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Mechanism of glutathionein improving depression-like behaviors in post-stroke depressed mice and chronic social defeat stress mice

Yuan ZHAO ; Sen LIN ; Qingwu YANG

Journal of Army Medical University.2024;46(8):775-785. doi:10.16016/j.2097-0927.202401108

Objective To investigate the potential pathogenesis of depression and the improving effect of glutathione (GSH)in the process.Methods Sixty male C57BL/6J mice (6-8 weeks old)were randomly divided into sham operation group (SHAM),post-stroke depression (PSD)group and chronic social defeat depression (CSDS)group,with 20 animals in each group.Open field test,elevated plus maze test,tail suspension test,and sucrose preference test were performed to identify whether they exhibited depressive-like behaviors.After the tissue samples of medial prefrontal cortex (mPFC)were harvested from the 2 models and control mice,neurotransmitter-targeted metabolomics sequencing was carried out.Principal component analysis (PCA)and correlation analysis were performed to explore the expression of metabolites in each group of mice to screen differential metabolites.Kyoto Encyclopedia of Genes and Genomes (KEGG)enrichment analysis was used to predict the metabolic pathways related to the depression models.The expression of key differential metabolites related to ferroptosis,including glutathione (GSH),malondialdehyde (MDA)and glutathione peroxidase 4 (GPX4).The effect of exogenous supplementation of GSH was observed by whether the depressive-like behavior of the model mice was improved.Results The results of neurotransmitter-targeted metabolomics analysis showed significant differences in the metabolic levels of the 3 groups of mice. Among the 38 metabolites detected in the mPFC,6 were specifically decreased in PSD and 4 were specifically decreased in CSDS.GSH,L-tryptophan,and L-lysine were significantly decreased in both PSD and CSDS groups (P<0.05 ).KEGG analysis indicated that the main pathways involved in the differential metabolites included GSH metabolism,beta-amino acid metabolism,and alanine,aspartate and glutamate metabolism. GSH/GSSG assay kit indicated that the ratio of reduced GSH/oxidized GSH (GSH/GSSG)in the mPFC of the 2 depression models was significantlly decreased (P<0.05 ),and the ferroptosis-related index test found that the 2 depression model mice had increased MDA level and significantly reduced GPX4-positive cells compared with the control group (P<0.05).Exogenous supplementation of GSH in the depression models extended the open arm time in the elevated plus maze test,increased the central zone time in the open field test (P<0.05),and reduced the immobility time in the tail suspension test (P<0.05),significantly improving the depressive-like behaviors.Conclusion There are 13 metabolite imbalances in the brain tissues of PSD and CSDS mice,and ferroptosis triggered by abnormal GSH metabolism may play an important role in the occurrence of depression.Exogenous supplementation of GSH improves depressive-like behaviors in mice.

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Effects of treadmill training on remyelination in hippocampus and cognitive function in rats exposed to acute plateau hypoxia

Qing ZHANG ; Yuan LIU ; Haodong LUO ; Hong SU ; Juan ZHONG ; Ce YANG ; Haiyan WANG ; Sen LI ; Ying YIN ; Botao TAN

Journal of Army Medical University.2024;46(8):786-795. doi:10.16016/j.2097-0927.202307080

