Research progress on the effects of 5-LOX and its inhibitors on tumors / 中国肿瘤生物治疗杂志

@#[摘 要] 脂氧合酶（LOX）家族蛋白在恶性肿瘤演进中发挥重要作用，其亚型5-LOX在多种肿瘤组织和细胞中呈高表达，且与淋巴结转移、患者的生存期有关，可通过诱导Src的磷酸化、选择性抑制p53依赖的促凋亡基因转录、促进肿瘤血管形成、刺激上皮-间质转化、形成转移前微环境等，促进肿瘤细胞增殖和转移、抑制凋亡，参与调控肿瘤的恶性演进过程。基于5-LOX及其激活蛋白ALOX5AP的促癌作用，已开发出直接、间接两类抑制剂及COX-2/5-LOX双重抑制剂，有望通过降低5-LOX活性或阻断5-LOX代谢途径发挥抗肿瘤作用。本文概述5-LOX在肿瘤中的调控作用以及5-LOX和其激活蛋白抑制剂的研究现状，以期为后续抗肿瘤药物的开发及临床应用提供参考及新的理论依据。

Construction and gene identification of myeloid specific Spi1 knockout mice / 安徽医科大学学报

Objective @#To construct myeloid specific Spi1 gene knockout mice and analyze their genotypes , so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets .@*Methods @#According to the principle of CRISPR/Cas9 technology and C re/LoxP system , sgRNA and Donor vectors were de signed and constructed . The transcript of Exon 2 ( Exon 2) was used as the knockout region , and Loxp elements were placed on both sides of Exon 2 . Cas9 protein , sgRNA and Donor vector were mixed and microinj ected into the fertilized eggs of C57BL/6J mice , the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice , and F0 generation was obtained after 19 ~ 20 days . Positive F0 mice were mated with C57BL/6J mice to ob tain stable F1 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/flox/Lyz2-cre - mice were used as wild type (WT) mice . DNA of WT and KO mice was extracted , and the genotypes were identified by agarose gel electro phoresis after PCR amplification . Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene , Spi - 1 /purine rich box - 1(PU . 1) in immune cells of WT and KO mice .@*Results@#The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1 flox/flox homozygote , and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre + . Western blot showed that compared with WT group , the protein PU . 1 was not expressed in bone marrow derived macropha ges (BMDMs ) and peritoneal macrophages (PM) in KO group (P < 0.01) . There was no significant difference of statistics in the expression level of PU . 1 in T cells between KO mice and WT mice . The results of PCR and West ern blot showed that myeloid specific Spi1 KO mice were successfully constructed . @*Conclusion @#The myeloid spe cific Spi1 gene KO mice are successfully constructed and identified , which provides animal model basis for further revealing the potential mechanism of PU . 1 inimmune regulation .

Levosimendan alleviates coronary microembolization-induced myocardial injury through LOX-1/p38 MAPK pathway / 中华急诊医学杂志

Objective:To study the effect of levosimendan on coronary microembolization (CME)-induced myocardial injury and LOX-1/p38MAPK pathway.Methods:Microspheres were injected into coronary anterior descending branch to construct swine CME model, swine was given levosimendan by continuous intravenous drip for 24 h before modeling, and myocardial-specific overexpression of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) was achieved through coronary artery injection of adeno-associated virus (AAVs) at 2 weeks before modeling. Then, echocardiography was used to measure cardiac function; HE staining and HBFP staining were used to observe the pathological changes of myocardium and myocardial microinfarction area, respectively; ELISA was used to detect the serum level of cTnI; TUNLE staining was used to detect cardiomyocyte apoptotic index; the LOX-1, Bax, caspase-3 p12, Bcl-2, and p-p38 MAPK protein in myocardial tissue was observed by immunofluorescence method.Results:Compared to the sham group, the LVEF, LVFS, and CO value in the CME group were decreased, while the LVEDd value was increased significantly (all P<0.05); the area of myocardial micro-infarction, serum cTnI level and cardiomyocyte apoptotic rate in the CME group were increased significantly (all P<0.05); the protein levels of Bax, caspase-3 p12, LOX-1, and p-p38 MAPK were increased significantly, while the Bcl-2 level was decreased significantly ( P<0.05). Levosimendan pretreatment significantly improved cardiac dysfunction, reduced the area of myocardial micro-infarction and serum cTnI level, alleviated cardiomyocyte apoptosis, and significantly reduced the LOX-1 and p-p38 MAPK protein expression levels following CME (all P<0.05); while pretreatment with levosimendan and LOX-1 overexpression AAVs simultaneously abolished the effects of pretreatment with levosimendan alone (all P<0.05). Conclusion:Levosimendan alleviates CME-induced myocardial injury through inhibiting cardiomyocyte apoptosis mediated by LOX-1/p38 MAPK signaling pathway.

