Experimental Animals最新文献

Genome editing technology is widely used in the field of laboratory animal science for the production of genetic disease models and the analysis of gene function. One of the major technical problems in genome editing is the low efficiency of precise knock-in by homologous recombination compared to simple knockout via non-homologous end joining. Many studies have focused on this issue, and various solutions have been proposed; however, they have yet to be fully resolved. In this study, we established a system that can easily determine the genotype at the mouse (Mus musculus) Tyr gene locus for genome editing both in vitro and in vivo. In this genome editing system, by designing the Cas9 cleavage site and donor template, wild-type, knockout, and knock-in genotypes can be distinguished by restriction fragment length polymorphisms of PCR products. Moreover, the introduction of the H420R mutation in tyrosinase allows the determination of knock-in mice with specific coat color patterns. Using this system, we evaluated the effects of small-molecule compounds on the efficiency of genome editing in mouse embryos. Consequently, we successfully identified a small-molecule compound that improves knock-in efficiency in genome editing in mouse embryos. Thus, this genome editing system is suitable for screening compounds that can improve knock-in efficiency.

The imbalance of bone resorption and bone formation causes osteoporosis (OP), a common skeletal disorder. Decreased osteogenic activity was found in the bone marrow cultures from N-acetylglucosaminyl transferase V (MGAT5)-deficient mice. We hypothesized that MGAT5 was associated with osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and involved in the pathological mechanisms of osteoporosis. To test this hypothesis, the mRNA and protein expression levels of MGAT5 were determined in bone tissues of ovariectomized (OVX) mice, a well-established OP model, and the role of MGAT5 in osteogenic activity was investigated in murine BMSCs. As expected, being accompanied by the loss of bone mass density and osteogenic markers (runt-related transcription factor 2, osteocalcin and osterix), a reduced expression of MGAT5 in vertebrae and femur tissues were found in OP mice. In vitro, knockdown of Mgat5 inhibited the osteogenic differentiation potential of BMSCs, as evidenced by the decreased expressions of osteogenic markers and less alkaline phosphatase and alizarin red S staining. Mechanically, knockdown of Mgat5 suppressed the nuclear translocation of β-catenin, thereby downregulating the expressions of downstream genes c-myc and axis inhibition protein 2, which were also associated with osteogenic differentiation. In addition, Mgat5 knockdown inhibited bone morphogenetic protein (BMP)/transforming growth factor (TGF)-β signaling pathway. In conclusion, MGAT5 may modulate the osteogenic differentiation of BMSCs via the β-catenin, BMP type 2 (BMP2) and TGF-β signals and involved in the process of OP.

Cerebral ischemia reperfusion (IR) injury as found in stroke is a complex and heterogeneous disorder and closely related to disability and death. Today, nutraceuticals and protective therapy to increase neuronal integrity and prevent pathological complication are common. We investigated the neuroprotective effect of betanin against cerebral IR injury in mice. Forty male institute of cancer research (ICR) mice were divided into Sham-veh, IR-veh, IR-Bet50 and IR-Bet100 groups. After 2 weeks of oral administration of normal saline (vehicle; veh) or 50 mg/kg or 100 mg/kg of betanin (Bet), mice were subjected to IR induction using 30-min bilateral common carotid artery occlusion, followed by 24 h of reperfusion. Brain infarction, oxidative status, cortical and hippocampal neurons and white matter pathologies were evaluated. Results showed that IR significantly increases brain infarction, Cornus Ammonis 1 (CA1) hippocampal and corpus callosum (CC) and internal capsule (IC) white matter degeneration (P<0.05). Brain oxidative status revealed significant elevation of malondialdehyde (MDA) together with a significant decrease in catalase (CAT) activity, induced by IR (P<0.05). Pretreatment with betanin 100 mg/kg led to a significant reduction in brain infarction and MDA, CA1 hippocampus, CC and IC white matter degeneration. Betanin also led to a significant increase in CAT activity (P<0.05), with enhancing effect on reduced glutathione levels (GSH, P<0.05). The present study revealed the neuroprotective efficacy of betanin against IR injury in mice's brains, including its inhibition of lipid peroxidation, and boosting of GSH and CAT activity.

