细菌脂多糖对人根尖牙乳头干细胞自噬作用研究

目的　研究细菌脂多糖（lipopolysaccharide,LPS）对人根尖牙乳头干细胞（stem cells from apical palilla,SCAP）自噬的作用及影响,以探讨年轻恒牙根尖周炎损伤与修复的分子机制。方法　原代分离培养人SCAP,采用不同质量浓度（0.05、0.5、5 μg/mL）的细菌LPS处理SCAP记为LPS处理组,未加入细菌LPS的记为对照组。Western blot检测各组自噬相关基因5（autophagy related gene 5,Atg5）及自噬调节蛋白P62的表达情况,透射电子显微镜观察细菌LPS对SCAP自噬泡形态及数量的影响,数据采用SPSS18.0软件进行统计学分析。结果　原代培养的人SCAP表达间充质干细胞表面标志物CD73、CD90、CD105,不表达造血干细胞表面标记物CD45,体外诱导培养具有成骨向分化潜能。Western blot结果显示,各组间Atg5和P62的表达总的比较差异具有统计学意义（F值分别为118.227、74.144,P < 0.05）。Atg5的表达随处理组细菌LPS质量浓度的增大而升高,其中0.05 μg/mL LPS处理组Atg5相对表达量低于对照组,5 μg/mL LPS处理组Atg5相对表达量高于对照组,差异均具有统计学意义（均P < 0.05）。与对照组相比,0.5、5 μg/mL LPS处理组P62的表达降低,其中5 μg/mL LPS组P62相对表达量最低,差异均有统计学意义（均P < 0.05）。透射电子显微镜观察发现,与对照组相比,LPS处理组自噬泡数量显著增加。结论　细菌LPS能够促进人SCAP自噬的发生,提示在炎性环境下自噬可能对人SCAP生物学性能的调控发挥重要作用。     

Tea polyphenols alleviate tri‐ortho‐cresyl phosphate‐induced autophagy of mouse ovarian granulosa cells

Tri‐ortho‐cresyl phosphate (TOCP), a widely used plasticizer in industry, can cause female reproductive damage. Tea polyphenols (TPs) have multiple health effects via inhibiting oxidative stress. However, the reproductive protection of TPs in TOCP‐induced female reproductive system damage is yet to be elucidated. In the study, TOCP inhibited cell viability and induced autophagy of mouse ovarian granulosa cells; while TPs could rescue the inhibition of viability and induction of autophagy. 3‐MA, an autophagy inhibitor, could also rescue the inhibition of cell viability. These results indicated that TPs played a protective role in TOCP‐induced autophagy. Furthermore, TPs could inhibit the induction of oxidative stress of the cells by TOCP, which implying that TPs might alleviate TOCP‐induced autophagy via inhibiting oxidative stress. Furthermore, TPs could rescue TOCP‐induced autophagy and oxidative stress in the mouse ovarian tissues. Taken together, these results indicated that TPs could protect TOCP‐induced ovarian damage via inhibiting oxidative stress.     

Protective effects of trehalose against Mn‐induced α‐synuclein oligomerization in mice: Involvement of oxidative stress and autophagy

Overexposure to manganese (Mn) is widely known to induce alpha‐synuclein (α‐Syn) oligomerization, which has been attributed to the oxidative damage of α‐Syn protein. Trehalose has been shown to induce autophagy and serve as a chemical chaperone, but little information has been reported about its effect on Mn‐induced α‐Syn oligomerization. In this study, we investigate whether trehalose can effectively interfere with Mn‐induced α‐Syn oligomerization, using different concentrations of trehalose (2% and 4% (g/vol [mL])) in a mouse model of manganism. After 6 weeks of exposure to Mn, both oxidative stress and autophagy were activated and resulted in α‐Syn oligomerization and neuronal cell damage in the mouse brain tissue. Our results also revealed that pretreatment with trehalose significantly reduced the oxidative damage to α‐Syn protein and increased autophagy activation. These findings clearly demonstrated that trehalose can relieve Mn‐induced α‐Syn oligomerization and neuronal cell damage through its anti‐oxidative and autophagy‐inducing effects.     