Objective To determine the effects of treadmill training on the structure of hippocampal myelin and cognitive function in rats exposed to acute plateau hypoxia.Methods With 30 SPF-grade female SD rats (aged 6-8 weeks,weighing 200-220 g),6 of them were used for observation of myelin structure after injury,and the remaining 24 rats were randomly divided into control group,hypobaric hypoxia group and treadmill training group (n=8).The rats in above experimental groups were placed in a low-pressure oxygen chamber at an altitude of 6000 m for 7 consecutive days,and the rats of the control group were placed in the confined chamber for the same period without hypoxia.Then,the rats of the treadmill training group received a 4-week treadmill training scheme since the day after hypoxia.Finally,all the rats were tested for cognitive function with open field test (OFT)and Morris water maze (MWM).Transmission electron microscopy (TEM) was used to observe the changes of demyelination in the hippocampus. The expression of oligodendrocyte transcription factor 2 (Olig2)and myelin basic protein (MBP )in the hippocampal CA1 and CA3 regions was measured by immunofluorescence staining and Western blotting.Results Behavioral tests showed that the number into the central area,total distance,distance ratio in OFT and the number of platform crossings and distance to the target area in MWM were reduced in the hypobaric hypoxia group than the control group (P<0.05 ),while these indexes were increased in the treadmill training group than in the hypobaric hypoxia group (P<0.05).Immunofluorescence staining indicated that the number of Olig2 positive cells per unit area and the mean fluorescence intensity of MBP in the CA1 and CA3 regions were significantly lessen in the hypobaric hypoxia group than the control group (P<0.05 ),while these indicators were higher in the treadmill training group than the hypobaric hypoxia group (P<0.05 ).Western blotting displayed that the expression levels of Olig2 and MBP in the hippocampus were obviously lower in the hypobaric hypoxia group than the control group (P<0.01 ),while the levels were increased in the treadmill training group than the hypobaric hypoxia group (P<0.01 ).Conclusion Treadmill training promotes the number of the oligodendrocyte spectrum cells in CA1 and CA3 regions,enhances the expression of myelin-related proteins and improves myelin repair in hippocampus of hypobaric hypoxia rats,and thereby ameliorates hypoxia-induced anxiety-like behaviors and memory dysfunction.

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Mechanism of glioma stem cells with high expression of PTPRZ1 inducing TAMs polarization to M2 immunosuppressive phenotype

Lele AN ; Ying YANG ; Qing LIU ; Feiyue DOU ; Lujing WANG ; Yue CHENG ; Chao WANG ; Qianying RUAN ; Lei ZHOU ; Haitao GUO ; Weikai KONG ; Xuegang LI ; Chuan LAN ; Fei LI ; Yu SHI

Journal of Army Medical University.2024;46(8):796-803. doi:10.16016/j.2097-0927.202401021

Objective To explore the effect of glioma stem cells with high expression of protein tyrosin phosphatase receptor type Z1 (PTPRZ1 )on the phenotypic polarization and phagocytosis of tumor-associated macrophages and its regulatory mechanism.Methods GSCs and non-stem tumor cells (NSTCs) were screened out from human glioblastoma (GBM) specimens using flow cytometry,and the PTPRZ1 expression in paired GSCs and NSTCs were detected.Human peripheral blood mononuclear cells (PBMC)-derived CD14+monocytes were exposed to the conditioned medium from glioma cells or recombinant chemokine C-C motif ligand 20 (CCL20)for TAM polarization.Stable PTPRZ1 knockout GSCs (PTPRZ1-KO GSCs) were constructed using CRISPR/Cas9. TAM phagocytosis to GSCs,NSTCs,PTPRZ1-Control GSCs (PTPRZ1-Ctrl GSCs)and PTPRZ1-KO GSCs and the expression of immunosuppressive phenotype (M2) polarization marker CD163 were examined using flow cytometry.Differentially expressed genes (DEGs ) between paired GSCs and NSTCs were determined using a bulk RNA-sequencing dataset (GSE54791 )from Gene Expression Omnibus (GEO).A gene set informing worse outcome of patients with GBM was generated using The Cancer Genome Atlas (TCGA)-GBM cohort.By intersecting the aforementioned gene set with the gene set that encodes for human membrance proteins,the PTPRZ1 gene is obtained.Gene set enrichment analysis (GSEA)was used for pathway enrichment analysis to compare the differentially regulated pathways between GBMs with high or low PTPRZ1 expression.Bulk RNA sequencing,qRT-PCR and Western blotting were used to identify the DEGs between PTPRZ1-KO GSCs and PTPRZ1-Ctrl GSCs.Results GSCs were more capable of escaping from TAM phagocytosis than NSTCs (P<0.05 )and had specifically up-regulated PTPRZ1 expression.PTPRZ1-KO significantly suppressed GSCs escaping from TAM phagocytosis (P<0.01 ). GBMs with high PTPRZ1 expression showed significant inhibition of pathways mediating phagocytosis (P<0.05).The expression of CCL20 as a M2 TAM polarization chemokine was significantly down-regulated in PTPRZ1-KO GSCs (P<0.05 ).Treatment with recombinant CCL20 up-regulated the expression of CD163 as a M2 TAM marker in TAM.Conclusion PTPRZ1+GSCs mediate M2 TAM polarization and inhibit TAM phagocytosis,which may be related to the up-regulation of CCL20 in PTPRZ1+GSCs.

