补肾解毒方抗脑老化机制的体外研究

摘要 目的：探讨补肾解毒方含药血清抗脑老化的体外分子机制。方法：采用过氧化氢诱导制备人脑胶质细胞衰老模型，使用补肾解毒方含药血清干预以期达到对人脑胶质细胞衰老模型的治疗效果，CCK-8法检测细胞活力，采用β-半乳糖苷酶染色法观察衰老阳性细胞比例，活性氧荧光探针法检测胞内活性氧水平，流式细胞术检测细胞凋亡比例，qPCR检测细胞中衰老相关基因CXCL1、FOXO1、P16、IL1A、IGFBP3、IL6的转录水平。结果：补肾解毒方含药血清可显著增强细胞活力（P＜0.05），显著减少过氧化氢诱导的衰老阳性细胞比例（P＜0.05），降低活性氧水平，显著减少凋亡细胞比例（P＜0.01），下调IL1A、IL6、CXCL1、IGFBP3、FOXO1转录水平。结论：补肾解毒方含药血清可显著抑制过氧化氢诱导的人脑胶质细胞衰老，其可能与增强细胞活力、抗炎、抗凋亡、降低胞内活性氧水平等有关。     

原花青素B2通过Akt/FoxO4通路拮抗内皮细胞衰老的实验研究

研究原花青素B2(PCB2)对内皮细胞衰老的调控作用,探讨其可能的分子机制。以棕榈酸(PA)50μmol/L处理胰岛内皮细胞MS-1建立细胞衰老模型,根据细胞活力、β-半乳糖苷酶(SA-β-Gal)染色以及细胞周期相关基因的表达来评估内皮细胞衰老;同时加入PCB2干预,检测衰老相关指标及Akt/FoxO4通路的蛋白表达水平的变化情况。MS-1细胞经PA处理后SA-β-Gal阳性细胞增加,细胞周期相关基因p53和p21上调显示细胞周期阻滞在G0/G1期,表明PA处理后能诱导内皮细胞衰老发生;经PCB2(50μg/mL)干预后能够有效改善PA诱导的细胞衰老。相比于正常组,经PA处理后抑制Akt活化,其表达由胞核转至胞浆,在胞核中表达明显减少;导致Akt下游信号FoxO4的磷酸化降低,使其在细胞核中明显增加;而应用PCB2(50μg/mL)处理后能有效逆转这一现象;PCB2对棕榈酸诱导的内皮细胞衰老有明显的保护作用,其可能通过Akt/FoxO4信号通路发挥调控作用。     

小檗碱对牙周炎大鼠的细胞因子水平、氧化应激和炎症性骨溶解的实验研究

摘要 目的：研究小檗碱对牙周炎大鼠的治疗效果，并探讨小檗碱治疗对牙周炎大鼠牙龈组织炎症细胞因子、氧化应激和炎症性骨溶解的影响。方法：45只SD大鼠被随机分为三组，即对照组、模型组和小檗碱治疗组。模型组和小檗碱治疗组大鼠通过涂抹牙周致病菌建立牙周炎模型，对照组大鼠涂抹生理盐水。小檗碱通过灌胃给予小檗碱进行治疗，对照组和模型组大鼠给予等量生理盐水。小檗碱治疗8周后，安乐死各组大鼠通过ELISA试剂盒测定各组大鼠牙龈组织中前列腺素E2（PEG2）、白介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、丙二醛（MDA）、过氧化氢脂质（LPO）、过氧化氢酶（CAT）、核因子-kB活化受体（RANKL）和骨保护素（OPG）含量。结果：与牙周炎模型组大鼠比较，小檗碱治疗组牙周炎大鼠牙龈指数和附着水平均显著降低（P<0.05）。小檗碱治疗8周后，牙周炎大鼠模型牙龈组织PEG2, IL-1β, TNF-α和IL-6等炎症细胞因子水平均显著下降（P<0.05），氧化应激指标MDA和LPO均显著下降（P<0.05），抗氧化应激指标CAT含量显著上升（P<0.05）。此外，小檗碱还显著降低牙周炎大鼠牙龈组织RANKL含量和RANKL/OPG比值（P<0.05），而显著提高牙周炎大鼠牙龈组织OPG含量（P<0.05）。结论：小檗碱对牙周炎大鼠具有较高的治疗效果，其机制可能与小檗碱治疗可降低牙周炎大鼠牙龈组织炎症细胞因子含量，降低氧化应激水平和缓解炎症性骨溶解有关。     

