CRISPR/Cas9 in the era of nanomedicine and synthetic biology

The CRISPR/Cas system was first discovered as a defense mechanism in bacteria and is now used as a tool for precise gene-editing applications. Rapidly evolving, it is increasingly applied in therapeutics. However, concerns about safety, specificity, and delivery still limit its potential. In this context, we introduce the concept of nanogenetics and speculate how the rational engineering of the CRISPR/Cas machinery could advance the biomedical field. In nanogenetics, the advantages of traditional approaches of synthetic biology could be expanded by nanotechnology approaches, enabling the design of a new generation of intrinsically safe and specific genome-editing platforms.     

利用CRISPR/Cas9技术构建斑马鱼sf3a1基因敲除突变体

目的 剪接因子SF3A (splicing factor 3a) 的三个亚基(SF3A1、SF3A2、SF3A3)对U2 snRNP的功能发挥和前剪接小体(prespliceosome)的组装至关重要。SF3A1突变会导致骨髓增生异常综合征(MDS)、慢性粒细胞白血病(CMML)和急性粒细胞白血病(AML)等血液系统疾病。为了研究SF3A1/Sf3a1在生物体中的详细生理功能及其调控机制,本文构建了斑马鱼sf3a1基因敲除突变体。方法 利用CRISPR/ Cas9技术构建斑马鱼sf3a1突变体,通过T7酶切及基因组测序筛选得到F0代及能够稳定遗传的F1代突变体,F1代自交后得到F2代,观察sf3a1纯合缺失对斑马鱼生长发育的影响。结果 基因型鉴定结果显示,本研究得到了2种不同类型的突变品系,分别为-7 bp和-2+13 bp。杂合sf3a1突变体正常存活且可育,而合子型纯合突变体斑马鱼胚胎发育出现明显异常,24 hpf时期胚胎细胞出现大量凋亡,胚胎发育迟缓,并于72 hpf之前死亡。结论 研究利用CRISPR/Cas9技术成功构建了斑马鱼sf3a1基因敲除突变体,为探索sf3a1对斑马鱼生长发育的影响提供了实验材料和依据。     

Characterization of organic anion transporting polypeptide 1b2 knockout rats generated by CRISPR/Cas9: a novel model for drug transport and hyperbilirubinemia disease

Organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1/3) as important uptake transporters play a fundamental role in the transportation of exogenous drugs and endogenous substances into cells. Rat OATP1B2, encoded by the Slco1b2 gene, is homologous to human OATP1B1/3. Although OATP1B1/3 is very important, few animal models can be used to study its properties. In this report, we successfully constructed the Slco1b2 knockout (KO) rat model via using the CRISPR/Cas9 technology for the first time. The novel rat model showed the absence of OATP1B2 protein expression, with no off-target effects as well as compensatory regulation of other transporters. Further pharmacokinetic study of pitavastatin, a typical substrate of OATP1B2, confirmed the OATP1B2 function was absent. Since bilirubin and bile acids are the substrates of OATP1B2, the contents of total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids in serum are significantly higher in Slco1b2 KO rats than the data of wild-type rats. These results are consistent with the symptoms caused by the absence of OATP1B1/3 in Rotor syndrome. Therefore, this rat model is not only a powerful tool for the study of OATP1B2-mediated drug transportation, but also a good disease model to study hyperbilirubinemia-related diseases.     

Akebia Saponin D ameliorated kidney injury and exerted anti-inflammatory and anti-apoptotic effects in diabetic nephropathy by activation of NRF2/HO-1 and inhibition of NF-KB pathway

