OBJECTIVE: To explore the mechanism of Dihuang Yinzi (DHYZ) in the treatment of Alzheimer's disease (AD) by integrating metabolomics and experimental verification. METHODS: Forty-eight male APP/PS1 mice were divided into model, high- (DHYZ-H), medium- (DHYZ-M), and low-dose DHYZ (DHYZ-L) groups (12 mice per group) according to a random number table. Mice in DHYZ groups were gavaged with DHYZ 6.34, 12.68, and 25.35 g/(kg·d), respectively. Twelve C57BL/6 mice were gavaged with distilled water as the blank group. Metabolomics was used to analyze differential metabolites in the brains of mice. Morris water maze test was used to detect the memory abilities of mice. The hematoxylin-eosin staining and transmission electron microscopy were used to observe the general morphology and ultrastructure of neurons. The enzyme-linked immunosorbent assay was used to detect the levels of superoxide dismutase (SOD), reactive oxygen species (ROS), and amyloid β -protein 1-42 (A β_1-42). The real-time quantitative polymerase chain reaction was used to detect the mRNA expressions of density-regulated protein 1 (DRP1), fission 1 (FIS1), mitofusin-1 (MFN1), and optic atrophy protein 1 (OPA1). Western blot was used to detect the protein expressions of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), cAMP response binding protein (CREB), brain-derived neurotrophic factor (BDNF), synapsin 1 (SYN1), synaptophysin (SYP), and postsynaptic density protein 95 (PSD95). RESULTS: A total of 82 differential metabolites were identified in the brains of APP/PS1 mice, among which 7 differential metabolites could be regulated by DHYZ. After DHYZ intervention, the memory abilities of mice significantly increased (P<0.05 or P<0.01), the number of synapses and neurons in the hippocampus increased, and the mitochondrial morphology and structure were relatively intact. The DHYZ groups exhibited a significant reduction in hippocampal ROS and A β_1-42 levels, along with a significant elevation in SOD level (P<0.05 or P<0.01). The mRNA expressions of DRP1 and FIS1 were reduced, while the mRNA expressions of MFN1 and OPA1 were increased after DHYZ treatment (P<0.05 or P<0.01). The cAMP/PKA/CREB-BDNF pathway was activated, and the expressions of SYN1, SYP and PSD95 proteins were significantly increased in the DHYZ-H group (P<0.05 or P<0.01). CONCLUSIONS DHYZ could improve mitochondrial dynamics and synaptic plasticity in APP/PS1 mice, inhibit oxidative stress, and thereby enhancing learning and memory abilities in APP/PS1 mice. Its mechanism might be related to activation of the cAMP/PKA/CREB-BDNF signaling pathway.
Animals ; Brain-Derived Neurotrophic Factor/metabolism* ; Male ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Brain/drug effects* ; Metabolomics ; Mice, Inbred C57BL ; Neuronal Plasticity/drug effects* ; Drugs, Chinese Herbal/therapeutic use* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Cyclic AMP/metabolism* ; Reactive Oxygen Species/metabolism* ; Amyloid beta-Protein Precursor/metabolism* ; Mice, Transgenic ; Mice ; Amyloid beta-Peptides/metabolism* ; Signal Transduction/drug effects* ; Alzheimer Disease/drug therapy* ; Superoxide Dismutase/metabolism*

