Interventional effect of hirudin on the expression of microtubule-associated protein 2 in peripheral tissue of hematom of model rats with acute intracerebral hemorrhage

BACKGROUND： It is suspected that dissociation, destruction or synthetic disorder of microtubule-associated protein 2 （MAP-2） may participate in secondary injury of intracerebral hemorrhage （ICH）, and the reason may be related to thrombin in high concentration after ICH; therefore, the mechanism should be studied further. OBJECTIVE： To explore the effect of hirudin on expression of MAP-2 in peripheral tissue of hematom after ICH and changes of water content in brain tissue and analyze pathogenesis of thrombin in secondary injury after ICH. DESIGN ： Completely randomized grouping design and controlled animal study SEn-ING ： Department of Neurology, the First Affiliated Hospital of Jilin University MATERIALS ： The experiment was carried out in the Neurological Laboratory of the First Affiliated Hospital of Jilin University from April 2003 to April 2004. A number of 80 healthy Wistar rats, of both genders, aged 3-4 months, weighing 250-350 g, were randomly divided into 8 groups： normal control group, 6-hour ICH group, 1-day ICH group, 2-day ICH group, 3-day ICH group, 7-day ICH group, 3-day hirudin group and 7-day hirudin group with 10 in each group. Five rats from each group were selected to measure their water content, and the others were undertaken immunohistochemical stain. Hirudin was produced by Sigma Company, USA, and MAP-2 rabbit-rat polyclonal antibody was provided by Fuzhou Maixin Biotechnology Company Limited. METHODS： ① Model establishing and grouping intervention： Rats in simple ICH group were collected their blood from tails and then inserted with 50 μL non-anticoagulant auto-arterial blood into the cauda of the putamen in right brain within 5 minutes. Rats in hirudin groups were inserted with 10 U hirudin （which was diluted with saline to 20 μL） into local hematom regions within 5 minutes, and the needle was pulled out after 10 minutes. Rats in normal control group were untouched. ② Water content in peripheral tissue of hematom： Based on the ratio between dry weight and wet weight, brain tissue at bleeding side and in right frontal lobe was selected to measure dry and wet weights so as to calculate the water content [（wet weight - dry weight） /wet weight] × 100%.③ Positive expression of MAP-2： Based on immunohistochemical stain, positive MAP-2 cells were regarded as neurons and they were buffy morphological. Positive rate of MAP-2 was calculated, i.e., percentage of positive cells in each sight to total cells in all sights. ④ Statistical analysis： Data among groups were compared with one-way analysis of variance, averages were compared with SNK-q test by each other, and relation between water content and MAP-2 was analyzed with linear regression technique. MAIN OUTCOME MEASURES： Changes of water content and MAP-2 expression in peripheral tissue of hematorn at various time points after ICH and intervention of hirudin. RESULTS： All 80 rats were involved in the final analysis. ①Water content： Water content was increased at day 1, reached peak at day 3 and decreased at day 7. It was （72.31±0.32）%, （77.42±0.53）%, （78.44±0.28）%, （74.10±0.13）%, （74.85±0.51）% and （70.07±0.36）%, respectively in 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was higher than that in normal control group （63.85±0.41, q=-4.684 3 to -7.262 0, P〈 0.05）; that in 2-day and 3-day ICH groups was higher than that in 7-day ICH group （q=-3.053 4, -3.727 0, P 〈 0.05）; and that in 3-day and 7-day ICH groups was higher than that in hirudin groups at the same time points （q=-2.965 6, -2.726 4, P 〈 0.05）. ②Positive expression of MAP-2： Positive expression of MAP-2 was decreased at 6 hours after ICH, reached the lowest value at day 3 and increased at day 7. Positive rate was （78.60±0.42）%, （60.56±0.74）%, （44.60±0.26）%, （25.45±0.85）%, （32.55±0.64）%, （37.69＋0.76）%, （41.75±0.68）%, respectively in 6-hour, 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was lower than that in normal control group [（96.50±0.33）%, q= -3.074 5 to -8.128 5, P 〈 0.05]. In addition, positive cells of MAP-2 disappeared plentifully at 3-7 days after ICH, stain of positive cells were light, and only stain of plasma was positive. That in 3-day and 7-day hirudin groups was higher than that in ICH groups at the same time points （q= -3.391 8, -2.967 9, P 〈 0.05）. Moreover, positive cells of MAP-2 was formed slightly but deeply stained. ③ Results of linear regression： Water content was negatively related to MAP-2 changes at 7 days after ICH （r= -0.894 9, P〈 0.01）, i.e., water content was increased with decrease of MAP-2 expression. CONCLUSION ： The deterioration of MAP-2 may be involved in the pathogenesis of thrombin within the first week after ICH, and the local administration of hirudin can protect neurons.

