A primary hallmark of pathological cardiac hypertrophy is excess protein synthesis due to enhanced translational activity. However, regulatory mechanisms at the translational level under cardiac stress remain poorly understood. Here we examined the translational regulations in a mouse cardiac hypertrophy model induced by transaortic constriction (TAC) and explored the conservative networks versus the translatome pattern in human dilated cardiomyopathy (DCM). The results showed that the heart weight to body weight ratio was significantly elevated, and the ejection fraction and fractional shortening significantly decreased 8 weeks after TAC. Puromycin incorporation assay showed that TAC significantly increased protein synthesis rate in the left ventricle. RNA-seq revealed 1,632 differentially expressed genes showing functional enrichment in pathways including extracellular matrix remodeling, metabolic processes, and signaling cascades associated with pathological cardiomyocyte growth. When combined with ribosome profiling analysis, we revealed that translation efficiency (TE) of 1,495 genes was enhanced, while the TE of 933 genes was inhibited following TAC. In DCM patients, 1,354 genes were upregulated versus 1,213 genes were downregulated at the translation level. Although the majority of the genes were not shared between mouse and human, we identified 93 genes, including Nos3, Kcnj8, Adcy4, Itpr1, Fasn, Scd1, etc., with highly conserved translational regulations. These genes were remarkably associated with myocardial function, signal transduction, and energy metabolism, particularly related to cGMP-PKG signaling and fatty acid metabolism. Motif analysis revealed enriched regulatory elements in the 5' untranslated regions (5'UTRs) of transcripts with differential TE, which exhibited strong cross-species sequence conservation. Our study revealed novel regulatory mechanisms at the translational level in cardiac hypertrophy and identified conserved translation-sensitive targets with potential applications to treat cardiac hypertrophy and heart failure in the clinic.
Animals ; Humans ; Cardiomegaly/physiopathology* ; Ribosomes/physiology* ; Protein Biosynthesis/physiology* ; Mice ; Cardiomyopathy, Dilated/genetics* ; Ribosome Profiling

To study the ameliorative effect of Eucommiae Cortex extract on spatial learning disabilities in APP/PS1 double-transgenic mice and explore its relationship with estrogen receptor β(ERβ)/c-Jun N-terminal kinase(JNK) signaling pathway, sixty 3-month-old male APP/PS1 mice were randomly divided into a model group, an anti-brain failure capsule group(0.585 g·kg~(-1)), a donepezil hydrochloride group(0.65 mg·kg~(-1)), and a Eucommiae Cortex extract group(1.3 g·kg~(-1)), and 15 C57BL/6 mice of the same genetic background were set as WT control group. The learning and memory ability of mice was assessed by the Morris water maze test(MWM), the passive avoidance test(PAT), and the novel object recognition test(NOR). The histomorphological and cellular ultrastructural features of the hippocampal region of the mice were observed by hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM); the molecular docking validation of the key active ingredients and the key targets was performed by using AutoDock Vina software, and the immunohistochemical method(IHC) was used to detect the ERβ expression in the dentate gyrus(DG) area of mouse hippocampus. Western blot(WB) was utilized to detect the expression of ERβ, p-JNK, and JNK in mouse hippocampal area. Compared with those in the WT control group, the results of behavioral experiments showed that the latency of the mice in the model group was significantly increased, the number of platform traversals, and the target quadrant residence time were significantly decreased in the MWM. The evasion latency was significantly reduced, and the number of errors was significantly increased in the PAT. The index of recognition of novel objects was significantly reduced in the NOR. The results of HE staining indicated that the hippocampal area of mice in the model group showed a decrease in the number of neurons, disorganization of pyramidal cell arrangement, nucleus consolidation, and other changes. TEM results showed that some neuronal nuclei in the hippocampal area had a consolidated state, slightly thickened and aberrant nuclear membranes, and fewer intracytoplasmic nidus bodies; the IHC results showed that the expression of ERβ in the hippocampal DG area of the mice was reduced. The WB results showed that the ERβ expression in the hippocampal tissue was decreased, and the p-JNK/JNK level was elevated. Compared with the model group, the Eucommiae Cortex extract group showed a significant decrease in latency, and increase in number of platform traversals and target quadrant residence time in the MWM, a significant increase in evasion latency and decrease in number of errors in the PAT, and a significant increase in the index of recognition of novel objects in the NOR. In addition, there was an increase in the number of neurons in the hippocampal area of mice. The pyramidal cells tended to be arranged in an orderly manner; the nuclei of neurons in the hippocampal area were in a better state; the expression of ERβ in the hippocampal DG area of the mice was elevated; the expression of ERβ in the hippocampal tissue was elevated, and the level of p-JNK/JNK was reduced. The effects of donepezil hydrochloride group and anti-brain failure capsule on APP/PS1 mice in terms of behavioral, HE, and TEM indexes were similar to those of Eucommiae Cortex extract, and there was no significant difference between donepezil hydrochloride group and the model group in IHC and WB experiments, and the results of molecular docking indicated that the estrogen-like components in Eucommiae Cortex extract were tightly bound to ERβ. In conclusion, the binding of Eucommiae Cortex extract to estrogen receptors, regulation of ERβ expression, and activation of ERβ/JNK signaling pathway may be one of the key mechanisms by which it improves the learning and memory ability of APP/PS1 mice.
Animals ; Male ; Mice ; Mice, Transgenic ; Memory/drug effects* ; Mice, Inbred C57BL ; Estrogen Receptor beta/genetics* ; Eucommiaceae/chemistry* ; Alzheimer Disease/psychology* ; Amyloid beta-Protein Precursor/metabolism* ; Presenilin-1/metabolism* ; Humans ; MAP Kinase Signaling System/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Hippocampus/metabolism* ; Maze Learning/drug effects* ; Learning/drug effects*