苦杏仁苷介导人肝癌细胞HuH-7凋亡的实验研究

摘要 目的：通过体内体外实验探讨苦杏仁苷在肝癌中的抗肿瘤作用。方法：MTT 法检测不同浓度的苦杏仁苷对肝癌 HuH-7细胞存活率的影响;DAPI染色法观察苦杏仁苷介导HuH-7细胞的凋亡形态学变化;流式细胞术检测苦杏仁苷干预后HuH-7细胞凋亡率变化;Western Blot法检测细胞凋亡相关蛋白Bax、Bcl-2的表达,计算Bax/Bcl-2的比值。建立裸鼠HuH-7细胞移植瘤模型,观察苦杏仁苷对荷瘤裸鼠移植瘤体积的影响。结果：体外实验结果证实苦杏仁苷能够诱导人肝癌HuH-7细胞凋亡的发生（P<0.05）。随着苦杏仁苷浓度的增加,HuH-7细胞的存活率降低,凋亡率升高,干预后Bax/Bcl-2比值明显升高（P<0.05）。体内实验结果表明,裸鼠HuH-7细胞移植瘤的体积增长速度减慢（P<0.05）。结论：苦杏仁苷能够诱导人肝癌HuH-7细胞和裸鼠HuH-7细胞移植瘤细胞发生凋亡,减缓肿瘤生长,从而发挥抗肿瘤作用。     

脂肪细胞衰老通过衰老相关分泌表型诱导微血管内皮细胞早衰和功能紊乱

摘要 目的：探讨衰老脂肪细胞对微循环内皮细胞（ECs）功能状态的影响,以及异常早衰在糖尿病肾病（DKD）中的潜在作用。方法：3T3-L1细胞被诱导分化为年轻和衰老的脂肪细胞。HMEC-1细胞分别培养在年轻、衰老脂肪细胞制成的条件培养基和对照培养基中。通过免疫荧光检测γH2AX和SA-β-半乳糖苷酶活性鉴定细胞衰老状态。通过qPCR、Western blot检测衰老相关分泌表型（SASP）、胰岛素受体底物1（IRS1）、Jun原癌基因（JUN）、组蛋白H3第4位赖氨酸二甲基化、三甲基化（H3K4me2、H3K4me3）等指标的表达水平。利用GEO数据库对衰老肾脏和早期糖尿病肾病的差异表达基因（DGE）进行生物信息学分析。结果：衰老脂肪细胞的SASP表达显著升高,其条件培养基成功诱导HMEC-1细胞衰老。与年轻HMEC-1细胞相比,诱导衰老的HMEC-1细胞中斯钙素1（STC1）表达上调,前炎症因子、JUN和H3K4me3均表达下调。与对照组相比,IRS1在年轻HMEC-1细胞中显著下调,在诱导衰老的HMEC-1细胞中无显著变化。生物信息学结果显示差异基因的交集仅存在于衰老肾脏的上调基因和早期糖尿病肾病的下调基因之间。PPI网络分析、GO及KEGG富集分析表明IL6-SOCS3-IRS1是异常早衰机制参与早期DKD发生的核心信号通路。结论：脂肪细胞衰老导致微循环内皮细胞早衰并损害其正常功能状态,异常早衰机制参与了DKD的发生发展。     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。     