Granulocyte colony-stimulating factor promotes growth of processes,growth associated protein 43 and microtubule-associated protein 2 expression in cultured rat retinal ganglion cells in vitro

Following granulocyte colony-stimulating factor （G-CSF） treatment,the growth of processes in cul-tured rat retinal ganglion cells （RGCs） in vitro,expression of growth associated protein 43,and expression of microtubule-associated protein 2 mRNA expression were significantly increased.In contrast,RhoA/Rock protein content was significantly reduced by G-CSF treatment.These results indicate that G-CSF promotes the growth of processes in RGCs and increases the expression of growth-associated protein 43 and microtubule-associated protein 2 mRNA by inhibiting the RhoA/Rock pathway,thereby benefiting axonal repair in RGCs exposed to hypoxia.

Expression changes of parvalbumin and microtubule-associated protein 2 induced by chronic constriction injury in rat dorsal root ganglia

Background Parvalbumin （PV）, as a mobile endogenous calcium buffer, plays an important role in affecting temporospatial characteristics of calcium transients and in modulating calcium homeostasis. PV is expressed in neurons in the dorsal root ganglion （DRG） and spinal dorsal horn and may be involved in synaptic transmission through regulating cytoplasm calcium concentrations. But the exact role of PV in peripheral sensory neurons remains unknown.Microtubule-associated protein 2 （MAP-2）, belonging to structural microtubule-associated protein family, is especially vulnerable to acute central nervous system （CNS） injury, and there will be rapid loss of MAP-2 at the injury site. The present study investigated the changes of PV expressing neurons and the MAP-2 neurons in the DRG after an operation for chronic constriction injury to the unilateral sciatic nerve （CCI-SN）, in order to demonstrate the possible roles of PV and MAP-2 in transmission and modulation of peripheral nociceptive information.Methods Seventy-two adult male Sprague-Dawley （SD） rats, weighing 180-220 g, were randomly divided into two groups （36 rats in each group）, the sham operation group and chronic constriction injury （CCI） group. Six rats in each group were randomly selected to receive mechanical and thermal sensitivity tests at one day before operation and 1,3, 5,7, and 14 days after surgery. After pain behavioral test, ipsilateral lumbar fifth DRGs were removed and double immunofluorescence staining was performed to assess the expression changes of PV and of MAP2 expressing neurons in the L5 DRG before or after surgery.Results The animals with CCI-SN showed obvious mechanical allodynia and thermal hyperalgesia （P＜0.05）. Both the thermal and mechanical hyperalgesia decreased to their lowest degree at 7 days after surgery compared to the baseline before surgery （P＜0.01）. In normal rats before surgery, a large number of neurons were MAP-2 single labeled cells, and just a small number of PV-expressed neurons were found. PV-positive neurons, PV-positive nerve fibers and PV-negative neurons, formed a direct or close contact for cross-talk. We used immunocytochemical staining to quantify the time course of changes to PV and MAP-2 expressing neurons in tissue, and found that the number of PV expressing neurons began to slightly decrease at 3 days after surgery, and had a significant reduction at CCI day 5, day 7 （P＜0.05）. But MAP-2 neurons significantly decreased on just the 3rd day after CCI （P＜0.05）. No changes in PV and MAP-2 expression were almost found in sham operated rats. The number of PV positive neurons, was positively correlated with the hyperalgesia threshold.Conclusions A sharp decline in MAP-2 neurons may be the early response to surgical injury, and PV positive neurons were much more effective at affecting the changes of pain behaviors, indicating that the down-regulation of PV protein could participate in, at least in part, the modulation of nociceptive transmission.

Protein tyrosine phosphatase nonreceptor 2:A New biomarker for digestive tract cancers

In this editorial,the roles of protein tyrosine phosphatase nonreceptor 2(PTPN2)in oncogenic transformation and tumor behavior and its potential as a therapeutic target in the context of gastrointestinal(GI)cancers are presented with respect to the article by Li et al published in ninth issue of the World Journal of Gastrointestinal Oncology.PTPN2 is a member of the protein tyrosine phosphatase family of signaling proteins that play crucial roles in the regulation of inflammation and immunity.Accordingly,early findings highlighted the contribution of PTPN2 to the pathogenesis of inflammatory and autoimmune disorders related to its dysfunction.On the other hand,recent studies have indicated that PTPN2 has many different roles in different cancer types,which is associated with the complexity of its regulatory network.PTPN2 dephosphorylates and inactivates EGFR,SRC family kinases,JAK1 and JAK3,and STAT1,STAT3,and STAT5 in cell type-and context-dependent manners,which indicates that PTPN2 can perform either prooncogenic or anti-oncogenic functions depending on the tumor subtype.While PTPN2 has been suggested as a potential therapeutic target in cancer treatment,to the best of ourknowledge,no clear treatment protocol has referred to PTPN2.Although there are only few studies that investigated PTPN2 expression in the GI system cancers,which is a potential limitation,the association of this protein with tumor behavior and the influence of PTPN2 on many therapy-related signaling pathways emphasize that PTPN2 could serve as a new molecular biomarker to predict tumor behavior and as a target for therapeutic intervention against GI cancers.In conclusion,more studies should be performed to better understand the prognostic and therapeutic potential of PTPN2 in GI tumors,especially in tumors resistant to therapy.

