Gamma-glutamyl transferase 5 overexpression in cerebrovascular endothelial cells improves brain pathology,cognition,and behavior in APP/PS1 mice

In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.

Qingyi decoction attenuates intestinal epithelial cell injury via the calcineurin/nuclear factor of activated T-cells pathway

BACKGROUND Recent studies have demonstrated that dysfunction of the intestinal barrier is a significant contributing factor to the development of severe acute pancreatitis(SAP).A stable intestinal mucosa barrier functions as a major anatomic and functional barrier,owing to the balance between intestinal epithelial cell(IEC)proliferation and apoptosis.There is some evidence that calcium overload may trigger IEC apoptosis and that calcineurin(CaN)/nuclear factor of activated Tcells(NFAT)signaling might play an important role in calcium-mediated apoptosis.AIM To investigate the potential mechanisms underlying the therapeutic effect of Qingyi decoction(QYD)in SAP.METHODS A rat model of SAP was created via retrograde infusion of sodium deoxycholate.Serum levels of amylase,tumor necrosis factor(TNF-α),interleukin(IL)-6,D-lactic acid,and diamine oxidase(DAO);histological changes;and apoptosis of IECs were examined in rats with or without QYD treatment.The expression of the two subunits of CaN and NFAT in intestinal tissue was measured via quantitative realtime polymerase chain reaction and western blotting.For in vitro studies,Caco-2 cells were treated with lipopolysaccharide(LPS)and QYD serum,and then cell viability and intracellular calcium levels were detected.RESULTS Retrograde infusion of sodium deoxycholate increased the severity of pancreatic and intestinal pathology and the levels of serum amylase,TNF-α,and IL-6.Both the indicators of intestinal mucosa damage(D-lactic acid and DAO)and the levels of IEC apoptosis were elevated in the SAP group.QYD treatment reduced the serum levels of amylase,TNF-α,IL-6,D-lactic acid,and DAO and attenuated the histological findings.IEC apoptosis associated with SAP was ameliorated under QYD treatment.In addition,the protein expression levels of the two subunits of CaN were remarkably elevated in the SAP group,and the NFATc3 gene was significantly upregulated at both the transcript and protein levels in the SAP group compared with the control group.QYD significantly restrained CaN and NFATc3 gene expression in the intestine,which was upregulated in the SAP group.Furthermore,QYD serum significantly decreased the LPS-induced elevation in intracellular free Ca^(2+)levels and inhibited cell death.CONCLUSION QYD can exert protective effects against intestinal mucosa damage caused by SAP and the protective effects are mediated,at least partially,by restraining IEC apoptosis via the CaN/NFATc3 pathway.

Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

AIM:To investigate the role of nuclear factor of activated T cell 2(NFAT2),the major NFAT protein in peripheral T cells,in sustained T cell activation and intractable inflammation in human ulcerative colitis(UC). METHODS:We used two-dimensional gel-electrophoresis, immunohistochemistry,double immunohistochemical staining,and confocal microscopy to inspect the expression of NFAT2 in 107,15,48 and 5 cases of UC, Crohn's disease(CD),non-specific colitis,and 5 healthy individuals,respectively. RESULTS:Up-regulation with profound nucleo- translocation/activation of NFAT2 of lamina propria mononuclear cells(LPMC)of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC,as compared to CD or NC(P<0.001,Kruskal- Wallis test).Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T,but was less prominent in CD4+T cells or CD20+B cells.It was strongly associated with the disease activity,including endoscopic stage (τ=0.2145,P=0.0281)and histologic grade(τ=0.4167, P<0.001). CONCLUSION:We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis.Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity.Since activation of NFAT2is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways,our results not only provide new insights into the mechanism for sustained intractable inflammation,but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

