Evaluation of mRNA expression level of the ATP synthase membrane subunit c locus 1 (ATP5G1) gene in patients with schizophrenia

BackgroundSchizophrenia is a serious, complex mental disorder. The impairment of oxidative phosphorylation has a detrimental consequence on CNS function. Different ATP synthase subunits have been involved in the pathological process of various neurodegenerative disorders. Our goal was to evaluate the mRNA expression level of the ATP synthase membrane subunit c locus 1 (ATP5G1, also named ATP5MC1) gene in patients with schizophrenia.MethodsDetermination of the expression levels of ATP5G1 in plasma and peripheral blood mononuclear cells (PBMCs) were performed by real-time PCR in 90 controls and 90 patients with schizophrenia.ResultsPatients had significantly decreased ATP5G1 mRNA expression levels in both plasma and PBMCs compared to controls. The receiver operating characteristic curve was applied to detect a cut-off value of ATP5G1 expression in plasma and PBMCs. The ATP5G1 relative expression in PBMCs had better performance with a cut-off value ≤ 21 (AUC = 0.892, P < 0.001), sensitivity of 94.44%, and specificity of 72.22% in discriminating between schizophrenic patients. ATP5G1 expression in PBMCs was an independent predictor in schizophrenia.ConclusionThis study revealed a down-regulation of ATP5G1 expression in schizophrenia, precisely expression in PBMCs. That might give insight into the role of ATP5G1 gene in the pathogenesis of schizophrenia.     

慢性牙周炎合并2型糖尿病患者龈沟液Sirtuin-1、Sirtuin-6的变化及临床价值研究

摘要 目的：探讨慢性牙周炎（CP）合并2型糖尿病（T2DM）患者龈沟液沉默信息调节因子-1（Sirtuin-1）、Sirtuin-6的变化和临床价值。方法：选择2020年3月至2023年3月中国人民解放军联勤保障部队第九七〇医院收治的147例CP合并T2DM患者（T2DM组）,128例单纯CP患者（CP组）和121例健康体检者（对照组）。根据牙周检查结果将T2DM组患者分为轻度组（n=49）、中度组（n=67）、重度组（n=31）。检测受试者龈沟液中Sirtuin-1、Sirtuin-6水平以及外周血单核细胞核苷酸结合寡聚化结构域样受体热蛋白结构域亚家族成员3（NLRP3）信使核糖核酸（mRNA）、程序性细胞死亡相关斑点样蛋白（ASC）mRNA、半胱氨酸蛋白酶1（Caspase-1）mRNA表达,并评估牙周临床指标。Pearson分析CP合并T2DM患者龈沟液Sirtuin-1、Sirtuin-6水平与牙周临床指标、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达的相关性。受试者工作特征（ROC）曲线分析龈沟液Sirtuin-1、Sirtuin-6诊断CP合并T2DM的价值。结果：T2DM组龈沟液Sirtuin-1、Sirtuin-6水平低于CP组和对照组（P＜0.05）,出血指数（SBI）、牙周袋探诊深度（PD）、牙龈指数（GI）、菌斑指数（PLI）、附着丧失（AL）、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于CP组和对照组（P＜0.05）。CP组龈沟液Sirtuin-1、Sirtuin-6水平低于和对照组（P＜0.05）,GI、SBI、PLI、PD、AL、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于对照组（P＜0.05）。重度组龈沟液Sirtuin-1、Sirtuin-6水平低于中度组和轻度组（P＜0.05）,GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于中度组和轻度组（P＜0.05）。中度组龈沟液Sirtuin-1、Sirtuin-6水平低于轻度组（P＜0.05）,GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于轻度组（P＜0.05）。CP合并T2DM患者龈沟液Sirtuin-1、 Sirtuin-6水平与GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达均呈负相关（P＜0.05）。龈沟液Sirtuin-1、 Sirtuin-6诊断CP合并T2DM的曲线下面积（AUC）为0.787、0.806,联合诊断AUC为0.912,高于单独诊断。结论：CP合并T2DM患者龈沟液中Sirtuin-1、Sirtuin-6水平降低,且与牙周组织破坏程度加重、NLRP3炎症小体激活有关。龈沟液Sirtuin-1联合Sirtuin-6在CP合并T2DM诊断中具有较高价值。     