紫丁香苷调控PI3K/Akt/mTOR信号通路诱导乳腺癌细胞凋亡的研究

目的 研究紫丁香苷的抗乳腺癌作用及分子机制，为紫丁香苷的临床应用提供理论依据。方法 MTT检测紫丁香苷对乳腺癌细胞增殖的抑制作用；台盼蓝、TUNEL和Annexin V-FITC/PI染色检测细胞的凋亡状况，Western bolt检测Caspase-3的活化情况，判断细胞凋亡是否发生；检测凋亡相关蛋白B淋巴细胞瘤2（Bcl-2）的表达，结合JC-1染色探讨紫丁香苷对线粒体凋亡途径的影响；运用PI3K激动剂Recilisib做对比，qRT-PCR和Western bolt检测紫丁香苷调控PI3K/Akt/mTOR通路诱导癌细胞凋亡的作用。结果 紫丁香苷对乳腺癌细胞的增殖具有时间和剂量依赖的抑制作用，能诱导癌细胞发生凋亡。进一步研究发现，紫丁香苷处理后，细胞内Caspase-3被激活，Bcl-2表达下降，线粒体膜电位明显丧失，PI3K、Akt和mTOR的mRNA与蛋白质水平表达无明显变化，但蛋白质磷酸化水平明显下降；Recilisib处理后部分抵消了紫丁香苷对乳腺癌细胞凋亡的作用。结论 紫丁香苷对乳腺癌细胞MDA-MB-231和MCF-7具有良好的抑制作用，其通过抑制PI3K/Akt/mTOR信号通路的活化来抑制细胞增殖并诱导细胞发生线粒体途径的凋亡。紫丁香苷是具有开发潜力的抗乳腺癌药物。     

研究报告: EIF2B5表达下调的人星形胶质细胞与人神经元在内质网应激后细胞存活及miRNA表达的差异

目的 探讨白质消融性白质脑病中胶质细胞选择性受累而神经元受累轻微的原因。方法 将EIF2B5-RNAi表达载体转染至人星形胶质细胞和人神经元,检测基础状态下及内质网应激（endoplasmic reticulum stress,ERS）后细胞凋亡和活力,检测参与ERS调控的已知和未知miRNA,筛选EIF2B5-RNAi人星形胶质细胞在ERS后miRNA变化。结果 与EIF2B5-RNAi人神经元相比,星形胶质细胞自发凋亡及细胞活力下降。较之神经元,更多miRNA参与星形胶质细胞ERS刺激后的调控,EIF2B5-RNAi组参与调控的miRNA数目显著减少。聚类分析发现,5条已知miRNA是通路连接的关键组分。结论 人星形胶质细胞在ERS后可能更加依赖众多促细胞增殖分化的miRNA修复,而EIF2B5-RNAi人星形胶质细胞存在自发凋亡,ERS后严重减少的miRNA可能导致细胞无法存活。     

小檗碱对幽门螺旋杆菌感染小鼠胃粘膜损伤和免疫功能的影响机制研究

摘要 目的：探讨小檗碱对幽门螺旋杆菌感染小鼠胃粘膜损伤和免疫功能的影响机制。方法：选择SPF雄性小鼠50只,随机分为5组,包括空白对照组、幽门螺杆菌模型组、小檗碱低剂量组（25 mg/mL,A组）、中剂量组（50 mg/mL,B组）和高剂量组（100 mg/kg/d,C组）。空白组大鼠腹腔注射等容量的生理盐水,其余组使用口服幽门螺杆菌的方法建立幽门螺旋杆菌感染小鼠模型。进行胃黏膜上皮细胞存活率和凋亡率检测,同时测定胃黏膜上皮细胞凋亡蛋白水平、炎症因子水平、免疫功能以及IL-4/STAT6 mRNA水平。结果：模型组、小檗碱低剂量组、中剂量组和高剂量组胃黏膜上皮细胞存活率均显著低于空白对照组（P<0.05）;且随着小檗碱剂量的增加,细胞存活率逐渐升高（P<0.05）;模型组、小檗碱低剂量组、中剂量组和高剂量组胃黏膜上皮细胞凋亡率均显著高于空白对照组（P<0.05）;且随着小檗碱剂量的增加,细胞凋亡率逐渐降低（P<0.05）;与模型组比较,小檗碱低、中、高剂量组小鼠胃黏膜上皮细胞凋亡蛋白Bax和Cl-caspase-3的水平、TNF-α、IFN-γ、IL-10和TGF-β的水平均显著降低（P<0.05）,且随着小檗碱剂量的增加,Bax和Cl-caspase-3的水平、TNF-α、IFN-γ、IL-10和TGF-β的水平逐渐降低（P<0.05）;与模型组比较,小檗碱低、中、高剂量组上清液中CD3⁺、CD4⁺和CD8⁺的水平、IL-4 mRNA和STAT6 mRNA均显著升高（P<0.05）,且随着小檗碱剂量的增加,CD3⁺、CD4⁺和CD8⁺水平、IL-4 mRNA和STAT6 mRNA逐渐升高（P<0.05）。结论：小檗碱能够减轻幽门螺旋杆菌感染小鼠胃粘膜损伤,提升免疫功能,可能与IL-4/STAT6信号通路有关。     

