing patient-derived xenograft models with CRPC characteristics using clinical surgical specimens is critical for CRPC research. In this article, we review the main phenotypic characteristics of a variety of CRPC patient- derived xenograft models, including hormone-independent phenotypes, androgen receptor-related phenotypes and neuroendocrine phenotypes, as well as pathophysiological characteristics, and we discuss potential experimental tools for studying the mechanisms of CRPC and for targeted drug screening for the disease.

Abstract:Pulmonary arterial hypertension (PAH) is a serious disease with poor prognosis, and its pathogenesis is still unclear. Current treatment method cannot cure the disease, and can only slow down its progression. Animal models are important tools for the study of PAH, and they play an extremely important role in the study of the pathophysiological mechanisms of the disease and in the evaluation of prevention and treatment strategies. In this review, we discuss the hemodynamic changes and pulmonary artery histological remodeling characteristics of classic PAH animal models (hypoxia and monocrotaline models) and PAH double-clip animal models, with the aim of providing a reference for the selection of animal models for the study of novel mechanisms and new targets for the disease.

Abstract:Heterogeneous transformation is not only the major clinical feature of prostate cancer, but also the main factor affecting therapeutic outcome. It is of great importance for the study of prostate cancer to establish patient-derived tumor xenograft ( PDX) transformation models. In this paper, we discuss the clinical heterogeneity of prostate cancer, review research progress in PDX models simulating clinical features, and focus on the urgently needed heterogeneous transformation model. We describe the ideal animal model needed for the study of prostate cancer transformation mechanisms and targeted drug screening.

Abstract:In the study of human diseases, animal models play a critical role in medical research. The immunological and physiological similarities between non-human primates and humans have prompted non-human primate models to be used to study virus pathogenesis, immunity, and the efficacy of vaccines and drugs. They play an indispensable role in the study of human viral infectious diseases, including emerging new viruses, such as SARS-CoV-2. In this article, we focus on the research progress in non-human primates as experimental animal models of several important human viral infectious diseases, including the important role of non-human primates in the study of SARS-CoV-2.

Abstract:Zika virus is an arbovirus of the Flaviviridae family that is transmitted by the bites of Aedes aegypti and Aedes albopictus, by blood and tears, as well as by sexual and vertical mother-to-child transmission. Once a pregnant woman is infected, there is a risk of infection to the fetus. Adults may have some mild clinical symptoms, but fetuses are more vulnerable to central nervous system involvement, which can lead to microcephaly, severe brain defects, and neuroinflammation. Therefore, it is extremely urgent to block the spread and infection of Zika virus. To better understand the mother-to-child transmission route of Zika virus, to study the pathogenic mechanisms, and to discover effective prevention, detection and treatment method , the establishment of reliable animal models is crucial. In this paper, we review the recent progress in mother-to-child transmission of Zika virus in various animal models.

Abstract:Osteoarthritis is a chronic degenerative disease characterized by cartilage degeneration and subchondral bone changes, and its prevalence has increased rapidly along with the aging of the population. There is still no drug or surgical treatment that completely resolves osteoarthritis. With the advent of cell therapy, the unique advantages of stem cells have been gradually uncovered, and their use in the treatment of osteoarthritis is increasingly studied. In this article, we review the mechanisms by which stem cells repair osteoarthritis. We describe the properties and function of mesenchymal stem cells and their therapeutic mechanisms of action in osteoarthritis in animal models and in the clinic setting.

Abstract:Neurodegenerative diseases are irreversible diseases caused by the gradual loss of neuronal structure or function, leading to cognitive or motor disorders. The pathological changes mainly include senile plaques in the central nervous system, neurofibrillary tangles and neuronal loss. In neurodegenerative diseases, olfactory dysfunction usually occurs earlier than classic motor and cognitive disorders. Using olfactory dysfunction as a clinical sign of neurodegenerative disease can help us to detect such diseases in the early stage. Investigation of the neuropathogenesis of the disease as well as screening and evaluation of therapeutic drugs require appropriate animal models. Therefore, in this paper, we review the main animal models of olfactory dysfunction in different neurodegenerative diseases.