补肾活血方对血管性痴呆大鼠海马神经细胞自噬的影响＊

目的 探究补肾活血方对血管性痴呆（Vascular Dementia， VD）大鼠模型自噬的影响。方法 52周龄SD雄性大鼠50只，随机分为假手术组（Sham组）、模型组（VD组）、模型+补肾活血方组（BSHX组）、模型+雷帕霉素组（Rap组）和模型+3-甲基腺嘌呤组（3-MA组），每组10只大鼠。除Sham组外其余各组采用两血管阻断法（2-VO）建立VD模型。BSHX组中药灌胃治疗28天；自噬干预组于造模前30 min侧脑室给药。运用Morris水迷宫测试各组大鼠的学习记忆能力；通过尼氏染色和透射电子显微镜（Transmission electron microscope，TEM）观察大鼠海马区病理形态及自噬变化；采用蛋白免疫印迹（Western blot）法和反转录实时荧光定量PCR（RT-qPCR）检测大鼠海马Beclin-1、P62以及微管相关蛋白1轻链3（LC3）蛋白及mRNA表达情况。结果 与VD组比较，BSHX组大鼠学习记忆能力显著提升（P<0.05），镜下观察BSHX组和3-MA组的细胞形态及数量均有改善，自噬小体鲜见，Beclin-1以及LC3蛋白和mRNA表达水平显著降低（P<0.05），P62蛋白和mRNA表达水平明显升高（P<0.05）结论 补肾活血方可以降低VD大鼠海马区Beclin-1和LC3蛋白及mRNA的表达，提高P62的表达，通过抑制自噬的发生，减轻对神经细胞的损伤，改善学习记忆能力。     

Anti-aging effect of DL-β-hydroxybutyrate against hepatic cellular senescence induced by D-galactose or γ-irradiation via autophagic flux stimulation in male rats

The present study aims to shed new light on anti-aging effect of DL-β-hydroxybutyrate (βOHB) against hepatic cellular senescence induced by d-galactose or γ-irradiation. The rats divided into 6 groups. Group 1, control, group 2, exposed to γ-ray (5 GY), group 3, injected by d-galactose (150 mg/kg) daily for consecutive 6 weeks, which regarded to induce the aging, group 4, injected intraperitoneal by β-hydroxybutyrate (βOHB) (72.8 mg/kg) daily for consecutive 14 days, group 5, exposed to γ-ray then treated with βOHB daily for consecutive 14 days, group 6, injected daily with d-galactose for consecutive 6 weeks, then treated with βOHB daily at the last two weeks of d-galactose. Aspartate amino transferase (AST), alanine amino transferase (ALT), Insulin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were estimated in serum. Moreover, protein expression of Microtubule-associated proteins 1A/1B light chain 3B (LC3-II/LC3-I) ratio, mechanistic target of rapamycin (mTOR), pAMPK, mRNA gene expression of 5′ AMP-activated protein kinase (AMPK), Nucleoporin p62 (p62), cyclin-dependent kinase inhibitor 1(P21^CIP1), cyclin-dependent kinase inhibitor 2A (p16^INK4a) and DNA fragmentation percentage were measured in liver tissue as a biomarker of cellular senescence. The results confirmed that βOHB modulated serum level of AST, ALT, insulin, IL-6 and TNF-α, protein expression of mTOR and LC3-II/LC3-I ratio, pAMPK and p62 in liver aging model induced by d-galactose or γ-irradiation. Histopathological examination results of liver tissue indicated coincidence with those recorded by molecular biochemical inspection. Taken together, these findings suggest that βOHB may be useful in combating hepatic cellular senescence induced by d-galactose or γ-irradiation via autophagy dependent mechanisms.     

Molecular mechanisms and cellular events involved in the neuroprotective actions of estradiol. Analysis of sex differences

Estradiol, either from peripheral or central origin, activates multiple molecular neuroprotective and neuroreparative responses that, being mediated by estrogen receptors or by estrogen receptor independent mechanisms, are initiated at the membrane, the cytoplasm or the cell nucleus of neural cells. Estrogen-dependent signaling regulates a variety of cellular events, such as intracellular Ca²⁺ levels, mitochondrial respiratory capacity, ATP production, mitochondrial membrane potential, autophagy and apoptosis. In turn, these molecular and cellular actions of estradiol are integrated by neurons and non-neuronal cells to generate different tissue protective responses, decreasing blood-brain barrier permeability, oxidative stress, neuroinflammation and excitotoxicity and promoting synaptic plasticity, axonal growth, neurogenesis, remyelination and neuroregeneration. Recent findings indicate that the neuroprotective and neuroreparative actions of estradiol are different in males and females and further research is necessary to fully elucidate the causes for this sex difference.     