靶向Bcl-2克服非小细胞肺癌EGFR-TKIs获得性耐药

摘要 目的：探讨靶向抗凋亡蛋白Bcl-2克服非小细胞肺癌EGFR-TKIs耐药的作用及重定位Bcl-2靶向抑制剂用于克服耐药的可行性。方法：通过药物浓度梯度递增法构建非小细胞肺癌多代EGFR-TKIs耐药株,根据亲本细胞和多代EGFR-TKIs耐药的非小细胞肺癌细胞的RNA-seq数据筛选出潜在耐药相关基因Bcl-2,通过Western blot 检测其在耐药细胞中的蛋白水平。为了探讨Bcl-2诱导耐药的作用,采用siRNA干扰和使用Bcl-2的抑制剂ABT199抑制Bcl-2,通过CCK8法、IncuCyte实时监测和Western blot等方法检测其对亲本和耐药细胞的细胞活力、药物敏感性、增殖和凋亡的影响。随后使用奥希替尼分别处理亲本及耐药细胞,通过Western Blot检测NRF2受到药物作用后及在耐药细胞中的蛋白水平,并以临床公共数据库分析辅助验证。使用siRNA干扰或NRF2抑制剂ML385敲低或抑制NRF2功能,借助Western Blot和CCK8法检测其对Bcl-2表达水平及对EGFR-TKI敏感性的影响;通过加入NRF2的激动剂Ki696探究其对Bcl-2的诱导作用、对EGFR-TKI敏感性的影响及取消靶向Bcl-2逆转耐药的作用。结果：Bcl-2在EGFR-TKIs耐药细胞中上调;敲低或抑制Bcl-2后,可选择性抑制耐药细胞的生长和活力,并诱导凋亡,且能逆转包括第三代药物奥希替尼在内的多代EGFR-TKIs耐药;EGFR-TKI可在敏感细胞中诱导NRF2的上调,且耐药细胞中上调的Bcl-2受NRF2调控。结论：EGFR-TKIs耐药细胞通过上调抗凋亡分子Bcl-2获得耐药,该分子的上调受NRF2的调控,靶向Bcl-2则可以逆转耐药。     

人脐带间充质干细胞条件培养基联合白藜芦醇对人滋养层细胞的作用

目的:探讨人脐带间充质干细胞条件培养基联合白藜芦醇对人绒毛膜外滋养层细胞凋亡的影响。方法:通过CCK8细胞活力检测试剂盒测定白藜芦醇及其与人脐带间充质干细胞条件培养基共同处理人绒毛膜外滋养层细胞HTR8后对细胞增殖及活性的影响;细胞迁移试验检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞迁移能力的影响;显微镜观察细胞形态,并用流式细胞仪检测细胞凋亡率的变化;Western blot检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞凋亡相关蛋白Bax、Bcl-2以及迁移相关蛋白MMP-9表达的影响。结果:白藜芦醇能够抑制HTR8细胞增殖,抑制细胞迁移及MMP-9蛋白的表达,改变Bax和Bcl-2蛋白表达诱导细胞凋亡的作用。而人脐带间充质干细胞条件培养基能够逆转白藜芦醇对细胞的抑制作用。结论:人脐带间充质干细胞条件培养基能够通过调控Bax、Bcl-2、MMP-9的蛋白表达逆转白藜芦醇对人绒毛膜外滋养层细胞的抑制作用。人脐带间充质干细胞条件培养基可作为潜在的治疗人绒毛膜外滋养层细胞功能障碍的临床手段,孕妇需要小心使用白藜芦醇。     