Diabetic nephropathy (DN), a common microvascular complication of type 2 diabetes mellitus (T2DM), causes increasing mortality and morbidity due to its high prevalence and severe consequences. Hence, it is urgent to search for effective agents that provide new insights into novel molecular therapeutic targets for DN. This study was designed to investigate the critical role of Akebia saponin D (ASD) in kidney damage, inflammation and apoptosis of renal tubular cells in DN. To probe the protective effects of ASD on DN in vivo, diabetes mellitus model was established by intraperitoneal (ip) injection of STZ (60 mg/kg) for 5 days consecutively. Besides, HG-induced human renal tubular cells (HK-2) were used to analyze the defined effects and underlying mechanism of ASD on DN in vitro. Blood glucose, insulin, serum creatinine (Scr), blood urea nitrogen (BUN), renal injury, inflammation, oxidative stress and apoptosis of renal tubular cells were respectively measured and evaluated. ASD prevented kidney damage, improved renal function and inflammatory reaction, ameliorated oxidative stress and inhibited apoptosis of renal tubular cells in DN mice via activation of NRF2/HO-1 pathway and inhibition of NF-KB pathway.     

刺五加注射液调控Nrf2/HO-1通路减轻大鼠肾缺血再灌注损伤作用

目的 探讨刺五加注射液对大鼠肾缺血再灌注损伤(RIRI)的保护作用及其机制.方法 将48只健康SD大鼠随机分为假手术组、模型组、刺五加注射液(30 mg/kg)组和刺五加注射液(30 mg/kg)+Nrf2抑制剂(鸦胆子苦醇,2 mg/kg)组,每组12只.除假手术组外,其他组采用夹闭双侧肾动脉45 min的方法复制R...     

Reactive Oxygen Species Mediate ET-1-Induced Activation of ERK1/2 Signaling in Cultured Feline Esophageal Smooth Muscle Cells

Reactive oxygen species (ROS) have been shown to play a critical role in propagating the signals of several growth factors, peptide hormones, and cytokines, such as epidermal growth factor, insulin, and interleukin-1. We investigated a possible role for ROS generation in mediating the action of ET-1 on activation of ERK1/2 in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated by 10nM ET-1; activation of ERK was examined by western blot analysis with phospho-specific antibodies of ERKs. ET-1 induced ERK1/2 phosphorylation in a dose- and time- dependent manner. ERK1/2 activation by ET-1 reached the maximal levels at 5min showing slight activation up to 20min, and then slowly declined. It was confirmed that the activation of ERK1/2 was reduced by MEK inhibitor PD98059. We observed the dose-dependent inhibitory effect of diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the ET-1-enhanced ERK1/2 phosphorylation in ESMC. Pretreatment of ESMC with N-acetylcysteine, a ROS scavenger, also attenuated the ET-1-induced ERK1/2 activation. In addition, DPI significantly inhibited the ET-1- induced ROS production when ROS was measured as a function of DCF fluorescence. The results suggest that ROS might be critical mediators of the ET-1-induced ERK1/2 signaling events in ESMC.     

Role of PI3K/Akt-Mediated Nrf2/HO-1 Signaling Pathway in Resveratrol Alleviation of Zearalenone-Induced Oxidative Stress and Apoptosis in TM4 Cells

Zearalenone (ZEA) is a common mycotoxin that induces oxidative stress (OS) and affects the male reproductive system in animals. Resveratrol (RSV) has good antioxidant activity and can activate nuclear factor erythroid 2-related factor (Nrf2) to protect cells through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. The objective of this study was to investigate the protective effect and the mechanism of RSV on OS and apoptosis in TM4 cells induced by ZEA. Prior to being exposed to ZEA, TM4 cells were pretreated with RSV or the PI3K/Akt inhibitor LY294002. Cell viability was measured by Cell Counting Kit-8 (CCK-8) assays. Flow cytometry was used to determine the level of apoptosis and intracellular reactive oxygen species (ROS). The expression of poly ADP-ribose polymerase (PARP), caspase-3, BCL2-associated X (Bax)/B-cell lymphoma-2 (Bcl-2), and PI3K/Akt-mediated Nrf2/heme oxygenase 1 (HO-1) signaling pathway-related proteins was evaluated by Western blotting. Nrf2 siRNA transfection and LY294002 treatment were used to investigate the role of the Nrf2/HO-1 and PI3K/Akt signaling pathways in RSV alleviation of ZEA-induced OS. The results showed that pretreatment with RSV significantly reduced the expression of apoptosis-related proteins and increased cell viability. Catalase (CAT) activity and glutathione (GSH) levels were also increased, whereas malondialdehyde (MDA) and ROS levels decreased (p < 0.05). RSV also upregulated Akt phosphorylation, Nrf2 nuclear translocation, and HO-1 expression under conditions of OS (p < 0.05). Transfection with Nrf2 siRNA abolished the protective effects of RSV against ZEA-induced cytotoxicity (p < 0.05), ROS accumulation (p < 0.05), and apoptosis (p < 0.05). LY294002 completely blocked the RSV-mediated increase in Nrf2 nuclear translocation (p < 0.05), HO-1 expression (p < 0.05), and cytoprotective activity (p < 0.05). Collectively, the above findings indicate that RSV can protect against ZEA-induced OS and apoptosis in TM4 cells by PI3K/Akt-mediated activation of the Nrf2/HO-1 signaling pathway.     