LKB1基因多态性与2型糖尿病相关性研究

目的:探讨陕西汉族人群中LKB1基因位点rs741765(380CT)及rs6510599(459GA)单核苷酸多态性(SNPs)与2型糖尿病遗传易感性及相关临床代谢指标的关系。方法:采用等位基因特异性引物PCR(SASP-PCR)对2型糖尿病患者130例及健康对照组100例进行LKB1基因内含子6 rs741765(380CT)及内含子1 rs6510599(459GA)两个位点进行基因多态性筛查,并测序鉴定,分析其基因多态性位点与2型糖尿病临床代谢指标关系。结果:rs741765(380CT)基因突变情况:2型糖尿病患者TT基因型频率显著高于健康对照组(P=0.023);TT基因2型糖尿病组中糖化血红蛋白水平及低密度脂蛋白胆固醇水平在型中明显升高(P=0.030;P=0.002);健康对照组中,空腹血糖水平在TT基因型中明显升高(P=0.011)。rs6510599(459GA)基因突变情况:AA基因型频率在2型糖尿病组及健康对照组间无显著性差异(P0.05);该基因位点与临床指标亦无相关性(P0.05)。结论:陕西汉族人群中LKB1基因内含子6 rs741765(380CT)及内含子1 rs6510599(459GA)存在基因多态性。LKB1基因内含子6 rs741765(380CT)基因多态性与2型糖尿病的发病有相关性。LKB1基因内含子1 rs6510599(459GA)基因多态性与2型糖尿病的发病无相关性。     

Interplay of LncRNAs NEAT1 and TUG1 in Incidence of Cytokine Storm in Appraisal of COVID-19 Infection

Background: In 2019, the coronavirus pandemic emerged, resulting in the highest mortality and morbidity rate globally. It has a prevailing transmission rate and continues to be a global burden. There is a paucity of data regarding the role of long non-coding RNAs (lncRNAs) in COVID-19. Therefore, the current study aimed to investigate lncRNAs, particularly NEAT1 and TUG1, and their association with IL-6, CCL2, and TNF-α in COVID-19 patients with moderate and severe disease.Methods: The study was conducted on 80 COVID-19 patients (35 with severe and 45 with moderate infection) and 40 control subjects. Complete blood count (CBC), D-dimer assay, serum ferritin, and CRP were assayed. qRT-PCR was used to measure RNAs and lncRNAs.Results: NEAT1 and TUG1 expression levels were higher in COVID-19 patients compared with controls (P<0.001). Furthermore, CCL2, IL-6, and TNF-α expressions were higher in COVID-19 patients compared to controls (P<0.001). CCL2 and IL-6 expression levels were significantly higher in patients with severe compared to those with moderate COVID-19 infection (P<0.001). IL-6 had the highest accuracy in distinguishing COVID-19 patients (AUC=1, P<0.001 at a cutoff of 0.359), followed by TUG1 (AUC=0.999, P<0.001 at a cutoff of 2.28). NEAT1 and TUG1 had significant correlations with the measured cytokines, and based on the multivariate regression analysis, NEAT1 is the independent predictor for survival in COVID-19 patients (P=0.02).Conclusion: In COVID-19 patients, significant overexpression of NEAT1 and TUG1 was observed, consistent with cytokine storm. TUG1 could be an efficient diagnostic biomarker, whereas NEAT1 was an independent predictor for overall survival.     