Lycium barbarum Polysaccharides Protect Human Lens Epithelial Cells against Oxidative Stress–Induced Apoptosis and Senescence

Objectives

We aimed to investigate the protective effect of Lycium barbarum polysaccharides (LBPs) against oxidative stress–induced apoptosis and senescence in human lens epithelial cells.

Methods

To study apoptosis, SRA01/04 cells, a human lens epithelial cell lines, were exposed to 200 µM hydrogen peroxide (H₂O₂) for 24 h with or without pretreatment with LBPs. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, intracellular reactive oxygen species (ROS), and the loss of mitochondria membrane potential (Δψm) were detected by flow cytometric analyses. Expression levels of Bcl-2 and Bax proteins were measured by western blot analysis. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were quantized using commercial enzymatic kits according to the manufacturer''s instructions. To study senescence, SRA01/04 cells were pre-incubated with LBPs and all cells were then exposed to 100 µM H₂O₂ for 96 h. Cellular senescence was assessed by morphologic examination and senescence-associated β-galactosidase (SA-β-gal) staining.

Results

LBPs significantly reduced H₂O₂-induced cell apoptosis, the generation of ROS, the loss of Δψm, and the levels of MDA. LBPs also inhibited H₂O₂-induced downregulated Bcl-2 and upregulated Bax proteins and increased the levels of SOD and GSH enzyme activity. Moreover, LBPs significantly attenuated H₂O₂-induced cellular senescence.

Conclusions

These findings suggested that LBPs protect human lens epithelial cells from H₂O₂-induced apoptosis by modulating the generation of ROS, loss of Δψm, Bcl-2 family, and antioxidant enzyme activity and attenuating cellular senescence.     

Echinacoside protects adenine-induced uremic rats from sciatic nerve damage by up-regulating α-Klotho

Objectives:To investigate the therapeutic effect of Echinacoside on uremia-induced sciatic nerve injury and explore the specific molecular mechanism and role of α-Klotho.Methods:SD rats were given continuous gavage of adenine to prepare a uremia-induced sciatic nerve injury model. The model was given either Echinacoside or α-Klotho by gavage. Histopathological changes of kidney and sciatic nerve were detected by H&E staining. The changes of creatinine, urea nitrogen, and urine protein were detected by biochemical detection. The changes of IL-1β and IL-18 were detected by ELISA. Nerve activity-related indicators were detected by biochemical detection. Changes in related mRNA and protein expression were detected by qPCR and western blot.Results:Creatinine, urea nitrogen, urine protein, and malondialdehyde (MDA) in the model group were significantly increased and inhibited by Echinacoside and α-Klotho treatment with Echinacoside dose-dependence. Meanwhile, the activities of ATP concentration, potassium adenosine triphosphate (Na+, K+ ATPase), succinate dehydrogenase (SDH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) showed opposite trends.Conclusions:Echinacoside can significantly relieve uremia-induced sciatic nerve injury in rats. Its specific molecular mechanism is related to the inhibition of the classical cellular pyroptosis pathway, which is likely achieved by promoting α-Klotho expression.     