Screening of phytocompounds, molecular docking studies, and in vivo anti-inflammatory activity of heartwood aqueous extract of Pterocarpus santalinus L.f

Objective:To evaluate the anti-inflammatory potential of aqueous extract of Pterocarpus santalinus L.f. heartwood using molecular docking and in vivo experiment. Methods:An aqueous extract of Pterocarpus santalinus heartwood was prepared using a Soxhlet apparatus. Phytocompounds in the extract were tentatively identified using high-resolution mass spectrometry. Molecular docking experiments were carried out to evaluate the binding affinity of selected compounds, phloridzin to cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostaglandin E synthase-1 (PGES-1) and 5-lipoxygenase (5-LOX). Anti-inflammatory potential was evaluated by carageenan induced paw edema model in rats. Results:The presence of major component phloridzin along with quercetin, parthenin, ginkgolide B, picrotoxinin, usnic acid, octopine, and epigallocatechin was detected in the extract. Molecular docking study showed that phloridzin inhibited COX-1, COX-2, PGES-1 and 5-LOX with more affinity than ibuprofen and paracetamol. Pterocarpus santalinus heartwood extract at 200 and 400 mg/kg BW showed significant reduction in carageenan-induced hind paw edema in a dose-dependent manner, but the effect was slow when compared with the standard ibuprofen (30 mg/kg p.o.). Conclusions:The study indicated that after clinical trials, the aqueous extract of Pterocarpus santalinus heartwood can be effectively used in phytotherapy to treat inflammation.

Study on visfatin-induced inflammation and necroptosis via LOX-1 in human umbilical vein endothelial cells / 生物医学工程学杂志

The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours' treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.

Lox Family and Their Role in Tumour Formation: An Overview

@#The lysyl oxidase family has five family members which are; Lysyl Oxidase (LOX), Lysyl Oxidase Like-1 (LOXL1), Lysyl Oxidase Like-2 (LOXL2), Lysyl Oxidase Like-3 (LOXL3), and Lysyl Oxidase Like-4 (LOXL4). These are amine oxidases which are copper (Cu) dependent. The main function of these secreted enzymes is covalently crosslinking extracellular collagens and elastins, making the extracellular matrix (ECM) stable. Association with LOX family enzymes has been found in various diseases including tumours, suggesting that it may be involved in the pathogenesis of the lesions. To add to the complexity, some of the LOX family members have been linked with tumour suppression while the other members were associated with tumour promotion, progression and metastasis. Thus, this review will explore further insight into the role of LOX family in tumour formation.

Effects of baicalin-gardenoside pairing on 5-lox/CysLTs/CysLT pathway of cerebral ischemic injury rats / 中成药

AIM To study the effects of baicalin and gardenoside pairing on 5-lox/CysLTs/CysLT pathway of rats with cerebral ischemia injury.METHODS SD rats randomly assigned into control group,model group,baicalin and gardenoside pairing groups (7 ∶ 3,dosed at 30,45,60 mg/kg,respectively) in their cerebral ischemia recovery period were simulated with permanent middle cerebral artery occlusion (pMCAO) if necessary.After a week of natural recovery,the drugs' pharmacodynamic effects were evaluated by the neurofunctional scoring and HE staining,their impact on the content of CysLTs was determined by ELISA,and the influence on the expression of 5-lox,CysLT1 and CysLT2 was detected by western blot.RESULTS One-week consecutive administration of baicalin and gardenoside pairing contributed to a reduction in infiltration and tissue edema of ischemic cells.If compared with pMCAO group,the baicalin and gardenoside pairing groups were observed with significantly lowered scores of neurologic functioning,inhibited microglia activation,decreased content of CysLTs and down-regulated expression of 5-LOX,CysLT1 and CysLT2 (P ＜ 0.01).CONCLUSION Baicalin and gardenia pairing's alleviating effect to the damage of cerebral ischemia may be associated with its inhibition on microglia activation and 5-lox/CysLTs/CysLT signaling pathway,and thus an inflammatory impairment reduction.