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Role of B cells in anti-PD-(L)1 therapy in tumor bearing mice

Junlei HOU ; Xuezhi YANG ; Fen DONG ; Haoran ZHA ; Fei YANG ; Bo ZHU

Journal of Army Medical University.2024;46(8):804-814. doi:10.16016/j.2097-0927.202401015

Objective To investigate the effect of tumor-infiltrating B cells on the therapeutic efficacy of programmed death ligand-1[PD-(L)1]inhibitors and elucidate the potential mechanisms.Methods Based on immunotherapy cohorts for melanoma patients in public databases,the relationship of B cells with progression-free survival (PFS) and response to immune checkpoint inhibitors treatment was analyzed.TC-1 and B16-OVA cells were implanted subcutaneously and in the liver in 6-8-week-old female C57BL/6 mice to establish tumor xenograft models.The effect of B cell clearance on PD-(L)1 therapy was compared.Flow cytometry was performed on the 15th day of TC-1 tumor microenvironment (TME)to confirm the number,function and phenotypic changes of T cells.Flow cytometry and quantitative real-time polymerase chain reaction (qPCR)were used to detect B cell surface molecules and cytokines.Results Based on ERP105482 data from the ICBatlas public database,high CD19 expression in the tumors was associated with longer PFS in melanoma patients (753 vs 95 d,HR=0.3,95％CI:0.13～0.65,P=0.003).B cells were significantly enriched in immunotherapy-responsive patients (P=0.01).In a mouse TC-1 liver-loaded tumor model,PD-(L)1 antibody treatment reduced tumor mass (P<0.01),whereas B-cell clearance attenuated the therapeutic efficacy.B cells enhanced PD-(L)1 antibody treatment by promoting T cell infiltration and function,and the treatment resulted in changes in B cell subsets,as evidenced by an increase in PD-1 low-expressing subsets (P<0.01).Conclusion After PD-(L)1 treatment,a decrease in PD-1 expression on B cell subsets might be one of the potential mechanisms by which B cells enhance the efficacy of PD-(L)1 therapy.

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Construction of a screening system for key intracellular survival proteins of macrophages of Staphylococcus aureus

Yaojia SHI ; Tian TIAN ; Tingrong XIONG ; Yu WANG ; Xiaokai ZHANG ; Quanming ZOU

Journal of Army Medical University.2024;46(8):815-821. doi:10.16016/j.2097-0927.202311091

Objective To establish a high-throughput screening system to obtain key Staphylococcus aureus (S.aureus)secretory proteins which required for S.aureus survival in macrophages.Methods Based on our validated eukaryotic expression vector library of S.aureus secretory proteins,DNA transfection was used to obtain an RAW264.7 macrophage array expressing S.aureus secretory proteins.After the RAW264.7 cells were infected with S.aureus,the extracellular bacteria were removed to observe the intracellular surviving situation of S.aureus.Finally,the screening results were validated by the overexpression and knockout S.aureus of corresponding secretory proteins.Results The optimal transfection dose (1.0 μg/well)of plasmids for RAW264.7,multiplicity of infection (MOI,1 .0 ),and infection time (4 h after removing extracellular bacteria of S.aureus ) were established respectively.To validate the screening results,the corresponding overexpression and knockout strains were constructed.And hypothetical protein and Serine protease E were found to promote the survival of intracellular S.aureus.Conclusion We successfully construct a screening system for key secreted secretory proteins which required for S.aureus surviving in macrophages,which may advance the study of the intracellular surviving mechanism of S.aureus.

