Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline, functional impairment, and neuropsychiatric symptoms. It represents the most prevalent form of dementia among the elderly population. Accumulating evidence indicates that oxidative stress plays a pivotal role in the pathogenesis of AD. Notably, elevated levels of oxidative stress have been observed in the brains of AD patients, where excessive reactive oxygen species (ROS) can cause extensive damage to lipids, proteins, and DNA, ultimately compromising neuronal structure and function. Amyloid β‑protein (Aβ) has been shown to induce mitochondrial dysfunction and calcium overload, thereby promoting the generation of ROS. This, in turn, exacerbates Aβ aggregation and enhances tau phosphorylation, leading to the formation of two pathological features of AD: extracellular Aβ plaque deposition and intracellular neurofibrillary tangles (NFTs). These events ultimately culminate in neuronal death, forming a vicious cycle. The interplay between oxidative stress and these pathological processes constitutes a core link in the pathogenesis of AD. The signaling pathways mediating oxidative stress in AD include Nrf2, RCAN1, PP2A, CREB, Notch1, NF‑κB, ApoE, and ferroptosis. Nrf2 signaling pathway serves as a key regulator of cellular redox homeostasis, exerts important antioxidant capacity and protective effects in AD. RCAN1 signaling pathway, as a calcineurin inhibitor, and modulates AD progression through multiple mechanisms. PP2A signaling pathway is involved in regulating tau phosphorylation and neuroinflammation processes. CREB signaling pathway contributes to neuroplasticity and memory formation; activation of CREB improves cognitive function and reduce oxidative stress. Notch1 signaling pathway regulates neuronal development and memory, participates in modulation of Aβ production, and interacts with Nrf2 toco-regulate antioxidant activity. NF‑κB signaling pathway governs immune and inflammatory responses; sustained activation of this pathway forms “inflammatory memory”, thereby exacerbating AD pathology. ApoE signaling pathway is associated with lipid metabolism; among its isoforms, ApoE-ε4 significantly increases the risk of AD, leading to elevated oxidative stress, abnormal lipid metabolism, and neuroinflammation. The ferroptosis signaling pathway is driven by iron-dependent lipid peroxidation, and the subsequent release of lipid peroxidation products and ROS exacerbate oxidative stress and neuronal damage. These interconnected pathways form a complex regulatory network that regulates the progression of AD through oxidative stress and related pathological cascades. In terms of therapeutic strategies targeting oxidative stress, among the drugs currently used in clinical practice for AD treatment, memantine and donepezil demonstrate significant therapeutic efficacy and can improve the level of oxidative stress in AD patients. Some compounds with antioxidant effects (such asα-lipoic acid and melatonin) have shown certain potential in AD treatment research and can be used as dietary supplements to ameliorate AD symptoms. In addition, non-drug interventions such as calorie restriction and exercise have been proven to exerted neuroprotective effects and have a positive effect on the treatment of AD. By comprehensively utilizing the therapeutic characteristics of different signaling pathways, it is expected that more comprehensive multi-target combination therapy regimens and combined nanomolecular delivery systems will be developed in the future to bypass the blood-brain barrier, providing more effective therapeutic strategies for AD.

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)method was developed for simultaneous analysis of 10 kinds of aphrodisiac drugs,including sildenafil,acetildenafil,nor-acetildenafil,homosildenafil,lodenafil carbonate,sildlenafil dimer impurity,vardenafil dimer,hydroxyacetildenafil,N-desmethylsildenafil and udenafil.By using different matrices(α-cyano-4-hydroxycinnamic acid(HCCA)and sinapic acid(SA)),the effects of solvents,amount of matrices,laser intensity and spotting methods on the peak strength and signal intensity of MALDI-TOF MS for the aphrodisiac drugs were investigated.As a result,SA was chosen as the matrix,and then dispersed in methanol-water(50∶50,V/V),and spotted by matrix firstly and sample secondly approach with the reflect linear positive mode and laser power of 60%.Under optimal conditions,the proposed MALDI-TOF MS method could obtain stable signal,high intensity and well-repeated mass spectrometric results.The results of method validation showed a linear range from 10 to 100 ng/mL,and the regression coefficients(r)were all above 0.985.The limits of detection(LOD)were 0.1-1.0 ng/mL,and the recoveries were in the range of 72.9%-109.9%for health wine,soft capsule and instant coffee samples,while the relative standard deviations(RSDs)ranged from 0.7%to 11.3%(n=3).The test results of 10 kinds of aphrodisiac drugs by MALDI-TOF MS were in accordance with the results of high-performance liquid chromatography-tandem mass spectrometry.This method exhibited high sensitivity,high accuracy,good anti-interference ability,low chemical solvents consumption and environmentally friendliness,and could be used to detect drugs without standards.The developed MALDI-TOF MS method could realize the non-target screening and detection of 10 kinds of illegally added aphrodisiac drugs in foods.

