Gabra3通过AKT/mTOR途径促进膀胱癌T24细胞侵袭和迁移

目的:探究γ-氨基丁酸受体亚基α-3(Gamma-aminobutyric acid receptor subunit alpha-3,Gabra3)基因对膀胱癌T24细胞凋亡、迁移和侵袭的作用及其机制。方法:TCGA数据库分析Gabra3基因在膀胱癌中的表达以及转移和生存的相关性。建立Gabra3过表达和Gabra3突变的T24细胞株,根据转染质粒不同,分为空白T24细胞(Control)、空pDONR223载体转染T24细胞(NC)、野生型Gabra3过表达T24细胞株(wt-Gabra3)、突变型Gabra3 T24细胞株(mut-Gabra3)。在回补实验中,分为转染突变型Gabra3 T24组(mut-Gabra3)、转染空pDONR223载体mut-Gabra3组(mut-Gabra3-NC)、转染野生型Gabra3过表达组(mut-Gabra3+wt-Gabra3)。用TUNEL染色检测T24细胞凋亡,细胞划痕和Transwell分别检测T24细胞迁移和侵袭,Western blot检测AKT/mTOR通路相关分子生物表达水平。结果:Gabra3基因在膀胱癌中显著高表达(P<0.05),生存曲线证实远处转移存在的患者生存期明显较短(P<0.05)。成功构建Gabra3过表达和突变的T24细胞株。与Control组相比,wt-Gabra3组细胞凋亡能力显著降低,迁移、侵袭能力显著增强(P<0.05),而mut-Gabra3组细胞凋亡显著增加,侵袭能力显著减弱(P<0.05);wt-Gabra3组细胞p-AKT、p-mTOR、p-4E-BP1、p-S6K蛋白表达均显示上凋(P<0.05),而mut-Gabra3组细胞上述蛋白无差异。在回补实验中,过表达wt-Gabra3的mut-Gabra3突变T24细胞凋亡能力受到抑制(P<0.05),迁移和侵袭能力显著增强(P<0.05),并且该组细胞AKT/mTOR通路相关分子显著激活(P<0.05)。结论:外源性的未编辑的Gabra3可以促进膀胱癌T24细胞的迁移、侵袭能力,并且可能与激活AKT/mTOR途径通路密切相关。

急性脑梗死患者血清miR-22-3p、NLRP3水平与炎性因子及预后不良的关系

目的探讨急性脑梗死(ACI)患者血清微小核糖核酸-22-3p(miR-22-3p)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)水平与炎性因子及预后不良的关系。方法选取2021年1月—2022年12月上海中医药大学附属第七人民医院神经内科收治ACI患者106例为ACI组,根据预后情况分为预后不良亚组37例和预后良好亚组69例,另选取同期体检健康者60例为健康对照组。采用实时荧光定量聚合酶链式反应检测血清miR-22-3p水平,酶联免疫吸附法检测血清NLRP3和炎性因子[白介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)]水平。通过Pearson相关性分析ACI患者血清miR-22-3p、NLRP3与IL-1β、IL-18、TNF-α的相关性。分析ACI患者预后不良的影响因素,绘制2项指标的受试者工作特征(ROC)曲线分析其预测预后的价值。结果与健康对照组比较,ACI组血清miR-22-3p水平降低,NLRP3、IL-1β、IL-18、TNF-α水平显著升高(t/P=18.698/<0.001、27.091/<0.001、30.154/<0.001、35.104/<0.001、39.834/<0.001)。Pearson相关性分析显示,ACI患者血清miR-22-3p与NLRP3、IL-1β、IL-18、TNF-α水平呈负相关(r/P=-0.733/<0.001、-0.719/<0.001、-0.683/<0.001、-0.680/<0.001),血清NLRP3与IL-1β、IL-18、TNF-α水平呈正相关(r/P=0.716/<0.001、0.715/<0.001、0.707/<0.001)。多因素Logistic回归分析显示,NIHSS评分、IL-1β、IL-18、TNF-α、NLRP3升高为ACI患者预后不良的独立危险因素[OR(95%CI)=1.244(1.034~1.497)、1.373(1.067~1.767)、1.047(1.011~1.086)、1.577(1.061~2.343)、1.084(1.022~1.149)],miR-22-3p升高为独立保护因素[OR(95%CI)=0.933(0.888~0.980)]。ROC曲线分析显示,血清miR-22-3p、NLRP3水平联合预测ACI患者预后不良的曲线下面积为0.875,大于两者单独预测的0.786、0.759(Z/P=2.405/0.016、2.517/0.012)。结论ACI患者血清miR-22-3p水平降低和NLRP3水平升高,与炎性因子水平升高和预后不良密切相关,血清miR-22-3p、NLRP3水平联合对ACI患者预后不良的预测价值较高。