Effects of newly introduced antidiabetic drugs on autophagy

Diabetes mellitus is a chronic metabolic disorder that has a complex molecular and cellular pathophysiology, resulting in its dynamic progression and that may show differing responses to therapy. The incidence of diabetes mellitus increases with age and requires additive therapeutic agents for its management. SGLT2i and DPP-4 inhibitors and GLP-1 receptor agonists (GLP-1RA) are newly introduced antidiabetic drugs that work through differing mechanisms; DPP-4 inhibitors maintain the endogenous level of GLP1; GLP-1RA result in pharmacological levels of GLP1, whilst SGLT2i act on the proximal tubules of the kidney. They have shown efficacy in the management of diabetes and in contrast to other antidiabetic drugs, do not inherently cause hypoglycemia in therapeutic doses. Autophagy as a highly conserved mechanism to maintain cell survival and homeostasis by degradation of damaged or aged organelles and components, and recognised to be increasingly important in diabetes. In the present review, we discuss the modulatory effects of these newly introduced antidiabetic drugs on the autophagy process.     

脉冲射频治疗对带状疱疹后神经痛模型大鼠脊髓背角自噬的影响

目的观察脉冲射频(PRF)治疗对带状疱疹后神经痛(PHN)模型大鼠疼痛的影响,探讨脊髓背角自噬改变的可能作用。方法雄性SD大鼠60只,体重200～240 g,随机分为五组:空白对照组(Blank组)、溶剂组(Solvent组)、PHN模型组(PHN组)、假治疗组(Sham组)和治疗组(PRF组),每组12只。PHN组、Sham组及PRF组腹腔注射树脂毒素(RTX)0.2 g/kg制备PHN模型。Blank组腹腔注射与PHN组相同体积的生理盐水。Solvent组腹腔注射相同体积的溶剂(10%吐温80、10%乙醇和80%生理盐水)。于制模前2 h、制模后1、4、7、10、14 d、PRF治疗后1、4、7、10、14、21、28、35和42 d测定大鼠机械缩足阈值(MWT)和热缩足潜伏期(TWL)。治疗后第42天处死大鼠,取L_(4-6)脊髓组织,采用Western blot法检测脊髓组织微管相关蛋白轻链Lc3Ⅱ和Lc3Ⅰ、Beclin-1和P62蛋白含量,计算Lc3Ⅱ和Lc3Ⅰ比值。RT-qPCR检测Lc3 mRNA和Beclin-l mRNA的表达量。结果与Blank组比较,PHN组、Sham组及PRF组RTX处理后大鼠MWT明显降低,TWL明显延长,脊髓组织Lc3Ⅱ/Lc3Ⅰ蛋白含量比值和Beclin-1蛋白含量明显升高,P62蛋白含量明显降低,Lc3 mRNA和Beclin-1 mRNA表达量明显增加(P均0.05)。与PHN组比较,PRF组大鼠在治疗7 d后MWT明显升高,TWL明显缩短,Lc3Ⅱ/Lc3Ⅰ蛋白含量比值明显降低,Beclin-1蛋白含量明显降低,P62蛋白含量明显增加,Lc3 mRNA和Beclin-1 mRNA表达量明显降低(P均0.05)。结论脉冲射频可能通过抑制脊髓背角组织自噬活性,改善线粒体功能,从而对PHN模型大鼠产生镇痛作用。     

狼疮肾炎患者自噬水平及其对足细胞相关蛋白表达水平的影响

目的探讨狼疮肾炎(lupus nephritis,LN)患者自噬水平及其对足细胞相关蛋白表达水平的影响。方法选择2017年5月至2019年5月于榆林市第一医院收治的69例LN患者为LN组,50例系统性红斑狼疮(systemic lupus erythematosus,SLE)患者为SLE组,50例肾切除手术患者为对照组,观察3组肾脏足细胞内自噬体数量,比较3组肾脏组织中自噬相关蛋白、微管相关蛋白轻链3(LC3)、B淋巴细胞瘤蛋白质-2-相互作用蛋白(Beclin1)的表达,以及足细胞相关蛋白,肾病蛋白(Nephrin)、足突蛋白(Podocin)的表达。分离狼疮肾炎患者肾脏足细胞,将足细胞分为自噬抑制组和自噬诱导组,自噬抑制组加入100 nmol/L 3-甲基腺嘌呤(3-MA),自噬诱导组加入100 nmol/L雷帕霉素(RAPA),比较3组足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin等蛋白的表达。结果LN组、SLE组肾脏足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于对照组(P<0.05);SLE组肾脏足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于LN组(P<0.05);自噬诱导组足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于正常对照组、自噬抑制组(P<0.05);正常对照组足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于自噬抑制组(P<0.05)。结论LN患者自噬水平呈升高状态,自噬水平升高可能通过上调足细胞相关蛋白Podocin、Nephrin水平而减轻足细胞损伤,抑制LN病情进展。     