Nrf2/HO-1 signaling pathway may be the prime target for chemoprevention of cisplatin-induced nephrotoxicity by lycopene

Cisplatin is used against various types of solid tumors. However, its use is limited by its nephrotoxicity, with about 25–35% patients experiencing a significant decline in renal function after a single dose of cisplatin. This study reports that lycopene mitigates the nephrotoxic effect of cisplatin in rat through Nrf2-mediated induction of heme oxygenase-1 (HO-1). Eight weeks old male rats (200–215 g) were supplemented with lycopene complex containing 6% lycopene, 1.5% tocopherols, 1% phytoene and phytofluene, and 0.2% β-carotene for 10 days at a dose level of 6 mg/kg bw, followed by a single i.p. injection of cisplatin (7 mg/kg bw). Western blot analysis of renal Nrf2, HO-1 and NF-κB p65 showed that cisplatin-induced decrease in the levels of Nrf-2 and HO-1 was counteracted by lycopene. On the other hand, cisplatin mediated increase in NF-κB p65 was brought down by lycopene. Lycopene supplementation is reported to significantly improve the changes associated with cisplatin nephrotoxicity, as also evident by increased level of antioxidant enzymes. The study suggests that Nrf2/HO-1 signaling pathway may be the prime target for chemoprevention of cisplatin-induced nephrotoxicity by lycopene, and reduces inflammation by inhibiting NF-κB. Correlation between NF-κB and Nrf2 is discussed.     

蛇床子素通过Nrf2/HO-1/NLRP3轴对大鼠卵巢储备功能减退的改善作用研究

目的：探讨蛇床子素(OST)对卵巢储备功能减退(DOR)大鼠的改善作用及其可能机制。方法：取80只大鼠,64只以50 mg·(kg·d)-1雷公藤多苷混悬液灌胃建立DOR大鼠模型,随机分为模型组、OST低剂量组、OST中剂量组、OST高剂量组,每组16只大鼠,其余16只为空白组。OST低、 中、高剂量组按照2 mL·d-1分别腹腔注射20、40和80 μg·mL-1 OST溶液,空白组、模型组注射等体积生理盐水,1次·d-1,给药21 d。比较各组大鼠卵巢组织学形态变化、脏器指数和血清中抗苗勒管激素(AMH)、雌二醇激素(E2)、促黄体生成激素(LH)、卵泡刺激素(FSH)、白细胞介素4(IL4)、γ干扰素(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白细胞介素10(IL-10)水平,以及卵巢组织中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、丙二醛(MDA)水平和转录因子核因子 E2相关因子2(Nrf2)、血红素氧合酶1(HO-1)、核苷酸结合寡聚化结构域受体3(NLRP3)蛋白表达水平。结果：HE染色结果显示,OST各剂量组卵巢组织卵泡和黄体数较模型组增加;与模型组比,OST 各剂量组AMH、E2、IL-4、IL-10水平,卵巢指数,子宫指数,SOD、CAT活性和Nrf2、HO-1蛋白表达水平均升高,LH、FSH、IFN-γ、TNF-α、MDA水平和NLRP3蛋白表达水平均降低(P＜0.05)。 AMH、E2、IL-4、IL-10水平,卵巢指数,子宫指数,SOD、CAT活性和Nrf2、HO-1蛋白表达水平与 OST呈剂量依赖性升高,LH、FSH、IFN-γ、TNF-α、MDA水平和NLRP3蛋白表达水平与OST呈剂量依赖性降低(P＜0.05)。结论：OST可有效改善DOR大鼠内分泌代谢,减轻卵巢炎症反应和氧化应激损伤,恢复卵巢储备功能,可能通过调控Nrf2/HO-1/NLRP3信号通路发挥作用。     