An oriental melon 9-lipoxygenase gene CmLOX09 response to stresses, hormones, and signal substances / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes-CmHPL and CmAOS-in the leaves of four-leaf stage seedlings of the oriental melon cultivar "Yumeiren" under wound, hormone, and signal substances. CmLOX09, CmHPL, and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA3); however, CmHPL and CmAOS showed differential responses to IAA and GA3. CmLOX09, CmHPL, and CmAOS were all induced by hydrogen peroxide (H2O2) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09, CmHPL, and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii. The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P. xanthii, the diseased leaves of the oriental melon were divided into four levels-levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P. xanthii through the allene oxide synthase (AOS) pathway to form JA.

Inhibition of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) by D-005 (A lipid extract of Acrocomia crispa fruits) / [Inhibición de la ciclooxigenasa (COX) y la 5-lipooxigenasa (5-LOX) por el D-005 (extracto lipidico del fruto de la Acrocomia crispa)

This study was aimed to investigate whether the a lipid extract from Acrocomia crispa fruits (D-005) inhibits COX and 5-LOX enzyme activities in vitro. This study demonstrates that D-005 inhibits markedly and in a dose dependent manner COX-2 and 5-LOX activities. The dual inhibition of COX-2 and 5-LOX supports further research on the potential anti-inflammatory effect of D-005.

El objetivo de este estudio fue investigar si el extracto lipídico de los frutos de Acrocomia crispa (D-005) inhibe in vitro las actividades de las enzimas COX y 5-LOX. Este estudio demuestra que el D-005 inhibe marcadamente y de manera dosis dependiente las actividades de la COX-2 y 5-LOX. La inhibición dual de la COX-2 y 5-LOX soportan futuras investigaciones sobre el potencial efecto anti-inflamatorio del D-005.

Effects of Gualou Xiebai Banxia decoction on blood lipid content, oxidative stress and ox-LDL/Lox-1 pathway in ApoE-/- mice / 中国中药杂志

To observe the functions of Gualou Xiebai Banxia decoction（GXBD） on regulating lipid metabolism, anti-oxidation, and interposing ox-LDL/Lox-1 pathway, and to explore its anti-atherosclerosis (AS) mechanisms. AS models were established by using 42 Apo-E-/- male mice with high fat diet. AS model mice were randomly divided into the model group, simvastatin group, and GXBD high and low dose groups. C57BL/6J male mice were used as the normal control group, n=10 and the treatment lasted for 8 weeks. The levels of TC, TG, LDL-C, HDL-C, SOD, MDA, GSH-px, and ox-LDL in blood serum were tested 24 h after the last administration. The changes of aortic tissues structure were observed by HE staining; the expression levels of Lox-1 protein and the expression levels of mRNA were detected by Western blot and PCR respectively.Results showed that the blood lipid levels and MDA, ox-LDL levels in blood serum of model group were significantly higher than those in the normal control group, but SOD, GSH-px levels were significantly lower than those in the normal control group, and the Lox-1 protein and mRNA expression levels were also significantly higher than those in the control group(P<0.05), namely aortic atherosclerosis lesions were obvious in model group.The levels of blood lipid and MDA, ox-LDL of GXBD high and low dose groups and simvastatin group were significantly lower than those in model group, while SOD, GSH-px levels were significantly higher than those in model group, and Lox-1 protein and mRNA expression levels were significantly lower than those in model group(P<0.05), namely the aortic atherosclerosis lesions were significantly relieved. The above results indicated that GXBD was capable of modulating blood lipid, anti-oxidation, and inhibiting the expression of Lox-1, and interposing ox-LDL/Lox-1 pathway in the AS model Apo-E-/- mice, which may be one of the mechanisms of anti-atherosclerosis.