Nampt及高糖高胰岛素微环境对人顺铂耐药肺癌细胞株增殖和转移的影响

摘要 目的：探究烟酰胺磷酸核糖转移酶（Nampt）及高糖高胰岛素（HG+HI）微环境对人顺铂耐药肺癌细胞增殖和转移的影响。方法：将人顺铂耐药细胞株A549/DDP分为6组（n=6）：对照组（control）、高糖高胰岛素干预组（HH，使用添加30 mmol/L的葡萄糖和500 mU/L的胰岛素的培养基培养72 h）、分别转染sh-NC和sh-Nampt组（sh-NC和sh-Nampt，使用Lipofectamine 2000将sh-NC和sh-Nampt分别转染到细胞中，转染时间为48 h）、HH干预sh-NC和sh-Nampt组（HH+sh-NC和HH+sh-Nampt）。每组6个重复样本。qRT-PCR检测转染效率，MTT法检测细胞增殖，流式细胞仪检测细胞凋亡，Transwell检测细胞迁移和侵袭，qRT-PCR检测Nampt mRNA，Western blot检测Nampt、Bcl-2、Bax、MMP-2、MMP-9、p-PI3K、PI3K、p-AKT和AKT蛋白表达。结果：与对照组和sh-NC组比较，sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，而细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。与对照组和sh-NC组比较，HH组和HH+sh-NC组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平升高，Bax蛋白表达水平降低（P<0.005）。与HH组和HH+sh-NC组比较，HH+sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。结论：高糖高胰岛素微环境可能通过上调Nampt/PI3K/AKT信号通路诱导人顺铂耐药肺癌细胞的增殖和转移。     

Forkhead box O3 protects the heart against paraquat‐induced aging‐associated phenotypes by upregulating the expression of antioxidant enzymes

Paraquat (PQ) promotes cell senescence in brain tissue, which contributes to Parkinson's disease. Furthermore, PQ induces heart failure and oxidative damage, but it remains unknown whether and how PQ induces cardiac aging. Here, we demonstrate that PQ induces phenotypes associated with senescence of cardiomyocyte cell lines and results in cardiac aging‐associated phenotypes including cardiac remodeling and dysfunction in vivo. Moreover, PQ inhibits the activation of Forkhead box O3 (FoxO3), an important longevity factor, both in vitro and in vivo. We found that PQ‐induced senescence phenotypes, including proliferation inhibition, apoptosis, senescence‐associated β‐galactosidase activity, and p16^INK4a expression, were significantly enhanced by FoxO3 deficiency in cardiomyocytes. Notably, PQ‐induced cardiac remolding, apoptosis, oxidative damage, and p16^INK4a expression in hearts were exacerbated by FoxO3 deficiency. In addition, both in vitro deficiency and in vivo deficiency of FoxO3 greatly suppressed the activation of antioxidant enzymes including catalase (CAT) and superoxide dismutase 2 (SOD2) in the presence of PQ, which was accompanied by attenuation in cardiac function. The direct in vivo binding of FoxO3 to the promoters of the Cat and Sod2 genes in the heart was verified by chromatin immunoprecipitation (ChIP). Functionally, overexpression of Cat or Sod2 alleviated the PQ‐induced senescence phenotypes in FoxO3‐deficient cardiomyocyte cell lines. Overexpression of FoxO3 and CAT in hearts greatly suppressed the PQ‐induced heart injury and phenotypes associated with aging. Collectively, these results suggest that FoxO3 protects the heart against an aging‐associated decline in cardiac function in mice exposed to PQ, at least in part by upregulating the expression of antioxidant enzymes and suppressing oxidative stress.     

松果菊苷对多柔比星心脏毒性保护作用及机制研究

目的:观察松果菊苷(ECH)能否减轻多柔比星(DOX)心脏毒性并初步阐明其作用机制。方法:通过单次腹腔注射大剂量多柔比星(15 mg/kg)建立急性心脏毒性小鼠模型,DOX处理后每日通过腹腔注射ECH(50 mg/kg/day)。实验分组如下:正常组(Control组);单纯松果菊苷处理组(ECH组);多柔比星处理组(DOX组);多柔比星+松果菊苷处理组(DOX+ECH组)。给药5天后检测左心室功能、心肌组织病理改变、氧化应激和心肌凋亡情况。结果:与Control组相比,DOX组小鼠心脏收缩和舒张功能明显减弱,心肌细胞出现空泡变性,心肌MDA含量、凋亡率以及促凋亡蛋白Bax和cleaved Caspase-3表达明显增加,而抑制凋亡蛋白Bcl-2表达量、SOD与GSH-Px活性明显下降。与DOX组相比,松果菊苷能明显改善心脏功能,缓解心肌空泡变性,降低MDA含量、凋亡率以及Bax和cleaved Caspase-3表达量,而提高Bcl-2表达量、SOD与GSH-Px活性(均P 0.05)。结论:松果菊苷可以通过抑制心肌组织氧化应激损伤和凋亡缓解多柔比星诱导的急性心脏毒性。     