Objective To explore the role of miR-429 in synovial mesenchymal stem cell-derived exosomes(SMSC-Exos)in repairing cartilage damage in temporomandibular joint osteoarthritis(TMJ OA)by extracting SMSC-Exos from human synovial tissue and screening differentially expressed microRNA(miRNA)through transcriptome sequencing.Methods Human synovial tissues were obtained from 6 patients who underwent surgery at the First Medical Center of the Chinese PLA General Hospital from June to December 2023,including 3 patients with osteoarthritis(OA group)and 3 control patients(control group),all of whom were male.SMSC-Exos were extracted from the synovial tissues for miRNA sequencing and differential expression analysis.Further,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed on the target genes of differentially expressed miRNA to identify key functional miRNA and construct miRNA-target gene regulatory networks and protein-protein interaction(PPI)networks of target genes.An in vitro model of rabbit condylar cartilage cell inflammatory microenvironment induced by interleukin-1β(IL-1β)was established,with the control group cultured in DMEM/F12 basic medium and the inflammation-induced group cultured in DMEM/F12 basic medium containing 10 ng/ml IL-1β.RT-qPCR was used to detect the effects of overexpressed target miRNA on the mRNA expression levels of cartilage phenotype factors such as type Ⅱ collagen α1 chain(Col2a1),aggrecan(Acan),as well as inflammatory factors including a disintegrin and metalloproteinase with thrombospondin motifs 5(Adamts5)and cyclooxygenase-2(Cox-2).Results(1)SMSC-Exos were successfully isolated,cultured,and identified.(2)miRNA sequencing of SMSC-Exos from OA and control groups revealed 16 differentially expressed miRNAs(|log2FC|>2,P<0.05).Compared with control group,7 miRNAs were up-regulated and 9 were down-regulated in OA group.GO and KEGG analysis indicated that the target genes of miR-429 were mainly involved in development process,anatomical structure development,system development,cell development and differentiation,and were enriched in inflammation-related pathways such as mitogen-activated protein kinase(MAPK)and phosphatidylinositol 3-kinase-protein kinase B(PI3K-Akt).(3)Functional validation of miR-429 in the rabbit condylar cartilage cell inflammatory model showed that overexpression of miR-429 increased the mRNA expression levels of Col2a1 and Acan(P<0.05)and decreased the mRNA expression levels of Adamts5 and Cox-2(P<0.05)in the inflammation-induced group.Conclusions miRNA sequencing of SMSC-Exos isolated and identified from human synovial tissues reveals a specific miRNA expression profile in OA patients,with miR-429 significantly down-regulated.Functional validation demonstrates that overexpression of miR-429 has reparative and anti-inflammatory effects on condylar cartilage cells in an inflammatory microenvironment.

OBJECTIVE: To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics. METHODS: Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR). RESULTS: The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method. CONCLUSION Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord/cytology* ; Cell Differentiation ; Female ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Angiopoietin-1/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Stem Cell Factor/metabolism* ; Flow Cytometry ; Pregnancy