5-LOX inhibitor MK886 suppresses the stemness of human colon cancer HT-29 cells / 医学研究生学报

Objective It remains a controversy whether 5-lipoxygenase (5-LOX) is associated with colon cancer stem cells.This study was to investigate the effect of the 5-LOX inhibitor MK886 in maintaining the stemness of the human colon cancer cell line HT-29.Methods Using CCK-8 assay, we examined the inhibitory effects of different concentrations of MK886 (12.5, 25, 50, 75, 100, and 200 μmol/L) on the colon cancer HT-29 cells cultured in vitro and calculated its half-inhibitory concentration (IC50).Then, we detected the effects of MK886 IC50 on the clone-and sphere-forming abilities of the cells, determined the mRNA expressions of the stemness markers CD133, Lgr5, Oct4 and Ascl2 by real-time PCR after 24 and 48 hours of MK886 IC50 intervention, and measured their protein expressions by Western blotting after 24, 48 and 72 hours of MK886 IC50 intervention.Results The inhibition rates of MK886 on the HT-29 cells at 24 and 48 hours were significantly increased in a time-and dose-dependent manner ([14.99±3.06] and [19.98±0.57]％ at 12.5 μmol/L, [20.46±1.14] and [34.97±6.02]％ at 25 μmol/L, [50.76±5.94] and [66.90±5.74]％ at 50 μmol/L, [66.84±1.77] and [73.11±2.48]％ at 75 μmol/L, [72.67±2.36] and [77.78±3.30]％ at 100 μmol/L, [83.67±0.24] and [84.69±2.24] ％ at 200 μmol/L) as compared with the blank control (0％ and 0％) (P<0.05).The clone-forming rate and number of spheres formed were remarkably lower in the MK886 intervention than in the control group ([10.60±1.71] vs [44.67±3.21]％, P<0.05;6.00±1.60 vs 19.07±2.89, P<0.05).After 24 and 48 hours of MK886 intervention, the mRNA expression of CD133 in the HT-29 cells was markedly up-regulated in comparison with that at 0 hour (0.72±0.10 and 0.39±0.07 vs 1.66±0.33, P<0.05), and so were those of Lgr5, Oct4 and Ascl2 (P<0.05).Conclusion The 5-LOX inhibitor MK886 can inhibit the proliferation and clone-and sphere-forming abilities of human colon cancer HT-29 cells by down-regulating the expressions of the stemness markers and thus suppressing the stemness of the colon cancer stem cells.

Efectos inflamatorios, oxidativos y aterogénicos del receptor endotelial de la lipoproteína de baja densidad oxidada-1 / Inflammatory, oxidative and atherogenic effects of endothelial lectin-like receptor of oxidized low density lipoprotein-1 / Efeitos inflamatórios, oxidativos e aterogênicos do receptor endotelial da lipoproteína de baixa densidade oxidativa-1

LOX-1 es un receptor endotelial de la familia de las lectinas. Su actividad biológica tiene un fuerte impacto en los fenómenos inflamatorios, oxidativos y aterogénicos endoteliales. Cuando se conoció el receptor de la lipoproteína de baja densidad (RLDL) y su regulación, se afirmó el papel aterogénico del colesterol transportado en esta lipoproteína (C-LDL). Este papel de las lipoproteínas fue la base de la denominación de dislipoproteinemias en reemplazo de dislipemias. En condiciones post-prandiales, las lipoproteínas ricas en triglicéridos, como quilomicrones y lipoproteínas de muy baja densidad (VLDL), son degradadas por la lipoproteína lipasa (LPL) de la pared vascular, produciéndose remanentes de quilomicrones (RQ) y lipoproteínas de densidad intermedia (IDL), respectivamente, que en conjunto se denominan lipoproteínas remanentes (RLPs). Dependiendo del estrés oxidativo las RLPs son oxidables y pueden unirse al LOX-1. También se liberan ácidos grasos que injurian células endoteliales y contribuyen a abrir brechas en el endotelio, que en condiciones fisiológicas es una barrera de células con uniones estrechas. El dominio intracelular de LOX-1 regula el reconocimiento de lipoproteínas de baja densidad oxidadas (LDLOX) y de RLPs. Además, posee un efecto dependiente de los radicales reactivos de oxígeno (ROS). Su dominio transmembrana actúa en el pasaje de LDLOX y monocitos al subendotelio. La inhibición de LOX-1 con anticuerpos específicos impide su unión con LDLOX, restableciendo la barrera entre el lumen vascular y el subendotelio. En cambio, las LDLOX unidas al dominio transmembrana, producen apoptosis de las células endoteliales y suprimen uniones estrechas intercelulares en el endotelio, facilitando la actividad de las moléculas de adhesión leucocitarias que promueven el pasaje al subendotelio de los elementos del lumen, tales como monocitos, plaquetas, LDLOX, RLPs oxidables y lipoproteínas (a) (Lp(a)) semejantes al plasminógeno. Las LDLOX subendoteliales aumentan la movilidad de células musculares lisas. Los monocitos subendoteliales se establecen como residentes, e incorporan LDLOX, convirtiéndose sucesivamente en macrófagos, células espumosas y lesiones aterogénicas. Sin embargo, desde Assmann G y su estudio PROCAM no puede ignorarse el papel de los triglicéridos y colesterol de lipoproteínas de alta densidad (C-HDL) como componentes del cuadro de riesgo en ECV.