n-3多不饱和脂肪酸加重FFAR4基因缺失小鼠结肠炎进程

炎症性肠病(inflammatory bowel disease, IBD)是一种慢性肠道疾病,额外营养支持有助于缓解IBD患者病情。研究表明膳食中n-3多不饱和脂肪酸具有良好的抗炎作用,而人群研究表明部分IBD患者额外补充n-3多不饱和脂肪酸无益于其疾病缓解。大规模测序发现n-3多不饱和脂肪酸受体基因FFAR4存在突变,n-3多不饱和脂肪酸在IBD治疗中的不确定结果是否与FFAR4基因的缺失有关,目前尚不清晰。通过对野生型(WT)小鼠和FFAR4基因缺失(FFAR4KO)小鼠进行结肠炎造模,并同时对两组小鼠额外补充n-3多不饱和脂肪酸,实验结果表明,在同时补充n-3多不饱和脂肪酸的情况下,相较于WT小鼠,FFAR4KO小鼠的结肠炎更加严重,表现为更多的体质量丢失、更高的疾病活动指数(disease activity index,DAI)、更高的结肠髓过氧化物酶(MPO)活性以及更高的结肠炎症因子转录水平。该研究为IBD患者得到精确营养干预提供了理论依据。

Dietary saturated fatty acid and polyunsaturated fatty acid oppositely affect hepatic NOD-like receptor protein 3 inflammasome through regulating nuclear factor-kappa B activation

AIM: To investigate the effect of different dietary fatty acids on hepatic inflammasome activation.METHODS: Wild-type C57BL/6 mice were fed either a high-fat diet or polyunsaturated fatty acid (PUFA)-enriched diet. Primary hepatocytes were treated with either saturated fatty acids (SFAs) or PUFAs as well as combined with lipopolysaccharide (LPS). The expression of NOD-like receptor protein 3 (NLRP3) inflammasome, peroxisome proliferator-activated receptor-&#x003b3; and nuclear factor-kappa B (NF-&#x003ba;B) was determined by real-time PCR and Western blot. The activity of Caspase-1 and interleukine-1&#x003b2; production were measured.RESULTS: High-fat diet-induced hepatic steatosis was sufficient to induce and activate hepatic NLRP3 inflammasome. SFA palmitic acid (PA) directly activated NLRP3 inflammasome and increased sensitization to LPS-induced inflammasome activation in hepatocytes. In contrast, PUFA docosahexaenoic acid (DHA) had the potential to inhibit NLRP3 inflammasome expression in hepatocytes and partly abolished LPS-induced NLRP3 inflammasome activation. Furthermore, a high-fat diet increased but PUFA-enriched diet decreased sensitization to LPS-induced hepatic NLRP3 inflammasome activation in vivo. Moreover, PA increased but DHA decreased phosphorylated NF-&#x003ba;B p65 protein expression in hepatocytes.CONCLUSION: Hepatic NLRP3 inflammasome activation played an important role in the development of non-alcoholic fatty liver disease. Dietary SFAs and PUFAs oppositely regulated the activity of NLRP3 inflammasome through direct activation or inhibition of NF-&#x003ba;B.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Gamma-aminobutyric acid promotes human hepatocellular carcinoma growth through overexpressed gamma-aminobutyric acid A receptor α3 subunit

