Low-Intensity Ultrasound Reduces High Glucose-Induced Nitric Oxide Generation in Retinal Pigment Epithelial Cells

Diabetic retinopathy (DR) is a severe micro-vascular complication of diabetes. High glucose (HG)-evoked nitric oxide (NO) production mediated by increased oxidative stress is a key factor in DR pathogenesis. In this study, we examined whether low-intensity ultrasound (LIUS) stimulation can reduce HG-induced NO generation. We determined that LIUS stimulation decreased the HG-induced NO generation possibly via inhibition of reactive oxygen species (ROS) and subsequently diminished the associated pro-inflammatory pathway involving the induced expression of inducible nitric oxide synthase, cyclooxygenase-2 and vascular endothelial growth factor. In addition, we determined that LIUS stimulation reduced the quantity of NO produced by N-acetylcysteine, which was not mediated by ROS. These results indicate that LIUS can inhibit both ROS-dependent and -independent NO generation processes in ARPE-19 cells. We envision LIUS as a potential therapeutic alternative to treat DR. Further studies are required to understand the underlying mechanism of the LIUS-induced reduction of NO generation for DR therapy.     

柚皮素对糖尿病视网膜病变大鼠氧化损伤、细胞凋亡及Nrf2-ARE的影响

目的探讨柚皮素对糖尿病视网膜病变(DR)大鼠氧化损伤、细胞凋亡及核因子E2相关因子2(Nrf2)-抗氧化反应元件(ARE)的影响。方法随机数字表法将健康雄性SD大鼠分为空白对照组、模型组、阳性对照组、低剂量柚皮素组、中剂量柚皮素组、高剂量柚皮素组。采用腹腔注射链脲佐菌素(STZ)法建立DR大鼠模型。阳性对照组灌胃150 mg·kg^-1·d^-1羟苯磺酸钙,低、中、高剂量柚皮素组分别灌胃25 mg·kg^-1·d^-1、50 mg·kg^-1·d^-1、100 mg·kg^-1·d^-1柚皮素,空白对照组和模型组灌胃1%二甲基亚砜(DMSO),连续灌胃60 d。取各组大鼠视网膜组织,试剂盒检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽还原酶(GSH-Px)活力,TUNEL染色法检测视网膜组织的神经节细胞凋亡情况,Western blot法检测核因子E2相关因子2(Nrf2)、醌氧化还原酶-1(NQO1)和血红素加氧酶-1(HO-1)蛋白表达。结果与空白对照组比,模型组大鼠视网膜组织MDA含量、细胞凋亡指数显著升高(P<0.05),SOD和GSH-Px活力、Nrf2、HO-1、NQO1蛋白表达显著降低(P<0.05)。与模型组比,中、高剂量柚皮素组和阳性对照组大鼠视网膜组织MDA含量、细胞凋亡指数显著降低(P<0.05),SOD和GSH-Px活力、Nrf2、HO-1、NQO1蛋白表达显著降低显著升高(P<0.05),但低剂量组各指标均无显著差异(P>0.05)。结论柚皮素可能通过激活Nrf2-ARE信号通路降低糖尿病视网膜病变大鼠氧化应激损伤,其作用呈剂量依赖性。     

莱菔硫烷诱导胰岛系INS-1细胞血红素氧合酶1的表达

背景:莱菔硫烷可用于氧化应激相关疾病的治疗,血红素氧合酶是一种催化血红素降解的应激蛋白,已经成为预防氧化攻击的首选研究靶标之一。目的:观察核因子E2相关因子2激动剂莱菔硫烷对大鼠胰岛细胞系INS-1细胞血红素氧合酶1蛋白表达作用及细胞保护机制。方法:体外培养INS-1细胞,先用3μmol/L莱菔硫烷进行干预培养3h,再分别加入不同的胰岛素抵抗诱导剂葡萄糖氧化酶、地塞米松和葡萄糖胺刺激建立胰岛素抵抗细胞模型。结果与结论:3μmol/L莱菔硫烷处理INS-1细胞中血红素氧合酶1的表达增加(P<0.05),其效应在4h后达到高峰(P<0.05)。3μmol/L莱菔硫烷的预处理可逆转由胰岛素抵抗诱导剂导致的血红素氧合酶1表达下调(P<0.05)。而且在葡萄糖胺处理的INS-1细胞中,莱菔硫烷对血红素氧合酶1的表达改善与磷酸化PKB的表达上调具有正相关性(P<0.05,r=0.23)。结果证实,莱菔硫烷可能通过诱导核因子E2相关因子2介导的血红素氧合酶1表达,增强胰岛细胞抗氧化防御功能和抗损伤信号系统,从而达到拮抗胰岛素抵抗诱导剂对胰岛细胞的损伤作用。     