LOX-1 is an endothelial receptor belonging to the family of lectins. Its biological activity has a strong impact on inflammatory, oxidative and atherogenic phenomena in endothelium. When Low Density Lipoprotein receptor (RLDL) and its regulation were known, the atherogenic role of the cholesterol transported in LDL (LDL-C) was confirmed. This lipoprotein role in atherosclerosis was the base to use the term dyslipoproteinemia instead of dyslipidemia. In post-prandial conditions, triglyceride-rich lipoproteins like chylomicrons and very low-density lipoproteins (VLDL), are degraded by lipoprotein lipase (LPL) on the vascular wall, with the resultant formation of chylomicron remnants (CR) and intermediate density lipoproteins (IDL) respectively, which as a whole are called remnant lipoproteins (RLPs). Depending on oxidative stress, RLPs are oxidized and then they can bind the LOX-1. In this process, fatty acids are also released, injuring endothelial cells and contributing to open gaps in endothelium, which under physiological conditions, is a barrier of cells with tight junctions. The intracellular domain of LOX-1 regulates the recognition of oxidized LDL (oxLDL) and RLPs, and its effect depends on reactive oxygen species (ROS). LOX-1 transmembrane domain acts in the passage of oxLDL and monocytes to the sub-endothelium. Inhibition of LOX-1 by specific antibodies prevents its binding with OxLDL, restoring the barrier between the vascular lumen and sub-endothelium. By contrast, the oxLDL, attached to the transmembrane domain, produce apoptosis of endothelial cells and the suppression of narrow intercellular junctions in the endothelium. Thus, enabling the activity of leucocyte adhesion molecules that promote the transfer to subendothelial elements lumen of monocytes, platelets, oxLDL, oxidized RLPs and lipoprotein (a) (Lp (a)), similar to plasminogen such as. Sub-endothelial OxLDL increase the mobility of smooth muscle cells. Sub-endothelial monocytes establish as resident, up-take oxLDL and successively become into macrophages, foam cells and atherosclerotic lesions. However, since Assman’s PROCAM study, the role of triglycerides and High Density Lipoprotein-cholesterol (HDL-C), as components of cardiovascular risk, cannot be ignored.

LOX-1 é um receptor endotelial da família das lectinas. Sua atividade biológica tem um importante impacto nos fenômenos inflamatórios, oxidativos e aterogênicos endoteliais. Quando foi conhecido o receptor da lipoproteína de baixa densidade (RLDL) e sua regulação, afirmou-se o papel aterogênico do colesterol transportado nesta lipoproteína (C-LDL). Este papel das lipoproteínas foi a base da denominação de dislipoproteinemias em substituição de dislipidemias. Em condições pós-prandiais, as lipoproteínas ricas em triglicérides como quilomícrons e Lipoproteínas de muito baixa densidade (VLDL) são degradadas pela lipoproteína lipase (LPL) da parede vascular, produzindo remanescentes de quilomícrons (RQ) e lipoproteínas de densidade intermediária (IDL) respectivamente, que em conjunto são chamadas lipoproteínas remanescentes (RLPs). Dependendo do estresse oxidativo, as RLPs são oxidáveis e podem se ligar ao LOX-1. Também são liberados ácidos graxos que injuriam células endoteliais e contribuem na abertura de fendas no endotélio, que em condições fisiológicas é uma barreira de células com uniões estreitas. O domínio intracelular de LOX-1 regula o reconhecimento de lipoproteínas de baixa densidade oxidativa (LDLOX) e de RLPs. Também possui um efeito dependente dos radicais reativos de oxigênio (ROS). Seu domínio transmembrana atua na passagem de LDLOX e monócitos para o subendotélio. A inibição de LOX-1 com anticorpos específicos impede sua união com LDLOX restabelecendo a barreira entre o lúmem vascular e o subendotélio. Entretanto, as LDLOX ligadas ao domínio transmembrana produzem apoptose das células endoteliais e suprimem estreitas junções intercelulares no endotélio, facilitando a atividade das moléculas de adesão leucocitária que promovem a passagem para o subendotélio de elementos do lúmem, tais como monócitos, plaquetas, LDLOX, RLPs oxidáveis e lipoproteínas (a) [Lp(a)] semelhantes ao plasminogênio. As LDLOX subendoteliais aumentam a mobilidade das células musculares lisas. Os monócitos subendoteliais se estabelecem como residentes, e incorporam LDLOX, virando sucessivamente em macrófagos, células espumosas e lesões aterogênicas. No entanto, desde Assman G e seu estudo PROCAM, não pode se ignorar o papel dos triglicérides e do colesterol de lipoproteínas de alta densidade (C-HDL) como componentes do evento de risco em ECV.

