PI3K/Akt信号通路在脓毒症肾脏损伤诱导的自噬中的调节作用*

 目的：研究脓毒症造成肾脏损伤时的自噬情况以及磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B(Akt)信号通路的调节作用。方法：对大鼠盲肠进行结扎与穿刺（CLP）,对肾脏组织切片进行HE染色,并测定血清尿素氮和肌酐。通过Western blotting定量分析CLP大鼠肾脏损伤发生后不同时点自噬相关分子微管相关蛋白轻链3（LC3）Ⅰ/Ⅱ、beclin-1和Akt蛋白磷酸化的表达情况;体外用LPS诱导人近端肾小管上皮细胞株HK-2发生自噬,检测不同浓度LPS和不同刺激时间自噬相关分子LC3Ⅰ/Ⅱ和Akt蛋白磷酸化的表达情况;进一步使用PI3K抑制剂、Akt抑制剂和LPS刺激HK-2细胞观察自噬相关蛋白的表达情况及细胞的凋亡水平。结果：同对照组相比,CLP大鼠显微镜下可见肾损伤的典型病理改变,血清尿素氮和肌酐均有上升。CLP肾脏损伤发生后,自噬相关蛋白LC3Ⅰ/Ⅱ、beclin-1含量及Akt磷酸化水平均有上升。LPS刺激HK-2细胞后,随着刺激浓度的增加,p-Akt(308)表达量逐渐提高,而LC3Ⅰ/Ⅱ及p-Akt(472)的表达量在10 mg/L LPS刺激组最高。随着刺激时间的延长,p-Akt(308)表达量逐渐提高;LC3Ⅰ/Ⅱ表达量同p-Akt(472)在刺激8 h时最高;使用PI3K抑制剂及Akt抑制剂后,LPS诱导的LC3表达显著下调,HK-2细胞凋亡明显增加。结论：CLP肾脏损伤发生时可以诱导自噬发生, PI3K／Akt信号通路在其中发挥重要调节作用。     

运动性心脏肥大：AT1受体、细胞自噬和miRNAs的调节

As a mechanical and exogenous stimulus, exercise training induces cardiac physiological hypertrophy, and the cardiac structure is changed slowly, steadily and coordinately. Simultaneously, energy metabolism and function of the cardiac muscle are also improved. These are positive adaptations in the heart when experiencing endurance exercise training. Recently, angiotensin Ⅱ type 1 (AT1) receptor, autophagy and miRNAs are all considered as important regulators to cardiac hypertrophy induced by exercise training at different molecular levels. Fully understanding the relations and the important role of AT1 receptor, autophagy and miRNAs in cardiac physiological hypertrophy will further enrich the signaling pathway of cardiac hypertrophy induced by exercise training.     

Cell death and autophagy in tuberculosis

Mycobacterium tuberculosis has succeeded in infecting one-third of the human race though inhibition or evasion of innate and adaptive immunity. The pathogen is a facultative intracellular parasite that uses the niche provided by mononuclear phagocytes for its advantage. Complex interactions determine whether the bacillus will or will not be delivered to acidified lysosomes, whether the host phagocyte will survive infection or die, and whether the timing and mode of cell death works to the advantage of the host or the pathogen. Here we discuss cell death and autophagy in TB. These fundamental processes of cell biology feature in all aspects of TB pathogenesis and may be exploited to the treatment or prevention of TB disease.     