FGFR1通过调控PI3K/AKT/mTOR信号通路介导非小细胞肺癌对吉非替尼的耐药

目的:探讨成纤维细胞生长因子受体1(FGFR1)诱导非小细胞肺癌(NSCLC)吉非替尼获得性耐药的机制。方法:用吉非替尼诱导PC9细胞构建耐药细胞株PC9/GR,用CCK-8、平板克隆形成、transwell技术检测细胞的增殖和迁移能力,用流式细胞术检测细胞的凋亡状况,qRT-PCR、免疫荧光和蛋白免疫印迹技术检测基因表达水平。进一步采用FGFR1抑制剂PD173074或si RNA-FGFR1处理PC9/GR细胞,检测细胞的增殖、迁移、克隆形成能力的变化及Akt、p-Akt、m TOR和p-mTOR表达的变化。结果:PC9/GR细胞的增殖、迁移及对吉非替尼的耐受能力显著增强;FGFR1在PC9/GR细胞中的表达水平显著升高;用PD173074处理PC9细胞后,其增殖、迁移能力及对吉非替尼的耐受能力显著下降;敲低FGFR1后Akt和m TOR的磷酸化水平显著下降。结论:FGFR1通过PI3K/AKT/mTOR信号通路介导非小细胞肺癌对吉非替尼的耐药。     

Vitamin D Deficiency—Prognostic Marker or Mortality Risk Factor in End Stage Renal Disease Patients with Diabetes Mellitus Treated with Hemodialysis—A Prospective Multicenter Study

BackgroundEnd stage renal disease (ESRD) patients on renal replacement therapy (RRT) with diabetes mellitus (DM) have a higher mortality rate and an increase prevalence of vitamin D deficiency compared to those without DM. It is still debated if vitamin D deficiency is a risk factor or a prognostic marker for mortality in these patients. This study investigated the prevalence of vitamin D deficiency and its impact on all-cause mortality in HD patients with DM.MethodsOur prospective non-interventional cohort study included 600 patients on hemodialysis therapy (HD) (median aged 56, interquartile range (19) years, 332 (55.3%) males) recruited from 7 HD centers, from all main geographical regions of Romania. The prevalence of DM was 15.3%. They were then followed regarding: dialysis duration, dialysis efficiency, renal anemia, CKD-MBD, inflammatory status and comorbidities: coronary artery disease (CAD), peripheral vascular disease (PVD) and stroke. The deficiency of 25-OH vitamin D was defined as a value lower than12 ng/mL.ResultsPatients were followed for 3 years. The overall 3 year mortality was 25.5% (153 individuals), being higher in patients with DM as compared to those without DM (33.7% vs. 24.0%; P = 0.049). The time-related prognosis was also influenced by the presence of DM, at the survival analysis resulting in a HR of 1.52 [1.03 to 2.26] 95% CI, P = 0.037, for death in dialyzed patients with DM. In DM patients, 25-OH vitamin D deficiency was significantly higher (37.0% compared to 24.0%, P = 0.009). Furthermore, in patients with DM we observed a shorter dialysis duration (2 vs. 3 years, P<0.001) and a lower intact parathyroid hormone (iPTH) (258.0 pg/ml vs. 441.9 pg/ml, P = 0.002). Regarding the presence of comorbidities at the inclusion in the study, the presence of diabetes in dialyzed patients was associated with increased prevalence of CAD (87.0% vs. 58.1%, P<0.001), PVD (67.4% vs. 17.3%, P<0.001) and history of stroke (29.3% vs. 14.0%, P<0.001). In patients with DM the presence of 25-OH vitamin D deficiency increased the probability of death (50.0% vs. 24.1%; P = 0.011). In multiple Cox proportional hazards analysis, vitamin D deficiency remained an independent predictor for mortality in dialysis patients with DM (HR = 1.71, 95% CI 1.21 to 2.43, P = 0.003). In the same time, multiple Cox proportional hazards analysis showed that age (HR = 1.02 per one year increase, P = 0.004), CAD (HR = 1.55, P = 0.046) and PVD (HR = 1.50, P = 0.029) were independent predictors for mortality in dialysis patients with DM.ConclusionsESRD patients with DM treated with HD have a higher overall mortality than non-DM patients. Vitamin D deficiency is significantly more prevalent in HD patients with DM. Low 25-OH vitamin D levels were associated with increased all-cause mortality in these patients. According to our data, in HD patients with DM, screening for vitamin D deficiency (and its correction) should be mandatory for an optimal risk reduction strategy.     