Association between serum retinol-binding protein and lower limb atherosclerosis risk in type 2 diabetes mellitus

BACKGROUND Serum retinol-binding protein(RBP)is the primary transport protein of circulating vitamin A.RBP has a crucial role in maintaining nutrient metabolism and physiologic homeostasis.Several studies have indicated that serum RBP participates in the progression of diabetes and diabetes-related complications.However,the impact of serum RBP on lower limb atherosclerosis has not been determined in individuals with type 2 diabetes mellitus(T2DM).AIM To determine the association between serum RBP and lower limb atherosclerosis in individuals with T2DM.METHODS This retrospective study enrolled 4428 eligible T2DM patients and divided the patients into non-lower limb atherosclerosis(n=1913)and lower limb atherosclerosis groups(n=2515)based on lower limb arterial ultrasonography results.At hospital admission,baseline serum RBP levels were assessed,and all subjects were categorized into three groups(Q1-Q3)based on RBP tertiles.Logistic regression,restricted cubic spline regression,subgroup analysis,and machine learning were used to assess the association between RBP levels and lower limb atherosclerosis risk.RESULTS Among 4428 individuals with T2DM,2515(56.80%)had lower limb atherosclerosis.Logistic analysis showed that lower limb atherosclerosis risk increased by 1%for every 1 unit rise in serum RBP level(odds ratio=1.01,95%confidence interval:1.00-1.02,P=0.004).Patients in the highest tertile group(Q3)had a higher lower limb atherosclerosis risk compared to the lowest tertile group(Q1)(odds ratio=1.36,95%confidence interval:1.12-1.67,P=0.002).The lower limb atherosclerosis risk gradually increased with an increase in RBP tertile(P for trend=0.005).Restricted cubic spline analysis indicated a linear correlation between serum RBP levels and lower limb atherosclerosis risk(non-linear P<0.05).Machine learning demonstrated the significance and diagnostic value of serum RBP in predicting lower limb atherosclerosis risk.CONCLUSION Elevated serum RBP levels correlate with an increased lower limb atherosclerosis risk in individuals with T2DM.

Synergistic inhibition of colorectal cancer progression by silencing Aurora A and the targeting protein for Xklp2

BACKGROUND Unraveling the pathogenesis of colorectal cancer(CRC)can aid in developing prevention and treatment strategies.Aurora kinase A(AURKA)is a key participant in mitotic control and interacts with its co-activator,the targeting protein for Xklp2(TPX2)microtubule nucleation factor.AURKA is associated with poor clinical outcomes and high risks of CRC recurrence.AURKA/TPX2 co-overexpression in cancer may contribute to tumorigenesis.Despite its pivotal role in CRC development and progression,the action mechanism of AURKA remains unclear.Further research is needed to explore the complex interplay between AURKA and TPX2 and to develop effective targeted treatments for patients with CRC.AIM To compare effects of AURKA and TPX2 and their combined knockdown on CRC cells.METHODS We evaluated three CRC gene datasets about CRC(GSE32323,GSE25071,and GSE21510).Potential hub genes associated with CRC onset were identified using the Venn,search tool for the retrieval of interacting genes,and KOBAS platforms,with AURKA and TPX2 emerging as significant factors.Subsequently,cell models with knockdown of AURKA,TPX2,or both were constructed using SW480 and LOVO cells.Quantitative real-time polymerase chain reaction,western blotting,cell counting kit-8,cell cloning assays,flow cytometry,and Transwell assays were used.RESULTS Forty-three highly expressed genes and 39 poorly expressed genes overlapped in cancer tissues compared to controls from three datasets.In the protein-protein interaction network of highly expressed genes,AURKA was one of key genes.Its combined score with TPX2 was 0.999,and their co-expression score was 0.846.In CRC cells,knockdown of AURKA,TPX2,or both reduced cell viability and colony number,while blocking G0/G1 phase and enhancing cell apoptosis.Additionally,they were weakened cell proliferation and migration abilities.Furthermore,the expression levels of B-cell lymphoma-2-Associated X,caspase 3,and tumor protein P53,and E-cadherin increased with a decrease in B-cell lymphoma-2,N-cadherin,and vimentin proteins.These effects were amplified when both AURKA and TPX2 were concurrently downregulated.CONCLUSION Combined knockdown of AURKA and TPX2 was effective in suppressing the malignant phenotype in CRC.Coinhibition of gene expression is a potential developmental direction for CRC treatment.