OBJECTIVES: Osteoarthritis (OA) is one of the most common chronic degenerative diseases, with chondrocyte apoptosis and extracellular matrix (ECM) degradation as the major pathological changes. The mechanical stimulation can attenuate chondrocyte apoptosis and promote ECM synthesis, but the underlying molecular mechanisms remain unclear. This study aims to investigate the role of primary cilia (PC) in mediating the effects of mechanical stimulation on OA progression. METHODS: In vivo, conditional knockout mice lacking intraflagellar transport 88 (IFT88^flox/flox IFT88 knockout; i.e., primary cilia-deficient mice) were generated, with wild-type mice as controls. OA models were established via anterior cruciate ligament transection combined with destabilization of the medial meniscus, followed by treadmill exercise intervention. OA progression was evaluated by hematoxylin-eosin staining, safranin O-fast green staining, and immunohistochemistry; apoptosis was assessed by TUNEL staining; and limb function by rotarod testing. In vitro, primary articular chondrocytes were isolated from mice and transfected with lentiviral vectors to suppress IFT88 expression, thereby constructing a primary cilia-deficient cell model. Interleukin-1β (IL-1β) was used to induce an inflammatory environment, while cyclic tensile strain (CTS) was applied via a cell stretcher to mimic mechanical loading on chondrocytes. Immunofluorescence and Western blotting were used to detect the protein expression levels of type II collagen α1 chain (COL2A1), primary cilia, IFT88, and caspase-12; reverse transcription polymerase chain reaction was performed to assess COL2A1 mRNA levels; and flow cytometry was used to evaluate apoptosis. RESULTS: In vivo, treadmill exercise significantly reduced Osteoarthritis Research Society International (OARSI) scores and apoptotic cell rates, and improved balance ability in wild-type OA mice, whereas IFT88-deficient OA mice showed no significant improvement. In vitro, CTS inhibited IL-1β-induced ECM degradation and apoptosis in primary chondrocytes; however, this protective effect was abolished in cells with suppressed primary cilia expression. CONCLUSIONS Mechanical stimulation delays OA progression by mediating signal transduction through primary cilia, thereby inhibiting cartilage degeneration and chondrocyte apoptosis.
Animals ; Chondrocytes/cytology* ; Apoptosis/physiology* ; Mice ; Cilia/metabolism* ; Osteoarthritis/pathology* ; Extracellular Matrix/metabolism* ; Mice, Knockout ; Disease Progression ; Interleukin-1beta ; Male ; Cells, Cultured

Loss-of-function variants of low-density lipoprotein receptor-related protein 5 (LRP5) can lead to reduced bone formation, culminating in diminished bone mass. Our previous study reported transcription factor osterix (SP7)‍-binding sites on the LRP5 promoter and its pivotal role in upregulating LRP5 expression during implant osseointegration. However, the potential role of SP7 in ameliorating LRP5-dependent osteoporosis remained unknown. In this study, we used mice with a conditional knockout (cKO) of LRP5 in mature osteoblasts, which presented decreased osteogenesis. The in vitro experimental results showed that SP7 could promote LRP5 expression, thereby upregulating the osteogenic markers such as alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and β‍-catenin (P<0.05). For the in vivo experiment, the SP7 overexpression virus was injected into a bone defect model of LRP5 cKO mice, resulting in increased bone mineral density (BMD) (P<0.001) and volumetric density (bone volume (BV)/total volume (TV)) (P<0.001), and decreased trabecular separation (Tb.‍Sp) (P<0.05). These data suggested that SP7 could ameliorate bone defect healing in LRP5 cKO mice. Our study provides new insights into potential therapeutic opportunities for ameliorating LRP5-dependent osteoporosis.
Animals ; Low Density Lipoprotein Receptor-Related Protein-5/metabolism* ; Osteoporosis/genetics* ; Mice ; Mice, Knockout ; Sp7 Transcription Factor/physiology* ; Osteogenesis ; Bone Density ; Osteoblasts/metabolism* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Mice, Inbred C57BL ; beta Catenin/metabolism*