归肾经方含药血清对阿霉素诱导的足细胞损伤及自噬相关蛋白的影响

目的：研究归肾经方对阿霉素（adriamycin,ADR）诱导的肾小球足细胞损伤标志蛋白nephrin及自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ、p-mTOR等表达的影响。 方法：以ADR体外诱导的方法制作足细胞损伤的细胞模型。除空白组外均以ADR诱导足细胞,分为5组：空白组（control group,10%空白血清）、模型组（model group,10%空白血清+ADR 0.5 μg·mL^-1）、中药高剂量组（H-TCM group,10%高剂量归肾经方含药血清+ADR 0.5 μg·mL^-1）、中药低剂量组（L-TCM group,10%低剂量归肾经方含药血清+ADR 0.5 μg·mL^-1）、替米沙坦组（TMST group,10%替米沙坦含药血清+ADR 0.5 μg·mL^-1）。CCK-8检测各组细胞活性改变,Western blot法检测各组足细胞标志蛋白nephrin、desmin及自噬相关蛋白beclin-1、LC3Ⅱ/Ⅰ等表达。 结果：①与空白组比较,模型组细胞活力显著降低（P<0.01）,与模型组比较,中药高剂量组及替米沙坦组细胞活性显著增高（P<0.05）;②Western blot检测结果显示,与control组比较,model组nephrin显著降低（P<0.01）,desmin、LC3Ⅱ/Ⅰ、Beclin-1、p-Akt、p-mTOR显著增高（P<0.01）;与Model组比较,H-TCM组及TMST组nehprin表达显著增高（P<0.05）,desmin表达显著降低（P<0.05）,H-TCM、L-TCM及TMST组Beclin-1、LC3Ⅱ/Ⅰ、p-Akt、p-mTOR表达显著降低（P<0.01）;与H-TCM组比较,L-TCM组LC3Ⅱ/Ⅰ表达显著增高（P<0.05）。结论：归肾经方可以修复ADR诱导的足细胞损伤,改善细胞内的自噬水平,与上调细胞内nephrin、Beclin-1、p-Akt、p-mTOR的表达有关。     

曲格列酮诱导人心肌细胞氧化应激和自噬的作用

目的研究曲格列酮的心肌细胞毒性特征,并从线粒体氧化应激和自噬角度探讨其潜在的毒作用机制。方法人源心肌细胞AC16给予不同浓度曲格列酮0~40μmol·L^-1孵育24 h。倒置显微镜观察细胞形态,CCK-8法检测细胞存活率,漏出法检测乳酸脱氢酶(LDH)释放量;荧光探针TMRM检测线粒体膜电位和CM-H2DCFDA检测全细胞活性氧的含量;Western印迹法检测微管相关蛋白Ⅱ/Ⅰ轻链3(LC3-Ⅱ/LC3-Ⅰ)比值和P62蛋白表达水平。结果与细胞对照组相比,曲格列酮可浓度依赖性地引起细胞质皱缩、细胞存活率下降(r=-0.928,P<0.05)和LDH释放量增加(r=0.746,P<0.05);曲格列酮10和20μmol·L^-1可明显降低细胞线粒体膜电位(P<0.05),增加全细胞活性氧含量(P<0.05);显著增加LC3-Ⅱ/LC3-Ⅰ比值,上调P62蛋白表达(P<0.05)。结论曲格列酮可引起心肌细胞损伤和线粒体功能障碍,其机制与线粒体氧化应激和自噬体降解受阻密切相关。     

Inhibition of autophagy increases apoptosis during re‐warming after cold storage in renal tubular epithelial cells

Prolonged cold storage and re‐warming (CS/REW) of kidneys are risk factors for delayed graft function (DGF). Studies in renal tubular epithelial cells (RTECs) have determined apoptosis and autophagy in models of either cold storage (CS) or re‐warming alone. The effect of both cold storage and re‐warming on apoptosis and autophagy, in RTECS is not known and is relevant to DGF as the kidney is subjected to both CS and re‐warming. We hypothesized that CS/REW of RTECs would induce autophagy that protects against apoptosis. In CS/REW, there was increased autophagic flux of RTECs. Autophagy inhibition using an Atg5 siRNA resulted in increased cleaved caspase‐3 and increased apoptotic cells (on both morphology and annexin V staining) during CS/REW. The effect of autophagy inhibition on necrosis in RTECs is unknown. There were increased necrosis and caspase‐1, a mediator of necrosis, during CS/REW, and the Atg5 siRNA had no effect on necrosis and caspase‐1. In a kidney transplant model, there was an increase in LC3 II, a marker of autophagy, in kidneys transplanted after cold storage. In summary, autophagic flux is increased during CS/REW. Autophagy inhibition resulted in increased cleaved caspase‐3 and increased apoptosis during CS/REW without an effect on necrosis or caspase‐1. In conclusion, autophagy inhibition in RTECs after CS/REW induces apoptotic cell death and may be deleterious as a therapy to decrease DGF.     

自噬抑制剂在内质网应激状态下对肝癌HepG2细胞和正常肝细胞L-02作用的差异

目的：研究自噬在内质网应激( ERS)状态下对肝癌HepG2细胞和正常肝细胞L-02作用的差异。方法体外常规培养的人肝癌HepG2细胞和正常肝细胞L-02,分别给予衣霉素( TM)单药和TM联合自噬抑制剂3-甲基腺嘌呤( TM+3-MA)或氯喹( TM+CQ)作用12、24、48 h后,采用噻唑蓝( MTT)法检测细胞活力变化,流式细胞术检测细胞凋亡率, Western blot法检测自噬蛋白LC3的变化。结果 TM可引起HepG2细胞和L-02细胞死亡并呈时间依赖关系,3-MA或CQ均可增加TM对HepG2细胞的生长抑制作用,24 h细胞存活率分别为TM+3-MA组(60%)、TM+CQ组(72%)、TM组(86%),差异有统计学意义(P<0．01);但对于L-02细胞,其存活率分别为83%、84%、83%,活力没有明显差异;流式细胞术显示TM+3-MA、TM+CQ和TM组对HepG2细胞的凋亡率分别为15%、11%、7%,差异有统计学意义( P<0．01),但对L-02细胞,凋亡率分别为16%、17%、16%,未见明显差异;Western blot法结果显示TM作用引起两种细胞自噬增加,自噬抑制剂3-MA与CQ可引起两种细胞自噬作用减弱。结论自噬抑制剂(3-MA 或 CQ)均可显著增加TM对肝癌HepG2细胞的生长抑制作用,但对正常肝细胞L-02的生长抑制作用差异无统计学意义。自噬在ERS状态下可对肝癌细胞的生存提供保护,但对正常肝细胞无保护作用。     

MiR-218 inhibits HMGB1-mediated autophagy in endometrial carcinoma cells during chemotherapy

Endometrial carcinoma is the most common gynecological malignancy among women worldwide. Although treatment for EC has improved with the introduction of Paclitaxel (Tax) chemotherapy, the majority of patients will develop resistance to the treatment, leading to poor prognosis. One of the causes of chemoresistance is the increased ability to undergo autophagy. In this study, we identified that miR-218 was significantly down-regulated in Tax-resistant EC cells compared to the non-drug resistant cell lines, and overexpression of miR-218 sensitized paclitaxel resistant EC cells to paclitaxel. Moreover, we demonstrated that miR-218 directly binds to the 3’-UTR of HMGB1 gene. HMGB1 was upregulated in paclitaxel resistant EC cells, it mediated autophagy and contributed to chemotherapy resistance in endometrial carcinoma in vitro. HMGB1-mediated autophagy could be suppressed by miR-218 overexpression in Tax resistant EC cells. In summary, we determined the targeting role of miR-218 to HMGB1 and the regulation of miR-218 on the HMGB1-mediated cell autophagy during chemotherapy resistance in endometrial carcinoma cells. These results reveal novel potential role of miR-218 against chemotherapy resistance during the treatment of endometrial carcinoma.     

Upregulation of cell surface estrogen receptor alpha is associated with the mitogen-activated protein kinase/extracellular signal-regulated kinase activity and promotes autophagy maturation

Recently, accumulating evidence has implicated the dysregulation of autophagy as underlying the pathophysiology of several neurodegenerative diseases. The human neuronal cell line SH-SY5Y was exposed to 1-Methyl-4-phenylpyridinium (MPP⁺). The mechanism is that the sustained activation of the MAPK/ERK pathway by MPP⁺ alters autophagy selectively at the maturation step, significant increasing in autophagy formation and delaying in autophagy degradation in SHSY5Y cells. In this study, we provided evidences that estrogen was capable of promoting SHSY5Y cells survival in MPP⁺-treated group. In particular, the up-regulation of mERα, but not mERβ, was associated with a rapid and transient activation of ERK phosphorylation compatible with promoting autophagy maturation. The up-regulation of mERα changed the sustained activation of ERK phosphorylation in MPP⁺-treated group into a temporary activation. Taken together, these findings strongly support that the expression of mERα promotes the maturation of autophagosomes into functional autolysosomes by regulating ERK, determining SHSY5Y cells survival.