葡萄籽原花青素通过调控ERK1/2通路促进H9C2心肌细胞增殖

摘 要 目的：研究葡萄籽原花青素（GSPE）通过调控ERK1/2通路促进H9C2心肌细胞增殖的可能机制。方法: 体外培养H9C2心肌细胞,MTT法测定葡萄籽原花青素对H9C2细胞增殖的影响;Western blot检测H9C2心肌细胞中ERK1/2、Bax、Bcl 2蛋白的表达;流式细胞术检测H9C2心肌细胞凋亡率。结果: 随着GSPE剂量增加,H9C2心肌细胞增殖率增加,与0 μg·ml-1GSPE组比较,差异有统计学意义（P<0.05）;与空白对照组比较,缺氧对照组和缺氧GSPE处理组ERK1/2、Bax、Bcl 2和Bax/Bcl 2、H9C2心肌细胞凋亡率均增高,GSPE处理组降低,差异均有统计学意义（P<0.05）;与缺氧对照组比较,GSPE处理组和缺氧GSPE处理组ERK1/2、Bax、Bcl 2和Bax/Bcl 2、H9C2心肌细胞凋亡率均降低,差异均有统计学意义（P<0.05）。结论: 体外培养条件下GSPE能促进H9C2心肌细胞的增殖,在缺氧环境中也能减少H9C2心肌细胞的凋亡,其作用机制可能与抑制H9C2心肌细胞的ERK1/2通路有关。     

电针调控Nrf2/HO-1通路对缺血缺氧性脑损伤大鼠小胶质细胞活化的影响

目的 探究电针调控核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)通路对缺血缺氧性脑损伤（HIBD）大鼠小胶质细胞活化的影响。方法 随机将40只SD大鼠均分为假手术组、模型组、电针组、电针+全反式维甲酸组。除假手术组外其余组均通过颈总动脉结扎及缺氧建立HIBD大鼠模型,假手术组大鼠仅暴露左侧颈总动脉及迷走神经,不进行结扎及缺氧处理。造模结束后,电针组于百会穴进行电针刺激;电针+全反式维甲酸组大鼠经电针刺激后腹腔注射全反式维甲酸（7 mg/kg）。通过Longa评分进行大鼠神经行为学评分;旷场及水迷宫实验检测大鼠自主活动能力和认知功能;TUNEL法检测大鼠神经细胞凋亡情况;免疫组化法检测大鼠脑组织中小胶质细胞标志蛋白离子钙结合衔接分子1(Iba1)表达;试剂盒检测大鼠脑组织中白细胞介素10(IL-10)、IL-1β、活性氧（ROS）及丙二醛（MDA）水平;Western blot检测脑组织CD68、诱导型一氧化氮合酶（iNOS）和精氨酸酶（Arginase）及Nrf2/HO-1通路相关蛋白表达。结果 与假手术组比较,模型组大鼠神经行为学评分、逃避潜伏期、大脑皮质神经细胞凋亡率...     

Cadmium-induced apoptosis in the BJAB human B cell line: involvement of PKC/ERK1/2/JNK signaling pathways in HO-1 expression

Heme oxygenase-1 (HO-1, EC 1.14.99.3) is a key enzyme in the cellular response to tissue injury and oxidative stress. It oxidizes heme, a pro-oxidant and toxic species, to biliverdin, CO, and free iron. Cytoprotection during the heat shock response is a complex phenomenon involving multiple inducible mechanisms. Several important pathways involving serine/threonine kinases mediate the induction of HO-1 in response to external stimuli.     