2型糖尿病合并骨质疏松患者血清miR-9-5p和NFAT5的表达及其与骨折的关系

目的:2型糖尿病(type 2 diabetes mellitus,T2DM)是一种多病因代谢性疾病,骨质疏松(osteoporosis,OP)和骨折是其常见并发症。本研究旨在探讨T2DM合并OP患者血清中微RNA(microRNA,miR)-9-5p和核转录因子5(nuclear factor of activated T-cells 5,NFAT5)的表达水平,以及其与骨折的关系。方法:收集郑州市第七人民医院就诊的T2DM合并OP患者184例(OP组),另纳入同时间段单纯T2DM患者184例(T2DM组)。实时聚合酶链反应检测血清miR-9-5p、NFAT5表达水平。随访2年,根据新发骨折情况,将T2DM合并OP患者分为骨折组(43例)与无骨折组(141例)。Pearson法分析血清miR-9-5p、NFAT5分别与空腹血糖(fasting plasma glucose,FPG)、I型前胶原N末端前肽(procollagen I N-terminal propeptide,PINP)、空腹胰岛素(fasting insulin,FINS)、胰岛素抵抗指数(insulin resistance index,HOMA-IR)、骨密度T值、I型胶原羧基端β降解产物(type I collagen hydroxy terminal peptideβdegradation products,β-CTX)相关性,以及miR-9-5p与NFAT5的相关性;采用受试者操作特征(receiver operator characteristic,ROC)曲线评估血清miR-9-5p、NFAT5对T2DM合并OP患者骨折的预测价值,多因素logistic回归分析T2DM合并OP患者骨折的影响因素。结果:OP组血清miR-9-5p水平高于T2DM组,NFAT5水平低于T2DM组(均P<0.05)。与无骨折组相比,骨折组患者糖尿病病程、FPG、HOMA-IR、β-CTX、miR-9-5p水平均升高,而PINP、NFAT5水平均降低(均P<0.05)。骨折患者血清miR-9-5p与NFAT5水平呈负相关(r=−0.716,P<0.05);miR-9-5p水平与FPG、HOMA-IR、β-CTX均呈正相关,与PINP呈负相关(均P<0.05),而血清NFAT5水平与FPG、HOMA-IR、β-CTX均呈负相关,与PINP呈正相关(均P<0.05)。血清miR-9-5p、NFAT5单一预测T2DM合并OP患者骨折风险的曲线下面积(area under curve,AUC)分别为0.878和0.868,联合预测的AUC为0.933。β-CTX、miR-9-5p为T2DM合并OP患者骨折的危险因素,PINP、NFAT5为T2DM合并OP患者骨折的保护因素(均P<0.05)。结论:T2DM合并OP患者血清miR-9-5p表达水平升高,NFAT5表达水平降低,两者与骨折发生均有一定关系,miR-9-5p联合NFAT5对骨折预测价值更高。

白藜芦醇激活细胞外信号调节激酶5信号蛋白促进小鼠MC3T3-E1细胞增殖

背景:细胞外信号调节激酶5信号蛋白对生物体的存活不可或缺,白藜芦醇能通过多种途径促进成骨细胞增殖,但其是否能通过细胞外信号调节激酶5信号蛋白调控成骨细胞功能还需进一步验证。目的:探究细胞外信号调节激酶5对MC3T3-E1细胞增殖以及相关分泌蛋白的调控作用,进一步验证白藜芦醇通过激活细胞外信号调节激酶5完成上述过程。方法:小鼠MC3T3-E1前成骨细胞分别用完全培养基、XMD8-92(细胞外信号调节激酶5抑制剂)、表皮生长因子(细胞外信号调节激酶5激活剂)和白藜芦醇单独干预及XMD8-92+表皮生长因子、白藜芦醇+XMD8-92干预后,通过Western blot检测各组细胞内细胞外信号调节激酶5、磷酸化细胞外信号调节激酶5蛋白,增殖相关蛋白Cyclin D1、CDK4、PCNA,以及成骨细胞分泌蛋白骨保护素、核因子κB受体活化因子配体的表达情况,使用细胞免疫荧光染色检测各组细胞外信号调节激酶5、骨保护素和核因子κB受体活化因子配体荧光强度,使用EdU染色检测各组细胞增殖情况。白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间由细胞形态学观察和CCK-8实验确定。结果与结论:①细胞外信号调节激酶5信号蛋白的激活能有效促进MC3T3-E1细胞增殖、上调骨保护素/核因子κB受体活化因子配体比值;②白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间为5μmol/L,24 h;③白藜芦醇可以激活细胞外信号调节激酶5信号蛋白,进而促进成骨细胞增殖,并上调骨保护素/核因子κB受体活化因子配体比值;④研究结果表明,白藜芦醇可以通过激活细胞外信号调节激酶5信号蛋白促进MC3T3-E1细胞增殖,并通过激活细胞外信号调节激酶5信号蛋白上调骨保护素/核因子κB受体活化因子配体比值。