稽留流产绒毛组织中APPL1，Nrf2 和HO-1 的水平表达与氧化应激的相关性研究

目的　探讨稽留流产绒毛组织中蛋白亮氨酸拉链基序1（leucine zipper motif 1, APPL1）、核因子E2 相关因子2（Nuclear factor-erythroid 2-related factor 2,Nrf2）和血红素加氧酶1（haem oxygenase 1,HO-1）的水平表达与氧化应激的相关性。方法　取46 例稽留流产并行人流术治疗者为实验组,取50 例同期正常早孕并行人工流产术者为对照组,收集两组绒毛组织标本,用放射免疫沉淀法裂解缓冲液(radio immunopreci pitation assay lysis buffer, RIPA) 提取绒毛组织蛋白。用免疫组织化学法检测绒毛组织中APPL1,Nrf2 和HO-1 蛋白表达;用Western blot 检测绒毛组织中APPL1,Nrf2 和HO-1 蛋白水平;酶联免疫吸附法（enzyme linked immunosorbent assay,ELISA）测定绒毛组织中氧化应激指标[ 超氧化物歧化酶（superoxide dismutase,SOD）、活性氧（reactive oxygen species,ROS）及丙二醛（malondialdehyde,MDA）] 水平。Pearson 检验分析实验组绒毛组织中APPL1,Nrf2 和HO-1 蛋白水平与氧化应激指标水平的相关性。结果　实验组绒毛组织中APPL1,Nrf2 和HO-1 表达阳性率分别为21.7%,32.6% 和26.1%,明显低于对照组的56.0%,64.0%,58.0%,差异均有统计学意义（χ2=9.442 ～ 11.759,均P＜ 0.05）;APPL1（3.2±0.9）,Nrf2（3.7±0.7）和HO-1（3.5±0.8）表达免疫组化评分亦明显低于对照组（4.6±1.4,5.5±1.7,5.1±1.3）,差异有统计学意义（t=5.772 ～ 7.187,均P ＜ 0.05）。实验组绒毛组织中APPL1（1.3±0.1）,Nrf2（0.7±0.1）和HO-1（1.1±0.1）蛋白表达水平明显低于对照组（1.6±0.2,1.1±0.0.1,1.4±0.1）,差异有统计学意义（t=9.171 ～ 19.579,均P ＜ 0.05）。实验组绒毛组织中ROS（8.4±1.5U/ml）,MDA（11.7±2.3nmol/ml） 水平明显高于对照组（3.6±0.5U/ml,5.6±0.8nmol/ml）,SOD（23.9±6.8U/ml）水平明显低于对照组（28.1±6.6U/ml）,差异均有统计学意义（t=3.070 ～ 21.381,均P ＜ 0.05）。实验组绒毛组织中APPL1,Nrf2 和HO-1 蛋白表达与ROS 水平呈负相关（r=-0.628,-0.510,-0.575,均P ＜ 0.05）;与MDA 水平呈负相关（r=-0.541,-0.489,-0.556,均P ＜ 0.05）;与SOD 水平呈正相关（r=0.359,0.423,0.408,均P ＜ 0.05）。结论　APPL1,Nrf2 和HO-1 在稽留流产绒毛组织中呈低表达,且与氧化应激指标ROS,MDA和SOD水平具有显著相关性,其三者异常表达可能通过介导氧化应激失衡进而诱导了稽留流产的发生。     

莱菔硫烷诱导胰岛系INS-1细胞血红素氧合酶1的表达

背景：莱菔硫烷可用于氧化应激相关疾病的治疗,血红素氧合酶是一种催化血红素降解的应激蛋白,已经成为预防氧化攻击的首选研究靶标之一。目的：观察核因子E2相关因子2激动剂莱菔硫烷对大鼠胰岛细胞系INS-1细胞血红素氧合酶1蛋白表达作用及细胞保护机制。方法：体外培养INS-1细胞,先用3μmol/L莱菔硫烷进行干预培养3h,再分别加入不同的胰岛素抵抗诱导剂葡萄糖氧化酶、地塞米松和葡萄糖胺刺激建立胰岛素抵抗细胞模型。结果与结论：3μmol/L莱菔硫烷处理INS-1细胞中血红素氧合酶1的表达增加（P〈0.05）,其效应在4h后达到高峰（P〈0.05）。3μmol/L莱菔硫烷的预处理可逆转由胰岛素抵抗诱导剂导致的血红素氧合酶1表达下调（P〈0.05）。而且在葡萄糖胺处理的INS-1细胞中,莱菔硫烷对血红素氧合酶1的表达改善与磷酸化PKB的表达上调具有正相关性（P〈0.05,r=0.23）。结果证实,莱菔硫烷可能通过诱导核因子E2相关因子2介导的血红素氧合酶1表达,增强胰岛细胞抗氧化防御功能和抗损伤信号系统,从而达到拮抗胰岛素抵抗诱导剂对胰岛细胞的损伤作用。     