Influence of acupuncture with exercise training on learning and memory functions, as well as microtubule-associated protein-2 and synaptophysin expression in the hippocampal CA3 region, in a rat model of cerebral infarction

The present study was designed to determine microtubule-associated protein-2 and synaptophysin expression in the hippocampal CA3 region in a rat model of middle cerebral artery occlusion. The rats were treated with acupuncture at Baihui （GV 20）, Qubin （GB 7）, and Qianding （GV 21） points, in addition to exercise training. Results were compared with rats undergoing exercise training only. The Y-maze method and immunohistochemistry revealed decreased error frequency of passing through Y-maze, as well as significantly increased microtubule-associated protein-2 and synaptophysin expression, in the acupuncture with exercise training group compared with the model and exercise training groups after 5 weeks. Microtubule-associated protein-2 and synaptophysin expressions negatively correlated with error frequency of passing through the Y-maze. These results suggested that acupuncture combined with exercise training improved learning and memory functions in a rat model of cerebral infarction. The mechanisms of action were hypothesized to be associated with dendritic or synaptic plasticity in the ipsilateral hippocampal CA3 region.

Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

C反应蛋白/白蛋白比值对2型糖尿病合并急性心肌梗死患者远期不良心脑血管事件的预测价值研究

背景急性心肌梗死(AMI)是威胁全球公众健康的主要原因之一。虽然已有相应的再灌注治疗策略,但AMI相关的主要不良心脑血管事件(MACCEs)仍然是全世界人口死亡的原因之一。尤其合并糖尿病的AMI患者,因冠状动脉病变复杂,病变程度严重,尽早发现和判断该部分患者远期预后相对困难,因此寻找相对简便、易获得的实验室指标,有利于为2型糖尿病(T2DM)合并AMI患者经皮冠状动脉介入(PCI)术后MACCEs的预测提供依据。目的探讨血清C反应蛋白(CRP)/白蛋白(Alb)比值(CAR)对T2DM合并AMI患者PCI术后远期MACCEs的预测价值。方法纳入2014—2019年就诊于宁夏医科大学总医院心血管内科1683例T2DM合并AMI患者为研究对象,收集患者的一般临床资料与检查结果。对所有患者进行电话或门诊随访,以全因死亡、非致死性心肌梗死、再发不稳定型心绞痛、非致死性脑卒中、新发心力衰竭或心力衰竭加重再入院、再次血运重建作为MACCEs。根据患者随访期间是否发生MACCEs分为MACCEs组(508例)和非MACCEs组(1175例)。采用单因素及多因素Logistic回归分析探讨T2DM合并AMI患者MACCEs事件的影响因素。采用Kaplan-Meier法绘制患者的生存曲线,生存曲线的比较采用Log-rank检验。采用受试者工作特征(ROC)曲线分析CAR对T2DM合并AMI患者远期发生MACCEs的预测效能,使用净重分类改善指标(NRI)和综合判别指数(IDI)评价CAR对T2DM合并AMI患者预后评估的改善效果。结果1683例患者中508例(30.18%)患者发生MACCEs。多因素Logistic回归分析显示高血压病[OR(95%CI)=1.994(1.142~3.483)]、冠状动脉植入支架长度[OR(95%CI)=1.031(1.002~1.062)]、CRP[OR(95%CI)=0.950(0.915~0.986)]、Alb[OR(95%CI)=0.933(0.880~0.989)]及CAR[OR(95%CI)=5.582(1.705~18.277)]是T2DM合并AMI患者PCI术后发生MACCEs的影响因素(P<0.05)。根据CAR中位表达水平(0.86),将患者分为CAR<0.86组和CAR≥0.86组,Log-rank检验结果显示,CAR≥0.86组MACCEs发生率高于CAR<0.86组(52.68%与22.92%;χ^(2)=65.65,P<0.001)。ROC曲线显示CAR预测T2DM合并AMI患者发生MACCEs的ROC曲线下面积为0.728(95%CI=0.702~0.754),最佳截断值为0.576,灵敏度为0.617,特异度为0.747。在基线模型基础上,与CRP、Alb相比,CAR能明显改善对患者发生MACCEs的预测效果(NRI=0.377,IDI=0.166,C指数=0.690;P<0.05)。结论CAR是T2DM合并AMI患者PCI术后远期MACCEs发生风险的有效预测指标。