Sphk1在1型糖尿病心肌病大鼠中的自噬作用的研究

目的:观察鞘氨醇激酶1(Sphk1)在糖尿病性心肌病(DCM)大鼠中的自噬作用,为DCM的防治寻求新的靶点。方法:雄性Wistar大鼠20只被随机分为正常对照组(n=10)、DCM组(n=10)。腹腔注射链脲佐菌素(STZ)70mg/kg建立DCM大鼠模型。每2周观察大鼠体重、空腹血糖,8周后,检测两组左室舒张末期内径(LVEDd)、左室收缩末期内径(LVESd)、左室射血分数(LVEF)等;取心脏组织,行HE染色观察心脏形态。应用蛋白质印迹法检测Sphk1、自噬相关蛋白LC3II/LC3I、自噬相关蛋白Beclin 1表达水平。结果:造模8周后,与正常对照组比较,DCM组体重[(351±27)g比(198±11)g]显著降低,血糖[(6.84±0.93)mmol/L比(32.45±4.27)mmol/L]显著升高(P均=0.001);LVEDd[(7.12±0.36)mm比(6.46±0.28)mm]显著减小、LVESd[(3.39±0.14)mm比(3.72±0.25)mm]显著增加,LVEF值[(82.69±3.37)%比(62.50±3.08)%]显著降低(P<0.05或<0.01);Sphk1[(0.30±0.02)比(1.12±0.20)]、LC3II/LC3I[(0.44±0.05)比(1.03±0.14)]、Beclin 1[(0.35±0.08)比(0.73±0.12)]蛋白表达显著升高(P<0.05或<0.01)。HE染色可见心肌细胞增生肥大、排列紊乱。结论:Sphk1通过自噬促使大鼠的糖尿病性心肌病的发生发展。     

ATG16L1 and NOD2 polymorphisms enhance phagocytosis in monocytes of Crohn’s disease patients

AIM:To investigate if the presence of relevant genetic polymorphisms has effect on the effectual clearance of bacteria by monocytes and granulocytes in patients with Crohn’s disease(CD).METHODS:In this study,we assessed the differential responses in phagocytosis by measuring the phagocytic activity and the percentage of active phagocytic monocytes and granulocytes in inflammatory bowel disease patients as well as healthy controls.As both autophagy related like 1(ATG16L1)and immunityrelated guanosine triphosphatase gene are autophagy genes associated with CD and more recently nucleo-tide-binding ligomerization domain-containing protein2(NOD2)has been identified as a potent inducer of autophagy we genotyped the patients for these variants and correlated this to the phagocytic reaction.The genotyping was done with restriction fragment length polymorphisms analysis and the phagocytosis was determined with the pHrodo?Escherichia coli Bioparticles Phagocytosis kit for flowcytometry.RESULTS:In this study,we demonstrate that analysis of the monocyte and granulocyte populations of patients with CD and ulcerative colitis showed a comparable phagocytic activity(ratio of mean fluorescence intensity)between the patient groups and the healthy controls.CD patients show a significantly higher phagocytic capacity(ratio mean percentage of phagocytic cells)compared to healthy controls(51.91%±2.85%vs 37.67%±7.06%,P=0.05).The extend of disease was not of influence.However,variants of ATG16L1(WT:2.03±0.19 vs homozygoot variant:4.38±0.37,P<0.009)as well as NOD2(C-ins)(heterozygous variant:42.08±2.94 vs homozygous variant:75.58±4.34(P=0.05)are associated with the phagocytic activity in patients with CD.CONCLUSION:Monocytes of CD patients show enhanced phagocytosis associated with the presence of ATG16L1 and NOD2 variants.This could be part of the pathophysiological mechanism resulting in the disease.     

Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells

AIM:To clarify whether histone deacetylase inhibitors histone deacetylase inhibitors(HDACIs)can sensitize hepatocellular carcinoma(HCC)cells to sorafenib treatment.METHODS:Bax,Bcl-2,ATG5-ATG12,p21,and p27protein levels in Hep3B,HepG2,and PLC/PRF/5 cells were examined by Western blot.CCK8 and a fluorometric caspase-3 assay were used to examine cellular viability and apoptosis levels.The effect of Beclin-1 on sensitization of HCC cells to sorafenib was examined by transfecting Beclin-1 siRNA into Hep3B,HepG2,and PLC/PRF/5 cells.RESULTS:Autophagy inhibition enhances the inhibitory effects of vorinostat and sorafenib alone or in combination on HCC cell growth.Vorinostat and sorafenib synergistically induced apoptosis and cell cycle alterations.Western blot data indicated that HDACIs and Beclin-1 knockdown increased the p53 acetylation level.The knockdown of Beclin-1 enhanced the synergistic effect of the combination of vorinostat with sorafenib.CONCLUSION:HDACIs can sensitize HCC cells to sorafenib treatment by regulating the acetylation level of Beclin-1.     