红景天苷调节AMPK/mTOR/ULK1信号通路对结肠癌SW480细胞裸鼠的肝脏损伤的影响

摘要 目的：探讨红景天苷（Sal）调节单磷酸腺苷活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）/Unc51样激酶1（ULK1）信号通路对结肠癌SW480细胞裸鼠肝脏损伤的影响。方法：通过皮下注射SW480细胞悬浮液建立肝转移裸鼠模型,将造模后的裸鼠随机分为模型组、Sal低剂量（Sal-L,50 mg/kg Sal）组、Sal中剂量（Sal-M,100 mg/kg Sal）组、Sal高剂量（Sal-H,200 mg/kg Sal）组,Sal-H+AMPK抑制剂（Compound C,200 mg/kg Sal+10 mg/kg Compound C）组,以未接种SW480细胞悬液的裸鼠作为对照组。腹部主动脉取血,检测裸鼠血清中丙氨酸氨基转移酶（AST）、天冬氨酸氨基转移酶（ALT）水平;处死裸鼠,检测肝转移瘤数目及肝脏重量;HE染色观察肝脏组织病理变化;qRT-PCR检测肝脏组织中AMPK、mTOR、ULK1 mRNA表达水平;Western blot检测肝脏组织中自噬（Beclin1、p62）蛋白及通路相关蛋白表达。结果：与对照组相比,模型组裸鼠组织中出现肝转移瘤,肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著增加（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著降低（P<0.05）;与模型组相比,Sal-L、Sal-M、Sal-H组肝转移瘤数目、肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著降低（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著增加（P<0.05）;与Sal-H组相比,Sal-H+Compound C组肝转移瘤数目、肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著增加（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著降低（P<0.05）。结论：Sal可通过减少裸鼠肝转移瘤形成,保护裸鼠肝脏,其机制可能与激活AMPK/mTOR/ULK1信号通路,促进肝脏自噬有关。     

Mechanistic target of rapamycin and an extracellular signaling-regulated kinases 1 and 2 signaling participate in the process of acetate regulating lipid metabolism and hormone-sensitive lipase expression

ObjectiveAcetate plays an important role in host lipid metabolism. However, the network of acetate-regulated lipid metabolism remains unclear. Previous studies show that mitogen-activated protein kinases (MAPKs) and mechanistic target of rapamycin (mTOR) play a crucial role in lipid metabolism. We hypothesize that acetate could affect MAPKs and/or mTOR signaling and then regulate lipid metabolism. The present study investigated whether any cross talk occurs among MAPKs, mTOR and acetate in regulating lipid metabolism.MethodsThe ceramide C6 (an extracellular signaling-regulated kinases 1 and 2 [ERK1/2] activator) and MHY1485 (a mTOR activator) were used to treat rabbit adipose-derived stem cells (ADSCs) with or without acetate, respectively.ResultsIt indicated that acetate (9 mM) treatment for 48 h decreased the lipid deposition in rabbit ADSCs. Acetate treatment decreased significantly phosphorylated protein levels of ERK1/2 and mTOR but significantly increased mRNA level of hormone-sensitive lipase (HSL). Acetate treatment did not significantly alter the phosphorylated protein level of p38 MAPK and c-Jun aminoterminal kinase (JNK). Activation of ERK1/2 and mTOR by respective addition in media with ceramide C6 and MHY1485 significantly attenuated decreased lipid deposition and increased HSL expression caused by acetate.ConclusionOur results suggest that ERK1/2 and mTOR signaling pathways are associated with acetate regulated HSL gene expression and lipid deposition.     