响应面法优化信号分子AI-2合成蛋白LuxS表达条件

群体感应是一种由自诱导物(autoinducers,AIs)介导的、细菌密度依赖的基因表达调控方式,已经被证实参与细菌多种生理功能的调控.信号分子AI-2介导的LuxS/AI-2型群体感应系统广泛存在于革兰氏阳性和阴性细菌,并备受关注.AI-2的合成依赖于S-核糖同型半胱氨酸酶(LuxS)的催化作用,而LuxS蛋白则是由luxS基因经过转录、翻译得到的.为了探索LuxS蛋白在表达菌株Escherichia coli BL21(DE3)中的最佳表达条件,从而获得较高产量且具有一定的生物活性的LuxS蛋白.首先对LuxS蛋白结构进行预测,确定E.coli BL21(DE3)作为表达菌株.其次,在单因素优化的基础上,进一步通过响应面法优化LuxS蛋白的表达条件.在菌液浓度OD 600为0.5、诱导温度为37℃、IPTG浓度为0.5 mmol/L、诱导时间为31 h条件下,获得LuxS蛋白的最高表达量.研究成功优化了LuxS蛋白在E.coli BL21(DE3)中的诱导表达条件,获得了具有生物活性的LuxS蛋白,并在后续的研究中成功合成了具有生物活性的信号分子AI-2,为体外合成AI-2奠定了基础.

内皮细胞特异性骨形态发生蛋白2对血管新生的影响:生物信息学分析和实验验证

背景:血管新生是心血管疾病的主要干预靶点,骨形态发生蛋白2具有调控血管新生作用,但内皮细胞特异性骨形态发生蛋白2对血管新生的调控作用不清楚。目的:探讨内皮细胞特异性骨形态发生蛋白2对血管新生的影响。方法:(1)生物信息学分析:通过Panglao DB公共基因表达数据库单细胞转录组荟萃分析观察骨形态发生蛋白2细胞群表达丰度和定位。血管新生小鼠和内皮(心内膜)过表达骨形态发生蛋白2小鼠转录组测序数据集探索内皮细胞骨形态发生蛋白2对血管新生信号通路的调控作用。(2)体内实验验证:建立小鼠后肢缺血模型,对比模型小鼠患侧与健侧缺血后肢7,14和21 d血流灌注情况,免疫荧光和免疫组织化学染色评估小鼠骨形态发生蛋白2和CD31的表达定位情况。(3)体外实验验证:体外培养人脐静脉内皮细胞,分为对照组、缺氧组和骨形态发生蛋白2抑制剂(Noggin蛋白)干预组,培养24 h,观察各组内皮细胞血管新生情况。结果与结论:(1)内皮细胞是表达骨形态发生蛋白2的重要细胞亚群,在血管新生内皮细胞和骨形态发生蛋白2过表达内皮细胞转录组再分析均发现骨形态发生蛋白2表达明显升高,血管新生通路明显激活。(2)缺血7 d小鼠新生血管周围骨形态发生蛋白2阳性血管明显增加(P<0.05),缺血2周骨形态发生蛋白2阳性血管明显减少(P<0.001)。(3)体外培养人脐静脉内皮细胞,缺氧干预后,内皮细胞迁移能力和血管出芽明显增加,血管新生因子血管内皮生长因子和血小板衍生生长因子的表达明显升高,Noggin明显减少了缺氧诱导的内皮细胞血管新生(P<0.001),并下调血管内皮生长因子和血小板衍生生长因子的表达(P<0.01)。(4)结果证实,内皮细胞特异性骨形态发生蛋白2具有调控血管新生作用,靶向性内皮细胞骨形态发生蛋白2可望改善血管新生。

N6-甲基腺苷甲基化相关基因IGF2BP3在肾透明细胞癌的作用研究

目的筛选肾透明细胞癌(ccRCC)中的关键N6-甲基腺苷(m^(6)A)甲基化相关基因,并研究其与ccRCC预后、ccRCC细胞的迁移和侵袭的关系。方法从癌症基因组图谱(TCGA)和基因型组织表达(GTEx)数据库中下载ccRCC和癌旁组织的RNA测序数据和临床数据,采用R4.1.1分析表达谱和预后,并筛选关键基因。收集10例ccRCC手术临床标本,采用定量PCR(qPCR)和免疫组织化学法分别检测基因mRNA和蛋白表达。在人ccRCC细胞系RCC23中,通过SiRNA敲减关键基因,并用CCK-8检测细胞的存活率,采用划痕试验和Transwell试验分别检测细胞的迁移和侵袭。结果19个m^(6)A甲基化相关基因中仅有胰岛素样生长因子ⅡmRNA结合蛋白3(IGF2BP3)在ccRCC组织中高表达,且IGF2BP3高表达与ccRCC患者预后不良呈正相关。通过qPCR和免疫组织化学法在临床标本中验证了IGF2BP3的高表达。通过小干扰RNA(siRNA)将IGF2BP3敲减后发现,RCC23细胞的存活率明显下降,且细胞的迁移和侵袭能力下降。结论IGF2BP3可能是预测ccRCC患者预后的生物标志物和潜在的药物治疗靶点。

