丹参酮生物合成相关SmERF108转录因子的鉴定及其靶基因分析＊

目的 鉴定与丹参酮合成相关转录因子AP2/ERF家族中SmERF108转录因子，并分析转录因子SmERF108的靶基因。方法 利用生物信息学在线分析平台如NCBI、PFAM等分析SmERF108的序列特征；MEGA-X软件用于构建SmERF108与不同功能转录因子的系统进化树；拟南芥原生质体转化法鉴定SmERF108蛋白的亚细胞定位；通过实时荧光定量PCR（Real-time qPCR）对SmERF108基因在丹参不同器官和组织差异表达进行检测；利用酵母体系对SmERF108的转录激活活性进行探究并用酵母单杂技术确定其靶基因。结果 SmERF108具有典型的AP2/ERF保守结构域，属于ERF-B3亚组，系统进化树和保守基序分析显示SmERF108与丹参中SmERF128、青蒿中AaERF2亲缘关系较近且保守基序分布一致；亚细胞定位显示SmERF108蛋白定位于细胞核；SmERF108基因在周皮中表达最高，且呈现周皮（R1）>韧皮部（R2）>木质部（R3）的规律；酵母自激活验证SmERF108具有转录激活活性，同时酵母单杂交确认其能与关键酶基因SmCPS1启动子结合。结论 鉴定到丹参中一个新转录因子SmERF108，生物信息学分析及差异表达分析预测与丹参酮合成相关，分子互作初步证实靶基因为SmCPS1二萜环化酶。     

健脾消癌方通过PI3K/Akt信号通路抑制结肠癌血管生成的研究＊

目的 观察结肠癌HCT116细胞健脾消癌方的条件培养液对HUVEC细胞管腔形成的影响，从PI3K/Akt生物轴调控角度探讨其作用机制。方法 培养HCT116细胞，细胞设3组：对照组，健脾消癌方组（加入15%健脾消癌方含药血清）及人参皂苷Rg3组；制备HCT116细胞健脾消癌方条件培养液（分组及制备方法见实验方法），用条件培养液干预HUVEC（脐静脉内皮细胞，Human Umbilical Vein Endothelial Cells），Matrigel基质胶法检测HCT116细胞健脾消癌方条件培养液对HUVEC小管形成的影响。随后采用蛋白免疫印迹法（Western blot）检测各组HCT116细胞磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、p-Akt、VEGF（血管内皮生长因子，Vascular endothelial growth factor）蛋白表达。最后在结肠癌HCT116荷瘤小鼠中验证健脾消癌方对肿瘤生长速度的影响，并经瘤组织VEGF蛋白表达、CD31免疫组化染色检测肿瘤内血管生成情况。结果 模型组HUVEC细胞管腔形成较空白血清组显著增加（P<0.05）；健脾消癌方组及人参皂苷Rg3组较模型组HUVEC细胞管腔形成显著减少（P<0.01）。p-Akt和VEGF蛋白表达水平模型组高于空白血清组（P<0.05），健脾消癌方组及人参皂苷Rg3组显著低于模型组（P<0.01）；PI3K、Akt蛋白表达量组间差异无统计学意义。与对照组比较，模型组荷瘤小鼠肿瘤体积显著性增大，瘤组织内VEGF表达、CD31阳性面积显著性增加，差异有统计学意义（P<0.05）；与模型组比较，健脾消癌方组及人参皂苷Rg3组荷瘤小鼠肿瘤体积显著减小，瘤组织内VEGF表达、CD31阳性面积降低，差异有统计学意义（P<0.05）。结论 健脾消癌方可抑制肿瘤的血管生成和生长，其作用机制可能与PI3K/Akt生物轴调控VEGF表达有关。     