AIM:To investigate the expression pattern of gamma-aminobutyric acid A(GABAA) receptors in hepatocellular carcinoma(HCC) and indicate the relationship among gamma-aminobutyric acid(GABA),gamma-aminobutyric acid A receptor α3 subunit(GABRA3) and HCC.METHODS:HCC cell line Chang,HepG2,normal liver cell line L-02 and 8 samples of HCC tissues and paired non-cancerous tissues were analyzed with semiquantitative polymerase chain reaction(PCR) for the expression of GABAA receptors.HepG2 cells were treated with gamma-aminobutyric acid(GABA) at serial concentrations(0,1,10,20,40 and 60 μmol/L),and their proliferating abilities were analyzed with the 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay,cell doubling time test,colon formation assay,cell cycle analysis and tumor planted in nude mice.Small interfering RNA was used for knocking down the endogenous GABRA3 in HepG2.Proliferating abilities of these cells treated with or without GABA were analyzed.RESULTS:We identified the overexpression of GABRA3 in HCC cells.Knockdown of endogenous GABRA3 expression in HepG2 attenuated HCC cell growth,suggesting its role in HCC cell viability.We determined the in vitro and in vivo effect of GABA in the proliferation of GABRA3-positive cell lines,and found that GABA increased HCC growth in a dose-dependent manner.Notably,the addition of GABA into the cell culture medium promoted the proliferation of GABRA3-expressing HepG2 cells,but not GABRA3-knockdown HepG2 cells.This means that GABA stimulates HepG2 cell growth through GABRA3.CONCLUSION:GABA and GABRA3 play important roles in HCC development and progression and can be a promising molecular target for the development of new diagnostic and therapeutic strategies for HCC.

STAT3 ameliorates truncated tau-induced cognitive deficits

Proteolytic cleavage of tau by asparagine endopeptidase(AEP)creates tau-N368 fragments,which may drive the pathophysiology associated with synaptic dysfunction and memory deterioration in the brain of Alzheimer’s disease patients.Nonetheless,the molecular mechanisms of truncated tau-induced cognitive deficits remain unclear.Evidence suggests that signal transduction and activator of transcription-3(STAT3)is associated with modulating synaptic plasticity,cell apoptosis,and cognitive function.Using luciferase reporter assays,electrophoretic mobility shift assays,western blotting,and immunofluorescence,we found that human tau-N368 accumulation inhibited STAT3 activity by suppressing STAT3 translocation into the nucleus.Overexpression of STAT3 improved tau-N368-induced synaptic deficits and reduced neuronal loss,thereby improving the cognitive deficits in tau-N368 mice.Moreover,in tau-N368 mice,activation of STAT3 increased N-methyl-D-aspartic acid receptor levels,decreased Bcl-2 levels,reversed synaptic damage and neuronal loss,and thereby alleviated cognitive deficits caused by tau-N368.Taken together,STAT3 plays a critical role in truncated tau-related neuropathological changes.This indicates a new mechanism behind the effect of tau-N368 on synapses and memory deficits.STAT3 can be used as a new molecular target to treat tau-N368-induced protein pathology.

MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.

Effect of ω-3 Polyunsaturated Fatty Acid on Toll-like Receptors in Patients with Severe Multiple Trauma

This study examined the effects of ω-3 polyunsaturated fatty acid（ω-3PUFA） on the expression of toll-like receptor 2（TLR2）,toll-like receptor 4（TLR4） and some related inflammatory factors in peripheral blood mononuclear cells（PBMCs） of patients with early-stage severe multiple trauma.Thirty-two patients who were admitted to the Department of Traumatic Surgery,Tongji Hospital（Wuhan,China） between May 2010 and November 2010,and diagnosed as having severe multiple trauma with a injury severity score（ISS） no less than 16,were enrolled in the study and divided into two groups at random（n=16 in each）：ω-3PUFA group and control group in which routine parenteral nutrition supplemented with ω-3PUFA or not was administered to the patients in two groups for consecutive 7 days.Peripheral blood from these patients was collected within 2 h of admission（day 0）,and 1,3,5 and 7 days after the nutritional support.PBMCs were isolated and used for detection of the mRNA and protein expression of TLR2 and TLR4 by using real-time PCR and flow cytometry respectively,the levels of NF-κB by quantum dots-based immunofluorescence assay,the levels of TNF-α,IL-2,IL-6 and COX-2 by ELISA,respectively.The results showed that the mRNA and protein expression of TLR2 and TLR4 in PBMCs was significantly lower in ω-3PUFA group than in control group 5 and 7 days after nutrition support（both P0.05）.The levels of TNF-α,IL-2,IL-6 and COX-2 were found to be substantially decreased in PBMCs in ω-3PUFA group as compared with control group at 5th and 7th day（P0.05 for all）.It was concluded that ω-3PUFA can remarkably decrease the expression of TLR2,TLR4 and some related inflammatory factors in NF-κB signaling pathway in PBMCs of patients with severe multiple trauma,which suggests that ω-3PUFA may suppress the excessive inflammatory response meditated by the TLRs/NF-κB signaling pathway.