Aim To study the difference in hepatotox-icity of psoralen on normal rats and Yin-deficiency rats from the perspective of lipid metabolism,so as to help explain the mechanism of psoralen cautiously used in patients with Yin deficiency recorded in ancient books.Methods SD rats were randomly divided into the nor-mal control group(carboxymethyl cellulose-Na,CMC-Na),normal administration group(CMC-Na+psor-alen),Yin-deficiency control group(CMC-Na+thy-roxine)and Yin-deficiency administration group(CMC-Na+thyroxine+psoralen).The model of Yin-deficiency was established by thyroxine(1 mg·kg-1)for ten days,and then psoralen(200 mg·kg-1)was given for three days.The serum indexes related to liver injury were detected by automatic biochemical analy-zer,the morphological changes of liver tissue were ob-served using HE and oil red O staining,and the relative transcription levels of lipid metabolism related enzymes and mRNA of transporter and endoplasmic reticulum stress related factors were detected using Real-time PCR.Results After intragastric administration of psoralen for three days,compared with the normal group,the levels of serum alanine aminotransferase(ALT),aspartate transaminase(AST),total bile acid(TBA)and triglyeride(TG)in Yin deficiency group increased more significantly,while TC,ALB and TP de-creased more significantly,and liver HE and oil red O staining showed more obvious lipid degeneration.TG synthesis factors adrenocortical carcinoma(ACC),fatty acid synthase(FASN)and sterolregulatory element binding protein-1(SREBP-1)were down-regulated more significantly,TG transport factors mili-total pro-tein(MTP)and lipoprotein pipase(LPL)were down-regulated more evidently,fatty acid β-oxidation related factors carnitine palmitoyltransferase 1A(CPT1A),carnitine/organic cation transporter 2(OCTN2)and peroxisome proliferators-activated receptors-alpha(PPARα)were down-regulated more apparently,TC transporter adenosine triphosphate binding cassette transporter G8(ABCG8)and bile acid receptor farne-soid X receptor(FXR)were down-regulated more ob-viously,and endoplasmic reticulum stress factor activa-ting transcription factor 4(ATF4)was up-regulated more significantly.Conclusions Psoralen can cause more severe hepatotoxicity in Yin deficiency rats than that in normal administration group,and its mechanism may be related to the disorder of hepatic lipid metabo-lism,aggravation of hepatic cholestasis and steatosis,and activation of endoplasmic reticulum stress re-sponse.

Aim To study the difference in hepatotox-icity of psoralen on normal rats and Yin-deficiency rats from the perspective of lipid metabolism,so as to help explain the mechanism of psoralen cautiously used in patients with Yin deficiency recorded in ancient books.Methods SD rats were randomly divided into the nor-mal control group(carboxymethyl cellulose-Na,CMC-Na),normal administration group(CMC-Na+psor-alen),Yin-deficiency control group(CMC-Na+thy-roxine)and Yin-deficiency administration group(CMC-Na+thyroxine+psoralen).The model of Yin-deficiency was established by thyroxine(1 mg·kg-1)for ten days,and then psoralen(200 mg·kg-1)was given for three days.The serum indexes related to liver injury were detected by automatic biochemical analy-zer,the morphological changes of liver tissue were ob-served using HE and oil red O staining,and the relative transcription levels of lipid metabolism related enzymes and mRNA of transporter and endoplasmic reticulum stress related factors were detected using Real-time PCR.Results After intragastric administration of psoralen for three days,compared with the normal group,the levels of serum alanine aminotransferase(ALT),aspartate transaminase(AST),total bile acid(TBA)and triglyeride(TG)in Yin deficiency group increased more significantly,while TC,ALB and TP de-creased more significantly,and liver HE and oil red O staining showed more obvious lipid degeneration.TG synthesis factors adrenocortical carcinoma(ACC),fatty acid synthase(FASN)and sterolregulatory element binding protein-1(SREBP-1)were down-regulated more significantly,TG transport factors mili-total pro-tein(MTP)and lipoprotein pipase(LPL)were down-regulated more evidently,fatty acid β-oxidation related factors carnitine palmitoyltransferase 1A(CPT1A),carnitine/organic cation transporter 2(OCTN2)and peroxisome proliferators-activated receptors-alpha(PPARα)were down-regulated more apparently,TC transporter adenosine triphosphate binding cassette transporter G8(ABCG8)and bile acid receptor farne-soid X receptor(FXR)were down-regulated more ob-viously,and endoplasmic reticulum stress factor activa-ting transcription factor 4(ATF4)was up-regulated more significantly.Conclusions Psoralen can cause more severe hepatotoxicity in Yin deficiency rats than that in normal administration group,and its mechanism may be related to the disorder of hepatic lipid metabo-lism,aggravation of hepatic cholestasis and steatosis,and activation of endoplasmic reticulum stress re-sponse.