Inhibiting 5-hydroxytryptamine receptor 3 alleviates pathological changes of a mouse model of Alzheimer's disease

Extracellular amyloid beta(Aβ) plaques are main pathological feature of Alzheimer’s disease.However,the specific type of neuro ns that produce Aβ peptides in the initial stage of Alzheimer’s disease are unknown.In this study,we found that 5-hydroxytryptamin receptor 3A subunit(HTR3A) was highly expressed in the brain tissue of transgenic amyloid precursor protein and presenilin-1 mice(an Alzheimer’s disease model) and patients with Alzheimer’s disease.To investigate whether HTR3A-positive interneurons are associated with the production of Aβ plaques,we performed double immunostaining and found that HTR3A-positive interneurons were clustered around Aβ plaques in the mouse model.Some amyloid precursor protein-positive or β-site amyloid precursor protein cleaving enzyme-1-positive neurites near Aβ plaques were co-localized with HTR3A interneurons.These results suggest that HTR3A-positive interneurons may partially contribute to the generation of Aβ peptides.We treated 5.0-5.5-month-old model mice with tro pisetron,a HTR3 antagonist,for 8 consecutive weeks.We found that the cognitive deficit of mice was partially reversed,Aβ plaques and neuroinflammation we re remarkably reduced,the expression of HTR3 was remarkably decreased and the calcineurin/nuclear factor of activated T-cell 4 signaling pathway was inhibited in treated model mice.These findings suggest that HTR3A interneurons partly contribute to generation of Aβ peptide at the initial stage of Alzheimer’s disease and inhibiting HTR3 partly reve rses the pathological changes of Alzheimer’s disease.

Role of nuclear factor κB in multiple sclerosis and experimental autoimmune encephalomyelitis

The transcription factor nuclear factor κB（NF-κB） plays major roles in inflammatory diseases through regulation of inflammation and cell viability.Multiple sclerosis（MS） is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system（CNS）.It has been shown that NF-κB is activated in multiple cell types in the CNS of MS patients,including T cells,microglia/macrophages,astrocytes,oligodendrocytes,and neurons.Interestingly,data from animal model studies,particularly studies of experimental autoimmune encephalomyelitis,have suggested that NF-κB activation in these individual cell types has distinct effects on the development of MS.In this review,we will cover the current literature on NF-κB and the evidence for its role in the development of MS and its animal model experimental autoimmune encephalomyelitis.

Blockage of PPARδ increases the expression of inflammatory factors in 3T3-L1 cells stimulated with TNFα

Objective: To investigate the role of peroxisome proliferator-activated receptors δ (PPARδ) in inflammatory reaction and its possible mechanism in adipocyte. Methods :Lentivirus-mediated RNA interference (RNAi) was used to block the expression of PPARS in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α(TNFα, 20 ng/ml) for 4 h. The expression of PPAR8, nuclear factor κB (NFκB) and C reactive protein (CRP) were determined by Western blot analysis. Results:The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα, but had no effect on normal cells. Conclusion: PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.