miR-21靶向Atg5对非小细胞肺癌A549细胞自噬调控促进细胞增殖、迁移和侵袭的实验研究

目的  探讨miR-21通过靶向作用自噬相关靶基因5（Atg5）调控非小细胞肺癌（NSCLC）自噬的作用机制及其在A549细胞增殖、迁移及侵袭中的作用。方法  无义核酸序列NC（NC组）、miR-21 模拟物（miR-21 mimics组）、miR-21抑制物（miR-21 抑制组）分别转染A549细胞， CCK-8检测细胞增殖情况；划痕实验检测细胞迁移能力； Transwell侵袭实验检测细胞侵袭能力。双荧光素酶报告实验验证miR-21和Atg5之间的靶向关系。Western blotting检测LC3B-II、p62和Atg5蛋白的表达。结果  与NC组比较，miR-21 mimics组细胞增殖、迁移、侵袭能力均上调，miR-21 抑制组细胞增殖、迁移、侵袭能力均下调（P＜0.05）。双荧光素酶报告实验结果显示，miR-21显著抑制野生型Atg5 3’-UTR质粒转染细胞的荧光素酶活性（P＜0.05），但对突变型Atg5 3’-UTR的基因报告质粒与miR-21 mimics共转染之后，并未对荧光素酶活性产生影响。NC组LC3B-II蛋白表达量为1.24±0.059，低于miR-21 mimics组的1.98±0.077，高于miR-21抑制组的0.52±0.021（P＜0.05）；NC组p62蛋白表达量为0.62±0.021，高于miR-21 mimics组的0.45±0.020，低于miR-21抑制组的0.79±0.031（P＜0.05）；NC组Atg5蛋白表达量为1.17±0.025，高于miR-21 mimics组的0.38±0.014，低于miR-21抑制组的1.40±0.039（P＜0.05）。与NC组比较， 3-MA处理降低miR-21 mimics转染诱导的A549细胞增殖能力（P＜0.05）；划痕实验和Transwell实验表明，3-MA处理抑制了miR-21mimics转染诱导的A549细胞的迁移和侵袭，差异有统计学意义（P＜0.05）。结论  miR-21靶向Atg5调控NSCLC自噬促进细胞增殖、迁移和侵袭。     

Synergism through combination of chemotherapy and oxidative stress-induced autophagy in A549 lung cancer cells using redox-responsive nanohybrids: A new strategy for cancer therapy

A combination of various therapeutic approaches has emerged as a promising strategy for cancer treatment. A safe and competent nano-delivery system is thus in urgent demand to facilitate the simultaneous transport of various therapeutic agents to cancer cells and a tumor region to achieve synergistic effect. Gold nanoparticles (GNPs) and mesoporous silica nanoparticle (MSNs) were fabricated herein as potential candidates for drug delivery. Serving as gatekeepers, GNPs (5 nm in diameter) were attached onto the amino-functionalized MSNs (denoted as NMSNs) via a relatively weak gold–nitrogen bonding. The resulting nanohybrids (denoted as GCMSNs) were uptaken by cells, and the detachment of GNPs and subsequent intracellular drug release from NMSNs were achieved by competitive binding of intracellular glutathione to GNPs. In addition to the function of gatekeeping, GNPs also play another role as the oxidative stress elicitor. Our in vitro studies revealed that GCMSNs induced higher oxidative stress in lung cancer cells (A549) than in normal cells (3T3-L1). This growth inhibitory effect found in the cancer cells was likely induced by mitochondria dysfunction originated from the GCMSN-induced, oxidative stress-triggered mitochondria-mediated autophagy. The redox-responsive nanohybrids were further loaded with camptothecin and the intensified synergistic therapeutic effects were observed associated with combined chemotherapy and oxidative stress strategy. The results clearly demonstrate that such unique nanohybrids hold great promise for selective and effective cancer treatments.     

Is the autophagy a friend or foe in the silver nanoparticles associated radiotherapy for glioma？

Malignant glioma is the most common intracranial tumor with a dismal prognosis. The radiosensitizing effect of silver nanoparticles (AgNPs) on glioma both in vitro and in vivo had been demonstrated in the previous studies of our group. However, the underlying mechanism is still unclear. Consistent with previous studies, a size and dose dependent antitumor effect and significant radiosensitivity enhancing effect of AgNPs were observed in our experiment system. We also found that cell protective autophagy could be induced by AgNPs and/or radiation, which was verified by the use of 3-MA. The mechanism through which had autophagy and the enhancement of radiosensitivity taken place was further investigated with inhibitors of ERK and JNK pathways. We demonstrated that ERK and JNK played pivotal roles in the radiosensitivity enhancement. Inhibiting ERK and JNK with U0126 and SP600125 respectively, we found that the autophagy level of the cells treated with AgNPs and radiation were attenuated. Moreover, SP600125 down-regulated the apoptosis rate of the co-treated cells significantly. Taken together, the present study would have important impact on biomedical applications of AgNPs and clinical treatment for glioma.