Intracerebral hemorrhage (ICH) is an incurable neurological disease. Microglia activation and its related inflammation contribute to ICH-associated brain damage. FERM domain containing kindlin 1 (FERMT1) is an integrin-binding protein that participates in microglia-associated inflammation, but its role in ICH is unclear. An ICH model was constructed by injecting 50 µl of autologous blood into the bregma of rats. FERMT1 siRNA was injected into the right ventricle of the rat for knockdown of FERMT1. A significant striatal hematoma was observed in ICH rats. FERMT1 knockdown reduced the water content of brain tissue, alleviated brain hematoma and improved behavioral function in ICH rats. FERMT1 knockdown reduced microglia activity, inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activity and decreased the expression of inflammatory factors including IL-1β and IL-18 in the peri-hematoma tissues. BV2 microglial cells were transfected with FERMT1 siRNA and incubated with 60 µM Hemin for 24 h. Activation of NLRP3 inflammasome induced by hemin were reduced in microglia when FERMT1 was knocked down, leading to decreased production of inflammatory factors IL-1β and IL-18. In addition, knockdown of FERMT1 prevented the activation of nuclear factor kappa B (NF-κB) signaling pathway in vivo and in vitro. Our findings suggested that down-regulation of FERMT1 attenuated microglial inflammation and brain damage induced by ICH via NLRP3/NF-κB pathway. FERMT1 is a key regulator of inflammatory damage in rats after ICH.

Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by cognitive dysfunction and neuropsychiatric symptoms, is the most prevalent form of dementia among the elderly. Amyloid aggregation, tau hyperphosphorylation, and neural cell loss are the main pathological features. Various hypotheses have been proposed to explain the development of AD. Some therapeutic agents have shown clinical benefits in patients with AD; however, many of these agents have failed. The degree of neural cell loss is associated with the severity of AD. Adult neurogenesis, which governs cognitive and emotional behaviors, occurs in the hippocampus, and some research groups have reported that neural cell transplantation into the hippocampus improves cognitive dysfunction in AD model mice. Based on these clinical findings, stem cell therapy for patients with AD has recently attracted attention. This review provides past and present therapeutic strategies for the management and treatment of AD.

Humanized mice are widely used to study the human immune system in vivo and investigate therapeutic targets for various human diseases. Immunodeficient NOD/Shi-scid-IL2rγ^null (NOG) mice transferred with human hematopoietic stem cells are a useful model for studying human immune systems and analyzing engrafted human immune cells. The gut microbiota plays a significant role in the development and function of immune cells and the maintenance of immune homeostasis; however, there is currently no available animal model that has been reconstituted with human gut microbiota and immune systems in vivo. In this study, we established a new model of CD34⁺ cell-transferred humanized germ-free NOG mice using an aseptic method. Flow cytometric analysis revealed that the germ-free humanized mice exhibited a lower level of human CD3⁺ T cells than the SPF humanized mice. Additionally, we found that the human CD3⁺ T cells slightly increased after transplanting human gut microbiota into the germ-free humanized mice, suggesting that the human microbiota supports T cell proliferation or maintenance in humanized mice colonized by the gut microbiota. Consequently, the dual-humanized mice may be useful for investigating the physiological role of the gut microbiota in human immunity in vivo and for application as a new humanized mouse model in cancer immunology.