Involvement of VLA-5 and VLA-6 in facilitating endothelium-oriented transmigration of hematopoietic stem/progenitor cells

目的 :研究 VLA- 5及 VLA- 6是否参与内皮细胞促进的造血干 /祖细胞定向移行。方法 :对纯化人 CD34+ 细胞进行体外移行及阻断实验 ,观察其穿移覆盖人脐静脉内皮细胞 ( HUVECs)滤膜的能力。应用四色荧光活化流式细胞术 ( FACS)检测 CD34+细胞其粘附分子及趋化因子受体CXCR- 4的表达谱。结果 :基质由来因子 ( SDF) - 1 α介导的动员外周血 ( m PB)及骨髓 ( BM)来源的CD34+ 细胞穿透覆盖 HUVECs滤膜百分率分别为 ( 5 6.6± 2 0 .1 ) %及 ( 1 5 .6± 1 .8) % ,显著高于其穿移未覆盖 HUVECs滤膜的比率。预先对 CD34+ 细胞进行抗 VLA- 5和 /或 VLA- 6中和抗体处理可消除这一促进效应。此外 ,BM来源的 CD34+细胞其穿移覆盖及未覆盖 HUVECs滤膜的能力均显著低于 m PB CD34+细胞 ,两者间穿移能力的差异与其 VLA- 5及 VLA- 6(而非 VLA- 4及趋化因子受体 CXCR- 4)抗原表达水平相关。结论 :VLA- 5和 VLA- 6参与 HUVECs促进 HS/PCs穿移能力。

川崎病患儿血清CaN、NFATc1水平与免疫球蛋白治疗反应的相关性

目的探讨川崎病患儿血清钙调神经磷酸酶(CaN)、活化T细胞核因子c1(NFATc1)水平与免疫球蛋白(IVIG)治疗反应的相关性,以期为临床改善治疗效果提供理论参考。方法采用前瞻性研究,选取平顶山市第一人民医院2018年1月至2023年1月100例川崎病患儿作为研究对象,检测入院时患儿的血清CaN、NFATc1水平,同时收集患儿的一般资料,根据IVIG治疗反应情况分为IVIG治疗敏感组与IVIG治疗无反应组。比较两组患儿的血清CaN、NFATc1水平及一般资料,采用点二列相关性检验血清CaN、NFATc1水平与IVIG治疗反应之间的关系,并采用logistic回归性检验二者对IVIG治疗反应的影响。结果100例患儿中有20例为IVIG治疗无反应,占比为20%(20/100)。IVIG治疗无反应组患儿入院时血清CaN、NFATc1及C反应蛋白(CRP)水平高于IVIG治疗敏感组(P<0.05)。经点二列相关性检验显示血清CaN、NFATc1水平与川崎病患儿IVIG治疗无反应存在正相关关系(r>0,P<0.05)。经logistic回归性分析检验显示高水平血清CaN、血清NFATc1是导致患儿IVIG治疗无反应的影响因素(P<0.05)。结论川崎病患儿IVIG治疗无反应发生风险较高,且与血清CaN、血清NFATc1存在关系,二者的高水平表达是导致IVIG治疗无反应的影响因素。

Inhibition of pacemaker activity in interstitial cells of Cajal by LPS via NF-κB and MAP kinase

AIM:To investigate lipopolysaccharide(LPS) related signal transduction in interstitial cells of Cajal(ICCs) from mouse small intestine.METHODS:For this study,primary culture of ICCs was prepared from the small intestine of the mouse.LPS was treated to the cells prior to measurement of the membrane currents by using whole-cell patch clamp technique.Immunocytochemistry was used to examine the expression of the proteins in ICCs.RESULTS:LPS suppressed the pacemaker currents of ICCs and this could be blocked by AH6809,a prostaglandin E2-EP2 receptor antagonist or NG-Nitro-Larginine Methyl Ester,an inhibitor of nitric oxide(NO) synthase.Toll-like receptor 4,inducible NO synthase or cyclooxygenase-2 immunoreactivity by specific antibodies was detected on ICCs.Catalase(antioxidant agent) had no action on LPS-induced action in ICCs.LPS actions were blocked by nuclear factor kB(NF-kB) inhibitor,actinomycin D(a gene transcription inhibitor),PD 98059(a p42/44 mitogen-activated protein kinases inhibitor) or SB 203580 [a p38 mitogen-activated protein kinases(MAPK) inhibitor].SB 203580 also blocked the prostaglandin E2-induced action on pacemaker currents in ICCs but not NO.CONCLUSION:LPS inhibit the pacemaker currents in ICCs via prostaglandin E2-and NO-dependent mechanism through toll-like receptor 4 and suggest that MAPK and NF-kB are implicated in these actions.