Free fatty acids receptors 2 and 3 control cell proliferation by regulating cellular glucose uptake

BACKGROUND Colorectal cancer(CRC)is a worldwide problem,which has been associated with changes in diet and lifestyle pattern.As a result of colonic fermentation of dietary fibres,short chain free fatty acids are generated which activate free fatty acid receptors(FFAR)2 and 3.FFAR2 and FFAR3 genes are abundantly expressed in colonic epithelium and play an important role in the metabolic homeostasis of colonic epithelial cells.Earlier studies point to the involvement of FFAR2 in colorectal carcinogenesis.AIM To understand the role of short chain FFARs in CRC.METHODS Transcriptome analysis console software was used to analyse microarray data from CRC patients and cell lines.We employed short-hairpin RNA mediated down regulation of FFAR2 and FFAR3 genes,which was validated using quantitative real time polymerase chain reaction.Assays for glucose uptake and cyclic adenosine monophosphate(cAMP)generation was done along with immunofluorescence studies to study the effects of FFAR2/FFAR3 knockdown.For measuring cell proliferation,we employed real time electrical impedancebased assay available from xCELLigence.RESULTS Microarray data analysis of CRC patient samples showed a significant down regulation of FFAR2 gene expression.This prompted us to study the FFAR2 in CRC.Since,FFAR3 shares significant structural and functional homology with FFAR2,we knocked down both these receptors in CRC cell line HCT 116.These modified cell lines exhibited higher proliferation rate and were found to have increased glucose uptake as well as increased level of glucose transporter 1.Since,FFAR2 and FFAR3 signal through G protein subunit(Gαi),knockdown of these receptors was associated with increased cAMP.Inhibition of protein kinase A(PKA)did not alter the growth and proliferation of these cells indicating a mechanism independent of cAMP/PKA pathway.CONCLUSION Our results suggest role of FFAR2/FFAR3 genes in increased proliferation of colon cancer cells via enhanced glucose uptake and exclude the role of PKA mediated cAMP signalling.Alternate pathways could be involved that would ultimately result in increased cell proliferation as a result of down regulated FFAR2/FFAR3 genes.This study paves the way to understand the mechanism of action of short chain FFARs in CRC.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist inhibits apoptosis of retinal ganglion cells in a rabbit model of optic nerve injury

A rabbit model of traumatic optic nerve injury, established by occlusion of the optic nerve using a vascular clamp, was used to investigate the effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist GYKI 52466 on apoptosis of retinal ganglion cells following nerve injury. Hematoxylin-eosin staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that retinal ganglion cells gradually decreased with increasing time of optic nerve injury, while GYKI 52466 could inhibit this process. The results demonstrate that following acute optic nerve injury, apoptosis of retinal ganglion cells is a programmed process, which can be inhibited by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist.

糖尿病视网膜病变患者血清miR-20b-3p、miR-192表达与NLRP3炎症小体和预后不良的关系

目的探讨糖尿病视网膜病变(DR)患者血清微小核糖核酸(miRNA)-20b-3p、miR-192表达与NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体和预后不良的关系。方法选取2021年1月至2022年3月我院收治的DR患者165例。根据临床分级标准分为增生型DR(PDR)组89例和非增生型DR(NPDR)组76例,另选取同期我院收治的100例单纯2型糖尿病(T2DM)患者纳入T2DM组及100例体检健康志愿者纳入对照组。检测并对比4组血清miR-20b-3p、miR-192和外周血单核细胞NLRP3 mRNA、半胱氨酸蛋白酶-1(Caspase-1)mRNA表达、白细胞介素(IL)-1β、IL-18水平。采用Pearson相关性分析DR患者血清miR-20b-3p、miR-192表达与NLRP3炎症小体相关指标的相关性。DR患者出院后随访1年,根据是否发生视力残疾分为预后不良组47例和预后良好组118例,比较预后不良组和预后良好组血清miR-20b-3p、miR-192水平。收集DR患者的临床资料,单因素和多因素Logistic回归分析影响DR患者预后不良的因素。结果对照组、T2DM组、NPDR组、PDR组血清miR-20b-3p、miR-192表达依次降低,外周血单核细胞NLRP3 mRNA、Caspase-1 mRNA和血清IL-1β、IL-18水平依次升高(P<0.05)。Pearson相关性分析显示,DR患者血清miR-20b-3p、miR-192表达与NLRP3 mRNA、Caspase-1 mRNA、IL-1β、IL-18水平呈负相关(P<0.05)。随访1年,165例DR患者视力残疾发生率为28.48%(47/165)。多因素Logistic回归分析显示,T2DM病程偏长、PDR占比偏高和糖化血红白蛋白(HbA1c)、NLRP3 mRNA、Caspase-1 mRNA、IL-1β、IL-18水平升高为影响DR患者预后的独立危险因素,miR-20b-3p、miR-192升高为独立保护因素(P<0.05)。结论DR患者血清miR-20b-3p、miR-192低表达,与NLRP3炎症小体激活和预后不良密切相关,可能成为DR患者病情和预后评估的指标。