Objective:To establish a mesenchymal stem cell(MSC)-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease(aGVHD).Methods:Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors,and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients.The recipient mouse received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1× 107/mouse)in 6-8 hours post irradiation to establish a bone marrow transplantation(BMT)mouse model(n=20).In addition,the recipient mice received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1 × 107/mouse)and spleen lymphocytes(2 × 106/mouse)in 6-8 hours post irradiation to establish a mouse aGVHD model(n=20).On the day 7 after modeling,the recipient mice were anesthetized and the blood was harvested post eyeball enucleation.The serum was collected by centrifugation.Mouse MSCs were isolated and cultured with the addition of 2％,5％,and 10％recipient serum from BMT group or aGVHD group respectively.The colony-forming unit-fibroblast(CFU-F)experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC.The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining.In addition,the expression of self-renewal-related genes including Oct-4,Sox-2,and Nanog in MSC was detected by real-time fluorescence quantitative PCR(RT-qPCR).Results:We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD.CFU-F assay showed that,on day 7 after the culture,compared with the BMT group,MSC colony formation ability of aGVHD serum concentrations groups of 2％and 5％was significantly reduced(P＜0.05);after the culture,at day 14,compared with the BMT group,MSC colony formation ability in different aGVHD serum concentration was significantly reduced(P＜0.05).The immunofluorescence staining showed that,compared with the BMT group,the proportion of MSC surface molecules CD29+and CD 105+cells was significantly dereased in the aGVHD serum concentration group(P＜0.05),the most significant difference was at a serum concentration of 10％(P＜0.001,P＜0.01).The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4,Sox-2,and Nanog was decreased,the most significant difference was observed at an aGVHD serum concentration of 10％(P＜0.01,P＜0.001,P＜0.001).Conclusion:By co-culturing different concentrations of mouse aGVHD serum and mouse MSC,we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability,which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Objective:To observe the inhibitory effect of dobutamine on proliferation of FLT3-ITD mutated acute myeloid leukemia(AML)cells and explore the feasibility of dobutamine as a monotherapy or in combination with quizartinib for the treatment of this type of AML.Methods:FLT3-ITD mutant cell lines MOLM13 and MV4-11 were cultured in vitro and divided into control group,dobutamine treatment group,quizartinib treatment group,and dobutamine combined with quizartinib treatment group.Cell viability,ROS levels,and apoptosis rate were detected by CCK-8,Flow cytometry,respectively,as well as the expression of YAP1 protein by Western blot.Results:Both dobutamine and quizartinib inhibited the proliferation of FLT3-ITD mutant AML cell lines.Compared with the control group,the dobutamine group exhibited a significant increase in ROS levels(P＜0.01),an increase in apoptosis rates(P＜0.05),and a decrease in YAP1 protein expression(P＜0.05).Compared with the dobutamine group,the combination of quizartinib and dobutamine significantly reduced cell viability(P＜0.05),increased ROS levels(P＜0.01),and decreased YAP1 expression(P＜0.05).Conclusion:Dobutamine as a monotherapy can inhibit the proliferation of FLT3-ITD mutated AML cells,inducing apoptosis.Additionally,the combination of quizartinib enhances the targeted inhibitory effect on FLT3-ITD mutated AML.The mechanism may involve the inhibition of YAP1 protein expression in AML cells of this type,leading to an increase in ROS levels and exerting its anti-tumor effects.