Role of osteoclasts in regulating hematopoietic stem and progenitor cells

Bone marrow(BM) cavities are utilized for hematopoiesis and to maintain hematopoietic stem cells(HSCs). HSCs have the ability to self-renew as well as to differentiate into multiple different hematopoietic lineage cells. HSCs produce their daughter cells throughout the lifespan of individuals and thus, maintaining HSCs is crucial for individual life. BM cavities provide a specialized microenvironment termed "niche" to support HSCs. Niches are composed of various types of cells such as osteoblasts, endothelial cells and reticular cells. Osteoclasts are unique cells which resorb bones and are required for BM cavity formation. Loss of osteoclast function or differentiation results in inhibition of BM cavity formation, an osteopetrotic phenotype. Osteoclasts are also reportedly required for hematopoietic stem and progenitor cell(HSPC) mobilization to the periphery from BM cavities. Thus, lack of osteoclasts likely results in inhibition of HSC maintenance and HSPC mobilization. However, we found that osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization by using three independent osteoclast-less animal models. In this review, I will discuss the roles of osteoclasts in hematopoietic stem cell maintenance and mobilization.

NFATc4在舌鳞状细胞癌神经侵犯诊断中的作用

目的:通过比较活化T细胞核因子胞质4(NFATc4)与S100钙结合蛋白(S100)、p75神经营养素受体(p75)对舌鳞状细胞癌(TSCC)神经侵犯(PNI)的免疫组织化学染色特点,探索NFATc4在TSCC PNI诊断中的作用。方法:收集59例TSCC病理标本,10例癌前病变为对照组,每个标本连续切片后采用免疫组织化学染色,观察NFATc4对TSCC以及神经的染色情况,并与S100和p75进行比较。结果:59例TSCC病理标本中,NFATc4阳性率为47.5%(28/59),PNI发生率为35.6%(21/59),NFATc4阳性表达组的PNI发生率高于NFATc4阴性表达组(P<0.05),NFATc4染色可见神经内膜淡染色,TSCC细胞胞质可见染色,NFATc4对神经的鉴别效果与S100和p75比较,无统计学差异(P>0.05)。结论:NFATc4的表达与PNI的发生存在关联,NFATc4能在染色神经束的同时,将TSCC组织染色,可以在同一个视野内直观地观察肿瘤与神经的关系,有利于提高PNI判读准确率,有望成为一个判断PNI的指标。

SIRT6过表达激活AMPK/Nrf2/HO-1通路抑制AngⅡ诱导的心肌细胞凋亡

[目的]探讨沉默调节蛋白6(SIRT6)过表达抑制血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞凋亡是否涉及腺苷酸活化蛋白激酶/核因子E2相关因子2/血红素加氧酶1(AMPK/Nrf2/HO-1)信号通路的激活。[方法]将实验分为4组:对照组、AngⅡ组、AngⅡ+SIRT6组和AngⅡ+空载体(EV)组,通过RT-PCR检测SIRT6的mRNA水平,MTT法检测细胞活性,流式细胞术检测细胞凋亡率,Western blot检测SIRT6、心肌细胞凋亡相关蛋白(Bax、cleaved Caspase-3、Bcl-2)、DNA损伤相关蛋白(γ-H2AX、p-ATM)及AMPK/Nrf2/HO-1信号通路相关蛋白(p-AMPK、Nrf2、HO-1)的表达水平,DCFH-DA染色法测定活性氧(ROS)含量,比较各组间上述指标的变化情况。[结果]与对照组相比,AngⅡ组SIRT6的mRNA、蛋白表达水平及细胞活性明显降低,细胞凋亡率增高,Bax、cleaved Caspase-3表达升高,Bcl-2表达降低,γ-H2AX、p-ATM蛋白表达升高,p-AMPK、Nrf2、HO-1蛋白表达降低,ROS活性增高(均P<0.01)。与AngⅡ+EV组相比,AngⅡ+SIRT6组SIRT6水平及细胞活性增高,细胞凋亡及Bax、cleaved Caspase-3表达降低,Bcl-2表达升高,γ-H2AX、p-ATM蛋白表达降低,p-AMPK、Nrf2、HO-1蛋白表达升高,ROS的活性降低(均P<0.01)。[结论]SIRT6过表达抑制AngⅡ诱导的心肌细胞凋亡与AMPK/Nrf2/HO-1信号通路的激活有关。

