萝卜硫素通过调节ALOX5/NF-κB信号通路调控巨噬细胞糖酵解抑制糖尿病肾病进展

目的探讨萝卜硫素(SFN)调节花生四烯酸5-脂氧合酶基因(arachidonic acid 5-lipoxygenase,ALOX5)/核因子kappa B(NF-κB)信号通路调节巨噬细胞糖酵解对糖尿病肾病(DN)进展的影响。方法生物信息学分析SFN治疗DN的靶基因。使用30 mmol/L高葡萄糖(HG)处理人近端肾小管上皮细胞系(HK-2细胞)诱导体外DN模型。将HK-2细胞分为如下组:正常糖(NG)组、HG组、HG+SFN(3 mmol/L)组、HG+ALOX5组、HG+SFN(3 mmol/L)+ALOX5组、HG处理的巨噬细胞+HK-2细胞组、HG+SFN(3 mmol/L)处理的巨噬细胞+HK-2细胞组、HG+ALOX5转染处理的巨噬细胞+HK-2细胞组、HG+SFN(3 mmol/L)+ALOX5转染处理的巨噬细胞+HK-2细胞组。CCK-8检测细胞活力,原位末端脱氧核苷酸转移酶标记(TUNEL)法检测细胞凋亡;葡萄糖和乳酸试剂盒检测各组细胞中葡萄糖和乳酸水平;Western blot检测各组细胞中ALOX5、NF-κB以及糖酵解相关蛋白己糖激酶-2(HK2)、丙酮酸激酶M2(PKM2)、葡萄糖转运蛋白1(GLUT1)的表达;使用链脲佐菌素(STZ)构建DN小鼠模型,DN小鼠给与SFN(0.5 mg/kg)治疗;检测小鼠各项生化指标,HE染色检测肾组织病理变化;Western blot检测小鼠肾脏巨噬细胞中糖酵解相关蛋白己糖激酶-2(HK2)、丙酮酸激酶M2(PKM2)、葡萄糖转运蛋白1(GLUT1)的表达。结果生物信息学分析结果显示ALOX5是SFN治疗DN的靶基因。与HG组相比,SFN处理增强HK-2细胞活力并抑制细胞凋亡(P<0.05);同时,SFN处理抑制HG诱导的巨噬细胞糖酵解相关蛋白的表达,减弱巨噬细胞介导的HK-2细胞损伤(P<0.05);Western blot结果表明SFN抑制ALOX5和NF-κB的表达(P<0.05);小鼠实验结果显示,SFN治疗改善DN小鼠肾功能和肾组织病理学改变,抑制肾组织中巨噬细胞糖酵解相关蛋白的表达(P<0.05)。结论SFN通过抑制ALOX5/NF-κB信号通路抑制巨噬细胞糖酵解从而改善DN进展。