血清Hcy、IGFBP-1联合预测多囊卵巢综合征患者孕早期自然流产的价值

目的探讨血清同型半胱氨酸(Hcy)、胰岛素样生长因子结合蛋白-1(IGFBP-1)联合检测对多囊卵巢综合征患者孕早期自然流产的预测价值。方法选取2018年3月—2020年5月行促排卵治疗并成功临床妊娠的多囊卵巢综合征165例,检测血清Hcy、IGFBP-1水平。根据孕早期自然流产发生情况分为孕早期自然流产组与未自然流产组,比较两组妊娠前一次促排卵后血清Hcy、IGFBP-1水平;多囊卵巢综合征患者孕早期自然流产的危险因素采用多因素Logistic回归分析,并采用受试者工作特征(ROC)曲线分析血清Hcy、IGFBP-1水平单项及联合检测对多囊卵巢综合征患者孕早期自然流产的预测价值。结果患者妊娠前一次促排卵后血清Hcy水平低于入院时,血清IGFBP-1水平高于入院时(P<0.01);自然流产组妊娠前一次促排卵后血清Hcy水平高于未自然流产组,血清IGFBP-1水平低于未自然流产组(P<0.01)。孕早期自然流产的发生率为28.48%。年龄≥35岁、多囊卵巢综合征分型、空腹胰岛素水平偏高、空腹血糖水平偏高、血清睾酮水平偏高、血清叶酸水平偏低、妊娠前一次促排卵后血清Hcy水平偏高及血清IGFBP-1水平偏低均是多囊卵巢综合征患者孕早期自然流产的危险因素(P<0.01)。妊娠前一次促排卵后血清Hcy水平联合血清IGFBP-1水平预测多囊卵巢综合征患者孕早期自然流产的敏感度、ROC曲线下面积均高于单独预测(P<0.01)。结论多囊卵巢综合征孕早期自然流产患者血清Hcy水平偏高,IGFBP-1水平偏低,二者均与多囊卵巢综合征患者孕早期自然流产密切相关,并对多囊卵巢综合征患者孕早期自然流产的发生具有较好的预测价值,联合检测时的预测效能更高。     

Interrelationships between Cellular Density,Mosaic Patterning,and Dendritic Coverage of VGluT3 Amacrine Cells

Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax-mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax-KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population.SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina.     

Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury

Apelin-13 is a novel endogenous ligand for an angiotensin-like orphan G-protein coupled receptor, and it may be neuroprotective against cerebral ischemia injury. However, the precise mechanisms of the effects of apelin-13 remain to be elucidated. To investigate the effects of apelin-13 on apoptosis and autophagy in models of cerebral ischemia/reperfusion injury, a rat model was established by middle cerebral artery occlusion. Apelin-13(50 μg/kg) was injected into the right ventricle as a treatment. In addition, an SH-SY5 Y cell model was established by oxygen-glucose deprivation/reperfusion, with cells first cultured in sugar-free medium with 95% N2 and 5% CO2 for 4 hours and then cultured in a normal environment with sugar-containing medium for 5 hours. This SH-SY5 Y cell model was treated with 10–7 M apelin-13 for 5 hours. Results showed that apelin-13 protected against cerebral ischemia/reperfusion injury. Apelin-13 treatment alleviated neuronal apoptosis by increasing the ratio of Bcl-2/Bax and significantly decreasing cleaved caspase-3 expression. In addition, apelin-13 significantly inhibited excessive autophagy by regulating the expression of LC3 B, p62, and Beclin1. Furthermore, the expression of Bcl-2 and the phosphatidylinositol-3-kinase(PI3 K)/Akt/mammalian target of rapamycin(mTOR) pathway was markedly increased. Both LY294002(20 μM) and rapamycin(500 nM), which are inhibitors of the PI3 K/Akt/mTOR pathway, significantly attenuated the inhibition of autophagy and apoptosis caused by apelin-13. In conclusion, the findings of the present study suggest that Bcl-2 upregulation and mTOR signaling pathway activation lead to the inhibition of apoptosis and excessive autophagy. These effects are involved in apelin-13-induced neuroprotection against cerebral ischemia/reperfusion injury, both in vivo and in vitro. The study was approved by the Animal Ethical and Welfare Committee of Jining Medical University, China(approval No. 2018-JS-001) in February 2018.     