参附注射液辅助治疗对急性心肌梗死大鼠NOD样受体蛋白3/半胱氨酸天冬氨酸特异性蛋白酶1介导的细胞焦亡信号通路以及炎性因子水平的影响

目的探讨参附注射液辅助治疗对急性心肌梗死(AMI)大鼠NOD样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸特异性蛋白酶1(Caspase-1)介导的细胞焦亡信号通路以及炎性因子水平的影响。方法40只大鼠随机分为假手术组、模型组、倍他乐克组(0.9 mg/kg)和联合组(倍他乐克0.9 mg/kg联合参附注射液6 mL/kg),每组10只,连续灌胃3周。检测造模前、造模后和治疗3周时大鼠血清肌钙蛋白I(cTnI)、肌酸激酶同工酶(CK-MB)、白细胞介素(IL)-6、IL-1β和肿瘤坏死因子-α(TNF-α)水平。造模后和治疗3周时,比较各组大鼠左心室射血分数(LVEF)、左心室收缩末期内径(LVESD)、左心室舒张末期内径(LVEDD)等心脏彩超参数以及心肌梗死面积。应用实时荧光定量聚合酶链式反应(qRT-PCR)及Western blot检测心肌梗死面积NLRP3、Caspase-1的mRNA及其蛋白表达水平。结果与假手术组相比,模型组造模后和治疗3周时cTnI、CK-MB、IL-6、IL-1β、TNF-α、心肌梗死面积、NLRP3 mRNA和Caspase-1 mRNA表达量均升高,差异有统计学意义(P<0.05);与模型组相比,倍他乐克组和联合组治疗3周时cTnI、CK-MB、IL-6、IL-1β、TNF-α、心肌梗死面积、NLRP3 mRNA和Caspase-1 mRNA表达量均降低,且联合组上述指标均低于倍他乐克组,差异有统计学意义(P<0.05)。结论参附注射液辅助治疗AMI能够进一步减轻心肌细胞损伤,缩小梗死面积,抑制炎症反应和细胞焦亡活性。

Role of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor regulation in stress-induced pain chronification

Persistent postsurgical pain is a serious issue in public health, which has received increased interest in recent years. Previous studies have reported that psychological factors promote the development of chronic postsurgical pain. However, it is unclear how chronification of postsurgical pain occurs. The α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor(AMPA) phosphorylation in the central nervous system plays a critical role in synaptic plasticity and contributes to central sensitization and chronic pain development. Here, we discuss the role of AMPA receptor regulation in stress-induced pain chronification after surgery.

豹皮菌中2－Amino－3－（1，2－dicarboxyethylthio）propanoic Acid非对映异构体的分离和鉴定

从豹皮菌中的新鲜子实体的乙醇提取物中分离得到一种ＮＭＤＡ受体格抗剂，经理化常数和波谱数据的测定鉴定为２－Ａｍｉｎｏ－３－（１，２－ｄｉｃａｒｂｏｘｙｅｔｈｙｌｔｈｉｏ）ｐｒｏｐａｎｏｉｃａｃｉｄ非对映异构体的混合物，该化合物是首次从豹皮菌中得到，经进一步分离，得到两种非对映体异构体。