Cyanidin-3-glucoside protects against high glucose-induced injury in human nucleus pulposus cells by regulating the Nrf2/HO-1 signaling

Cyanidin-3-glucoside (C3G) is a well-known natural anthocyanin with antioxidant and anti-inflammatory properties. In this study, we explored the role and action mechanism of C3G in high glucose (HG)-induced damage of human nucleus pulposus cells (HNPCs). Cell viability was assessed by CCK-8 assay. TUNEL assay was performed for detecting apoptotic rate. Western blot was performed to determine the expression levels of cl-caspase-3, caspase-3, Bax, Bim, collagen II, aggrecan, MMP-3, MMP-13, and ADAMTS5. Reactive oxygen species (ROS) generation was analyzed using DCFH-DA staining. The Nrf2 was knocked down or overexpressed in HNPCs through transfection with si-Nrf2 or pcDNA3.0-Nrf2. C3G treatment (12.5, 25, and 50 μM) improved cell viability of HNPCs under HG condition. HG-induced cell apoptosis of HNPCs was attenuated by C3G with decreased apoptotic rate and relative levels of cl-caspase-3/caspase-3, Bax, and Bim. C3G treatment caused significant increase in expression levels of collagen II and aggrecan and decrease in the relative levels of MMP-3, MMP-13, and ADAMTS5. After treatment with C3G, ROS generation in HNPCs was markedly reduced. Treatment with N-acetylcysteine (NAC) reversed HG-induced cell apoptosis and extracellular matrix (ECM) degradation. C3G treatment induced the expression of Nrf2 and HO-1 in HG-induced HNPCs. Moreover, knockdown of Nrf2 reversed the inhibitory effect of C3G on ROS production. Summarily, C3G exerted a protective effect on ROS-mediated cellular damage in HNPCs under HG condition, which was attributed to the induction of the Nrf2/HO-1 signaling pathway.     

藏药三果汤调节Nrf2/HO-1信号通路干预高脂血症大鼠的作用机制研究

目的：基于Nrf2/HO-1信号通路探讨藏药三果汤对高脂血症大鼠保护作用及相关作用机制。方法：雄性SD大鼠48只,随机分为6组,即正常对照组、模型对照组、辛伐他汀组（3.5 mg/kg）,藏药三果汤低、中、高剂量组（0.43 g/kg、0.86 g/kg、1.72 g/kg）,每组8只。正常组给予基础饲料喂养,其余各组给予H00016高脂饲料喂养,制备高脂血症大鼠模型,造模的同时,各给药组每天一次给予相应药物灌胃,正常对照组、模型对照组给予等体积的生理盐水（1次/d）,连续灌胃6周。实验期间每周固定时间称取各组大鼠体重一次,6周后试剂盒检测血清中血脂（TC、TG、LDL与HDL）及氧化指标（MAD、SOD与GSH）的水平。Western Blot法测定肝组织中Nrf2、HO-1、Keap1、NQO1蛋白表达,采用person相关性分析分析血脂与氧化指标之间的相关性。结果：与正常对照组比较,模型对照组的体重明显增加,血清中的TC、TG、LDL、MDA含量明显升高,而血清中HDL含量明显减低,SOD、GSH活力明显减低（P<0.05或P<0.01）;与模型对照组比较,各给药组的体重减轻,血清中的TC、TG、LDL、MDA含量明显减低,血清中的HDL含量明显升高,SOD、GSH活力明显升高（P<0.05或P<0.01）。与空白对照组比较,模型对照组Keap1蛋白水平表达明显上调,Nrf2、HO-1与NQO1蛋白水平表达明显下调（P<0.05或P<0.01）,与模型对照组比较,三果汤低、高剂量肝组织中的Keap1蛋白水平表达明显下调,Nrf2、HO-1与NQO1蛋白水平表达明显上调（P<0.05或P<0.01）。相关性分析可见TG与SOD、HO-1及NQO1呈负相关,与Keap1呈正相关,TC与SOD、HO-1、GSH及Nrf2呈负相关,与Keap1及MDA呈正相关。结论：藏药三果汤可改善高脂血症大鼠体重及血脂水平,其机制可能与调节Nrf2/HO-1信号通路,改善氧化应激有关。     

Finasteride Increases the Expression of Hemoxygenase-1 (HO-1) and NF-E2-Related Factor-2 (Nrf2) Proteins in PC-3 Cells: Implication of Finasteride-Mediated High-Grade Prostate Tumor Occurrence

A number of naturally-occurring or synthetic chemicals have been reported to exhibit prostate chemopreventive effects. Synthetic 5α-reductase (5-AR) inhibitors, e.g. finasteride and durasteride, gained special interests as possible prostate chemopreventive agents. Indeed, two large-scale epidemiological studies have demonstrated that finasteride or durasteride significantly reduced the incidence of prostate cancer formation in men. However, these studies have raised an unexpected concern; finasteride and durasteride increased the occurrence of aggressive prostate tumor formation. In the present study, we have observed that treatment of finasteride did not affect the growth of androgen-refractory PC-3 prostate cancer cells. Finasteride also failed to induce apoptosis or affect the expression of proto-oncogenes in PC-3 cells. Interestingly, we found that treatment of finasteride induced the expression of Nrf2 and HO-1 proteins in PC-3 cells. In particular, basal level of Nrf2 protein was higher in androgen-refractory prostate cancer cells, e.g. DU-145 and PC-3 cells, compared with androgen-responsive prostate cancer cells, e.g. LNCaP cells. Also, treatment of finasteride resulted in a selective induction of Nrf2 protein in DU-145 and PC-3 cells, but not in LNCaP cells. In view of the fact that upregulation of Nrf2-mediated phase II cytoprotective enzymes contribute to attenuating tumor promotion in normal cells, but, on the other hand, confers a selective advantage for cancer cells to proliferate and survive against chemical carcinogenesis and other forms of toxicity, we propose that finasteride-mediated induction of Nrf2 protein might be responsible, at least in part, for an increased risk of high-grade prostate tumor formation in men.     

Implication of Nrf2/HO-1 pathway in the coloprotective effect of coenzyme Q10 against experimentally induced ulcerative colitis

Purpose

Given the increased incidence of ulcerative colitis worldwide, the current study was designed to investigate the coloprotective potential of CoQ10 against experimentally induced ulcerative colitis (UC) and specify the implicated mechanisms.

Methods

Ulcerative colitis was induced by intracolonic instillation of [2 ml, 3% v/v acetic acid (AA)]. Rats in the different experimental groups received CoQ10 (10 or 30 and 100 mg/kg, orally) for eight consecutive days, either in a protective or curative regimen.

Results

Intracolonic AA instillation significantly increased colon/body weight index, colon weight/colon length ratio, clinical evaluation and macroscopic scoring of UC, serum LDH, C-reactive protein and decreased the serum total antioxidant capacity. Colon MDA, TNF-α and calcium content significantly increased as well, with concomitant reduction in colon GSH, SOD, CAT, Nrf2 and HO-1 contents. Moreover, immunohistochemical staining of colon specimen revealed increased expression of caspase-3 with significant histopathological changes. Coenzyme Q10 suppressed the release of inflammatory biomarkers and restored oxidants/antioxidants hemostasis. In a dose-dependent manner, CoQ10 significantly decreased colon/body weight index, colon weight/colon length ratio, clinical evaluation and macroscopic scoring of UC, serum LDH, C-reactive protein, colon MDA, TNF-α, caspase-3 expression and increased the serum total antioxidant capacity. Colon GSH, SOD, CAT, Nrf2 and HO-1 contents significantly increased. Moreover, coenzyme Q10 significantly preserved tissue histopathological architecture. It appears that the coloprotective effect of CoQ10 was calcium-independent.

Conclusion

Coenzyme Q10 dose-dependently protects against AA-induced UC mainly via modulation of Nrf2/HO-1 and caspase-3 pathways. Antioxidant, anti-inflammatory and anti-apoptotic properties of CoQ10 are implicated in its observed therapeutic benefit.