基于5-LOX/LTB4信号通路探讨黄芩苷/栀子苷对PM_(2.5)暴露致血管内皮功能障碍的干预作用及其机制

目的基于5-LOX/LTB4信号通路探究黄芩苷/栀子苷(BC/GD)对PM_(2.5)诱导的血管内皮功能障碍的改善作用及其机制。方法利用离体肌张力描记技术测定BC/GD对不同状态下血管环张力的影响。将32只大鼠随机分为对照组、PM_(2.5)组、30 mg·kg^(-1)BC/GD组和60 mg·kg^(-1)BC/GD组,利用气管滴注法构建PM_(2.5)暴露模型,持续2个月,造模1个月后灌胃给予对应药物,持续1个月,末次给药后,HE染色观察肠系膜动脉血管内皮状态;化学发光法检测肠系膜动脉活性氧(ROS)水平;硝酸还原酶法检测一氧化氮(NO)水平;ELISA法检测血清炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、转化生长因子β1(TGF-β1)、白三烯B4(LTB4)水平;Western blot法检测5-脂氧酶(5-LOX)、内皮细胞一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)蛋白表达。结果BC/GD对基础状态的肠系膜动脉血管环张力无明显影响,但能明显舒张由5-HT预收缩的血管环;一氧化氮合酶抑制剂L-NAME、环氧合酶抑制剂INDO或L-NAME+INDO均能明显抑制BC/GD的血管舒张作用,但L-NAME的抑制作用更明显;50、100 mg·mL^(-1)PM_(2.5)能降低血管环对乙酰胆碱(ACh)的舒张血管效应,而BC/GD能明显改善PM_(2.5)诱导的舒张效应减弱。与对照组相比,PM_(2.5)组大鼠肠系膜动脉内皮完整性严重受损、内皮皱缩,大鼠血清中TNF-α、IL-1β、IL-6、LTB4水平显著升高、TGF-β1水平显著降低,肠系膜动脉组织中5-LOX、iNOS蛋白表达明显增加,eNOS蛋白表达降低,ROS水平升高,NO水平降低。与PM_(2.5)组相比,BC/GD能改善内皮损伤程度和完整性,可显著降低血清中TNF-α、IL-1β、IL-6、LTB4水平,升高TGF-β1水平;明显降低肠系膜动脉5-LOX、iNOS表达,升高eNOS蛋白表达;降低ROS水平,升高NO水平。结论BC/GD可通过抑制5-LOX/LTB4信号通路表达,从而降低ROS水平,抑制炎性损伤,上调eNOS表达,下调iNOS表达,升高血管中NO水平,改善PM_(2.5)诱导的血管内皮功能障碍。

Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Lipoxygenase （LOX, EC1.13.11.12） is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a（＋） and transformed into Escherichia coil BL21 （DE3）. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside （IPTG） and the purified recombinant protein was obtained by His.Bind Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 （acetate buffer） and the optimum temperature was 30℃ for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

外源茉莉酸对脂氧合酶基因LOX3敲除的粳稻防御响应的影响

【目的】揭示外源茉莉酸(Jasmonic acid,JA)对脂氧合酶基因LOX3敲除水稻株防御响应的影响,为利用外源JA等激发子提高农作物因抗性相关基因丧失导致的水稻防御响应降低问题提供基础数据。【方法】以感病粳稻丽江新团黑谷(LTH)及其LOX3敲除水稻株系(Δlox3#1、Δlox3#10和Δlox3#20)为材料,开展水稻株高、分蘖数和百粒重等表型分析、过氧化物酶(POD)、活性氧(ROS)和木质素等抗菌化合物活性(含量)测定,以及防御相关基因表达分析;进一步对供试水稻进行稻瘟菌接种,调查供试水稻稻瘟病症状,检测稻瘟菌接种水稻中抗菌化合物活性(含量)及防御相关基因表达等,并观察稻瘟菌在水稻中的侵染进程;在此基础上,使用外源JA处理稻瘟菌接种36和48 hpi时的Δlox3敲除水稻株和LTH,分析JA对水稻稻瘟病症状及稻瘟菌侵染的水稻中抗菌化合物活性(含量)和防御相关基因表达等的影响,同时观察JA对稻瘟菌侵染进程的影响。【结果】Δlox3敲除水稻株种子发芽速度快于野生型,但株高、分蘖数和百粒重等与野生型相近;稻瘟菌接种的Δlox3敲除水稻株稻瘟病症状较稻瘟菌接种的野生型水稻严重,且胼胝质数、POD活性及木质素含量降低,ROS含量和死细胞数增加,OsWRKY45、OsPR1a和OsPOX1基因下调幅度和稻瘟菌菌丝定殖量明显高于稻瘟菌接种野生型水稻;外源JA可有效减缓稻瘟菌接种36和48 hpi的Δlox3敲除水稻株稻瘟病症状,且JA提高了稻瘟菌接种36和48 hpi的Δlox3敲除水稻株中胼胝质数、POD活性和木质素含量以及防御相关基因OsWRKY45、OsPR1a和OsPOX1表达水平,而降低了OsRbohB表达量及ROS含量和细胞死亡数,同时限制了稻瘟菌菌丝扩展;其中JA对稻瘟菌接种48 hpi的Δlox3敲除水稻株稻瘟病症状减轻效果优于JA对稻瘟菌接种36 hpi的Δlox3敲除水稻株,主要体现在JA使稻瘟菌接种48 hpi的Δlox3敲除水稻株中抗菌化合物增加幅度、防御相关基因表达水平、细胞死亡数和稻瘟菌菌丝扩展受限等优于JA处理稻瘟菌接种36 hpi的Δlox3敲除水稻株。【结论】LOX3基因敲除提升了水稻种子发芽速度而降低了水稻抗瘟性,外源JA则减缓LOX3敲除导致的水稻防御响应降低,提高了水稻抗瘟性。JA在减缓农作物因抗性基因持久性短而造成的抗性降低或丧失方面具有潜在的应用前景。

结肠癌中ALOX12表达与临床病理特征及预后的关系分析

目的:探讨花生四烯酸12-脂氧合酶(arachidonate 12-lipoxygenase,ALOX12)在结肠癌患者癌组织中的表达及意义。方法:回顾性收集257例结肠癌患者肿瘤及瘤旁组织,免疫组化法检测ALOX12的表达,比较肿瘤及瘤旁组织内表达差异并分析其表达与患者临床病理特征的相关性。随访并探讨ALOX12表达与患者5年总生存率的关系,并采用Kaplan-Meier法绘制生存曲线。运用单因素及多因素Cox回归综合分析影响结肠癌患者5年总生存率的因素。结果:与瘤旁组织相比,ALOX12在肿瘤中阳性表达率更高(63.8%vs.33.1%),差异具有统计学意义(P<0.001)。ALOX12表达与患者年龄、性别、肿瘤部位均无明显相关性(P>0.05),而与TNM分期、淋巴结转移有显著相关性(P<0.05)。ALOX12高表达患者的5年总生存率明显低于低表达患者(P<0.001)。此外,单因素和多因素Cox回归分析结果显示年龄、TNM分期、淋巴结转移、ALOX12表达是影响患者5年总生存率的独立影响因素(P<0.05)。结论:ALOX12高表达与结肠癌TNM分期、淋巴结转移相关,可作为预测患者病情及预后的潜在指标。

Variants of the arachidonate 5-lipoxygenase-activating protein (ALOX5AP) gene and risk of ischemic stroke in Han Chinese of eastern China

Variants of the arachidonate 5-1ipoxygenase-activating protein （ALOX5AP） gene have been suggested to play an important role in the pathogenesis of atherosclerosis and ischemic stroke. This study was aimed to explore the association of ALOX5AP variants with ischemic stroke risk in Han Chinese of eastern China. A total of 690 ischemic stroke cases and 767 controls were recruited. The subjects were further subtyped according to the Trial of Org 10172 in Acute Stroke Treatment （TOAST） criteria. On the basis of that, two polymorphisms of the ALOX5AP gene （rs10507391 and rs12429692） were determined by TaqMan genotyping assay. In addition, plasma leukotriene B4 （LTB4） levels were analyzed in these subjects. There was no evidence of association between the two variants of ALOX5AP and the risk of ischemic stroke or its TOAST-subtypes. Haplotype analysis and stratification analysis according to sex, age, body mass index, hypertension, and diabetes also showed negative association. Analysis of LTB4 levels in a subset of cases and controls revealed that LTB4 levels were significantly higher in ischemic stroke cases than in the controls （70.06± 14.75 ng/L vs 57.34±10.93 ng/L; P = 0.000） and carriers of the T allele of the rs10507391 variant were associated with higher plasma LTB4 levels （P = 0.000）. The present study suggests there is no association of the two polymorphisms in the ALOX5AP gene with ischemic stroke risk in Han Chinese of eastern China.

JA-mediated MYC2/LOX/AOS feedback loop regulates osmotic stress response in tea plant

Osmotic stress caused by low-temperature,drought and salinity was a prevalent abiotic stress in plant that severely inhibited plant development and agricultural yield,particularly in tea plant.Jasmonic acid(JA)is an important phytohormone involving in plant stress.However,underlying molecular mechanisms of JA modulated osmotic stress response remains unclear.In this study,high concentration of mannitol induced JA accumulation and increase of peroxidase activity in tea plant.Integrated transcriptome mined a JA signaling master,MYC2 transcription factor is shown as a hub regulator that induced by mannitol,expression of which positively correlated with JA biosynthetic genes(LOX and AOS)and peroxidase genes(PER).CsMYC2 was determined as a nuclei-localized transcription activator,furthermore,ProteinDNA interaction analysis indicated that CsMYC2 was positive regulator that activated the transcription of CsLOX7,CsAOS2,CsPER1 and CsPER3via bound with their promoters,respectively.Suppression of CsMYC2 expression resulted in a reduced JA content and peroxidase activity and osmotic stress tolerance of tea plant.Overexpression of CsMYC2 in Arabidopsis improved JA content,peroxidase activity and plants tolerance against mannitol stress.Together,we proposed a positive feedback loop mediated by CsMYC2,CsLOX7 and CsAOS2 which constituted to increase the tolerance of osmotic stress through fine-tuning the accumulation of JA levels and increase of POD activity in tea plant.

CgLOX4基因在长牡蛎幼虫发育中的表达特征

为研究长牡蛎(Crassostrea gigas)LOX4基因(CgLOX4)在幼虫发育中的表达特征,采用PCR技术扩增CgLOX4的全长cDNA序列,利用荧光定量PCR和整体免疫荧光技术检测CgLOX4在幼虫不同发育时期的表达特征。结果表明:CgLOX4的cDNA全长为1 862 bp,开放阅读框为834 bp,可编码277个氨基酸,等电点为8.36,预测其氨基酸序列只含有一个保守的HOX结构域;CgLOX4在长牡蛎成体各组织中均有表达,其中在闭壳肌中的表达量显著高于肝胰腺、鳃、血淋巴、性腺和唇瓣组织(P<0.05);CgLOX4在所检测的早期胚胎中均有表达,其中在受精卵和4细胞期的表达量相对较高;CgLOX4阳性信号主要分布在担轮幼虫壳形成区、D型幼虫内脏团和壳顶幼虫消化腺中。研究表明,CgLOX4是长牡蛎早期发育过程中较早激活的转录因子之一,可能在调控幼虫壳形成和消化器官形成等方面发挥重要作用。

钩吻脂氧合酶基因GeLOX1的鉴定及低温胁迫表达分析

近年来,钩吻(Gelsemium elegans)的药用和饲用价值日益凸显,但钩吻在生长过程中不耐低温,挖掘其低温响应基因,为钩吻的抗寒育种研究奠定基础。植物中,脂氧合酶(lipoxygenase, LOX)在种子老化、抗逆境胁迫等方面的生理生化过程中有重要影响。基于课题组构建的钩吻转录组数据库,挖掘响应低温胁迫的钩吻LOX基因,运用RT-PCR技术,从中克隆到一条GeLOX1的cNDA全长序列,对其进行生物信息学、亚细胞定位、基因表达、原核表达及平板胁迫等分析。结果显示,GeLOX1所编码蛋白的氨基酸长度为761 aa,蛋白相对分子质量为87.00 kD,预测为不稳定的亲水性蛋白,含有28个丝氨酸磷酸化位点,22个苏氨酸磷酸化位点和9个酪氨酸磷酸化位点。进化树分析结果表明,GeLOX1属于9-LOX家族的成员。亚细胞定位检测结果显示,GeLOX1蛋白定位于细胞质中。实时荧光定量PCR分析发现,GeLOX1在钩吻的根中高表达,且其在4℃低温胁迫下的表达量呈现下调的趋势。经原核表达诱导后,GeLOX1的重组蛋白在约111 kD处出现目标条带,且重组蛋白的积累量在诱导8 h时达到峰值。此外,平板胁迫试验表明,GeLOX1的原核表达菌株相较于对照组对低温胁迫更敏感。钩吻GeLOX1能够应答低温胁迫。

12/15-Lipoxygenase inhibition counteracts MAPK phosphorylation in mouse and cell culture models of diabetic peripheral neuropathy

Background: Increased mitogen-activated protein kinase (MAPK) phosphorylation has been detected in peripheral nerve of human subjects and animal models with diabetes as well as high-glucose exposed human Schwann cells, and have been implicated in diabetic peripheral neuropathy. In our recent studies, leukocytetype 12/15-lipoxygenase inhibition or gene deficiency alleviated large and small nerve fiber dysfunction, but not intraepidermal nerve fiber loss in streptozotocin-diabetic mice. Methods: To address a mechanism we evaluated the potential for pharmacological 12/15-lipoxygenase inhibition to counteract excessive MAPK phosphorylation in mouse and cell culture models of diabetic neuropathy. C57Bl6/J mice were made diabetic with streptozotocin and maintained with or without the 12/15-lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC). Human Schwann cells were cultured in5.5 mMor30 mMglucose with or without CDC. Results: 12(S) HETE concentrations (ELISA), as well as 12/15-lipoxygenase expression and p38 MAPK, ERK, and SAPK/JNK phosphorylation (all by Western blot analysis) were increased in the peripheral nerve and spinal cord of diabetic mice as well as in high glucose-exposed human Schwann cells. CDC counteracted diabetes-induced increase in 12(S)HETE concentrations (a measure of 12/15-lipoxygenase activity), but not 12/15-lipoxygenase overexpression, in sciatic nerve and spinal cord. The inhibitor blunted excessive p38 MAPK and ERK, but not SAPK/ JNK, phosphorylation in sciatic nerve and high glucose exposed human Schwann cells, but did not affect MAPK, ERK, and SAPK/JNK phosphorylation in spinal cord. Conclusion: 12/15-lipoxygenase inhibition counteracts diabetes related MAPK phosphorylation in mouse and cell culture models of diabetic neuropathy and implies that 12/15-lipoxygenase inhibitors may be an effective treatment for diabetic peripheral neuropathy.

Effects of Lipoxygenase Null Genes of Soybean in Controlling Beany-flavor of Soymilk and Soyflour

The flavor of the soymilk and soyflour obtained from the lipoxygenase mutant isolines was concentrated by simultaneous distillation and extraction (SDE), and its constituents were identified by gas chro-matography (GC) and gas chromatography-mass spectrometry. Results showed that the same 24 flavor constituents were isolated in both soymilk and soyflour, and most of them were aldehydes and alcohols. Lox2 was most responsible for the production of the volatile and beany-flavor components, and Lox1 less responsible. Lox3 was least responsible and can reduce the yield of hexanal. Either Lx1 or Lx2 could significantly reduce the volatile and beany-flavor, and Lx3 could significantly increase the yield of hexanal. Primary and secondary interactions existed among the null mutant genes, and the major effects and interactions could be affected by processing conditions. The isoline with triple lipoxygenase null genes yielded the least volatile and beany-flavor components, and the isoline without the lipoxygenase gene Lx3 produced the greatest amount of the volatile and beany-flavor components. The amounts of volatile and beany-flavor components produced by the other isolines were between that of the isoline with triple lipoxygenase null genes and the isoline without lipoxygenase gene Lx3. According to the correlation analysis, the hexanal amount could be used as an index in evaluating the importance of lipoxygenase isozymes in the yield of beany-flavor compounds, and the effects of the different types of lipoxygenase null mutants in controlling beany-flavor compounds. The cultivars with triple lipoxygenase null genes will be a quality raw material for soy food processing.

Generation of seed lipoxygenase-free soybean using CRISPR-Cas9

Beany flavor induced by three lipoxygenases(LOXs, including LOX1, LOX2, and LOX3)restricts human consumption of soybean. It is desirable to generate lipoxygenase-free new mutant lines to improve the eating quality of soybean oil and protein products. In this study, a pooled clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPRassociated protein 9(Cas9) strategy targeting three GmLox genes(GmLox1, GmLox2, and GmLox3) was applied and 60 T_0 positive transgenic plants were generated, carrying combinations of sg RNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1 gmlox2 gmlox3 triple mutants and GmLox-40 was a gmlox1 gmlox2 double mutant.Sequencing of T_1 mutant plants derived from GmLox-28, GmLox-60, and GmLox-40 showed that mutation in the GmLox gene was inherited by the next generation. Colorimetric assay revealed that plants carrying different combinations of mutations lost the corresponding lipoxygenase activities. Transgene-free mutants were obtained by screening the T_2 generation of lipoxygenase-free mutant lines(GmLox-28 and GmLox-60). These transgeneand lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.

Effect of Ethylene on Polygalacturonase,Lipoxygenase and Expansin in Ripening of Tomato Fruits

Fruit ripening is a complex process and is regulated by many factors.Ethylene and polygalacturonase(PG),lipoxygenase(LOX),expansin(EXP) are all critical regulating factors in fruit ripening and softening process.With antisense ACS tomato,Nr mutant tomato and cultivated tomato as materials,Northern blot hybridization showed that PG,LeEXP1 and LOX expressed differently in different parts of cultivated tomato fruit during ripening,which was related to fruit ripening.The ripening process of columella and radial pericarp was faster than pericarp.In both Nr mutant and antisense ACS transgenic tomato fruit,expression levels of PG,LeEXP1 and LOX were generally lower than those in cultivated fruit but still related to fruit ripening.The expression levels of PG,LeEXP1 and LOX increased in the mature green tomato fruits after 0.5 h treatment with ethylene(100 μL/L).These results indicate that gene expression of PG,LeEXP1 and LOX were positively regulated by ethylene.The time and cumulative effect of the concentration exists in the expression of PG regulated by ethylene.The regulation of LOX expression mainly depended on the fruit development after great amount of ethylene was produced.PG played a major role in ripening and softening of tomato fruit,and cooperated with the regulation of EXP and LOX.

高油酸低脂氧合酶(FAD-LOX)多基因编辑大豆优异种质创制

大豆是植物油和蛋白质的主要来源。不饱和脂肪酸的含量是影响植物油品质的主要因素,油酸是不饱和脂肪酸中含量最丰富的成分之一,具有较强的氧化稳定性和热稳定性。增加油酸的含量可以提高大豆油的耐储存性,也有利于人体健康。GmFAD2-1A和GmFAD2-1B是大豆编码脂肪酸去饱和酶,调控油酸向亚油酸转化的关键基因。脂氧合酶是大豆种子中的抗营养因子之一。它在酶促氧化的条件下产生豆腥味,限制了大豆产品的应用。为满足不同人群的需要和降低加工成本,有必要培育无脂氧合酶的突变品系。大豆中GmLOX基因(包括GmLOX1、GmLOX2、GmLOX3)编码脂氧合酶。脂氧酶也能催化亚油酸氧化生成脂质氢过氧化物,然后形成豆腥味。因此,降低亚油酸的含量有助于减少豆腥味的形成。同时编码脂氧酶也能延缓豆油的酸败。本研究利用CRISPR/Cas9对中吉602的GmFAD2-1A、GmFAD2-1B、GmLOX1、GmLOX2和GmLOX3基因进行编辑。通过农杆菌介导的大豆遗传转化,在T2代分离到7个具有不同等位变异的优异新种质。与中吉602相比,突变体种子中脂氧合酶活性显著降低,油酸含量提高至81.7%~83.5%。本研究为有效开发大豆种质资源以满足不断变化的市场需求提供了新材料。

Inhibition of 12-lipoxygenase reduces proliferation and induces apoptosis of hepatocellular carcinoma cells in vitro and in vivo

BACKGROUND:12-lipoxygenase(12-LOX) has been reported to be an important gene in cancer cell proliferation and survival,and tumor metastasis.However,its role in hepatocellular carcinoma(HCC) cells remains unknown.METHODS:Expression of 12-LOX was assessed in a diethylnitrosamine-induced rat HCC model,and in SMMC-7721,HepG2 and L-02 cells using immunohistochemical staining and reverse transcriptase-polymerase chain reaction(RT-PCR).GST-π and Ki-67 were determined in vivo by immunohistochemical staining.Apoptosis was evaluated by TUNEL assay.Cell viability and apoptosis were determined by MTT assay and flow cytometry,respectively.Apoptosis-related proteins in SMMC-7721 and HepG2 cells were detected by Western blotting.RESULTS:Immunohistochemical staining and RT-PCR showed that 12-LOX was over-expressed in rat HCC and two HCC cell lines,while the expression was inhibited by baicalein,a specific inhibitor of 12-LOX.Baicalein inhibited cell proliferation and induced apoptosis in rat HCC and both cell lines in a dose-and time-dependent manner.Our in vivo study demonstrated that baicalein also reduced neoplastic nodules.Mechanistically,baicalein reduced Bcl-2 protein expression coupled with a slight increase of the expression of Bax and activation of caspase-3.Furthermore,baicalein inhibited the activation of ERK-1/2(phosphorylated).Interestingly,the effects of baicalein were reversed by 12(S)-HETE,a metabolite of 12-LOX.CONCLUSIONS:Inhibition of 12-LOX leads to reduced numbers of HCC cells,partially caused by increased apoptosis.12-LOX may be a potential molecular target for HCC prevention and treatment.

Cloning and Sequence Analysis of Lipoxygenase Gene cDNA from Cucumber Fruit (Cucumis sativus L.)

Lipoxygenases are nonheme-iron-containing dioxygenases that catalyze the hydroperoxidation of unsatrated fatty acids containing a cis, cis-1,4-pentadiene structure producing hydroperoxy acids with conjugated dienes.

Involvement of eicosanoids in the pathogenesis of pancreatic cancer: the roles of cyclooxygenase-2 and 5-lipoxygenase

The interplay between inflammation and cancer progression is a growing area of research. A combination of clinical, epidemiological, and basic science investigations indicate that there is a relationship between inflammatory changes in the pancreas and neoplastic progression. Diets high in ω-6 polyunsaturated fatty acids provide increased substrate for arachidonic acid metabolism by cyclooxygenase-2(COX-2) and 5-lipoxygenase(5-LOX) to form eicosanoids. These eicosanoids directly contribute to pancreatic cancer cell proliferation. Both COX-2 and 5-LOX are upregulated in multiple cancer types, including pancreatic cancer. In vitro studies using pancreatic cancer cell lines have demonstrated upregulation of COX-2 and 5-LOX at both the mRNA and protein levels. When COX-2 and 5-LOX are blocked via a variety of mechanisms, cancer cell proliferation is abrogated both in vitro and in vivo.The mechanism of COX-2 has been shown to include effects on apoptosis as well as angiogenesis. 5-LOX has been implicated in apoptosis. The use of COX-2 and 5-LOX inhibitors in clinical studies in patients with pancreatic cancer has been limited. Patient enrollment has been restricted to those with advanced disease which makes evaluation of these drugs as chemopreventive agents difficult. COX-2 and 5-LOX expression have been shown to be present during the early neoplastic changes of pancreatic cancer, well before progression to invasive disease. This indicates that the ideal role for these interventions is early in the disease process as preventive agents, perhaps in patients with chronic pancreatitis or hereditary pancreatitis.

Expression of 5-Lipoxygenase in human colorectal cancer

AIM: To evaluate the 5-lipoxygenases (Loxs) expression level in human colorectal cancer specimens in order to determine its clinicopathologic significance in human tumorigenesis. METHODS: The relative quantity of 5-Lox mRNA in paired 91 colorectal tumor and adjacent normal mucosa samples was determined by real time quantitative PCR. Additionally, the expression of 5-Lox and cyclooxygenase (Cox)-2 proteins was also examined using immunohistochemical staining methods. RESULTS: There was a marked increase in 5-Lox mRNA levels in the tumor compared with paired normal mucosa samples (P < 0.0001). Sixty six (72.5%) tumors showed high 5-Lox mRNA levels. The positivity rate of 5-Lox and Cox-2 protein expression was 68.7% and 79.1% respectively. There was a significant association between tumoral 5-Lox mRNA level and tumor size (Rho = 0.392, P = 0.0002), depth or vessel invasion. CONCLUSION: These results suggest that 5-Lox is up-regulated in colorectal cancer and that inhibition of its expression might be valuable in the prevention and treatment of colorectal cancer.

LOX1在胃癌中的表达及其促肿瘤生长的机制探讨

目的:探讨血凝素样氧化型低密度脂蛋白受体1(lectin-like oxidized low density lipoprotein receptor-1,LOX1)在胃癌中的表达及与预后相关性,同时明确其促胃癌生长作用及潜在机制。方法:利用人类癌症基因组图谱(TCGA)、基因表达汇编(GEO)和癌症细胞系百科全书(CCLE)数据库分析LOX1在胃癌组织和细胞中的表达水平;利用多种统计方法分析LOX1表达水平与胃癌患者预后及临床特征的相关性;通过质粒转染敲低LOX1,进一步应用细胞功能实验(CCK-8、克隆形成、EdU实验)探究LOX1对胃癌细胞增殖能力的调控作用;通过Western blot、qPCR、免疫荧光分析LOX1对NF-κB信号通路激活的调控作用。结果:LOX1在胃癌组织和细胞中显著高表达;高水平LOX1提示胃癌患者预后较差且肿瘤较易发生转移;敲低LOX1显著抑制肿瘤细胞增殖;敲低LOX1显著抑制胃癌细胞中NF-κB信号通路的激活。结论:LOX1通过激活NF-κB信号通路对胃癌细胞增殖能力具有调控作用;LOX1可作为生物标记物提示患者预后;靶向LOX1可作为潜在胃癌治疗新方案。

15-Lipoxygenase inhibition of Commelina benghalensis,Tradescantia fluminensis,Tradescantia zebrina

Objective:To evaluate the 15-lipoxygenase inhibitory activity of the methanol leaf extracts of Commelina benghalensis,Tradescantia fluminensis(T.fluminensis)and Tradescantia zebrina.Method:The inhibitory activity was evaluated using a spectrophotometric assay by observing the increase in absorbance at 234 nm due to the formation of the product 13-hydroperoxyoctadecadienoic acid.The extracts were also tested for the presence of terpenoids,saponins,tannins,flavonoids,steroids,phenolic compounds,alkaloids and cardiac glycosides.Results:All the extracts inhibited the action of 15-lipoxygenase at a concentration of 0.2μg/mL.T.fluminensis and Tradescantia zebrina exhibited higher than 50%inhibition with T.fluminensis at 87.2%.T.fluminensis was partitioned with ethyl acetate and hexane and their IC_(50)values were determined at 8.72μg/mL and 98.04μg/mL,respectively.Conclusions:T.fluminensis is a potentially good source of 15-lipoxygenase inhibitors.