Our previous study has shown that p66^Shc plays an important role in the process of myocardial regeneration in newborn mice, and p66^Shc deficiency leads to weakened myocardial regeneration in newborn mice. This study aims to explore the role of p66^Shc protein in myocardial injury repair after myocardial infarction in adult mice, in order to provide a new target for the treatment of myocardial injury after myocardial infarction. Mouse myocardial infarction models of adult wild-type (WT) and p66^Shc knockout (KO) were constructed by anterior descending branch ligation. The survival rate and heart-to-body weight ratio of two models were compared and analyzed. Masson's staining was used to identify scar area of injured myocardial tissue, and myocyte area was determined by wheat germ agglutinin (WGA) staining. TUNEL staining was used to detect the cardiomyocyte apoptosis. The protein expression of brain natriuretic peptide (BNP), a common marker of myocardial hypertrophy, was detected by Western blotting. The results showed that there was no significant difference in survival rate, myocardial scar area, myocyte apoptosis, and heart weight to body weight ratio between the WT and p66^ShcKO mice after myocardial infarction surgery. Whereas the protein expression level of BNP in the p66^ShcKO mice was significantly down-regulated compared with that in the WT mice. These results suggest that, unlike in neonatal mice, the deletion of p66^Shc has no significant effect on myocardial injury repair after myocardial infarction in adult mice.
Animals ; Mice ; Body Weight ; Cicatrix/metabolism* ; Mice, Knockout ; Myocardial Infarction/genetics* ; Oxidative Stress ; Shc Signaling Adaptor Proteins/metabolism* ; Src Homology 2 Domain-Containing, Transforming Protein 1/metabolism*

Objective To investigate the long non-coding RNA(lncRNA) MRAK08838 regulates macrophage function to influence the development of asthmatic airway inflammation. Methods MRAK088388 gene knockout (MRAK088388^-/-) mouse model was prepared and allergic asthma was induced by dust mite protein Dermatophagoides farinae 1 (Der f1). The mice were sacrificed after 28 days of modeling, and serum was collected to measure IgE and IgG. The FinePointe RC system was used to measure airway hyperresponsiveness and evaluate lung function in mice. Lung tissue was taken for HE staining, and periodic acid-Schiff (PAS) staining was used to evaluate inflammatory infiltration and mucus secretion in mouse lungs. Fluorescence quantitative PCR was used to detect the expression level of lncRNA MRAK08838 in bronchoalveolar lavage fluid (BALF) cells and lung tissue of asthmatic mice. ELISA was used to detect the levels of inflammatory cytokines IFN-γ, IL-4, IL-5, IL-13, IL-10 and IL-17A. Flow cytometry was used to evaluate the phenotype of macrophages in BALF and lung tissue, as well as the proportion of neutrophils, eosinophils, and alveolar macrophages. The changes of the above indicators were detected in mice by adoptive transfer of bone marrow-derived macrophages (BMDM). Results Under the challengle of Der f1, MRAK088388^-/- mice showed reduced allergic airway inflammation, including reduced eosinophils in BALF and reduced production of IgE and IgG1. In addition, Der f1-treated MRAK088388^-/- mice had fewer M2 macrophages than wild-type asthmatic mice. Wild-type mouse BMDM (M0) and Der f1-treated MRAK088388^-/- mice also showed mild inflammatory response. Conclusion Knockout of MRAK088388 alleviates airway inflammation in asthmatic mice by inhibiting M2 polarization of airway macrophages.
Animals ; Mice ; Mice, Knockout ; RNA, Long Noncoding/genetics* ; Asthma/genetics* ; Macrophages ; Immunoglobulin E