Low-density lipoprotein receptor-related protein 2(LRP2)is required for lipid export in the midgut of the migratory locust,Locusta migratoria

Low-density lipoprotein receptor-related protein 2(LRP2)is a multifunctional endocytic receptor expressed in epithelial cells.In mammals,it acts as an endocytic receptor that mediates the cellular uptake of cholesterol-containing apolipoproteins to maintain lipid homeostasis.However,little is known about the role of LRP2 in lipid homeostasis in insects.In the present study,we investigated the function of LRP2 in the migratory locust Locusta migratoria(LmLRP2).The mRNA of LmLRP2 is widely distributed in various tissues,including integument,wing pads,foregut,midgut,hindgut,Malpighian tubules and fat body,and the amounts of LmLRP2 transcripts decreased gradually in the early stages and then increased in the late stages before ecdysis during the nymphal developmental stage.Fluorescence immunohistochemistry revealed that the LmLRP2 protein is mainly located in cellular membranes of the midgut and hindgut.Using RNAi to silence LmLRP2 caused molting defects in nymphs(more than 60%),and the neutral lipid was found to accumulate in the midgut and surface of the integument,but not in the fat body,of dsLmLRP2-treated nymphs.The results of a lipidomics analysis showed that the main components of lipids(diglyceride and triglyceride)were significantly increased in the midgut,but decreased in the fat body and hemolymph.Furthermore,the content of total triglyceride was significantly increased in the midgut,but markedly decreased in the fat body and hemolymph in dsLmLRP2-injected nymphs.Our results indicate that LmLRP2 is located in the cellular membranes of midgut cells,and is required for lipid export from the midgut to the hemolymphand fat body in locusts.

血清MMP-2、T-AOC、CRP对腹股沟疝患者TEP术后复发的预测价值

目的探究血清金属蛋白酶-2(MMP-2)、总抗氧化能力(T-AOC)、C反应蛋白(CRP)与腹股沟疝(IH)患者腹腔镜下全腹膜外修补术(TEP)术后复发的关系及预测价值。方法选取2019年1月至2022年12月在西安交通大学第一附属医院确诊并进行TEP的122例IH患者作为观察组,选取同期122例体检健康者作为对照组;对患者进行为期10个月的随访,根据患者的复发情况将其分为复发组(n=37)和痊愈组(n=85)、采用全自动生化分析仪检测血清MMP-2水平,采用化学比色法检测T-AOC水平,采用酶联免疫吸附法测定CRP的水平;IH患者TEP术后复发的影响因素采用Logistic回归分析;血清MMP-2、T-AOC、CRP对IH患者TEP术后复发的预测价值采用受试者工作特征(ROC)曲线进行分析。结果观察组血清MMP-2、CRP水平显著高于对照组(P<0.05),T-AOC水平显著低于对照组(P<0.05);复发组MMP-2、CRP显著高于痊愈组(P<0.05),T-AOC水平低于痊愈组(P<0.05);血清MMP-2、T-AOC、CRP水平预测IH患者TEP术后复发的曲线下面积(AUC)分别为0.775、0.804、0.731,三者联合预测的AUC为0.887,三者联合优于各指标单独预测(Z=2.597、1.983、3.275,P=0.009、0.047、0.001)。结论TEP术后复发IH患者血清MMP-2、T-AOC、CRP水平显著升高,可作为预测IH患者TEP术后复发的有效指标,且三者联合预测效能更高。