山东省青岛市城阳区糖尿病患者慢性并发症现状调查

目的对山东省青岛市城阳区糖尿病(DM)患者的慢性并发症现状进行横断面调查。方法对2019年3—12月青岛市城阳区参与“三高共管”项目的8个辖区街道的DM患者进行随机抽样调查,按有无慢性并发症将患者分为2组,对患者的人口学资料、人体测量学等指标进行问卷调查,比较2组DM患者的特点,并对DM慢性并发症的影响因素进行Logistic回归分析。结果本研究共随机抽取DM患者26090例进行调查,其中男12560例,女13530例,有慢性并发症者18950例,无慢性并发症者7140例;肥胖、文化程度低、血糖控制欠佳、遵医嘱行为差、合并基础疾病的DM患者更容易出现并发症。亚组分析表明,女性DM患者的心血管疾病、神经病变和眼科疾病的发生率高于男性,差异有统计学意义(P<0.05或P<0.01);糖化血红蛋白(HbA1c)水平越高,DM病程越长,DM患者的慢性并发症发生率均越高,差异有统计学意义(P<0.05或P<0.01)。患者慢性并发症越多,则人均治疗费用越高,年住院次数越多,住院时间越长,差异有统计学意义(P<0.05或P<0.01)。多因素Logistic回归分析显示,性别、年龄、体质量指数(BMI)、DM病程、HbA1c、空腹血糖(FBG)、低密度脂蛋白(LDL)、尿白蛋白肌酐比值(UACR)、24 h尿蛋白定量、文化程度、规律服药程度、规律测量血糖程度均是DM慢性并发症的影响因素(P<0.05或P<0.01)。结论青岛市城阳区DM患者慢性并发症发生率较高,且影响因素较多,尽早明确危险因素有助于防治DM慢性并发症。     

The endogenous inflammatory reflex inhibits the inflammatory response to different immune challenges in mice

The splanchnic anti-inflammatory pathway, the efferent arm of the endogenous inflammatory reflex, has been shown to suppress the acute inflammatory response of rats to systemic lipopolysaccharide (LPS). Here we show for the first time that this applies also to mice, and that the reflex may be engaged by a range of inflammatory stimuli. Experiments were performed on mice under deep anaesthesia. Half the animals were subjected to bilateral section of the splanchnic sympathetic nerves, to disconnect the splanchnic anti-inflammatory pathway, while the remainder underwent a sham operation. Mice were then challenged intravenously with one of three inflammatory stimuli: the toll-like receptor (TLR)-4 agonist, LPS (60 µg/kg), the TLR-3 agonist Polyinosinic:polycytidylic acid (Poly I:C, 1 mg/kg) or the TLR-2 and -6 agonist dipalmitoyl-S-glyceryl cysteine (Pam2cys, 34 µg/kg). Ninety minutes later, blood was sampled by cardiac puncture for serum cytokine analysis. The splanchnic anti-inflammatory reflex action was assessed by comparing cytokine levels between animals with cut versus those with intact splanchnic nerves. A consistent pattern emerged: Tumor necrosis factor (TNF) levels in response to all three challenges were raised by prior splanchnic nerve section, while levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were reduced. The raised TNF:IL-10 ratio after splanchnic nerve section indicates an enhanced inflammatory state when the reflex is disabled. These findings show for the first time that the inflammatory reflex drives a coordinated anti-inflammatory action also in mice, and demonstrate that its anti-inflammatory action is engaged, in similar fashion, by inflammatory stimuli mimicking a range of bacterial and viral infections.     

Elevated serum uric acid is a predictor of contrast associated acute kidney injury in patient with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention

BackgroundContrast associated-acute kidney injury (CA-AKI) has been associated with adverse outcomes after ST-segment elevation myocardial infarction (STEMI). However, early markers of CA-AKI are still needed to improve risk stratification. We investigated the association between elevated serum uric acid (eSUA) and CA-AKI in patients with STEMI treated with primary percutaneous coronary intervention (pPCI).Methods and resultsSerum creatinine (Scr) was measured at admission and 24, 48 and 72 h after pPCI. CA-AKI was defined as an increase of 25% (CA-AKI 25%) or 0.5 mg/dl (CA-AKI 0.5) of Scr level above the baseline after 48 h following contrast administration. Multivariable analyses to investigate CA-AKI predictors were performed by binary logistic regression and multivariable backward logistic regression model.In the 3023 patients considered, CA-AKI was more frequent among patients with eSUA as compared with patients with normal SUA levels, considering both CA-AKI definitions (CA-AKI25%: 20.8% vs 16.2%, p < 0.012; CA-AKI 0.5: 10.1% vs 5.8%, p < 0.001). The association between eSUA and CA-AKI was confirmed at multivariable analyses (CA-AKI 25%: odd ratio 1.32, 95% CI 1.03–1.69, p = 0.027; CA-AKI 0.5: odd ratio 1.76, 95% CI 1.11–2.79, p = 0.016).ConclusionElevated serum uric acid is associated with CA-AKI after reperfusion in patients with STEMI treated with pPCI.