Effects of hyperoxia exposure on the expression of Nrf2 and heme oxygenase-1 in lung tissues of premature rats

Bronchopulmonary dysplasia (BPD) is a chronic lung disease with long-term sequelae including neurodevelopmental delay. Although the precise mechanism of BPD is not well defined, oxidative stress is thought to be involved in the pathogenesis process of BPD. Nrf2 (Nuclear factor erythroid 2-related factor 2)-Keap1 (Kelch-like ECH associated protein 1)-ARE (Antioxidant Reaction Elements) signaling pathway is one of the main protective mechanisms of BPD, which can induce cytoprotective gene expression, such as heme oxygenase‐1 (HO-1), nicotinamide quinone oxidoreductase 1 (NQO1) and so on. We exposed premature rats to hyperoxia and identified lung developmental retardation in preterm rats, with similar pathological changes as BPD. The expression of Nrf2 and HO-1 in premature rats was significantly higher after hyperoxia exposure. To explore the changes of Nrf2 and HO-1 in premature rats and enhance their beneficial functions may provide new treatment strategies for infants at risk of BPD.     

Nrf2-mediated haeme oxygenase-1 up-regulation induced by cobalt protoporphyrin has antinociceptive effects against inflammatory pain in the formalin test in mice

This study investigated the effect of haeme oxygenase-1 (HO-1) in nociception induced by formalin injection in the mice hind paw. Intraperitoneal (i.p.) administration of cobalt protoporphyrin (CoPP, an HO-1 inducer, 5mg/kg) 24h before the test, inhibited the nociceptive response during the second phase, but not during the first phase of the formalin test. The effect of CoPP was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO-1 activity) administered either by i.p. (25mg/kg, 30 min before the test) or intraplantar (400 nmol/paw, 5 min before the test) routes. Human embryonic kidney (HEK) 293T cells treated with 10 microM CoPP expressed 20-fold higher HO-1 levels when compared to controls; this effect was suppressed by transfection with the dominant negative for the nuclear factor-erythroid 2-related factor 2 (Nrf2). Western blot analysis also revealed that CoPP treatment induced a similar 20-fold increase in HO-1 expression in the paw; this effect was attenuated in knockout mice for Nrf2. CoPP treatment of wild-type, but not in Nrf2 knockout mice, resulted in a striking increase of HO-1 stained cells surrounding the muscular tissues of the hind limbs. HO-1 positive cells were scarce in wild-type and in Nrf2 knockout untreated mice. CoPP-induced HO-1 expression in Nrf2 knockout mice was lost and correlated with the loss of antinociceptive effects. In conclusion, Nrf2-mediated HO-1 expression induced an antinociceptive effect at peripheral sites. These results suggest that HO-1 modulates the inflammatory pain pathways. Hence, the development of drugs that could raise peripheral HO-1 could be relevant in inflammatory pain treatment.     

S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及功能

背景：S100a7蛋白在细胞增殖、血管形成等病理生理过程中发挥重要作用,但在视网膜色素上皮细胞中的作用少有报道。目的：探讨S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及作用。方法：体外培养ARPE-19细胞株,通过免疫荧光及Western blot技术,检测S100a7在ARPE-19细胞中的表达;不同稀释度（1∶1000,1∶10000,1∶100000）S100a7蛋白抗体与ARPE-19细胞共培养,通过MTT技术检测S100a7蛋白在人视网膜色素上皮细胞增殖过程中的作用。结果与结论：免疫荧光及Westernblot技术证实该蛋白在ARPE-19细胞中表达;MTT实验结果显示,人视网膜色素上皮细胞加入S100a7抗体72h后,各浓度处理组的吸光度值较对照组显著均降低（P〈0.05）。结果证实S100a7蛋白在人视网膜色素上皮细胞中的表达,并明显促进人视网膜色素上皮细胞增殖。     

Puerarin protects human umbilical vein endothelial cells against high glucose-induced apoptosis by upregulating heme oxygenase-1 and inhibiting calpain activation