三结构域蛋白21对非酒精性脂肪性肝病的影响及机制研究

目的探讨三结构域蛋白21(tripartite motif containing protein 21,TRIM21)对游离脂肪酸(free fatty acid,FFA)诱导的肝细胞脂质沉积的影响,并探讨核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)及相关基因在该过程中的作用。方法用TRIM21敲低慢病毒及TRIM21过表达质粒分别感染及转染细胞,构建TRIM21基因干扰体外模型并使用qRT-PCR和Western blotting实验进行模型验证;验证FFA诱导非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)体外模型方法的可行性;干扰TRIM21表达后建立NAFLD体外模型,油红O染色检测细胞脂质沉积情况;DCFH-DA荧光探针检测细胞内活性氧水平;qRT-PCR检测Nrf2、Keap1、HO-1 mRNA表达水平;Western blottingting检测Nrf2、Keap1蛋白表达水平;免疫荧光检测Nrf2蛋白在细胞内的分布情况。结果(1)油红O染色结果表明:敲低TRIM21表达减少FFA诱导的肝细胞内脂质沉积(P<0.05),过表达TRIM21增加FFA诱导的肝细胞内脂质沉积(P<0.05)。(2)细胞内ROS水平检测结果显示:敲低TRIM21表达,胞内ROS水平降低(P<0.05),TRIM21过表达时胞内ROS水平升高(P<0.05)。(3)qRT-PCR检测结果显示:敲低TRIM21表达,Nrf2、Keap1、HO-1 mRNA表达增加(P<0.05),TRIM21过表达时,Nrf2 mRNA表达增加(P<0.01),Keap1、HO-1表达减少(P<0.05)。(4)Western blotting结果显示:敲低TRIM21表达,Nrf2蛋白表达减少(P<0.01),Keap1蛋白表达增加(P<0.001),TRIM21过表达时Nrf2蛋白表达增加(P<0.05),Keap1蛋白表达减少(P<0.0001)。(5)敲低TRIM21表达Nrf2免疫荧光染色平均荧光强度(细胞核/细胞质)较对照组增加(P<0.001),TRIM21过表达时Nrf2平均荧光强度较对照组减少(P<0.001)。结论干扰TRIM21表达可能通过调节Nrf2及相关蛋白来影响FFA诱导的细胞脂质沉积,进而影响NAFLD发生发展过程。

血清AQP4、HMGB1、FGL2水平联合颅内压和脑组织氧分压监测在创伤性脑损伤患者预后中的价值

目的探讨血清通道蛋白4(AQP4)、高迁移率族蛋白B1(HMGB1)、纤维蛋白原样蛋白2(FGL2)水平联合颅内压和脑组织氧分压(PbtO_(2))监测在创伤性脑损伤(TBI)患者预后中的价值。方法选取2022年5月—2024年5月上海交通大学附属第六人民医院南院/上海市奉贤区中心医院重症医学科诊治的TBI患者128例为研究对象,根据患者治疗后随访3个月预后情况,将其分为预后不良组(n=38)、预后良好组(n=90)。采用ELISA法检测血清AQP4、HMGB1、FGL2水平;Spearman法分析TBI不同预后患者颅内压、PbtO_(2)、血清AQP4、HMGB1、FGL2与格拉斯哥昏迷量表(GCS)评分的相关性;运用ROC曲线分析颅内压、PbtO_(2)联合血清AQP4、HMGB1、FGL2对TBI患者预后的预测价值。结果预后不良组患者颅内压高于预后良好组,GCS评分、PbtO_(2)值显著低于预后良好组(t/P=7.491/<0.001、9.882/<0.001、7.215/<0.001)。预后不良组血清AQP4、HMGB1、FGL2水平明显高于预后良好组(t/P=7.106/<0.001、7.642/<0.001、7.383/<0.001);患者PbtO_(2)与GCS评分呈显著正相关(r/P=0.523/<0.001),而颅内压、血清AQP4、HMGB1、FGL2与GCS评分呈显著负相关(r/P=-0.515/<0.001、-0.492/<0.001、-0.617/<0.001、-0.569/<0.001);血清AQP4、HMGB1、FGL2、颅内压、PbtO_(2)及五者联合预测TBI患者预后的曲线下面积(AUC)分别为0.882、0.876、0.817、0.825、0.756、0.969,五者联合优于各自单独预测TBI患者预后的价值(Z/P=2.803/0.005、2.769/0.006、3.543/<0.001、3.269/0.001、3.956/<0.001)。结论TBI患者颅内压、血清AQP4、HMGB1、FGL2水平显著升高,PbtO_(2)显著降低,与患者预后有着紧密联系,联合检测对TBI患者预后有更高的预测价值。

Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis

BACKGROUND Recently,type 2 diabetic osteoporosis(T2DOP)has become a research hotspot for the complications of diabetes,but the specific mechanism of its occurrence and development remains unknown.Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP.Polycytosine RNA-binding protein 1(PCBP1),an iron ion chaperone,is considered a protector of ferroptosis.AIM To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes.METHODS A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose(HG)and/or ferroptosis inhibitors at different concentrations and times.Transmission electron microscopy was used to examine the morpho-logical changes in the mitochondria of osteoblasts under HG,and western blotting was used to detect the expression levels of PCBP1,ferritin,and the ferroptosis-related protein glutathione peroxidase 4(GPX4).A lentivirus silenced and overex-pressed PCBP1.Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin(OPG)and osteocalcin(OCN),whereas flow cytometry was used to detect changes in reactive oxygen species(ROS)levels in each group.RESULTS Under HG,the viability of osteoblasts was considerably decreased,the number of mitochondria undergoing atrophy was considerably increased,PCBP1 and ferritin expression levels were increased,and GPX4 expression was decreased.Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1,increased the expression levels of ferritin,GPX4,OPG,and OCN,compared with the HG group.Flow cytometry results showed a reduction in ROS,and an opposite result was obtained after silencing PCBP1.CONCLUSION PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment.Moreover,PCBP1 may be a potential therapeutic target for T2DOP.