Structural optimization and biological evaluation of 1,5-disubstituted pyrazole-3-carboxamines as potent inhibitors of human 5-lipoxygenase

Human 5-lipoxygenase (5-LOX) is a well-validated drug target and its inhibitors are potential drugs for treating leukotriene-related disorders. Our previous work on structural optimization of the hit compound 2 from our in-house collection identified two lead compounds, 3a and 3b, exhibiting a potent inhibitory profile against 5-LOX with IC50 values less than 1 µmol/L in cell-based assays. Here, we further optimized these compounds to prepare a class of novel pyrazole derivatives by opening the fused-ring system. Several new compounds exhibited more potent inhibitory activity than the lead compounds against 5-LOX. In particular, compound 4e not only suppressed lipopolysaccharide-induced inflammation in brain inflammatory cells and protected neurons from oxidative toxicity, but also significantly decreased infarct damage in a mouse model of cerebral ischemia. Molecular docking analysis further confirmed the consistency of our theoretical results and experimental data. In conclusion, the excellent in vitro and in vivo inhibitory activities of these compounds against 5-LOX suggested that these novel chemical structures have a promising therapeutic potential to treat leukotriene-related disorders.

Expression and significance of 12-lipoxygenase in human glioma / 中国肿瘤临床

Objective:To investigate the expression and clinical significance of 12-lipoxygenase (12-LOX) in glioma. Methods:12-LOX expression in 40 glioma cases and 10 normal human-brain tissues was assayed by immunohistochemistry. Clinicopathological data were analyzed to reveal the association between 12-LOX expression and prognosis of glioma patients. Results:12-LOX was weak-ly expressed in the normal human brain tissues, whereas 12-LOX was strongly expressed (72.5%) in glioma tissues (P<0.05). The strong 12-LOX expression was correlated with the histopathological grading of glioma (P=0.012), whereas 12-LOX expression was not correlated with factors such as patient gender and age, tumor size, and Karnofsky Performance Score. Median survival time was longer in the group with low 12-LOX expression (25.6 months) than in the group with high 12-LOX expression (13.2 months) (P<0.05). Con-clusion:Abnormal 12-LOX expression is implicated in glioma. 12-LOX expression was correlated with the histopathological grading of glioma and was closely associated with patient prognosis.

Lectin-Like Oxidized LDL Receptor 1 Mediates the Uptake of the C-Terminal Domain of Hsp70 (A Promising Immune Adjuvant Molecule) and Antigen Peptide Complexes.