ObjectiveThis study aimed to observe the impact of sinomenine hydrochloride on the proliferation of fibroblasts and the mRNA expression of related genes in knee joint adhesion and contracture in rabbits. Additionally, we sought to explore its potential mechanisms in combating knee joint adhesion and contracture. MethodsFibroblasts were cultured in vitro, and experimental groups with varying concentrations of sinomenine hydrochloride were established alongside a control group. Cell proliferation was assessed using the CCK-8 assay. Changes in the mRNA expression of fibroblast-related genes following sinomenine hydrochloride treatment were evaluated using RT-qPCR. The impact of the drug on serum levels of inflammatory cytokines was determined using the ELISA method, and the expression of related proteins was assessed using Western blot. ResultsSinomenine hydrochloride was found to inhibit fibroblast viability, with viability decreasing as the concentration of sinomenine hydrochloride increased. The effects of sinomenine hydrochloride in all experimental groups were highly significant (P<0.05). At the mRNA expression level, compared to the control group, sinomenine hydrochloride led to a significant downregulation of inflammatory cytokines in all groups (P<0.05). Additionally, the expression levels of apoptosis-related proteins significantly increased, while Bcl-2 mRNA expression decreased (P<0.05). The mRNA expression levels of the PI3K/mTOR/AKT3 signaling pathway also decreased (P<0.05). At the protein expression level, in comparison to the control group, the levels of inflammatory cytokines IL-6, IL-8, IL-1β, and TGF-β were significantly downregulated in the middle and high-dose sinomenine hydrochloride groups (P<0.05). The expression levels of cleaved-PARP, cleaved caspase-3/7, and Bax increased and were positively correlated with the dose, while the expression levels of the anti-apoptotic protein Bcl-2 and the PI3K/AKT3/mTOR signaling pathway were negatively correlated with the dose. Sinomenine hydrochloride exhibited a significant inhibitory effect on the viability of rabbit knee joint fibroblasts, which may be associated with the downregulation of inflammatory cytokines IL-6, IL-8, and IL-1β, promotion of apoptosis-related proteins cleaved-PARP, cleaved caspase-3/7, and Bax, suppression of Bcl-2 expression, and inhibition of gene expression in the downstream PI3K/AKT3/mTOR signaling pathway. ConclusionSinomenine hydrochloride can inhibit the inflammatory response of fibroblasts in adhesive knee joints and accelerate fibroblast apoptosis. This mechanism may offer a novel approach to improving and treating knee joint adhesion.

Magnetic cell sorting technology is a highly specific and rapid cell sorting technology using superparamagnetic nanocomposites for cell sorting, which is widely used in immunology, stem cytology, oncology, clinical medicine and other fields. Magnetic cell sorting technology is divided into positive isolation, negative isolation/untouched cell isolation, depletion, multi-step isolation and automated cell separation systems. In this review, we firstly give a brief introduction to the classification and application of magnetic cell sorting technology, then discuss several new techniques and challenges based on magnetic cell sorting in recent years, such as improving the sorting efficiency by improving the structure of magnetic materials and magnetic field structure. The necessity of biological evaluation of magnetic cell sorting products was emphatically analyzed. Through the biological evaluation, the advantages and disadvantages of magnetic cell sorting products can be understood, and the research and development ability could be improved. Therefore, 10 biological evaluation technical parameters related to magnetic cell sorting products were proposed: yield, purity, sterility, cytotoxicity, cell morphology, viability, light scattering characteristics of cells, fluorescent antibody labeling ability of cells, cell activation and cell proliferation. The 10 biological evaluation technical parameters play an important role in promoting the standardized application of magnetic cell sorting.