The aim of this study was to investigate whether puerarin protects against high glucose (HG)-induced apoptosis by suppressing calpain activation in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to normal glucose (NG) (5.5 mm) or HG (33 mm) for 48 h; then, apoptosis and caspase-3 activity were determined. The expression of heme oxygenase-1 (HO-1) mRNA was evaluated by RT-PCR analysis. The activation of calpain and HO activity were also assessed. Compared with the NG group, exposure of HUVECs to HG for 48 h resulted in significant increases in calpain and caspase-3 activity as well as apoptosis, which were prevented by co-incubation with puerarin (1-100 μm) in a concentration-dependent manner. HO-1 mRNA expression and HO activity were decreased in HUVECs treated with HG for 48 h. Compared with the group exposed to HG alone, co-incubation of HUVECs with puerarin and HG induced increases in HO-1 mRNA expression and HO activity. The HO-1 inhibitor protoporphyrin IX zinc (II) abolished the inhibitory effect of puerarin on HG-induced calpain and caspase-3 activation, as well as apoptosis. The data show that puerarin protects against HG-induced endothelial cell apoptosis by a mechanism involving upregulation of HO-1 expression and inhibition of calpain activity.     

MiR-873-5p regulated LPS-induced oxidative stress via targeting heme oxygenase-1 (HO-1) in KGN cells

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. Increasing evidence reveals that PCOS may be associated with an increased level of oxidative stress. This study aimed to explore the role and potential mechanism of microRNA-873-5p (miR-873-5p) in PCOS. Quantitative real time-PCR (qRT-PCR) analysis was performed to evaluate the miR-873-5p levels in both clinical follicular fluid samples from patients with PCOS and cultured human ovarian granulosa cell-like KGN cells. The results indicated that miR-873-5p was up-regulated in the follicular fluid from patients with PCOS, as well as in the LPS-induced KGN cells. MTT assay showed that miR-873-5p inhibitor attenuated the LPS-induced inhibition of KGN cell viability. Flow cytometry indicated that miR-873-5p inhibitor suppressed cell apoptosis in LPS-induced KGN cells. Besides, miR-873-5p inhibitor resulted in a decrease in oxidative stress, which was evidenced by the reduced production of reactive oxygen species (ROS) and malondialdehyde (MDA). Further luciferase reporter assay proved that miR-873-5p directly targeted the 3′UTR of HO-1 mRNA. Small-interfering RNA (siRNA) targeting heme oxygenase-1 (HO-1) attenuated the effect of miR-873-5p inhibitor on oxidative stress in KGN cells. Besides, we also found that miR-873-5p inhibitor activated the p38/Nrf2/HO-1 signaling pathway in KGN cells. The findings may provide insightful evidence for preventing and treating PCOS by targeting miR-873-5p.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women.     

脑蛋白水解物对急性脑卒中患者Nrf2氧化应激信号通路的影响

目的 探讨急性脑卒中患者应用脑蛋白水解物对核因子-E2相关因子2为核心的 Keap1-Nrf2/ARE(Nrf2)氧化应激信号通路的影响。方法 选取我院2017年10月至2020年10月收治的急性脑卒中患者195例,按照治疗方法分为研究组(n=98)与对照组(n=97)。对照组予改善脑循环、改善脑功能、抗血小板聚集等常规药物治疗,研究组基于对照组方案,再加用脑蛋白水解物治疗,两组均治疗14 d。观察对比两组疗效。观察对比两组治疗前后国立美国卫研院卒中量表(NIHSS)评分及日常生活活动能力(Barthel)指数的改变情况。观察对比两组治疗前后血清炎性因子肿瘤坏死因子-α(TNF-α)、白介素-8(IL-8)、白介素-19(IL-19)水平的改变情况。观察对比两组治疗前后氧化应激指标血清歧化酶-超氧化物(SOD)、丙二醛(MDA)水平的改变情况。观察对比两组治疗前后血管内皮功能血清脂联素(APN)、血管性因子-假血友病(vWF)的改变情况。观察对比两组治疗前后 Nrf2氧化应激信号通路 Keap1、NQO1、ARE、Nrf2蛋白表达的改变情况。结果 研究组总有效率达到91.84%,对照组总有效率60.82%,差异有统计学意义(P<0.01)。研究组治疗后NIHSS评分显著低于对照组,Barthel指数显著高于对照组(P<0.01)。研究组治疗后血清炎性因子TNF-α、IL-8、IL-19、MDA、vWF及Keap1水平显著低于对照组,血清 SOD、APN、NQO1、ARE及Nrf2水平显著高于对照组,差异有统计学意义(P<0.01)。结论 急性脑卒中患者应用脑蛋白水解物治疗,对Nrf2氧化应激信号通路相关蛋白能够有效调节,对血管内皮功能的改善、炎症及氧化应激的控制、神经功能及生活质量的提高,均具有显著效果。     