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

LncRNA NEAT1通过调节miR-125b-5p/IGFBP5轴对血管瘤内皮细胞增殖、凋亡、迁移的实验研究

目的 探讨长链非编码RNA(long non-coding RNA,LncRNA)核富集转录本1(nuclear enriched abundant transcript 1,NEAT1)通过调节微小核糖核酸(micro RNAs,miR)-125b-5p/胰岛素样生长因子结合蛋白5(insulinlike growth factor binding protein 5,IGFBP5)轴对血管瘤内皮细胞增殖、凋亡和迁移的影响。方法 实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)、蛋白免疫印迹(Western blot)分别检测血管瘤组织(2016年3月~2019年3月收集,n=18)、瘤旁组织样本(2016年3月~2019年3月收集,n=18)以及人脐静脉内皮细胞HUVES,人血管瘤内皮细胞HemECs,HDEC中NEAT1,miR-125b-5p及IGFBP5蛋白表达。构建沉默NEAT1,同时沉默NEAT1和miR-125b-5p的HemECs细胞系,通过细胞活力检测试剂盒(cell counting kit-8,CCK-8)、台盼蓝染色、流式细胞术、划痕愈合实验、Western blot分别观察NEAT1和miR-125b-5p对HemECs细胞增殖、凋亡、迁移及IGFBP5,增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白表达的影响;双荧光素酶报告基因实验检测NEAT1与miR-125b-5p,mi R-125b-5p与IGFBP5的关系。结果 与瘤旁组织比较,血管瘤组织中NEAT1(2.87±0.22 vs 1.00±0.00),IGFBP5蛋白(1.45±0.14 vs 0.27±0.02)表达水平升高,miR-125b-5p(0.24±0.02 vs 1.00±0.00)表达水平降低,差异具有统计学意义(t=35.400~161.220,均P <0.05);与HUVES细胞比较,HemECs,HDEC细胞中NEAT1(2.76±0.24,1.78±0.13 vs 1.00±0.00),IGFBP5蛋白(1.31±0.15,0.78±0.06 vs 0.24±0.02)表达升高,miR-125b-5p表达(0.19±0.02,0.45±0.04 vs 1.00±0.00)降低,差异具有统计学意义(t=17.320~99.204,14.697~33.680,均P<0.05),且HemECs细胞中NEAT1和IGFBP5蛋白表达量最高,miR-125b-5p表达量最低,因此,选取HemECs细胞为研究对象;与si-NC组比较,si-NEAT1组NEAT1(0.32±0.02 vs 1.01±0.12)表达、A值(0.45±0.04 vs 1.13±0.11)、细胞生长率(32.28%±2.79%vs 99.41%±0.22%)、划痕愈合率(20.33%±1.23%vs 49.24%±2.43%)及IGFBP5(0.41±0.04 vs 1.31±0.20),PCNA(0.36±0.04 vs 1.27±0.14),Bcl-2(0.48±0.04 vs 1.39±0.16)和MMP-9(0.21±0.02 vs 1.09±0.10)蛋白表达降低,mi R-125b-5p(1.87±0.15 vs 1.02±0.10)表达、细胞凋亡率(45.58%±3.34%vs 12.36%±1.07%)升高,差异具有统计学意义(t=10.809~58.755,均P <0.05);下调miR-125b-5p减弱了沉默NEAT1对HemECs细胞增殖、迁移的抑制及对细胞凋亡的促进作用(t=9.218~15.010,均P <0.05);NEAT1与miR-125b-5p,miR-125b-5p与IGFBP5存在靶向调控关系。结论 沉默NEAT1通过上调miR-125b-5p来抑制IGFBP5表达,从而抑制HemECs细胞增殖、迁移,并促进细胞凋亡。