Brain size abnormality is correlated with an increased frequency of autism spectrum disorder (ASD) in offspring. Genetic analysis indicates that heterozygous mutations of the WD repeat domain 62 (WDR62) are associated with ASD. However, biological evidence is still lacking. Our study showed that Wdr62 knockout (KO) led to reduced brain size with impaired learning and memory, as well as ASD-like behaviors in mice. Interestingly, Wdr62 Nex-cKO mice (depletion of WDR62 in differentiated neurons) had a largely normal brain size but with aberrant social interactions and repetitive behaviors. WDR62 regulated dendritic spinogenesis and excitatory synaptic transmission in cortical pyramidal neurons. Finally, we revealed that retinoic acid gavages significantly alleviated ASD-like behaviors in mice with WDR62 haploinsufficiency, probably by complementing the expression of ASD and synapse-related genes. Our findings provide a new perspective on the relationship between the microcephaly gene WDR62 and ASD etiology that will benefit clinical diagnosis and intervention of ASD.
Mice ; Animals ; Microcephaly/genetics* ; Autistic Disorder/metabolism* ; Autism Spectrum Disorder/metabolism* ; Nerve Tissue Proteins/metabolism* ; Brain/metabolism* ; Mice, Knockout ; Cell Cycle Proteins/metabolism*

The present study was aimed to investigate whether Gasdermin D (GSDMD)-mediated pyroptosis participated in lipopolysaccharide (LPS)-induced sepsis-associated acute kidney injury (AKI), and to explore the role of caspase-1 and caspase-11 pyroptosis pathways in this process. The mice were divided into four groups: wild type (WT), WT-LPS, GSDMD knockout (KO) and KO-LPS. The sepsis-associated AKI was induced by intraperitoneal injection of LPS (40 mg/kg). Blood samples were taken to determine the concentration of creatinine and urea nitrogen. The pathological changes of renal tissue were observed via HE staining. Western blot was used to investigate the expression of pyroptosis-associated proteins. The results showed that the concentrations of serum creatinine and urea nitrogen in the WT-LPS group were significantly increased, compared with those in the WT group (P < 0.01); whereas serum creatinine and urea nitrogen in the KO-LPS group were significantly decreased, compared with those in the WT-LPS group (P < 0.01). HE staining results showed that LPS-induced renal tubular dilatation was mitigated in GSDMD KO mice. Western blot results showed that LPS up-regulated the protein expression levels of interleukin-1β (IL-1β), GSDMD and GSDMD-N in WT mice. GSDMD KO significantly down-regulated the protein levels of IL-1β, caspase-11, pro-caspase-1, caspase-1(p22) induced by LPS. These results suggest that GSDMD-mediated pyroptosis is involved in LPS-induced sepsis-associated AKI. Caspase-1 and caspase-11 may be involved in GSDMD cleavage.
Animals ; Mice ; Acute Kidney Injury ; Caspase 1 ; Caspases/metabolism* ; Creatinine ; Lipopolysaccharides ; Mice, Knockout ; Nitrogen ; Sepsis ; Urea ; Gasdermins/metabolism*

OBJECTIVE: To investigate the changes and roles of reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) related antioxidases during erythroid development. METHODS: Flow cytometry was used to detect the sensibility of peripheral red blood cells of wild-type mice to a strong oxidant hydrogen peroxide (H₂O₂). Erythroid cells from different developmental stages in bone marrow (BM) were obtained using fluorescence-activated cell sorter and the ROS levels were detected by flow cytometry. RT-qPCR was used to detect the changes of expression levels of Nrf2 and related antioxidases in erythroid cells from different developmental stages in BM. The ROS levels of the peripheral blood and BM nucleated erythrocytes in Nrf2 knockout mice were further examined. The expression level of Nrf2 in erythroid precursors isolated from 14.5 d embryonic liver of wild-type mice during differentiation and culture in vitro was detected. RESULTS: In the peripheral blood of wild-type mice, the ROS level of reticulocytes and mature erythrocytes treated with H₂O₂ increased about 4 times and 7 times, respectively (P<0.01). In BM erythrocytes, the ROS level gradually decreased as the cells matured (r=0.85), while the expression level of Nrf2 and its related anti-oxidative genes increased (r=0.99). The ROS levels in peripheral blood erythrocytes and BM nucleated erythrocytes of Nrf2 knockout mice were significantly increased compared with wild-type mice (P<0.01). The expression of Nrf2 increased during the early erythroid development after embryonic liver cell sorting (P<0.01). CONCLUSION The expression levels of Nrf2 and its related factors vary during erythropoiesis. Nrf2 at physiological level plays an important antioxidant role during the erythroid development.
Animals ; Mice ; Hydrogen Peroxide ; Mice, Knockout ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress ; Reactive Oxygen Species/metabolism*