Imprinted genes play a key role in regulating mammalian placental and embryonic development. Here, we generated glutaminyl-peptide cyclotransferase-knockout (Qpct^-/-) mice utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform and identified Qpct as a novel anti-angiogenic factor in regulating mouse placentation. Compared with Qpct^+/+ mice, placentae and embryos (Qpct^-/+ and Qpct^-/-) showed significant overgrowth at embryonic Day 12.5 (E12.5), E15.5, and E18.5. Using single-cell transcriptome analysis of 32 309 cells from Qpct^+/+ and Qpct^-/- mouse placentae, we identified 13 cell clusters via single-nucleus RNA sequencing (snRNA-seq) (8880 Qpct^+/+ and 13 577 Qpct^-/- cells) and 20 cell clusters via single-cell RNA sequencing (scRNA-seq) (6567 Qpct^+/+ and 3285 Qpct^-/- cells). Furthermore, we observed a global up-regulation of pro-angiogenic genes in the Qpct^-/- background. Immunohistochemistry assays revealed a notable increase in the number of blood vessels in the decidual and labyrinthine layers of E15.5 Qpct^-/+ and Qpct^-/- mice. Moreover, the elevation of multiple pairs of ligand-receptor interactions was observed in decidual cells, endothelial cells, and macrophages, promoting angiogenesis and inflammatory response. Our findings indicate that loss of maternal Qpct leads to altered phenotypic characteristics of placentae and embryos and promotes angiogenesis in murine placentae.
Animals ; Female ; Pregnancy ; Mice ; Placentation/genetics* ; Single-Cell Analysis ; Gene Expression Profiling ; Mice, Knockout ; Transcriptome ; Placenta/blood supply* ; Neovascularization, Pathologic/genetics* ; Genomic Imprinting ; Single-Cell Gene Expression Analysis ; Angiogenesis

OBJECTIVES: To explore the effect of quercetin for mitigating HIV-1 gp120-induced astrocyte neurotoxicity and its underlying mechanism. METHODS: Primary rat astrocytes were isolated and treated with quercetin, HIV-1 gp120, or gradient concentrations of quercetin combined with HIV-1 gp120. The formation of stress granules (SGs) in the treated cells was observed with immunofluorescence assay, and the levels of oxidative stress markers and protein expressions were measured using specific assay kits and Western blotting. HIV-1 gp120 transgenic mice were treated with quercetin (50 mg/kg) by gavage for 4 weeks, and the changes in cognitive functions and oxidative stress levels were examined by behavioral assessments, oxidative stress index analysis in serum, and immunohistochemical and Western blotting of the brain tissue. RESULTS: In primary rat astrocytes, treatment with quercetin significantly reduced HIV-1 gp120-induced SG formation, increased the levels of antioxidant indexes, decreased the levels of oxidative substances, and up-regulated protein level associated with SG depolymerization. In the transgenic mouse models, quercetin obviously improved the cognitive function of the rats, reduced oxidative stress levels, and promoted the expression of proteins associate with SG depolymerization in the brain tissues. CONCLUSIONS Quercetin mitigates HIV-1 gp120-induced astrocyte neurotoxicity and cognitive function impairment by inhibiting oxidative stress, enhancing expressions of SG depolymerization-related proteins, and promoting SG disassembly, suggesting the value of quercetin as a potential therapeutic agent for neuroprotection in HIV-associated neurocognitive disorders.
Animals ; Quercetin/pharmacology* ; Astrocytes/metabolism* ; HIV Envelope Protein gp120 ; Oxidative Stress/drug effects* ; Rats ; Stress Granules/drug effects* ; Mice ; Mice, Transgenic ; Rats, Sprague-Dawley ; Cells, Cultured