Effects of P2Y_1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways

Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein （GFAP） production and glial cell line-derived neurotrophic factor （GDNF） secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion （tMCAO） and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）, Western blotting, enzyme linked immunosorbent assay （ELISA） were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium （MRS2179） reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein （CREB）, and reduction of phosphorylated Janus kinase2 （JAK2） and signal transducer and activator of transcription3 （STAT3, Ser727）. After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 （inhibitor of JAK2） reduced phosphorylation of STAT3 （Ser727） as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase （PI3-K）, decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 （MEK 1/2） U0126, an important molecule of Ras/extracellular signal- regulated kinase （ERK） signaling pathway, decreased the phosphorylation of JAK2, STAT3 （Ser727）, Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

熊果酸对脂多糖诱导胃癌细胞增殖、炎症反应的影响及其与NLRP3炎症小体的关系

目的观察熊果酸(UA)对脂多糖(LPS)诱导的胃癌细胞增殖及炎症反应的影响,并探讨该作用是否与NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体有关。方法取人胃癌细胞系HGC-27、SGC-7901、BGC-823、MGC-803、AGS及正常人胃黏膜上皮细胞株GES-1,RT-q PCR法检测各细胞系中NLRP3表达。将NLRP3表达最高的胃癌细胞BGC-823分为空白对照组、LPS组、LPS+MCC950组及LPS+UA组,LPS组加入100 ng/m L的LPS、LPS+MCC950组加入50μmol/L的NLRP3抑制剂MCC950及100 ng/m L的LPS、LPS+UA组加入50μmol/L的UA及100 ng/m L的LPS,空白对照组不做任何处理。采用CCK-8法观察各组细胞增殖能力,Western blotting、RT-q PCR法检测各组细胞NLRP3蛋白和m RNA,RT-q PCR法检测各组细胞炎症因子白细胞介素1β(IL-1β)、IL-6、IL-18、肿瘤坏死因子α(TNF-α)m RNA。结果各时点细胞存活率LPS组>空白对照组>LPS+MCC950组、LPS+UA组,NLRP3蛋白和m RNA表达LPS组>LPS+MCC950组、LPS+UA组>空白对照组,IL-1β、IL-6、IL-18、TNF-αm RNA表达LPS组>LPS+MCC950组、LPS+UA组>空白对照组(P均<0.05)。结论UA可抑制LPS诱导的胃癌细胞BGC-823细胞增殖及炎症反应,其机制可能与抑制NLRP3炎症小体的激活有关。

红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化

目的探讨小鼠实验性自身免疫性脑脊髓炎(EAE)模型中,外源性和内源性红细胞膜相关蛋白(ERMAP)通过白细胞介素6/信号转录及转录激活因子3(IL-6/STAT3)/维甲酸相关孤核受体γt/(ROR-γt)信号通路对辅助性T细胞17(Th17)细胞分化的影响。方法流式细胞术验证不同浓度ERMAP-Ig融合蛋白功能;琼脂糖凝胶电泳鉴定ERMAP基因敲除小鼠。流式细胞术检测ERMAP-Ig融合蛋白在体外对Th17细胞分化影响。40只6周龄普通C57BL/6小鼠随机分为2组建立EAE模型:对照(control)-Ig和ERMAP-Ig组,每组20只;记录临床评分;流式细胞术检测EAE小鼠体内Th17细胞分化情况。40只6周龄已鉴定的野生型和ERMAP基因敲除小鼠分为2组建立EAE模型:ERMAP^(+/+)和ERMAP^(-/-)组,每组20只。记录临床评分;脊髓HE和固蓝(LFB)染色并进行组织学半定量评分。流式细胞术检测IL-17A+CD4+T细胞百分比;Western blotting检测IL-6、白细胞介素17(IL-17)、STAT3、磷酸化STAT3(p-STAT3)、ROR-γt蛋白水平表达;Real-time PCR检测IL-17、肿瘤坏死因子α(TNF-α)、IL-6、STAT3和ROR-γt的mRNA表达;ELISA检测细胞水平IL-17和TNF-α的表达。结果1.外源性ERMAP-Ig融合蛋白抑制体内外Th17细胞分化且减轻小鼠EAE症状。2.与对照组相比,ERMAP^(-/-)组EAE小鼠炎性浸润和脱髓鞘症状加重,Th17分泌IL-17A增加。3.内源性ERMAP基因敲除后,IL-17、TNF-α、IL-6、STAT3及ROR-γt表达均增加。差异均有统计学意义(P<0.05)。结论ERMAP可能通过IL-6/STAT3/ROR-γt信号通路调控Th17细胞分化,参与小鼠EAE发生发展。

The transient receptor potential melastatin 2:a new therapeutical target for Parkinson's disease?