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Establishment and stability assessment of mouse cervical heterotopic heart transplantation model with "Anchoring Node"

Zixuan LI ; Yibing FANG ; Wei CHENG

Journal of Army Medical University.2024;46(8):822-828. doi:10.16016/j.2097-0927.202312083

Objective To optimize the operational steps and processes in mouse cervical heterotopic heart transplantation by modifying the conventional cuff technique for vascular anastomosis and consequently establish a more stable cervical heterotopic heart transplantation model in mice.Methods C57BL/6 male mice (6～8 weeks old,weighing 20～24 g)were categorized into control (conventional cuff technique)and experimental groups (our"Anchoring Node"technique).Time for each surgical step,frequencies of vascular everting and vascular trimming,and the reasons for failure were recorded and compared between the 2 groups. Postoperative survival of heart allograft was determined by daily observation and touching,and the mice with the survival time>48 h were considered as successful model establishment.On the 7th and 14th days after surgery,HE staining was used to observe the pathologic changes in the vascular tissues at anastomosis.The expression of troponin T (cTnT)in the heart on the 7th day was detected with immunofluorescence assay. Results ① In the 25 hearts from each group,2 hearts from the experimental group and 8 from the control group failed,and the survival rate of heart allografts was 92％and 68％,respectively.In the experimental group,arterial and venous everting occurred at an average of 1 .16 times,with an average frequency of trimming of 0.04 times,while in the control group,arterial and venous everting was 2.00 and 2.28 times,respectively,with an average frequency of trimming of 0.21 and 0.46 times,respectively.② Significant differences were observed in the overall duration for cervical heterotopic heart transplantation (77.22±3.82 vs 87.49±8.01 min),vascular separation plus cannulation (30.06±2.68 vs 36.50±6.67 min),and cervical anastomosis (7.31±1 .08 vs 12.34±2.58 min)between the experimental and control groups (all P<0.05).③HE staining displayed that vascular patency was observed in the experimental group on the 7th and 14th days after surgery.④cTnT staining indicated no massive myocardial necrosis was seen in both groups. Conclusion Based on conventional cuff technique for mouse cervical heterotopic heart transplantation,our modified"Anchoring Node"technique ensures the stability and efficiency of one-man microscopic operation with controllable quality,with the advantages of longer postoperative survival of heart allograft,high patency rate of anastomotic vessels,good cardiac function,and fewer postoperative complications.

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Protective effect of TLR2/TLR9 agonists on pulmonary Acinetobacter baumannii infection in mice

Hao CHENG ; Yun YANG ; Hongwu SUN ; Yan DENG ; Guocheng LI ; Jingwen CAO ; Jing WEI ; Yu CHI ; Haibo LI

Journal of Army Medical University.2024;46(8):829-836. doi:10.16016/j.2097-0927.202401052

Objective To investigate the protective effect of Toll-like receptor (TLR)2/TLR9 agonists,Pam2 CSK4(Pam)and CpG ODN (CpG)on mice infected with Acinetobacter baumannii (Ab)in the lungs.Methods Female C57 mice (6～8 weeks old)were randomly divided into PBS,Pam,CpG and Pam+CpG groups.In 24 h after intranasal immunization with different doses of the corresponding agonists,the mice were given a lethal dose of Ab infection in the lungs,and the survival rates of the mice were observed.A sublethal dose lung infection model of Ab was then established,and the bacterial colonization in the blood,lungs,liver,kidneys and spleen was measured respectively in the mice after infection.HE staining was used to observe the pathological damages in the lungs and kidneys.The protective effect of the agonists in the immunized mice against Ab was examined at 1,3 and 7 d after immunization to explore the protective time window.Pam+CpG was used to stimulate A549 cells and RAW264.7 cells to investigate the killing or phagocytic effects on Ab.Results Compared to PBS,Pam+CpG treatment significantly improved the survival rate of the mice after a lethal dose of Ab lung infection (P<0.05,P<0.01 ),reduced bacterial colonization in the blood (P<0.01 ),lungs (P<0.01 ),liver (P<0.01 ),kidneys (P<0.01 )and spleen (P<0.01 )in the mice after sublethal challenge,and alleviated pathological damage caused by infection. Immunization at 1 or 3 d before infection significantly improved the survival rate (P<0.05 ),and the protective effect was the best in 3 d after immunization.Furthermore,compared to single PBS,Pam and CpG immunization,Pam+CpG significantly promoted the killing and phagocytic effects of A549 epithelial cells and RAW264.7 cells,respectively,against Ab (P<0.01 ).Conclusion Combined application of TLR2/TLR9 agonists exerts a significant protective effect on both lethal and sublethal infections of Ab,which might be by its promoting the killing or phagocytic effect of lung epithelial cells and macrophages against Ab.

