CD8+ T cell survival in lethal fungal sepsis was ameliorated by T-cell-specific mTOR deletion

Lethal fungal sepsis causes high morbidity and mortality in intensive care patients. Fungal infections have an immunological basis, and it has been shown in recent studies that decreased CD8⁺ T-cell count in fungal infections is related to prognosis, while the underlying mechanism is still unclear. Here, a lethal fungal sepsis model induced by candidemia was created and we found a decreased CD8⁺ T-cell count and exaggerated apoptosis. Simultaneously, expression of light chain (LC)3B in CD8⁺ T cells increased, along with increased autophagosomes and accumulation of p62 in infected mice. We regulated the activity of the mammalian target of rapamycin (mTOR) pathway using T-cell-specific mTOR/ TSC1 deletion mice. We observed increased number of autophagosomes and expression of LC3B in CD8⁺T cells after T-cell-specific mTOR knockout, while accumulation of p62 was not ameliorated, and there was no increase in the number of autolysosomes. Apoptosis rate and expression of BIM, a pro-apoptotic gene, decreased in CD8⁺ T cells in mTOR-deletion mice but increased in TSC1-deletion mice. Our results showed increased CD8⁺ T-cell death in spleen of lethal fungal sepsis mice, and decreased expression of mTOR ameliorated CD8⁺ T-cell survival. mTOR may be a possible target to reverse CD8⁺ T-cell immune dysfunction in lethal fungal sepsis.     

自噬对高胆固醇饮食大鼠下颌骨结构改变的影响研究

目的    探讨自噬对高胆固醇饮食大鼠下颌骨结构改变的影响及辛伐他汀的治疗效果。方法　将30只SD大鼠随机分为正常饮食组（NC组）10只、高胆固醇组20只;喂养24周后,高胆固醇组再随机分成两组,每组10只,一组继续喂养高胆固醇饲料（HC组）,另一组在高胆固醇饲料中加入5 mg/kg辛伐他汀（HC+SIM组）。喂养32周后,取大鼠空腹血进行生化检测;处死大鼠后对下颌骨进行骨组织形态分析;实时荧光定量PCR（qRT-PCR）及免疫组织化学染色检测各组自噬相关基因Lc3及p62的mRNA和蛋白表达水平。结果　（1）3组总胆固醇和低密度脂蛋白水平总的比较,差异均有统计学意义（F值分别为21.539、16.596,均P < 0.05）;其中NC组和HC+SIM组总胆固醇和低密度脂蛋白水平均低于HC组（均P < 0.05）,而NC组和HC+SIM组组间比较,差异无统计学意义（P > 0.05）。（2）与NC组比较,HC组大鼠骨体积分数、骨小梁厚度、骨小梁数目明显下降,而骨小梁间隔增加,Lc3及p62 mRNA表达水平明显升高,差异均有统计学意义（均P < 0.05）;与HC组比较,HC+SIM组骨体积分数、骨小梁厚度、骨小梁数目均明显升高,骨小梁间隔减小,p62 mRNA和蛋白表达水平明显下降,差异均有统计学意义（均P < 0.05）;而NC组和HC+SIM组这些指标组间比较,差异均无统计学意义（均P > 0.05）。结论    自噬可能参与调控高胆固醇饮食大鼠下颌骨结构的改变,辛伐他汀能够改善高胆固醇饮食导致的大鼠下颌骨微结构的破坏     

低氧对C2C12细胞分化不同阶段自噬和肌细胞生成素表达的影响

目的:研究低氧对C2C12细胞分化不同阶段自噬相关蛋白Beclin1、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3B)和肌细胞生成素(myogenin)表达的影响。方法:C2C12细胞诱导分化1、2、3、4 d,在常氧和低氧状态下观察细胞分化情况。免疫细胞化学染色法检测自噬相关蛋白LC3B和Beclin1的表达。Western blot检测Beclin1、LC3B和myogenin的蛋白表达水平,采用SPSS 22.0软件对数据进行统计学分析。结果:1.与常氧组比较,低氧组分化形成的肌管数量较少,形态细小;2.免疫细胞化学染色结果显示,分化1、2、3、4 d低氧组细胞LC3B和Beclin1荧光强度均低于常氧组;3.Western blot结果显示分化前中期低氧组Beclin1、LC3B和myogenin蛋白表达水平显著低于常氧组(P<0.05)。随着分化时间的增加,低氧状态下LC3B的表达水平进一步降低,而myogenin表达水平回升。结论:低氧抑制C2C12细胞分化;低氧能降低细胞分化前中期Beclin1、LC3B和myogenin蛋白表达水平。     

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。