小胶质细胞自噬和炎症及其交互作用对脑出血调控机制的研究进展

脑出血好发于中老年人,对中老年人健康威胁极大。我国脑出血发病率仅次于脑梗死,是第二大脑卒中类型,且脑出血患者在脑卒中患者中所占比例远高于西方发达国家,这对已步入老龄化的中国社会及患者家庭造成了沉重的负担。目前,临床尚缺乏确切有效的脑出血治疗手段。作为大脑组织的主要吞噬细胞,小胶质细胞在脑出血后脑损伤过程中发挥着重要作用,如通过自噬及调节炎症发挥中枢神经系统保护作用、减轻脑损伤等。本文综述了小胶质细胞自噬和炎症及其交互作用对脑出血的调控机制,旨在为脑出血的诊疗、新治疗靶点的发现及预后改善等提供参考。     

敲除Sirt3后激活自噬对阿尔茨海默病有保护作用

目的: 探究Sirt3在阿尔茨海默病（AD）发生发展中的作用及可能机制。方法: 体内实验以C57BL/6野生型小鼠和Sirt3基因敲除小鼠为对象,采用腹腔注射D-半乳糖联合脑定位注射β-淀粉样蛋白（Aβ）_1-40建立AD小鼠模型。莫里斯（Morris）水迷宫实验、自发活动开场实验和悬尾实验观察造模前后小鼠学习记忆和焦虑抑郁状态的改变,免疫荧光染色观察脑内海马区域Aβ沉积情况,蛋白质印迹法检测小鼠脑组织中自噬和凋亡相关蛋白表达。体外实验选小鼠皮层原代细胞作为对象,给予Aβ_1-40建立AD体外模型,MTT法检测细胞活性。结果: 体内实验结果显示,野生型小鼠诱发AD后平台潜伏期延长（P < 0.05）,穿越平台次数减少、目标象限停留时间缩短（均P < 0.05）,提示造模后小鼠的学习记忆能力下降;而Sirt3敲除能够缓解AD所致的学习记忆障碍（均P < 0.05）。相较于野生型小鼠,Sirt3基因敲除小鼠脑内海马区域的Aβ沉积减少（P < 0.05）,凋亡蛋白cleaved caspase 3表达减少（P < 0.05）。另外,野生型小鼠诱发AD后LC3-Ⅱ和P62蛋白表达增加（均P < 0.05）,提示自噬流受阻;而Sirt3基因敲除小鼠诱发AD后LC3-Ⅱ蛋白表达增加,P62蛋白表达减少（均P < 0.05）,提示自噬被激活。小鼠皮层原代细胞AD模型中,Sirt3基因敲除的细胞较野生型细胞死亡减少（P < 0.05）;加用自噬抑制剂氯喹后,Sirt3基因敲除的保护作用消失（P < 0.05）。结论: Sirt3敲除对于D-半乳糖联合Aβ_1-40诱发的AD有保护作用,这种保护作用可能与自噬流活化有关。     

Tim-3在药物性急性肾损伤动物模型中的表达及作用机制

目的 探索T细胞免疫球蛋白黏蛋白分子3(Tim-3)在急性肾损伤(AKI)中的作用,并研究其作用机制。 方法 将6~8周C57雄性小鼠随机分为对照组(CTRL组)、顺铂组(AKI组)及顺铂+阻断Tim-3组(RMT组),每组4只,AKI组使用顺铂(20 mg/kg)腹腔注射,构建AKI动物模型,检测肾脏损伤情况与Tim-3表达情况。RMT组使用抗体RMT3-23(200 μg/只)阻断Tim-3分子,建立AKI模型,检测AKI组与RMT组肾脏损伤情况以及Tim-3、自噬相关分子的表达情况。 结果 肾脏近端小管出现损伤,Western blotting 结果显示 AKI组Tim-3表达水平较CTRL组增加(t=3.876,P=0.008 2)。与AKI组相比,RMT组的NGAL和P62表达水平均增高,但差异无统计学意义(t_NGAL=1.664,P=0.157 0;t_P62=1.991,P=0.103 1),LC3II表达下降,差异有统计学意义(t=5.901,P=0.002 0),自噬受到抑制且肾脏小管损伤加重(P=0.010 1)。 结论 Tim-3可以通过调节肾小管上皮细胞自噬活性,减轻顺铂诱导的AKI肾损伤。