     

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals

High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO₂), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200 mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals.     

右美托咪定对高氧诱导急性肺损伤小鼠的保护作用及Nrf2/HO-1通路的影响

摘 要 目的：探讨右美托咪定对高氧诱导急性肺损伤小鼠的保护作用及核转录因子红细胞系2p45相关因子2（Nrf2）/血红素加氧酶 1（HO-1）通路的影响。 方法: 将100只C57BL/6小鼠随机分为5组：正常对照组、模型组、地塞米松组（100 mg·kg-1）、右美托咪定低（40 mg·kg-1）、高（80 mg·kg-1）剂量组。除正常对照组外,其余各组建立高氧诱导急性肺损伤模型,并于造模成功后腹腔注射相应药物,正常对照组和模型组给予等体积生理盐水,qd,持续7 d。实验结束后,检测小鼠肺/体比值、肺损伤评分、血氧分压（PaO2）,小鼠肺组织中Nrf2、HO-1 mRNA及蛋白表达水平,胱天蛋白酶 1（caspase 1）、白细胞介素 1β（IL 1β）、白细胞介素 18（IL 18 ）蛋白水平。制作肺部HE染色切片,观察肺损伤情况。 结果: 与正常对照组比较,模型组肺/体比值、肺损伤评分、Nrf2 mRNA及蛋白表达水平、IL 1β、IL 18、caspase 1蛋白表达水平明显升高,PaO2、HO-1 mRNA及蛋白表达水平明显降低（P<0.05）;与模型组比较,右美托咪定组肺/体比值、肺损伤评分、Nrf2 mRNA及蛋白表达水平、IL 1β、IL 18、caspase 1蛋白表达水平明显降低,PaO2、HO-1 mRNA及蛋白表达水平明显升高（P<0.05）,且呈剂量相关性;右美托咪定低剂量组与地塞米松组比较差异有统计学意义（P<0.05）。正常对照组肺泡组织结构正常,无炎症细胞浸润、无胶原蛋白沉淀;模型组、右美托咪定低剂量组肺泡壁明显增厚、破裂,可见中性细胞及少量嗜酸性细胞浸润,肺间质可见较多胶原蛋白沉淀;右美托咪定高剂量组及地塞米松组肺泡结构基本正常,有少量中性细胞侵润,几乎无胶原蛋白沉淀。 结论: 右美托咪定对高氧诱导急性肺损伤小鼠的保护作用与抑制Nrf2 mRNA及蛋白表达水平表达,进而抑制IL 1β、IL 18、caspase 1蛋白表达有关。     

Regulation of lysophosphatidic acid-induced COX-2 expression by ERK1/2 activation in cultured feline esophageal epithelial Cells

Lysophosphatidic acid (LPA), a potent bioactive phospholipid, mediates diverse cellular responses by binding to specific G protein-coupled receptors (GPCRs). We investigated the signaling mechanisms underlying LPA-induced COX-2 expression in primary cultures of feline esophageal epithelial cells. The identity of the cultures was confirmed by immunocytochemistry using a cytokeratin antibody. Western blot analysis revealed a concentration-and time-dependent induction of COX-2 in response to LPA. Of the three major MAPKs, only ERK1/2 was activated by LPA in a time-dependent manner. LPA-induced COX-2 expression was significantly attenuated by the MEK inhibitor, PD98059, but not by the JNK inhibitor, SP600125, or the p38 MAPK inhibitor, SB212090. LPA-induced COX-2 expression was repressed by pertussis toxin, GF109204X, and Ki16425, indicating the involvements of PTX-sensitive G_i/o protein, PKC, and the LPA_1/3 receptor, respectively. Our data suggest that in esophageal epithelial cells, LPA-induced COX-2 expression requires activation of PKC and ERK1/2 downstream of the LPA_1/3 receptor, Understanding the regulation of COX-2 expression induced by LPA in esophageal epithelial cells might provide a new therapeutic strategy for esophageal inflammatory diseases.