Atractylodis rhizoma water extract attenuates fructose-induced glomerular injury in rats through anti-oxidation to inhibit TRPC6/p-CaMK4 signaling

BackgroundAtractylodis rhizoma, an aromatic herb for resolving dampness, is used to treat Kidney-related edema in traditional Chinese medicine for thousands years. This herb possesses antioxidant effect. However, it is not yet clear how Atractylodis rhizoma prevents glomerular injury through its anti-oxidation.PurposeBased the analysis of Atractylodis rhizoma water extract (ARE) components and network pharmacology, this study was to explore whether ARE prevented glomerular injury via its anti-oxidation to inhibit oxidative stress-driven transient receptor potential channel 6 (TRPC6) and its downstream molecule calcium/calmodulin-dependent protein kinase IV (CaMK4) signaling.MethodsLiquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ARE components. Network pharmacology analysis was preliminarily performed. Male Sprague-Dawley rats were given 10% fructose drinking water (100 mL/d) for 16 weeks. ARE at 720 and 1090 mg/kg was orally administered to rats for the last 8 weeks. Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity in rat kidney cortex were detected, respectively. In rat glomeruli, redox-related factors forkhead box O3 (FoxO3), SOD2 and catalase (CAT), podocyte slit diaphragm proteins podocin and nephrin, cytoskeleton proteins CD2-associated protein (CD2AP) and α-Actinin-4, as well as TRPC6, p-CaMK4 and synaptopodin protein levels were analyzed by Western Blotting. SOD2 and CAT mRNA levels were detected by qRT-PCR.Results36 components were identified in ARE. Among them, network pharmacology analysis indicated that ARE might inhibit kidney oxidative stress. Accordingly, ARE up-regulated nuclear FoxO3 expression, and then increased SOD2 and CAT at mRNA and protein levels in glomeruli of fructose-fed rats. It reduced H₂O₂ and MDA levels, and increased SOD activity in renal cortex of fructose-fed rats. Subsequently, ARE down-regulated TRPC6 and p-CaMK4, and up-regulated synaptopodin in glomeruli of fructose-fed rats. Furthermore, ARE increased podocin and nephrin, as well as CD2AP and α-Actinin-4, being consistent with its reduction of urine albumin-to-creatinine ratio and improvement of glomerular structure injury in this animal model.ConclusionsThese results suggest that ARE may prevent glomerular injury in fructose-fed rats possibly by reducing oxidative stress to inhibit TRPC6/p-CaMK4 signaling and up-regulate synaptopodin expression. Therefore, ARE may be a promising drug for treating high fructose-induced glomerular injury in clinic.     

MiR-181a-5p is involved in the cardiomyocytes apoptosis induced by hypoxia–reoxygenation through regulating SIRT1

ABSTRACT

MiR-181a-5p’s mechanism in hypoxia–reoxygenation (H/R)-induced cardiomyocytes apoptosis has not been clarified. This study verified that SIRT1 was the target of miR-181a-5p. MiR-181a-5p expression was up-regulated or down-regulated in H/R-induced cardiomyocytes, and SIRT1 was transfected into cells alone or in combination with miR-181a-5p. Cell viability, apoptosis, levels of released lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as the Bcl-2, Bax, and Caspase 3 levels in treated cells were tested. On the one hand, down-regulated miR-181a-5p promoted cell viability, reduced released LDH and MDA, and increased SOD level in H/R-induced cardiomyocytes. On the other hand, miR-181a-5p inhibited apoptosis and elevated Bcl-2 expression while decreasing the expressions of Bax and Caspase 3 in treated cells, but the effects of miR-181a-5p could be rescued by SIRT1. In conclusion, miR-181a-5p involved in H/R-induced cardiomyocytes apoptosis through regulating SIRT1, which might become a novel direction for related diseases.     