Dialysis membrane-induced oxidative stress: role of heme oxygenase-1

Dialysis membranes have been reported to induce monocyte apoptosis. We studied the role of oxidative stress in the induction of dialysis membrane-induced monocyte apoptosis. Superoxide dismutase, a superoxide scavenger, prevented dialysis membrane-induced monocyte apoptosis. Similarly, other antioxidants also inhibited dialysis membrane- induced apoptosis. In addition, the interaction of dialysis membranes with monocytes was associated with the generation of molecules leading to oxidative stress such as superoxide and TBARS. Interestingly, pre-induction of heme oxygenase (HO)-1 by hemin prevented dialysis membrane-induced monocyte apoptosis, whereas inhibition of HO-1 activity (treatment with tin protoporphyrin, SN-P) enhanced dialysis membrane-induced monocyte apoptosis. We suggest that oxidative injury associated with dialysis membrane and monocyte interaction plays a role in monocyte injury. Pre-induction of HO-1 may attenuate dialysis membrane-induced monocyte apoptosis.     

原发性胆汁性胆管炎患者血清核因子相关因子2、血红素加氧酶1的含量与熊去氧胆酸短期应答的相关性

目的观察核因子相关因子2(Nrf2)、血红素加氧酶1(HO-1)在原发性胆汁性胆管炎(PBC)患者治疗前后血清中的水平变化,并探讨二者与熊去氧胆酸(UDCA)短期疗效的相关性。方法纳入山西医科大学第一医院PBC患者及健康对照者各80例。收集PBC患者治疗前后的临床资料及血清样本,采用ELISA法检测样本中Nrf2及HO-1的含量,硫代巴比妥酸法、黄嘌呤氧化酶法分别检测丙二醛(MDA)及超氧化物歧化酶(SOD)的含量,并进一步分析UDCA治疗前后PBC患者血清中Nrf2、HO-1的水平变化及临床意义。结果治疗前PBC患者血清Nrf2、HO-1含量分别为(626.07±103.95)U/L、(16.62±5.06)U/L显著高于健康对照者[分别为(164.45±35.12)U/L、(11.74±2.0)U/L],差异均有统计学意义(P均<0.001);与治疗前比较,UDCA治疗后1个月PBC患者血清中Nrf2[(754.30±104.36)U/L]、HO-1[(22.60±5.51)U/L]含量显著增加(P均<0.001),肝功能各项指标也得到改善(P均<0.001)。患者治疗前血清中Nrf2水平(r=0.751,P=0.012)、HO-1水平(r=0.621,P=0.038)与治疗效果均呈正相关。以治疗前Nrf2=586.17 U/L作为阈值,预测的UDCA短期疗效的敏感度为84.6%,特异度为77.5%,曲线下面积为0.824(P<0.05);以治疗前HO-1=14.92 U/L作为阈值,预测的UDCA短期疗效的敏感度为88.5%,特异度为75.0%,曲线下面积为0.861(P<0.05)。结论Nrf2、HO-1在PBC患者疾病过程中发挥重要作用,二者在血清中的基线水平及动态变化同UDCA疗效相关,可以提示PBC患者对UDCA短期治疗的应答情况。     

加味茵陈蒿汤影响Nrf2/HO-1信号通路抗大鼠皮肤光老化作用

目的研究加味茵陈蒿汤对UV照射引起的皮肤光老化作用研究,并探讨其作用机制。方法将SD大鼠随机分成4组,随机分为空白组和药物组(加味茵陈蒿汤低浓度组和加味茵陈蒿汤高浓度组)。采用紫外线辐射诱导光老化模型,每天一次,持续10周。光照结束后,通过生化测定各组大鼠皮肤组织中抗氧化酶(CAT、SOD)活性和MDA含量;Western blot法检测p-Nrf2和HO-1蛋白表达。结果给予加味茵陈蒿汤灌药组可明显升高抗氧化酶(CAT、SOD)活力,降低MDA的含量,升高Nrf2/HO-1通路中p-Nrf2和HO-1蛋白表达。结论加味茵陈蒿汤能显著降低光老化模型大鼠氧化应激压力,降低MDA的含量和炎症因子的表达,增加Nrf2/HO-1抗炎途径,促进光老化皮肤的修复,保护皮肤免受紫外线辐射的损害。