Circulating expression levels of CircHIPK3 and CDR1as circular-RNAs in type 2 diabetes patients

Background

Recent investigations suggested that deregulated levels of Circular RNAs (circRNAs) could be associated with type 2 diabetes mellitus (T2DM) pathogenesis. Accordingly, this study aimed to determine the expression levels of circulating CircHIPK3, CDR1as and their correlation with biochemical parameters in patients with T2DM, pre-diabetes and control subjects.

Methods and results

The expression of circRNAs in peripheral blood was determined using QRT-PCR in 70 patients with T2DM, 60 pre-diabetes and in 69 age and sex matched healthy controls. Moreover, bioinformatics tools were applied to explore and predict the potential interactions between circRNAs and other non-coding RNAs (ncRNAs). Our analysis revealed that the expression level of CircHIPK3 was significantly elevated in T2DM patients compared to healthy participants (P?<?0.001) and pre-diabetes subjects (P?=?0.018). In addition, ROC analysis suggested that at the cutoff value of 0.24 and the sensitivity and specificity of 50% and 88.4%, respectively, CircHIPK3 could distinguish between T2DM patients and control subjects. Furthermore, it was observed that the expression level of CDR1as is higher in pre-diabetic individuals than healthy individuals (P?=?0.004). Finally, Spearman correlation analysis showed that there was a significant correlation between CircHIPK3 and CDR1as expression levels and clinical and anthropometrical parameters such as BMI, systolic and diastolic blood pressure, HbA1c and fasting blood glucose (P?<?0.005).

Conclusions

The data of this study provided evidence that the expression levels of CircHIPK3, CDR1as increased in T2DM and pre-diabetes subjects, respectively.

     

冠状动脉CTA联合血清HCY、Cys-C、ApoB/ApoA1比值对2型糖尿病患者合并冠状动脉病变的诊断价值

摘要 目的：探究冠状动脉CT血管造影（CTA）联合血清同型半胱氨酸（HCY）、胱抑素C（Cys-C）、载脂蛋白B/载脂蛋白A1（ApoB/ApoA1）比值对2型糖尿病（T2DM）患者合并冠状动脉病变的诊断价值。方法：回顾性选取2018年8月到2021年8月间我院收治的358例T2DM患者,均行常规生化指标、CTA检查、冠状动脉造影（CAG）检查,根据CAG检查结果为金标准将T2DM患者分为未合并冠脉病变组（190例）和合并冠脉病变组（168例）,比较两组血清HCY、Cys-C、ApoB/ApoA1比值,分析CTA与CAG诊断冠脉狭窄结果的一致性,应用受试者工作特征（ROC）曲线评估冠状动脉CTA联合血清HCY、Cys-C、ApoB/ApoA1比值对T2DM合并冠状动脉病变的诊断价值。结果：与未合并冠脉病变组比较,合并冠脉病变组血清HCY、Cys-C、ApoB、ApoB/ApoA1比值水平明显更高（P＜0.05）,ApoA1明显更低（P＜0.05）。以CAG为金标准,CTA诊断冠脉狭窄程度与CAG一致性较高（Kappa值0.748）。ROC曲线评估冠状动脉CTA诊断T2DM合并冠脉病变的AUC、灵敏度、特异度、准确度依次为0.802、74.40%、83.71%、79.11%。三项血清指标联合AUC、准确度显著优于单一指标（P＜0.05）。冠状动脉CTA联合血清HCY、Cys-C、ApoB/ApoA1比值诊断T2DM合并冠脉病变的价值显著优于各项指标单一诊断或三项血清指标联合诊断（P＜0.05）。结论：冠状动脉CTA联合血清HCY、Cys-C、ApoB/ApoA1比值诊断T2DM患者合并冠状动脉病变的价值较高,相较各项指标单一应用而言更具优势。     

Insulin resistance,intramyocellular lipid content,and plasma adiponectin in patients with type 1 diabetes