Objective:To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells(MSCs).Methods:Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with 60Co,and the radiation dose rate was 0.98 Gy/min.Bulk RNA-seq was performed on control and irradiated MSCs.The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis.Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro,and real-time fluorescence quantitative PCR(qPCR)was used to detect the expression differences of key regulatory factors Cebpa,Lpl and Pparg after radiation treatment.At the same time,qPCR and Western blot were used to detect the effect of inhibition of Nrf2,a key factor of antioxidant stress pathway,on the expression of key regulatory factors of adipogenesis.Moreover,the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR.Results:Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways.The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs,with high expression of Cebpa,Lpl and Pparg,as well as oxidative stress-related gene Nrf2.Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation.Notably,the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs.In addition,irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs.Conclusion:Ionizing radiation promotes adipogenesis of MSCs in mice,and oxidative stress pathway participates in this effect,blocking Nrf2 further promotes the adipogenesis of MSCs.Additionally,irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.

Objective:To establish an in vitro cell model simulating acute graft-versus-host disease(aGVHD)bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells(MSCs).Methods:The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model,respectively.Bone marrow transplantation(BMT)mouse model(n=20)was established by being injected with bone marrow cells(1 × 10'per mouse)from donor mice within 4-6 hours after receiving a lethal dose(8.0 Gy,72.76 cGy/min)of y ray general irradiation.A mouse model of aGVHD(n=20)was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells(1 × 107 per mouse)and spleen lymphocytes(2 × 106 per mouse).The blood was removed from the eyeballs and the mouse serum was aspirated on the 7th day after modeling.Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2％,5％and 10％BMT mouse serum and aGVHD mouse serum in the medium,respectively.The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining.The expression levels of related proteins PPARy and CEBPα were detected by Western blot.The expression differences of key adipogenic transcription factors including PPARy,CEBPα,FABP4 and LPL were determined by real-time quantitative PCR(RT-qPCR).Results:An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established.Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10％compared with BMT serum.Western blot experiments showed that adipogenesis-related proteins PPARy and CEBPα expressed in MSCs were down-regulated.Further RT-qPCR assay showed that the production of PPARy,CEBPα,FABP4 and LPL,the key transcription factors for adipogenic differentiation of MSC,were significantly reduced.Conclusion:The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.

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.

Traumatic supraorbital fissure syndrome (TSOFS) is a symptom complex caused by nerve entrapment in the supraorbital fissure after skull base trauma. If the compressed cranial nerve in the supraorbital fissure is not decompressed surgically, ptosis, diplopia and eye movement disorder may exist for a long time and seriously affect the patients′ quality of life. Since its overall incidence is not high, it is not familiarized with the majority of neurosurgeons and some TSOFS may be complicated with skull base vascular injury. If the supraorbital fissure surgery is performed without treatment of vascular injury, it may cause massive hemorrhage, and disability and even life-threatening in severe cases. At present, there is no consensus or guideline on the diagnosis and treatment of TSOFS that can be referred to both domestically and internationally. To improve the understanding of TSOFS among clinical physicians and establish standardized diagnosis and treatment plans, the Skull Base Trauma Group of the Neurorepair Professional Committee of the Chinese Medical Doctor Association, Neurotrauma Group of the Neurosurgery Branch of the Chinese Medical Association, Neurotrauma Group of the Traumatology Branch of the Chinese Medical Association, and Editorial Committee of Chinese Journal of Trauma organized relevant experts to formulate Chinese expert consensus on the diagnosis and treatment of traumatic supraorbital fissure syndrome ( version 2024) based on evidence of evidence-based medicine and clinical experience of diagnosis and treatment. This consensus puts forward 12 recommendations on the diagnosis, classification, treatment, efficacy evaluation and follow-up of TSOFS, aiming to provide references for neurosurgeons from hospitals of all levels to standardize the diagnosis and treatment of TSOFS.