Extracellular heat shock protein 70 (Hsp70) is an adjuvant molecule that stimulates the immune system. The C-terminal domain of Hsp70 (C70), without the ATPase domain, is sufficient for antigen cross-presentation. However, the mechanism by which the receptor mediates the uptake of C70–peptide complex remains unclear. We therefore aimed to determine the process by which the receptor mediates the uptake of antigenic peptide-bound C70. Methodology: Hsp70 and C70 individually cloned into pET28a were expressed in Escherichia coli BL21 (DE3) and were purified on Ni-NTA agarose and MonoQ HR5/5. Hsp70 and C70 were labeled with Alexa 555 and Alexa 633, respectively, to detect cellular binding. HEK293 cells stably expressing lectin-like oxidized LDL receptor-1 (LOX 1) and KG-1 human dendritic-like cells were incubated with Alexa-labeled Hsp70 and C70 individually or with C70 and antigenic complexes and were observed using fluorescence microscopy. The affinity of LOX-1 toward Hsp70 and C70 was analyzed by chip assay using surface plasmon resonance, which immobilized LOX-1 ligand recognition domain. Results: HEK293 cells stably expressing LOX-1 and KG-1 cells accepted the C70– peptide and Hsp70–peptide complexes. Anti-LOX-1-neutralizing antibody inhibited the uptake of the C70–peptide complexes by KG-1 cells. The dissociation constant (KD) of C70 toward the LOX-1 extracellular domain, measured by surface plasmon resonance, was 4.02 × 10−7 M and that of the C70–peptide complex was 6.6 × 10−8 M. C70 increased the LOX-1 affinity by forming a complex with the antigen peptide. Conclusion: Our findings suggest that LOX-1 is the primary receptor for the C70–peptide and the Hsp70–peptide complexes. C70 is a promising adjuvant molecule that is internalized via LOX-1. In addition, it is convenient to prepare C70 using an E. coli expression system and C70 is more stable than full-length Hsp70.

In vitro Pharmacological Investigations of Using Leaf Callus Extracts of Canthium parviflorum Lam.

Aims: The Canthium parviflorum Lam. is an important medicinal plant extensively used in traditional oriental herbal medicines. It is important to screen for the novel biological activities and novel chemical constituents to further validate the use of this plant extract. This was mainly intended to screen in vitro pharmacological activities with callus extracts of Canthium parviflorum. Experiments were designed according to the standard methods and processes. Place and Duration of Study: The Pharmacology Department of Laila Research Centre in Vijayawada, Andhra Pradesh, India between September-October 2012. Methodology: Callus was induced from leaf explants of Canthium parviflorum on MS medium supplemented with 2,4-Dichlorophenoxyacetic acid and the compounds were extracted from dried callus using methanol solvent with Soxhlet apparatus. Results: The Graph pad Prism Version‑5 software was used to analyze data in the form of figures. The callus extract was potentially inhibited the 5- Lipoxygenase inhibitory enzyme at significantly less IC50 value that was comparable with the standard drug inhibition. In addition, this extract was exhibited remarkable cytotoxicity with less LC50 value. However, this callus extract was shown very low potency in inhibiting the enzymes of acetyl cholinesterase, tyrosinase, alpha-glucosidase. Conclusion: The study demonstrated that callus extract of Canthium parviflorum shown more potent inhibition of 5-LOX and also remarkable cytotoxicity to be further screened for in vivo anticancer and anti-inflammatory activity.

Efecto in vitro del policosanol (alcoholes de la cera de Saccharum officinarum L) sobre la enzima 5-lipooxigenasa / In vitro effects of policosanol (Saccharum officinarum L wax alcohols) on the 5-lipooxygenase enzyme

INTRODUCTION: policosanol, a mixture of high molecular weight aliphatic alcohols purified from sugarcane with octacosanol as the main component, shows cholesterol-lowering and antiplatelet effects in addition to an inhibitory effect on type I cicloxygenase. OBJECTIVE: to determine whether policosanol may inhibit 5-LOX enzyme activity in vitro. METHODS: effects on 5-LOX enzyme activities were assessed in rat blood polymorphonuclear leukocytes. Vehicle or Policosanol suspensions (0.6 to 6 000 µg/mL) were added to tubes containing the reaction mix and then absorbance changes at 234 nm were measured. RESULTS: added Policosanol inhibited in vitro 5-LOX activity by 30 percent, which was not a significant figure but depended on the concentration(r= 0.992; p< 0.05); it was 1 250 µg/mL. CONCLUSIONS: policosanol did not significantly inhibit 5-LOX enzyme activity in rat PMNL preparations, so that it does not seem to be a dual inhibitor of COX and-LOX enzymes. This result differs from that found for beeswax alcohols and underlines the different effects of the mixtures of long-chain fatty alcohols purified from the sugarcane and the beeswax(AU).