Insulin resistance is a key pathogenic factor of type 2 diabetes (T2DM); in contrast, in type 1 diabetes (T1DM) it is considered a secondary alteration. Increased intramyocellular lipid (IMCL) content accumulation and reduced plasma adiponectin were suggested to be pathogenic events of insulin resistance in T2DM. This study was designed to assess whether IMCL content and plasma adiponectin were also associated with the severity of insulin resistance in T1DM. We studied 18 patients with T1DM, 7 older and overweight/obese patients with T2DM, and 15 nondiabetic, insulin-resistant offspring of T2DM parents (OFF) and 15 healthy individuals (NOR) as appropriate control groups matched for anthropometric features with T1DM patients by means of the euglycemic hyperinsulinemic clamp combined with the infusion of [6,6-2H2]glucose and 1H magnetic resonance spectroscopy of the calf muscles. T1DM and T2DM patients showed reduced insulin-stimulated glucose metabolic clearance rate (MCR: 5.1 +/- 0.6 and 3.2 +/- 0.8 ml x kg(-1) min(-1)) similar to OFF (5.3 +/- 0.4 ml x kg(-1) x min(-1)) compared with NOR (8.5 +/- 0.5 ml x kg(-1) min(-1), P < 0.001). Soleus IMCL content was increased in T1DM (112 +/- 15 AU), T2DM (108 +/- 10 AU) and OFF (82 +/- 13 AU) compared with NOR (52 +/- 7 AU, P < 0.05) and the result was inversely proportional to the MCR (R2 = 0.27, P < 0.001); an association between IMCL content and Hb A1c was found only in T1DM (R2 = 0.57, P < 0.001). Fasting plasma adiponectin was reduced in T2DM (7 +/- 1 microg/ml, P = 0.01) and OFF (11 +/- 1 microg/ml, P = 0.03) but not in T1DM (25 +/- 6 microg/ml), whose plasma level was increased with respect to both OFF (P = 0.03) and NOR (16 +/- 2 microg/ml, P = 0.05). In conclusion, in T1DM, T2DM, and OFF, IMCL content was associated with insulin resistance, demonstrating that IMCL accretion is a marker of insulin resistance common to both primary genetically determined and secondary metabolic (chronic hyperglycemia) alterations. The increased adiponectin levels in insulin-resistant patients with T1DM, in contrast to the reduced levels found in patients with T2DM and in OFF, demonstrated that the relationship of adiponectin to insulin resistance in humans is still unclear.     

miR-142通过靶向HMGB1对宫颈癌细胞生物学行为影响的实验研究

摘要 目的：通过实验探究miR-142靶向高迁移率族蛋白1（high-mobility group box 1 protein,HMGB1）对宫颈癌（cervical cancer,CC）细胞生物学行为的影响及其潜在的作用机制。方法：采用实时荧光定量PCR（RT-PCR）和蛋白质免疫印迹法（Western Blot）检测CC组织和正常组织中miR-142和HMGB1 mRNA及蛋白表达水平,采用荧光素酶报告实验分析miR-142与HMGB1的靶向关系,CCK-8法检测CC细胞生存能力,克隆形成实验检测CC细胞增殖能力,划痕修复实验检测CC细胞迁移能力,基质胶侵袭实验检测CC细胞侵袭能力。结果：CC组miR-142 mRNA和蛋白表达水平显著低于正常组（P<0.05）,HMGB1 mRNA和蛋白表达水平显著高于正常组（P<0.05）,且CC癌组织中miR-142和HMGB1 mRNA和蛋白表达水平均呈显著负相关（r=-0.399,P=0.002;r=-0.429,P=0.001）;miR-142与HMGB1存在靶向关系;CCK-8法实验、克隆形成实验、划痕修复实验和基质胶侵袭实验结果显示,miR-142 mimic组细胞生存、增殖、迁移和侵袭能力显著低于miR-NC组（P<0.05）,miR-142 inhibitor组细胞生存、增殖、迁移和侵袭能力显著高于miR-NC组;Western Blot实验结果显示,HMGB1过表达时miR-142 mimic+plasmid组HMGB1蛋白表达水平显著高于miR-142 mimic+control plasmid组（P<0.05）,显著低于miR-NC+plasmid组（P<0.05）;CCK-8法实验、克隆形成实验、划痕修复实验和基质胶侵袭实验结果显示,HMGB1过表达时miR-142 mimic+plasmid组细胞生存、增殖、迁移和侵袭能力显著高于miR-142 mimic+control plasmid组（P<0.05）,显著低于miR-NC+plasmid组（P<0.05）。结论：miR-142可通过靶向负调控HMGB1表达,进而抑制CC细胞生存、增殖、迁移和侵袭。     