α_(1A)-肾上腺素能受体与增强型绿色荧光蛋白标记的活化T细胞核因子2稳定共表达细胞的构建

目的建立α_(1A)-肾上腺素能受体(α_(1A)-AR)与增强型绿色荧光蛋白(EGFP)标记的活化T细胞核因子2(NFAT2)稳定共表达细胞。方法①将pcDNA3.1-α_(1A)-AR-3×FLAG重组质粒转染至U2OS-EGFPNFAT2细胞,经潮霉素B(Hygro-B)200 mg·L^(-1)压力筛选后加入α_(1A)-AR激动剂去甲肾上腺素(NE,10μmol·L^(-1))孵育30 min,通过高内涵筛选系统检测细胞核内绿色荧光强度,验证EGFP-NFAT2核转位,筛选得到稳定表达α_(1A)-AR的U2OS-EGFP-NFAT2-α_(1A)-AR细胞。②采用实时荧光定量PCR(RT-qPCR)和Western印迹法检测该细胞和对照细胞U2OS-EGFP-NFAT2中α_(1A)-AR mRNA和蛋白的表达水平。③将U2OS-EGFP-NFAT2-α_(1A)-AR细胞接种于96孔板,分别加入NE(10^(-8)~10^(-5) mol·L^(-1))或α2-AR激动剂右美托咪定(DMED,10^^(-8.8)~10^(-5) mol·L^(-1))孵育30 min,通过高内涵筛选系统检测EGFP-NFAT2核转位。④将U2OS-EGFP-NFAT2-α_(1A)-AR细胞分为溶剂对照组、α1-AR拮抗剂萘派地尔(1μmol·L^(-1))组、NE(1μmol·L^(-1))组、萘派地尔+NE(各1μmol·L^(-1)共孵育)组、α2-AR拮抗剂阿替美唑(0.1μmol·L^(-1))组、DMED(0.1μmol·L^(-1))组、阿替美唑+DMED(各0.1μmol·L^(-1)共孵育)组和萘派地尔+DMED(萘派地尔1μmol·L^(-1)与DMED 0.1μmol·L^(-1)共孵育)组,药物孵育时间均为30 min,通过高内涵筛选系统检测EGFP-NFAT2核转位,验证该细胞α_(1A)-AR功能的特异性。结果①Hygro-B压力筛选得到58株U2OS-EGFP-NFAT2-α_(1A)-AR细胞,NE 10μmol·L^(-1)孵育后,其中50号细胞核内绿色荧光强度最强,故选定其为稳定共表达α_(1A)-AR和EGFPNFAT2的U2OS-EGFP-NFAT2-α_(1A)-AR细胞。②Western印迹法结果显示,U2OS-EGFP-NFAT2-α_(1A)-AR细胞可明显表达α_(1A)-AR蛋白,而对照细胞U2OS-EGFP-NFAT2中未见α_(1A)-AR蛋白表达。RT-qPCR结果显示,该细胞在传代5~20代内α_(1A)-AR mRNA均稳定表达,其表达水平为对照细胞的500~800倍。③NE或DMED使U2OS-EGFP-NFAT2-α_(1A)-AR细胞中EGFP-NFAT2核转位明显增加,半数有效浓度(EC50)分别为5.94×10^(-7)和6.15×10^(-8) mol·L^(-1)。④与溶剂对照组和萘派地尔组比较,NE组U2OS-EGFP-NFAT2-α_(1A)-AR细胞EGFP-NFAT2核转位明显增强(P<0.01),而萘派地尔+NE组EGFP-NFAT2核转位较NE组明显减弱(P<0.01)。与溶剂对照组和阿替美唑组比较,DMED组EGFP-NFAT2核转位明显增强(P<0.01),阿替美唑+DMED组EGFP-NFAT2核转位与DMED组比较无明显差别,而萘派地尔+DMED组EGFP-NFAT2核转位较DMED组明显减弱(P<0.01)。结论成功构建稳定共表达α_(1A)-AR和EGFP-NFAT2的U2OS-EGFPNFAT2-α_(1A)-AR细胞,可用于靶向α_(1A)-AR化合物筛选和受体分子机制研究。