OBJECTIVE: To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology. METHODS: 20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×10⁷/mice, n=20) and spleen nucleated cells (5×10⁶/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5⁺ and Clu⁺ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings. RESULTS: The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5⁺ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu⁺ cells were observed in the 5% group. The Lgr5⁺ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu⁺ cells in the 20% group significantly increased. CONCLUSION The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.
Mice ; Female ; Animals ; Mice, Inbred C57BL ; Bone Marrow Transplantation ; Graft vs Host Disease ; Stem Cells ; Organoids

OBJECTIVES: To study the clinical characteristics and prognosis of SARS-CoV-2 Omicron variant infection-associated acute necrotizing encephalopathy (ANE) in children . METHODS: A retrospective analysis was conducted on the medical data of 12 children with SARS-CoV-2 Omicron variant infection-associated ANE who were admitted to the Pediatric Intensive Care Unit, Qingdao Women and Children's Hospital from December 18 to 29, 2022. The children were divided into two groups based on outcomes: death group (7 cases) and survival group (5 cases). The clinical manifestations and auxiliary examination results were compared between the two groups. RESULTS: The median age of the 12 patients was 30 months, with a male-to-female ratio of 1:1. All patients presented with persistent high fever, with a median highest body temperature of 41℃. The median time from fever onset to seizure or consciousness disturbance was 18 hours. The death group had a higher proportion of neurogenic shock, coagulation dysfunction, as well as elevated lactate, D-dimer, interleukin-6, interleukin--8, and interleukin-10 levels compared to the survival group (P<0.05). CONCLUSIONS Children with SARS-CoV-2 Omicron variant infection-associated with ANE commonly present with persistent high fever, rapidly progressing disease, and have a high likelihood of developing consciousness disorders and multiorgan dysfunction within a short period. The occurrence of neurogenic shock, coagulation dysfunction, and significantly elevated cytokine levels suggests an increased risk of mortality.
Humans ; Female ; Child ; Male ; Infant ; SARS-CoV-2 ; Retrospective Studies ; COVID-19/complications* ; Brain Diseases/etiology* ; Prognosis ; Fever ; Blood Coagulation Disorders

AIM: To analyze the effect of full-femtosecond small incision lenticule extraction(SMILE)on the treatment of high myopia based on propensity score matching.METHODS: A total of 48 cases(48 eyes)of high myopia patients who underwent SMILE surgery in our hospital from May 2019 to May 2021 were selected as the observation group, and 48 cases(48 eyes)of high myopia patients who underwent FS-LASIK surgery were matched using propensity score matching as the control group. Follow up for 6mo after surgery, the changes in cylindrical, central corneal thickness, uncorrected visual acuity(UCVA), corneal endothelial cell related indicators [percentage of hexagonal endothelial cells(6A), coefficient of variation(CV)of endothelial cell area, central corneal endothelial cell density(ECD)] and corneal biomechanical indicators [simulated Goldman intraocular pressure(IOPg), corneal hysteresis(CH), corneal resistance factor(CRF), corneal compensated intraocular pressure(IOPcc)] between the two groups were compared, and the incidence of complications in both groups of patients was recorded.RESULTS: Both groups of patients showed significant improvements in cylindrical and UCVA at 3 and 6mo after surgery, as well as decreased central corneal thickness, corneal endothelial cells, and corneal biomechanics related indicators. The changes in the observation group were more significant(all P<0.05). During the follow-up period, there was no significant difference in the incidence of complications between the observation group and the control group(8% vs. 17%, P>0.05).CONCLUSION: SMILE has a definite effect on patients with high myopia and is helpful to improve visual acuity.

Aim To explore the effects of corilagin on non-alcoholic fatty liver disease induced by high-fat and high-sugar diet in mice via regulating AMPK-autophagy signaling. Methods Healthy 8-week-old male C57BL/6J mice were randomly divided into control group, model group and corilagin group. The mice of model group and corilagin group were fed with a high-fat and high-sugar diet for four weeks at the age of eight weeks. The corilagin group mice were also intraperitoneally injected with corilagin (20 mg • k g