The transient receptor potential melastatin 2 is a calcium-permeable cation channel member of the TRP family. Also known as an oxidative stress-activated channel, the transient receptor potential melastatin 2 gating mechanism is dependent on reactive oxygen species. In pathological conditions, transient receptor potential melastatin 2 is overactivated, leading to a Ca~(2+) influx that alters cell homeostasis and promotes cell death. The role of transient receptor potential melastatin 2 in neurodegenerative diseases, including Alzheimer's disease and ischemia, has already been described and reviewed. However, data on transient receptor potential melastatin 2 involvement in Parkinson's disease pathology has emerged only in recent years and the issue lacks review studies that focus specifically on this topic. The present review aims to elucidate the role of the transient receptor potential melastatin 2 channel in Parkinson's disease by reviewing, summarizing, and discussing the in vitro, in vivo, and human studies published until August 2022. Here we describe fourteen studies that evaluated the transient receptor potential melastatin 2 channel in Parkinson's disease. The Parkinson's disease model used, transient receptor potential melastatin 2 antagonist and genetic approaches, and the main outcomes reported were discussed. The studies described transient receptor potential melastatin 2 activation and enhanced expression in different Parkinson's disease models. They also evidenced protective and restorative effects when using transient receptor potential melastatin 2 antagonists, knockout, or silencing. This review provides a literature overview and suggests where there is a need for more research. As a perspective point, this review shows evidence that supports transient receptor potential melastatin 2 as a pharmacological target for Parkinson's disease in the future.

基于TLR4/MyD88/NF-κB信号通路探讨积雪草酸对急性肺损伤大鼠氧化应激和NLRP3炎症小体的影响

目的 观察积雪草酸对脂多糖(LPS)诱导的急性肺损伤大鼠氧化应激和核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体相关蛋白的影响,并探讨其相关分子机制。方法 将60只SD雄性大鼠随机分为对照组、模型组、积雪草酸低剂量组、积雪草酸高剂量组和地塞米松组,每组12只。除对照组外,其余组大鼠均采用LPS气管滴注法建立急性肺损伤模型。模型建立成功后第2天,积雪草酸低、高剂量组大鼠分别腹腔注射25 mg/kg和75 mg/kg积雪草酸溶液,地塞米松组大鼠腹腔注射2 mg/kg地塞米松注射液,对照组和模型组大鼠腹腔注射等量生理盐水,均1次/d,连续注射8周。HE染色观察大鼠肺组织病理形态,称重法计算肺组织湿/干重比值,TUNEL染色检测大鼠肺组织细胞凋亡情况,试剂盒检测大鼠支气管肺泡灌洗液中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)含量,Western blot法检测肺组织中NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、Toll样受体4(TLR4)、髓样分化蛋白88(MyD88)、核转录因子-κB(NF-κB)和磷酸化NF-κB(p-NF-κB)表达情况。结果 与对照组比较,模型组大鼠肺组织可见明显病理损伤,肺组织湿/干重比值、细胞凋亡率、支气管肺泡灌洗液中MDA含量和肺组织中NLRP3、ASC、Caspase-1、TLR4、MyD88蛋白相对表达量及p-NF-κB/NF-κB比值均显著升高(P均<0.05),支气管肺泡灌洗液中SOD、GSH-Px含量均显著降低(P均<0.05)。与模型组比较,积雪草酸低、高剂量组和地塞米松组大鼠肺组织病理损伤显著改善,肺组织湿/干重比值、细胞凋亡率、支气管肺泡灌洗液中MDA含量和肺组织中NLRP3、ASC、Caspase-1、TLR4、MyD88蛋白相对表达量及p-NF-κB/NF-κB比值均显著降低(P均<0.05),支气管肺泡灌洗液中SOD、GSH-Px含量均显著升高(P均<0.05),且积雪草酸高剂量组和地塞米松组大鼠上述指标变化均显著优于积雪草酸低剂量组(P均<0.05)。结论 积雪草酸可通过抑制氧化应激和NLRP3炎症小体激活减轻LPS诱导的急性肺损伤,其作用机制可能与调控TLR4/MyD88/NF-κB信号通路有关。