皮肤鳞状细胞癌患者中STAT3、NF-κB p65表达及与其分化程度的关系研究

目的探讨皮肤鳞状细胞癌(cSCC)患者信号传导与转录活化因子3(STAT3)、核因子-κB(NF-κB)p65表达与分化程度的关系。方法选取行手术治疗的58例cSCC患者,取手术切除cSCC组织标本作为研究组,距离肿瘤边缘5 cm以上的正常皮肤组织为对照组。采用免疫组化法检测STAT3、NF-κB p65表达情况,分析两者阳性表达与cSCC临床病理特征的关系。结果研究组STAT3、NF-κB p65阳性表达率较对照组高,差异有统计学意义(P<0.05);有淋巴转移者STAT3、NF-κB p65阳性表达率高于无淋巴转移者,Ⅲ~Ⅳ期STAT3、NF-κB p65阳性表达率高于Ⅰ~Ⅱ期者,且分化程度越低者STAT3、NF-κB p65阳性表达率越高,且肿瘤最大直径≥4 cm者STAT3、NF-κB p65阳性表达率高于<4 cm者,差异有统计学意义(P<0.05);STAT3、NF-κB p65阳性表达率与cSCC患者年龄、性别无关(P>0.05);STAT3、NF-κB p65阳性表达率与肿瘤最大直径、临床分期呈负相关(P<0.05),与分化程度、淋巴结转移呈正相关(P<0.05)。结论STAT3、NF-κB p65在cSCC中呈高表达,其阳性表达率与肿瘤最大直径、临床分期、分化程度、淋巴结转移密切相关,可作为判断患者病情和治疗的新靶点。

血清ANGPTL3、NFATc1水平与脑梗死患者病情严重程度、预后的关系

目的探究血管生成素样蛋白-3(ANGPTL3)、活化T细胞核因子c1(NFATc1)在脑梗死患者血清中的表达以及与脑梗死患者病情严重程度、预后的关系。方法收集唐山工人医院2021年1月至2023年1月期间进行治疗的180例脑梗死患者(脑梗死组)作为研究对象;根据NIHSS评分将患者分为轻度组(n=68),中度组(n=76),重度组(n=36);根据患mRS评分将患者分为预后良好组(n=117)和预后不良组(n=63);另选取180例同期门诊健康体检者作为对照组。比较各组血清ANGPTL3、NFATc1水平;多因素logistic回归分析脑梗死患者预后的影响因素;ROC曲线分析血清ANGPTL3、NFATc1对脑梗死患者预后的预测价值。结果脑梗死组血清ANGPTL3、NFATc1水平高于对照组(P<0.05);轻度组、中度组、重度组血清ANGPTL3、NFATc1水平依次显著升高(P<0.05);预后不良组脑梗死患者脑梗死体积、白细胞计数、ANGPTL3、NFATc1水平显著高于预后良好组(P<0.05)。回归分析显示脑梗死体积、白细胞计数、ANGPTL3、NFATc1是脑梗死患者预后的影响因素(P<0.05)。ANGPTL3、NFATc1二者联合预测脑梗死患者预后效能优于各自单独预测(Z联合检测-ANGPTL3=3.345、Z联合检测-NFATc1=2.898,P=0.001、0.004)。结论脑梗死患者血清ANGPTL3、NFATc1水平显著升高,且随着病情严重程度的增加而显著升高,二者联合对脑梗死患者预后有较高的预测价值。