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.
Animals ; Mice ; Mice, Knockout ; CRISPR-Cas Systems ; Blood Pressure ; Gene Knockout Techniques ; Zygote

OBJECTIVE: To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC). METHODS: Mouse models of VC were established in ApoE-deficient (ApoE^-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with N^ε-carboxymethyl-lysine for 16 weeks. ApoE^-/- mice (control group), ApoE^-/- diabetic mice (VC group), ApoE^-/- diabetic mice with BI-1 overexpression (VC + BI-1^TG group), and ApoE^-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1^TG+OPA1^-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L β-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining. RESULTS: ApoE^-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007). CONCLUSION BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.
Animals ; Apolipoproteins E/metabolism* ; Calcium/metabolism* ; Caspase 3/metabolism* ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Diabetes Mellitus, Experimental/pathology* ; GTP Phosphohydrolases/metabolism* ; Membrane Proteins/metabolism* ; Mice ; Mice, Knockout ; Muscle, Smooth, Vascular/pathology* ; Myocytes, Smooth Muscle/pathology* ; Optic Atrophy, Autosomal Dominant/pathology* ; Osteogenesis ; Vascular Calcification/pathology* ; bcl-2-Associated X Protein/metabolism*

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

In mammals, the piezoelectric protein, Prestin, endows the outer hair cells (OHCs) with electromotility (eM), which confers the capacity to change cellular length in response to alterations in membrane potential. Together with basilar membrane resonance and possible stereociliary motility, Prestin-based OHC eM lays the foundation for enhancing cochlear sensitivity and frequency selectivity. However, it remains debatable whether Prestin contributes to ultrahigh-frequency hearing due to the intrinsic nature of the cell's low-pass features. The low-pass property of mouse OHC eM is based on the finding that eM magnitude dissipates within the frequency bandwidth of human speech. In this study, we examined the role of Prestin in sensing broad-range frequencies (4-80 kHz) in mice that use ultrasonic hearing and vocalization (to >100 kHz) for social communication. The audiometric measurements in mice showed that ablation of Prestin did not abolish hearing at frequencies >40 kHz. Acoustic associative behavior tests confirmed that Prestin-knockout mice can learn ultrahigh-frequency sound-coupled tasks, similar to control mice. Ex vivo cochlear Ca²⁺ imaging experiments demonstrated that without Prestin, the OHCs still exhibit ultrahigh-frequency transduction, which in contrast, can be abolished by a universal cation channel blocker, Gadolinium. In vivo salicylate treatment disrupts hearing at frequencies <40 kHz but not ultrahigh-frequency hearing. By pharmacogenetic manipulation, we showed that specific ablation of the OHCs largely abolished hearing at frequencies >40 kHz. These findings demonstrate that cochlear OHCs are the target cells that support ultrahigh-frequency transduction, which does not require Prestin.
Animals ; Cochlea/metabolism* ; Hair Cells, Auditory, Outer/metabolism* ; Hearing ; Humans ; Mammals/metabolism* ; Mice ; Mice, Knockout ; Molecular Motor Proteins/metabolism*

OBJECTIVE: To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice. METHODS: In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H^-/--Thy1⁺(KO-GFP) and CACNA1H^+/+-Thy1⁺ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed. RESULTS: In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F_{1, 14}=95.086, P < 0.05; Post-Hoc: P < 0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F_{1, 14}=18.062, P < 0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P < 0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P < 0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P < 0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P < 0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P < 0.05), without any significant difference in spine maturity (t=-1.935, P>0.05). CONCLUSION CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.
Animals ; Autistic Disorder/genetics* ; Calcium Channels, T-Type/genetics* ; Gene Knockout Techniques ; Hippocampus ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons