AIM:To investigate the effects of aerobic and anti-resistance exercise on skin aging in SAMP8 mice via interleukin-18(IL-18)/IL-18 receptor(IL-18R)pathway and provide new concepts for anti-skin aging and pre-vention and treatment of chronic inflammatory skin-related diseases.METHODS:Twenty-four healthy male 28-week-old rapidly aging SAMP8 mice were randomly divided into model group,aerobic exercise group,and resistance exercise group,with 8 mice in each group.Eight same-aged rapidly aging SAMR1 mice were used as control group.The mice in aerobic exercise group underwent running table training at a constant speed(1 time per day,5 times per week)for 8 weeks,and those in model group underwent incremental weight-bearing ladder training for 8 weeks(1 time every other day,3 times per week).The HE staining was performed to observe the structural changes of the epidermis and dermis,and Weigert staining was conducted to observe the changes in skin elastic fibers.Immunohistochemistry was performed to observe the expression of skin aging markers P16 and P21.The ELISA was used to detect the concentrations of serum and skin inflammatory factors IL-18,IL-1β and tumor necrosis factor-α(TNF-α)and the levels of dermal IL-18R and IL-18 binding protein(IL-18BP).Double immunofluorescence staining of IL-18 and IL-18R was conducted.The RT-qPCR was used to detect the mRNA expression levels of IL-18 and IL-18R.RESULTS:Compared with control group,the mice in model group exhibited obvious signs of aging.The results of HE staining revealed indications such as rough skin texture and skin thinning.Weigert staining revealed the following:the elastic fibers were disordered,separated,thickened,shortened,twisted,and broken.The expression levels of P16 and P21 proteins in the epidermis and dermis were elevated(P<0.01).Serum and skin IL-1β,TNF-α and IL-18 levels were significantly higher.Skin IL-18R content was significant-ly higher,and IL-18BP content was significantly lower(P<0.05 or P<0.01).Colocalization phenomenon existed between IL-18 and IL-18R.Skin IL-18 and IL-18R mRNA expression levels were significantly higher(P<0.01).Compared with model group,the skin appearance of the aerobic exercise and resistance exercise groups was significantly improved(P<0.05 or P<0.01).The thickness of the epidermis(P<0.05)and dermis increased significantly in resistance exercise group(P<0.01),and the elastic fibers were arranged in a more neat and orderly manner.The expression of P16 protein in the skin was decreased in aerobic exercise group(P<0.05).The levels of serum TNF-α,and serum and skin IL-18 were decreased in aerobic exercise group(P<0.05).The levels of serum and skin IL-1β,TNF-α and IL-18 in the resistance ex-ercise group were decreased(P<0.05 or P<0.01).Skin IL-18R content was significantly decreased in both aerobic and re-sistance exercise groups(P<0.01).IL-18BP content was significantly increased(P<0.01),and colocalized expression levels of IL-18 and IL-18R were significantly decreased.The mRNA expression of IL-18 and IL-18R was significantly de-creased in both aerobic and resistance exercise groups(P<0.01).CONCLUSION:Both aerobic and resistance exercise delay age-increased skin aging in rapidly aging mice,and the IL-18/IL-18R signaling pathway down-regulated by exercise may be the pathway of this effect.
Mesenchymal stem cells (MSCs) primarily secrete physiologically functional exosomes via paracrine effects that act on various adjacent and distant cells, thus exerting their therapeutic effects. In recent years, hypoxic preconditioning, as a novel MSC culture mode, has emerged as a research hotspot. Many previous studies have shown the role and underlying regulatory mechanisms of hypoxic preconditioning in various diseases, which has provided sufficient reference materials for the MSC research field. Therefore, this review summarizes the progress in application of hypoxia-preconditioned MSC-derived exosomes that substantially increases and improves the biological activity of specific molecules, such as microRNA.
目的:探讨淫羊藿苷(ICA)对高糖(HG)诱导的人肾小管上皮细胞上皮-间充质转化(EMT)的影响及机制.方法:将人肾小管上皮HK-2细胞分为正常对照(NC)组(5.5 mmol/L D-葡萄糖)、高渗(OSM)组(5.5 mmol/L D-葡萄糖+24.5 mmol/L甘露醇)、HG组(30 mmol/L D-葡萄糖)、低剂量ICA(L-ICA)组(30 mmol/L D-葡萄糖+10 µmol/L ICA)、高剂量ICA(H-ICA)组(30 mmol/L D-葡萄糖+40 µmol/L ICA)、H-ICA+SC79[蛋白激酶B(PKB/AKT)激活剂]组(30 mmol/L D-葡萄糖+40 µmol/L ICA+10 µmol/L SC79)和LY294002[磷脂酰肌醇3-激酶(PI3K)抑制剂]组(30 mmol/L D-葡萄糖+10 µmol/L LY294002).CCK-8法检测细胞活力;倒置显微镜观察细胞形态;RT-qPCR检测细胞α-平滑肌肌动蛋白(α-SMA)和上皮钙黏素(E-cadherin)的mRNA表达水平;Western blot检测细胞中α-SMA、E-cadherin、PI3K、p-PI3K、AKT和p-AKT蛋白水平;划痕实验检测细胞的迁移能力.结果:与NC组比较,HG组HK-2细胞活力下降,迁移能力增强,细胞失去原有形态,部分细胞呈长梭状,E-cadherin的mRNA和蛋白表达水平显著下降,α-SMA的mRNA和蛋白表达水平及p-PI3K/PI3K和p-AKT/AKT比值均显著升高(P<0.05).相较于NC组,OSM组细胞形态无明显改变,其它相关上述指标变化均无统计学意义(P>0.05),提示高渗透压对HK-2细胞无显著影响.与HG组比较,不同剂量ICA组细胞形态由长梭状逐渐趋向正常形态,细胞活力增强,迁移能力降低,E-cad-herin的mRNA和蛋白表达水平显著升高,α-SMA的mRNA和蛋白表达水平及p-PI3K/PI3K和p-AKT/AKT比值均显著降低(P<0.05);LY294002组细胞E-cadherin蛋白表达水平显著升高,α-SMA蛋白表达水平及p-PI3K/PI3K和p-AKT/AKT比值均显著降低(P<0.05).与H-ICA组相比,H-ICA+SC79组细胞中E-cadherin蛋白表达水平显著下降,α-SMA蛋白表达水平及p-AKT/AKT比值均显著升高(P<0.05).结论:ICA能够抑制HG诱导的HK-2细胞EMT,可能与其抑制PI3K/AKT信号通路活化有关.
目的:探讨钙网蛋白(CALR)在胃癌组织中的表达水平及其调节AGS、HGC-27细胞转移的作用机制.方法:收集65例胃癌患者肿瘤组织样本和16例大网膜转移组织样本,采用免疫组织化学法检测胃癌及大网膜转移组织中CALR的表达水平;通过小干扰RNA(siRNA)下调AGS和HGC-27细胞中CALR的表达,通过慢病毒转染技术过表达AGS和HGC-27细胞中的CALR,采用Transwell实验和细胞划痕实验检测CALR表达对AGS、HGC-27细胞侵袭和迁移的影响;采用活性氧(ROS)和线粒体三磷酸腺苷(ATP)荧光探针检测AGS、HGC-27细胞ROS水平和ATP荧光强度;采用线粒体膜电位检测试剂盒(JC-1)检测AGS、HGC-27细胞线粒体膜电位水平;Western blot法检测CALR、肌浆/内质网Ca2+转运ATP酶A2(ATP2A2)、ATP酶Na+/K+转运亚基Beta 3(ATP1B3)和ATP合酶-偶联因子6(ATP5J)的蛋白表达水平;建立胃癌裸鼠腹腔移植瘤模型,探究CALR在体内对HGC-27细胞增殖的影响.结果:与癌旁组织比较,CALR在胃癌组织和大网膜转移组织中显著过表达(P<0.01);CALR过表达显著促进AGS、HGC-27细胞侵袭和迁移,而siRNA CALR则抑制胃癌细胞的侵袭和迁移(P<0.01);pcDNA3.1-CALR组AGS、HGC-27细胞中ATP荧光强度、线粒体膜电位水平均显著升高,ROS水平则降低;而siRNA-CALR组ATP荧光强度、线粒体膜电位水平均显著降低,ROS水平则升高(P<0.01);此外,与阴性对照组比较,pcDNA3.1-CALR组AGS、HGC-27细胞中CALR、ATP2A2、ATP1B3和ATP5J蛋白表达水平均显著升高,而siRNA-CALR组CALR、ATP2A2、ATP1B3和ATP5J蛋白表达水平均显著降低(P<0.01);在裸鼠模型中,敲减CALR可显著抑制肿瘤组织的生长,降低肿瘤组织中ATP2A2、ATP1B3和ATP5J蛋白的表达水平.结论:CALR在人胃癌组织及大网膜转移组织中过表达,CALR可通过调节线粒体膜电位和ATP合酶介导线粒体功能,影响胃癌AGS和HGC-27细胞的转移.
目的:本研究拟观察过表达沉默信息调节因子3(silent information regulator 3,Sirt3)对高糖(high glucose,HG)诱导的肾小管上皮细胞应激性衰老的影响,并进一步探究过表达Sirt3对Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)/核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)通路活性的影响,探讨其可能的作用机制.方法:本研究以HK-2细胞为模型,应用质粒转染过表达Sirt3,采用ML385(Nrf2特异性抑制剂)阻断Keap1/Nrf2通路,按照预定细胞分组给予不同刺激,Western blot法检测Sirt3、Keap1、Nrf2、衰老相关蛋白P16和P21蛋白的表达,DCHF-DA标记法检测细胞内活性氧(reactive oxygen species,ROS)的积聚,衰老相关β-半乳糖苷酶(senescence-associated β-galactosidase,SA-β-Gal)染色法检测细胞衰老状况.结果:(1)与正常糖对照组相比,高糖刺激48 h后,衰老相关蛋白P16和P21的水平显著升高(P<0.05);与高糖+空载质粒转染对照组相比,过表达Sirt3后,P16和P21的表达明显降低(P<0.05),SA-β-Gal阳性细胞减少,且显著逆转高糖环境下HK-2细胞内ROS的蓄积.(2)与正常糖对照组相比,高糖环境下HK-2细胞中Nrf2、醌氧化还原酶1(NADPH quinone oxidore-ductase 1,NQO1)蛋白水平降低,Keap1蛋白表达水平升高(P<0.05);与高糖+空载质粒转染对照组相比,转染Sirt3质粒后,Nrf2及其下游蛋白NQO1的表达增加,同时核Nrf2的表达显著增加(P<0.05).(3)进一步应用ML385抑制Keap1/Nrf2通路活性,结果显示,与高糖+转染Sirt3质粒组相比,加入ML385后Nrf2、NQO1和血红素加氧酶1(heme oxygenase-1,HO-1)蛋白表达降低,衰老相关蛋白P16和P21的表达明显升高(P<0.05).结论:高糖环境下,过表达Sirt3可通过上调Keap1/Nrf2通路的活性,促进Nrf2向细胞核易位,减少细胞内ROS蓄积,从而减轻应激性衰老.