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Characterization of CD8+T cell subsets in male and female non-obese diabetic mice

Shushu YANG ; Dong LIU ; Jie LI ; Mengjun ZHANG ; Li WANG

Journal of Army Medical University.2024;46(8):837-848. doi:10.16016/j.2097-0927.202312058

Objective To compare and analyze the differences in CD8+naive,effector,memory,exhausted and regulatory T cells in order to investigate the impact of gender on the differentiation fate of CD8+T cells in the context of type 1 diabetes (T1D)based on female and male non-obese diabetic (NOD)mice and healthy Institute for Cancer Research (ICR)mice.Methods The frequencies and phenotypes of CD8+T cell differentiation subsets including naive T cells (TN),central memory T cells (TCM),effector T cells(TEFF),effector precursor T cells (TEP),exhausted T cells (TEX),precursor exhausted T cells (TPEX)and regulatory T cells (Tregs)in the spleen,pancreatic draining lymph nodes (pLN)and pancreas infiltrating lymphocytes (PIL)of male and female NOD mice were detected by flow cytometry.Results The frequencies of IFN-γ+,CD107a+and CCL5+CD8+TEFF in pLN and PIL of female NOD mice were significantly higher than those of male NOD mice.However,the frequencies of CD8+TN,CD8+TCM,CD8+TEX,CD8+TPEX and CD122+CD8+Tregs subsets in the spleen were significantly decreased.While there were no significant differences in the above CD8+T cell subsets except CD8+Tregs between female and male ICR mice. Conclusion Androgen may inhibit the differentiation of memory T cells into effector T cells and promote the exhaustion of effector T cells,leading to the difference in morbidity between the male and female mice.

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Role of macrophages in pulmonary blood-air barrier impairment induced by PM2.5 exposure

Mengfei YAO ; Guozhen WANG ; Xiaonan HOU ; Duo TANG ; Zijia LIU ; Chao SHENG ; Yuchen ZHENG ; Qi ZONG ; Wenke LI ; Zhixiang ZHOU

Journal of Army Medical University.2024;46(8):849-858. doi:10.16016/j.2097-0927.202402002