Torulene and torularhodin,protects human prostate stromal cells from hydrogen peroxide-induced oxidative stress damage through the regulation of Bcl-2/Bax mediated apoptosis

The current study was designed to elucidate the cytoprotective effects and possible mechanisms of torulene and torularhodin on hydrogen peroxide (H₂O₂)-induced oxidative stress damage in human prostate stromal cells (WPMY-1). After treated with H₂O₂, a notable decrease was appeared in cell viability, yet the decrease was attenuated when cells were pretreated with torulene and torularhodin (0.5–10?μM) as evaluated by WST-1 assay. Pretreatment with these two carotenoids significantly attenuated H₂O₂-induced apoptosis in WPMY-1 cells through the inhibition of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction, as well as the activation of the activities in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Finally, pretreatment of cells with carotenoids resulted in the regulation of the mRNA and protein expression of Bcl-2 and Bax in H₂O₂-exposed prostate stromal cells. The present results indicate that both torulene and torularhodin can protect human prostate stromal cells from oxidative stress damage via Bcl-2/Bax mediated apoptosis.     

紫檀芪调节Keap-1/Nrf2/HO-1信号通路对非酒精性脂肪肝大鼠氧化应激和细胞凋亡的影响

摘要 目的：研究紫檀芪调节Kelch样ECH关联蛋白1（Keap-1）/核因子E2相关因子2（Nrf2）/血红素加氧酶-1（HO-1）信号通路对非酒精性脂肪肝（NAFLD）大鼠氧化应激和细胞凋亡的影响。方法：将60只SD大鼠随机分为对照组、模型组、紫檀芪低剂量组（30 mg/kg）、紫檀芪高剂量组（60 mg/kg）、紫檀芪（60 mg/kg）+N-（4-（2,3-二氢-1-（2''-甲基苯甲酰）-1H-吲哚-5-基）-5-甲基-2-噻唑基）-1,3-苯并二氧唑-5-乙酰胺（ML385）（30 mg/kg）组，每组12只。模型组与药物干预组大鼠以高脂饲料饲养诱导NAFLD模型，对照组大鼠以普通饲料饲养，各组连续喂养12周。以紫檀芪和ML385分组处理14 d后（对照组以等剂量生理盐水处理），检测各组大鼠脂代谢指标[三酰甘油（TG）、总胆固醇（TC）及游离脂肪酸（FFA）水平]、肝指数、肝功能指标[谷丙转氨酶（ALT）及谷草转氨酶（AST）]水平、血清白细胞介素（IL）-17、IL-6、IL-10、氧化应激指标[丙二醛（MDA）、超氧化物歧化酶（SOD）及过氧化氢酶（CAT）]水平；原位末端标记法（TUNEL）染色检测各组大鼠肝细胞凋亡率；蛋白免疫印迹法检测各组大鼠肝组织凋亡相关蛋白及Keap-1/Nrf2/HO-1通路相关蛋白表达。结果：与对照组相比，模型组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平显著降低（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平显著升高（P＜0.05）。与模型组相比，紫檀芪低、高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平均升高（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1、Bax表达水平均降低（P＜0.05）；与紫檀芪低剂量组相比，紫檀芪高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平升高（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平降低（P＜0.05）；与紫檀芪高剂量组相比，紫檀芪+ML385组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平降低（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Bax表达水平升高（P＜0.05）。结论：紫檀芪可能通过激活Keap-1/Nrf2/HO-1信号通路，改善NAFLD大鼠脂代谢水平，调节炎症反应及氧化应激，减轻肝组织脂肪变性及细胞凋亡。