1型糖尿病小鼠前额叶皮层小胶质细胞数量和Iba1表达水平的变化

本文旨在研究1型糖尿病(type 1 diabetes mellitus,T1DM)小鼠前额叶皮层小胶质细胞的激活情况以及与神经退行性疾病相关的小胶质细胞新亚型(disease-associated microglia,DAM)标志性分子表达的变化。60只健康成年雄性C57BL/6J小鼠,随机分为正常对照(CON)组和T1DM组,每组30只。T1DM组小鼠腹腔注射链脲佐菌素(streptozocin,STZ)建立T1DM模型。模型构建成功后第8周通过Morris水迷宫检测小鼠空间学习记忆能力,通过免疫荧光染色法和Western blot检测小鼠前额叶皮层小胶质细胞数量及激活情况,通过RT-FQ-PCR法检测多个DAM标志性分子mRNA水平的变化。结果显示,与CON组相比,T1DM组小鼠空腹血糖明显升高,体重显著降低,水迷宫逃避潜伏期明显延长,差异均有统计学意义(P<0.05)。与CON组相比,T1DM组小鼠前额叶皮层Iba1蛋白表达水平上调,且小胶质细胞数目明显增多(P<0.05),胞体增大,呈现激活状态。同时,T1DM组小鼠前额叶皮层多个DAM标志性分子mRNA水平显著升高(P<0.05)。以上结果提示,T1DM小鼠前额叶皮层小胶质细胞被激活,且向DAM型转化。     

PI3K/AKt/mTOR信号通路介导黄芩苷抑制增生性瘢痕组织成纤维细胞的增殖

黄芩苷作为一种黄酮类成分可通过抑制细胞增殖、促进凋亡发挥抗肿瘤作用,但它是否对异常增生的瘢痕具有抑制增生的作用尚不清楚.本研究探讨黄芩苷抑制人增生性瘢痕组织成纤维细胞增殖的分子机制. 采用MTT比色法检测不同浓度的黄芩苷（2.24×10-2 ～ 2.24×102 mmol/L）对体外培养的增生性瘢痕组织成纤维细胞增殖的抑制作用.发现浓度为2.24×100～2.24×102 mmol/L黄芩苷处理组明显抑制增生性瘢痕组织成纤维细胞的增殖（P<0.05）.转染后的荧光素酶报告基因活性检测、RT-PCR及Western印迹分析技术检测其mRNA水平及细胞的帽状依赖翻译的表达.2.24×102 mmol/L黄芩苷处理后,黄芩苷作用组的mRNA水平并无明显差异（P>0.05）;增生性瘢痕成纤维细胞的帽状依赖结构的翻译明显被黄芩苷所抑制.采用Western印迹分析检测被黄芩苷干预的增生性瘢痕组织成纤维细胞的增殖相关的蛋白的表达;m7GTP琼脂糖珠沉淀结合蛋白4E-BP1与eIF4E的变化.发现增殖相关的蛋白mTOR、p70S6K、S6、4EBP1、eIF4E及其上游的AKT表达明显下调（P<0.05）,而PTEN表达明显上调.p-AKT(Ser473)、p-mTOR(Ser2448)、p-S6(Ser235/236)、p-4EBP1(Thr37/ 46)、p-PTEN（T380/S382/383）磷酸化水平下降（P<0.05）.在黄芩苷作用下的增生性成纤维细胞中的游离的4E-BP1明显减少（P<0.05）,而与eIF4E结合的4E-BP1明显增加（P<0.05）黄芩苷诱导游离的4E-BP1与eIF4E结合,从而抑制帽状依赖蛋白翻译.以上结果说明,黄芩苷可通过抑制PI3K/AKT/mTOR信号通路抑制人增生性瘢痕组织成纤维细胞的增殖.     