Objective To investigate the role of macrophages in the process of fine particulate matter (PM2.5)exposure induced damage to pulmonary blood-air barrier.Methods Eighteen male BALB/C mice (aged of 10 weeks,weighing 24～27 g)were randomly divided into control group and low-and high-dose PM2.5 exposure groups (receiving 1 .8 and 16.2 mg/kg,respectively),with 6 mice in each group.The control group received tracheal instillations of normal saline on days 1,4,and 7,whereas the exposure groups were administered corresponding dose of PM2.5 exposure at the same time points.In 24 h after last exposure,pathological changes in the lung tissues were observed,and the contents of total protein (TP ),lactate dehydrogenase (LDH ),and alkaline phosphatase (AKP ) in bronchoalveolar lavage fluid (BALF ),and F4/80 protein level in lung tissue were measured to evaluate the blood-air barrier damage and macrophage infiltration within the lung tissues.Additionally,an in vitro model of the blood-air barrier was established using A549 alveolar epithelial cells and EA.hy926 vascular endothelial cells.In combination with a THP-1 macrophage model,the supernatant PM2.5 supernatant,macrophage supernatant,and PM2.5-macrophage supernatant were incubated with the barrier model for 24 h,respectively.Transmembrane electrical resistance (TEER),sodium fluorescein permeability of the barrier model,and LDH release from the barrier cells were measured to ascertain the extent of macrophage-mediated enhancement in barrier damage induced by PM2.5 exposure.Furthermore,the expression of inflammatory cytokines,such as TNF-α,IL-1β,IL-6,and IL-8 in the macrophages after PM2.5 exposure was analyzed with quantitative real-time PCR (qPCR)and enzyme-linked immunosorbent assay (ELISA).Results PM2.5 exposure induced lung tissue damage in mice in a dose-dependent manner,significantly elevated the contents of TP,LDH and AKP in the BALF and caused marked infiltration of macrophages into the lung tissue,especially the high-dose exposure when compared with the mice from the control group (P<0.01 ).In vitro barrier model exposure experiments showed that in comparison with the treatment of 150 and 300 μg/mL PM2.5 and macrophage supernatant,the same doses of PM2.5-macrophage supernatant resulted in notably decreased TEER and significantly enhanced permeability in the barrier model (P<0.01 ),and markedly increased LDH release from epithelial and endothelial barrier cells (P<0.01 ).Additionally,the exposure of 150 and 300μg/mL PM2.5 led to a significant up-regulation of TNF-α,IL-1β,IL-6,and IL-8 in the macrophages (P<0.01 ).Conclusion Macrophages deteriorate PM2.5-induced functional impairment of the pulmonary blood-air barrier.

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Construction and validation of a Klotho-based machine learning model for predicting all-cause mortality in chronic kidney disease

Yating WANG ; Jiachuan XIONG ; Jinghong ZHAO

Journal of Army Medical University.2024;46(8):859-867. doi:10.16016/j.2097-0927.202312065

Objective To develop and validate a machine learning (ML)model based on serum Klotho protein that can accurately predict all-cause mortality in chronic kidney disease (CKD ) patients.Methods A retrospective cohort trial was conducted on all the non-dialysis adult patients diagnosed with CKD stages 1～5 in our department from February 7,2012 to October 18,2019.They were assigned into a training set and an internal validation set in a ratio of 7:3.A total of 47 clinical features,including serum Klotho protein level,were used as variables to inform these models.Based on the training set,univariate Cox regression model was employed to screen out the possible risk factors for all-cause mortality,and Lasso-Cox regression model was further applied for the screening.Then multivariate Cox stepwise regression analysis was conducted to develop a nomogram risk prediction model for all-cause mortality,and the model performance was evaluated through internal validation.Results There were totally 400 patients enrolled in this trial,and 280 of them (including 52 dead and 228 survival)were assigned into the training set and other 120 (including 21 dead and 99 survival)into the validation set.For the constructed 5-year all-cause mortality risk prediction model,the area under the curve (AUC)value was 0.760 (95％CI:0.676～0.844)in the training set and 0.788 (95％CI:0.679～0.897)in the validation set,and the overall C-index was 0.755 (95％CI:0.685～0.826)and 0.720 (95％CI:0.614～0.826),respectively in the 2 sets.Univariate Cox regression analysis showed that age,history of cardiovascular disease(CVD),cystatin C(Cys-C),alkaline phosphatase (ALP),albumin,eosinophil (EOS)count,hemoglobin (Hb),complement C3,calcium,C-reactive protein (CRP),TNF-α and serum Klotho protein may be predictive factors for all-cause mortality (P<0.05).Multivariate Cox stepwise regression analysis finally screened age,albumin,complement C3 and serum Klotho protein as independent predictors (P<0.05).Based on these 4 predictors,a risk prediction model for all-cause mortality was constructed and validated.Conclusion A Klotho-based risk ML model for predicting all-cause mortality in CKD patients is successfully developed and validated.Advanced age is a risk factor,and higher albumin,complement C3 and serum Klotho protein levels are protective factors for all-cause mortality in CKD patients.

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