OBJECTIVES: To explore the effect of A. muciniphila gavage on intestinal microbiota and gut-brain interaction disorders (DGBIs) in gp120tg transgenic mouse models of HIV-associated neurocognitive disorder (HAND). METHODS: Intestinal microbiota was detected by 16S rRNA gene sequencing in 6-, 9-, and 12-month-old wild-type (WT) mice and gp120tg transgenic mice. The 12-month-old WT and transgenic mice were divided into 2 groups for daily treatment with PBS or A.muciniphila gavage (2×10⁸ CFU/mouse) for 6 weeks. After the treatment, immunohistochemistry, ELISA and qPCR were used to detect changes in colonic expression levels of glycosylated mucins, MBP and IL-1β, eosinophil infiltration, serum lipopolysaccharide (LPS) levels, and colonic expressions of occludin, ZO-1, IL-10, TNF-α and INF-γ mRNA. Morris water maze test and immunofluorescence assay were used to assess learning and spatial memory abilities and neuronal damage of the mice. RESULTS: Compared with WT mice, the transgenic mice exhibited significantly lowered Simpson's diversity of the intestinal microbiota with reduced abundance of Akkermansia genus, increased serum LPS levels and decreased colonic expression of glycosylated mucin. A.muciniphila gavage obviously ameliorated the reduction of glycosylated mucin in the transgenic mice without causing significant changes in body weight. The 12-month-old gp120tg mice had significantly decreased cdonic expressions of Occludin and ZO-1 with increased eosinophil infiltration and TNF-β, INF-γ and IL-1β levels and obviously lowered IL-10 level; all these changes were significantly mitigated by A.muciniphila gavage, which also improved cognitive impairment and neuronal loss in the hippocampus and cortex of the transgenic mice. CONCLUSIONS The gp120tg mice have lower intestinal microbiota richness and diversity than WT mice. The 12-month-old gp120tg mice have significantly reduced Akkermansia abundance with distinct DGBIs-related indexes, and A. muciniphila gavage can reduce intestinal barrier injury, colonic inflammation and eosinophil activation, cognitive impairment and brain neuron injury in these mice.
Animals ; Mice, Transgenic ; Gastrointestinal Microbiome ; Mice ; Brain ; HIV Envelope Protein gp120/genetics* ; Akkermansia ; Disease Models, Animal

OBJECTIVES: To study the effect of CDC20 knockdown on proliferation, migration and invasion of cervical cancer cells and its underlying mechanism. METHODS: CDC20 expression in cervical cancer tissues was analyzed using the TCGA database, and the protein expressions of CDC20 and β-Catenin in clinical specimens of cervical cancer and adjacent tissues were detected using immunohistochemistry. A dual target sgRNA2&7 sequence for CDC20 gene was designed for CDC20 gene knockdown in cervical cancer C33A cells using CRISPR/Cas9 technology, and CDC20 mRNA and protein expression levels in the transfected cells were detected using qRT-PCR and Western blotting. The changes in proliferation, cell cycle, apoptosis, migration and invasiveness of the transfected cells were evaluated using colony-forming assay, fluorescence activated cell sorting (FACS) and Transwell assay. In the animal experiment, naïve C33A cells and the cells with CDC20 knockdown were injected subcutaneously into the left and right axillae of nude mice (n=5) to observe tumor growth. The expressions of CDC20 and β-Catenin proteins in transfected cells and the xenograft were analyzed using Western blotting, and their interaction was confirmed by co-immunoprecipitation (CoIP) and immunofluorescence co-localization assays. RESULTS: Cervical cancer tissues expressed significantly higher CDC20 and β‑Catenin levels than the adjacent tissues. C33A cells with CDC20 knockdown showed reduced proliferation, increased apoptosis, and lowered migration and invasion abilities. CDC20 knockdown significantly suppressed the growth of C33A cell xenograft in nude mice, and the tumor-bearing mice did not exhibit obvious body mass changes. CDC20 and β-Catenin levels were both significantly lowered in C33A cells with CDC20 knockdown. Co-immunoprecipitation and co-localization assays confirmed the interaction between CDC20 and β‑Catenin. CONCLUSIONS CDC20 is highly expressed in cervical cancer tissues, and CDC20 knockdown can suppress proliferation, invasion, and metastasis while enhancing apoptosis of C33A cells, which is closely related with the regulation of the Wnt/β-Catenin signaling pathway.
Humans ; Uterine Cervical Neoplasms/metabolism* ; Female ; Cdc20 Proteins/genetics* ; Cell Proliferation ; Animals ; Cell Movement ; Neoplasm Invasiveness ; Apoptosis ; Mice, Nude ; beta Catenin/metabolism* ; CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis

Autism Spectrum Disorders (ASDs) are reported as a group of neurodevelopmental disorders. The structural changes of brain regions including the hippocampus were widely reported in autistic patients and mouse models with dysfunction of ASD risk genes, but the underlying mechanisms are not fully understood. Here, we report that deletion of Trio, a high-susceptibility gene of ASDs, causes a postnatal dentate gyrus (DG) hypoplasia with a zigzagged suprapyramidal blade, and the Trio-deficient mice display autism-like behaviors. The impaired morphogenesis of DG is mainly caused by disturbing the postnatal distribution of postmitotic granule cells (GCs), which further results in a migration deficit of neural progenitors. Furthermore, we reveal that Trio plays different roles in various excitatory neural cells by spatial transcriptomic sequencing, especially the role of regulating the migration of postmitotic GCs. In summary, our findings provide evidence of cellular mechanisms that Trio is involved in postnatal DG morphogenesis.
Animals ; Dentate Gyrus/metabolism* ; Mice ; Morphogenesis/physiology* ; Neurons/pathology* ; Cell Movement ; Mice, Inbred C57BL ; Autism Spectrum Disorder/pathology* ; Mice, Knockout ; Neural Stem Cells ; Male ; Neurogenesis ; Autistic Disorder/genetics*

IL-33 and its receptor ST2 play crucial roles in tissue repair and homeostasis. However, their involvement in optic neuropathy due to trauma and glaucoma remains unclear. Here, we report that IL-33 and ST2 were highly expressed in the mouse optic nerve and retina. Deletion of IL-33 or ST2 exacerbated retinal ganglion cell (RGC) loss, retinal thinning, and nerve fiber degeneration following optic nerve (ON) injury. This heightened retinal neurodegeneration correlated with increased neurotoxic astrocytes in Il33^-/- mice. In vitro, rIL-33 mitigated the neurotoxic astrocyte phenotype and reduced the expression of pro-inflammatory factors, thereby alleviating the RGC death induced by neurotoxic astrocyte-conditioned medium in retinal explants. Exogenous IL-33 treatment improved RGC survival in Il33^-/- and WT mice after ON injury, but not in ST2^-/- mice. Our findings highlight the role of the IL-33/ST2 axis in modulating reactive astrocyte function and providing neuroprotection for RGCs following ON injury.
Animals ; Interleukin-33/genetics* ; Interleukin-1 Receptor-Like 1 Protein/genetics* ; Optic Nerve Injuries/pathology* ; Retinal Ganglion Cells/pathology* ; Astrocytes/pathology* ; Mice ; Mice, Knockout ; Mice, Inbred C57BL ; Neuroprotection/physiology*

Anxiety disorder is a major symptom of autism spectrum disorder (ASD) with a comorbidity rate of ~40%. However, the neural mechanisms of the emergence of anxiety in ASD remain unclear. In our study, we found that hyperactivity of basolateral amygdala (BLA) pyramidal neurons (PNs) in Shank3 InsG3680 knock-in (InsG3680^+/+) mice is involved in the development of anxiety. Electrophysiological results also showed increased excitatory input and decreased inhibitory input in BLA PNs. Chemogenetic inhibition of the excitability of PNs in the BLA rescued the anxiety phenotype of InsG3680^+/+ mice. Further study found that the diminished control of the BLA by medial prefrontal cortex (mPFC) and optogenetic activation of the mPFC-BLA pathway also had a rescue effect, which increased the feedforward inhibition of the BLA. Taken together, our results suggest that hyperactivity of the BLA and alteration of the mPFC-BLA circuitry are involved in anxiety in InsG3680^+/+ mice.
Animals ; Prefrontal Cortex/metabolism* ; Basolateral Nuclear Complex/metabolism* ; Mice ; Anxiety/metabolism* ; Nerve Tissue Proteins/genetics* ; Male ; Gene Knock-In Techniques ; Pyramidal Cells/physiology* ; Mice, Transgenic ; Neural Pathways/physiopathology* ; Mice, Inbred C57BL ; Microfilament Proteins