补肾活血针刺法对SAMP8小鼠认知功能及海马神经元沉默信息调节因子2调控作用的研究

目的探究补肾活血针刺法对加速衰老的小鼠模型SAMP8小鼠认知功能和海马神经元沉默信息调节因子2(SIRT2)介导的内质网蛋白4B(RTN4B)/β淀粉样前体蛋白裂解酶1(BACE1)通路的影响。方法6只SAMR1小鼠为正常对照组,18只SAMP8小鼠随机分为模型对照组、针刺治疗组、非针刺治疗组,各6只。针刺治疗组血海、膈俞施捻转泻法,肾俞、百会施捻转补法,运针1 min,留针10 min;非针刺治疗组在非经非穴点进行抓捉刺激;均干预8周。通过Morris水迷宫试验评估小鼠认知功能。采用免疫组织化学染色和酶联免疫吸附试验评估海马神经元小胶质细胞的激活和β淀粉样蛋白(Aβ)沉积。通过蛋白质免疫印迹检测SIRT2途径相关蛋白SIRT2、RTN4B、BACE1和β淀粉样前体蛋白C-末端片段(APP-CTF)的表达水平。结果与正常对照组比较,模型对照组逃避潜伏期延长,目标象限停留时间减少,穿越平台次数减少(P<0.05),海马组织Aβ42和CD68阳性面积增大(P<0.01),血清Aβ42水平增高,海马组织中SIRT2、BACE1和APP-CTF表达明显增加,RTN4B表达降低(P<0.05)。与模型对照组及非针刺治疗组比较,针刺治疗组逃避潜伏期缩短[第1天:(37.80±10.42)s比(49.80±6.14)s、(44.60±7.40)s,P<0.05;第2天:(36.80±12.69)s比(48.80±5.97)s、(44.20±7.72)s,P<0.05;第3天:(38.60±9.71)s比(51.20±5.54)s、(43.60±6.46)s,P<0.05;第4天:(36.00±11.20)s比(46.40±5.81)s、(45.20±7.36)s,P<0.05;第5天:(36.60±11.37)s比(47.80±5.31)s、(43.80±9.44)s,P<0.05],目标象限停留时间增加[(7.83±0.98)s比(1.00±0.63)s、(3.33±0.52)s,P<0.05],穿越平台次数增加[(13.33±1.03)次比(3.17±1.17)次、(7.33±0.52)次,P<0.05];海马组织Aβ42和CD68阳性面积减少(P<0.01),血清Aβ42水平显著降低[(11.38±1.57)μg/mL比(23.14±2.41)μg/mL、(17.16±1.27)μg/mL,P<0.05];海马组织中SIRT2[(1.98±0.19)比(4.21±0.31)、(3.22±0.23),P<0.05或P<0.01]、BACE1[(1.81±0.14)比(2.80±0.19)、(2.43±0.13),P<0.05或P<0.01]和APP-CTF[(2.56±0.26)比(4.53±0.33)、(3.48±0.25),P<0.05或P<0.01]表达降低,RTN4B[(0.79±0.06)比(0.27±0.03)、(0.46±0.05),P<0.05或P<0.01]表达增高。结论补肾活血针刺法通过抑制SIRT2介导的RTN4B/BACE1途径,改善SAMP8小鼠的认知功能和Aβ沉积。

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Role of Microtubule-associated Protein Tau Phosphorylation in Alzheimer's Disease

As a major microtubule-associated protein, tau plays an important role in promoting microtubule assembly and stabilizing microtubules. In Alzheimer’s disease(AD) and other tauopathies, the abnormally hyperphosphorylated tau proteins are aggregated into paired helical filaments and accumulated in the neurons with the form of neurofibrillary tangles. An imbalanced regulation in protein kinases and protein phosphatases is the direct cause of tau hyperphosphorylation. Among various kinases and phosphatases, glycogen synthase kinase-3β(GSK-3β) and protein phosphatase 2A(PP2A) are the most implicated. Accumulation of the hyperphosphorylated tau induces synaptic toxicity and cognitive impairments. Here, we review the upstream factors or pathways that can regulate GSK-3β or PP2A activity mainly based on our recent findings. We will also discuss the mechanisms that may underlie tau-induced synaptic toxicity.