Pancreatic fibrosis (PF) is a hallmark of chronic pancreatitis (CP), but its molecular mechanism remains unclear. This study was conducted to explore the role of Kruppel-like factor 4 (KLF4) in PF in CP mice. The CP mouse model was established using caerulein. After KLF4 interference, pathological changes in pancreatic tissues and fibrosis degree were observed by hematoxylin-eosin staining and Masson staining, and levels of Collagen I, Collagen III, and alpha-smooth muscle actin, inflammatory cytokines, KLF4, signal transducer and activator of transcription 5A (STAT5) in pancreatic tissues were measured by enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blot assay, and immunofluorescence. The enrichment of KLF4 on the STAT5 promoter and the binding of KLF4 to the STAT5 promoter were analyzed. The rescue experiments were performed by co-injection of sh-STAT5 and sh-KLF4 to confirm the regulatory mechanism of KLF4. KLF4 was upregulated in CP mice. Inhibition of KLF4 effectively attenuated pancreatic inflammation and PF in mice. KLF4 was enriched on the STAT5 promoter and enhanced the transcriptional and protein levels of STAT5. Overexpression of STAT5 reversed the inhibitory role of silencing KLF4 in PF. In summary, KLF4 promoted the transcription and expression of STAT5, which further facilitated PF in CP mice.

Diabetic retinopathy (DR), a common complication of diabetes, involves excessive proliferation and inflammation of Muller cells and ultimately leads to vision loss and blindness. SRY-box transcription factor 9 (SOX9) has been reported to be highly expressed in Müller cells in light-induced retinal damage rats, but the functional role of SOX9 in DR remains unclear. To explore this issue, the DR rat model was successfully constructed via injection with streptozotocin (65 mg/kg) and the retinal thicknesses and blood glucose levels were evaluated. Müller cells were treated with 25 mmol/l glucose to create a cell model in vitro. The results indicated that SOX9 expression was significantly increased in DR rat retinas and in Müller cells stimulated with a high glucose (HG) concentration. HG treatment promoted the proliferation and migration capabilities of Müller cells, whereas SOX9 knockdown reversed those behaviors. Moreover, SOX9 knockdown provided protection against an HG-induced inflammatory response, as evidenced by reduced tumor necrosis factor-α, IL-1β, and IL-6 levels in serum and decreased NLRP3 inflammasome activation. Notably, SOX9 acted as a transcription factor that positively regulated thioredoxin-interacting protein (TXNIP), a positive regulator of Müller cells gliosis under HG conditions. A dual-luciferase assay demonstrated that SOX9 could enhance TXNIP expression at the transcriptional level through binding to the promoter of TXNIP. Moreover, TXNIP overexpression restored the effects caused by SOX9 silencing. In conclusion, these findings demonstrate that SOX9 may accelerate the progression of DR by promoting glial cell proliferation, metastasis, and inflammation, which involves the transcriptional regulation of TXNIP, providing new theoretical fundamentals for DR therapy.

Autoimmune hepatitis (AIH) is a kind of autoimmune disease mediated by T cells, and its incidence is gradually increasing in the world. Oroxylin A (OA) is one of the major bioactive flavonoids that has been reported to inhibit inflammatory. Here, an AIH model of mouse was induced by Concanavalin A (Con A). It found that serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were decreased in mice with the treatment of OA. Hematoxylin-eosin staining showed that the liver injury was attenuated by OA, and TUNEL staining indicated that the cells apoptosis of liver was weakened in mice with OA treatment. ELISA analysis of cytokines and chemokines suggested that OA reduced the expression of IL-6, IL-17A, chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 1 (CXCL1) and CXCL10, but promoted the expression of IL-10 and TGF-β in mice. The mRNA levels of Il-17a in liver and spleen tissues were also significantly decreased, on the contrary, the mRNA levels of Il-10 in liver and spleen tissues were increased. The proportion of Treg/Th17 detected by flow cytometry revealed that OA promoted the differentiation of Treg and inhibited the differentiation of Th17 both in the liver and spleen. The results of this study demonstrated the inhibitory effects of OA on AIH-induced liver injury and the inflammatory response of AIH, and revealed that OA affected the balance of Treg/Th17 and shifted the balance toward Treg differentiation. It provided new potential drugs for the prevention of AIH.