利用慢病毒介导的shRNA在GT1-7细胞中敲低Srsf1及其对GnRH、Kiss1的影响

目的:探讨慢病毒介导短发夹RNA(shRNA)敲低丝氨酸/精氨酸富集剪接因子1(Srsf1)基因对小鼠GT1-7细胞中性发育相关基因促性腺激素释放激素(GnRH)、Kiss1的影响。方法:实验分为3组,即空白对照组、阴性对照组及shRNA干扰组。用Srsf1 shRNA慢病毒稳转GT1-7细胞,Real-time PCR和Western blot检测GT1-7细胞中Srsf1在mRNA和蛋白水平的变化,并检测稳转株中GnRH、Kiss1基因的表达情况。结果:慢病毒介导的shRNA成功感染了GT1-7细胞,与空白对照组及阴性对照组相比,shRNA干扰组中Srsf1在mRNA水平降低了45%(P0.001),在蛋白水平敲低了42%(P0.05)。在稳定低表达Srsf1的GT1-7细胞中,GnRH、Kiss1基因在mRNA水平也显著降低(P0.05)。结论:成功的构建了Srsf1基因敲低的细胞株。在GT1-7细胞中敲低Srsf1基因会抑制GnRH、Kiss1基因的表达。     

C-Reactive Protein Inhibits Survivin Expression via Akt/mTOR Pathway Downregulation by PTEN Expression in Cardiac Myocytes

C-reactive protein (CRP) is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.     

Association of TRIM22 with the type 1 interferon response and viral control during primary HIV-1 infection

Type 1 interferons (IFNs) induce the expression of the tripartite interaction motif (TRIM) family of E3 ligases, but the contribution of these antiviral factors to HIV pathogenesis is not completely understood. We hypothesized that the increased expression of select type 1 IFN and TRIM isoforms is associated with a significantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection. We measured IFN-α, IFN-β, myxovirus resistance protein A (MxA), human TRIM5α (huTRIM5α), and TRIM22 mRNA levels in peripheral blood mononuclear cells (PBMCs) of high-risk, HIV-1-uninfected participants and HIV-1-positive study participants. Samples were available for 32 uninfected subjects and 28 infected persons, all within 1 year of infection. HIV-1-positive participants had higher levels of IFN-β (P = 0.0005), MxA (P = 0.007), and TRIM22 (P = 0.01) and lower levels of huTRIM5α (P < 0.001) than did HIV-1-negative participants. TRIM22 but not huTRIM5α correlated positively with type 1 IFN (IFN-α, IFN-β, and MxA) (all P < 0.0001). In a multivariate model, increased MxA expression showed a significant positive association with viral load (P = 0.0418). Furthermore, TRIM22 but not huTRIM5α, IFN-α, IFN-β, or MxA showed a negative correlation with plasma viral load (P = 0.0307) and a positive correlation with CD4(+) T-cell counts (P = 0.0281). In vitro studies revealed that HIV infection induced TRIM22 expression in PBMCs obtained from HIV-negative donors. Stable TRIM22 knockdown resulted in increased HIV-1 particle release and replication in Jurkat reporter cells. Collectively, these data suggest concordance between type 1 IFN and TRIM22 but not huTRIM5α expression in PBMCs and that TRIM22 likely acts as an antiviral effector in vivo.