IINTRODUCCIÓN: el policosanol es una mezcla de alcoholes alifáticos aislados y purificados de la caña de azúcar cuyo componente mayoritario es el octacosanol, con efecto sobre la reducción de colesterol y antiagregante plaquetario, además inhibe la ciclooxigenasa (COX) tipo 1. OBJETIVO: determinar el poder de inhibición del policosanol en la actividad de la enzima 5-LOX in vitro. MÉTODOS: el efecto sobre la actividad de la enzima 5-LOX se determinó en leucocitos polimorfonucleares obtenidos de sangre de ratas. Se añadieron vehículo o suspensiones de policosanol (0,6 a 6 000 µg/mL) a tubos que contenían la mezcla de reacción y se medió el cambio de absorbancia a 234 nm. RESULTADOS: la adición de policosanol inhibió in vitro la actividad de la 5-LOX en un 30 por ciento que no fue significativo pero sí dependiente de la concentración (r= 0,992; p< 0,05), inhibición esta que alcanzó 1 250 µg/mL. CONCLUSIÓN: el policosanol no inhibió significativamente la actividad de la enzima 5-LOX en preparación de polimorfonucleares de ratas, por lo que no es un inhibidor dual de las enzimas. Este resultado difiere del encontrado para los alcoholes de la cera de abeja y subraya la diferencia de los efectos hallados entre las mezclas de alcoholes alifáticos de cadenas largas purificados de la caña de azúcar y la cera de abeja(AU)

In vitro Antioxidant, Lipoxygenase and Xanthine Oxidase inhibitory activity of fractions and macerate from Pandiaka angustifolia (vahl) Hepper.

Pandiaka angustifolia (Vahl) Hepper, a species belonging to Amaranthaceae’s family is used in traditional medicine of Burkina Faso for the treatment of several diseases. The aim of this study was to evaluate the phenolics and flavonoids content, the antioxidant capacity, the lipoxygenase and xanthine oxidase inhibition potentials of Pandiaka angustifolia hydroacetonic macerate and fractions. Assay revealed that the Dichloromethane Fraction (DCMF) possess the best inhibitory activity of lipoxygenase, a key enzyme involved in inflammatory process with 83.84±1.89% of inhibition, as regards Xanthine Oxidase (XO) inhibiting percentage, n-hexane fraction (n-HF) exhibited the highest percentage of inhibition with 76.22 ± 4.78% at a concentration of 100μg/ml. Phenolics and flavonoids content in fractions and hydroacetonic macerate evaluation showed that hydroacetonic macerate possess the highest rate in total phenolic and the Dichloromethane fraction (DCMF) the highest rate in flavonoids content. This study showed that the apolar fractions of P. angustifolia is a potential natural source for the treatment of oxidative stress and inflammatory related diseases.

Effect of Tangshenning on Renal Function and LOX-1 mRNA Expression in Early DN Rats Model / 中国药师

Objective:To observe the effect of Tangshenning on renal function and LOX-1 mRNA expression in the kidney of early diabetic nephropathy rats model. Methods:Early diabetic nephropathy rats model was made with high fat diet, STZ and unilateral ne-phregtomy. After the 12-week drug intervention, the rats were sacrificed, the kidneys were removed and the mRNA expressions of LOX-1 in the kidney were observed by the method of RT-PCR. Results:Early diabetic nephropathy rats model was successfully made by the triple-modeling method. The mRNA expression of LOX-1 in the kidney was significantly increased, and Tangshenning could low-er the expression of LOX-1 in the kidney in a dose-dependent manner. Conclusion:The abnormal expression of LOX-1 could be one of the mechanisms for diabetic nephropathy. Tangshening has good kidney protective effect through decreasing LOX-1 abnormal expression in the kidneys, which deserves further research.

Ox-LDL increases OX40L in endothelial cells through a LOX-1-dependent mechanism

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis, and it can stimulate the expression of a variety of inflammatory signals. As a new and highly sensitive inflammation index, OX40L may be a key to understanding the mechanisms that regulate interactions between cells within the vessel wall and inflammatory mediators during the development of atherosclerosis. To investigate whether Ox-LDL regulates OX40L expression through an oxidized LDL-1 receptor (LOX-1)-mediated mechanism, we investigated the effect of different concentrations of Ox-LDL (50, 100, 150 µg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells, and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression.