All-trans Retinoic Acid Diminishes Collagen Production in a Hepatic Stellate Cell Line via Suppression of Active Protein-1 and c-Jun N-terminal Kinase Signal

Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood.The influence of retinoids on HSCs and hepatic fibrosis remains controversial.The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation,mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),fibrolytic genes (MMP-3,MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G).Cell proliferation was evaluated by measuring BrdU incorporation.The mRNA expression levels of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and fibrolytic genes (MMP-3,MMP-13) were quantitatively detected by using real-time PCR.The mRNA expression of JNK and AP-1 was quantified by RT-PCR.The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)] and profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1.These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal,then decrease the mRNAs expression of profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly induce the mRNA expression of MMP-3 and MMP-13.

c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed "lipoapoptosis," in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet(HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12(AML12) cells treated with palmitate(PA) were used as an in vitro model. RESULTS: LC3-Ⅱ, p62, and Run domain Beclin-1 interacting and cysteine-rich containing(Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase(JNK) inhibition at both the m RNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-Ⅱ and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown.CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide antifibrosis in the NASH liver.

c-Jun N-terminal kinase 3 deficiency protects axotomized retinal ganglion cells via affecting mitochondria involved apoptosis pathway

AIM: To illustrate the isoform-specific role and mechanism of c-Jun N-terminal kinases(JNKs) in mouse optic nerve axotomy induced neurotrauma. METHODS: We firstly investigated the expression of JNK1, JNK2, and JNK3 in the retinal ganglion cells(RGCs) by double-immunofluorescent staining. Then we created optic nerve axotomy model in wild type as well as JNK1, JNK2, JNK3, isoform specific gene deficiency mice. With that, we checked the protein expression profile of JNKs and its active form, and quantified the survival RGCs number by immunofluorescence staining. We further explored the molecules underlying isoform specific protective effect by real-time polymerase chain reaction(PCR) and Western blotting assay. RESULTS: We found that all the three isoforms of JNKs were expressed in the RGCs. Deficiency of JNK3, but not JNK1 or JNK2, significantly alleviated optic nerve axotomyinduced RGCs apoptosis. We further established that expression of Noxa, a pro-apoptotic member of BH3 family, was significantly suppressed only in JNK3 gene deficiency mice. But tumor necrosis factor receptor 1(TNFR1) and Fas, two key modulators of death receptor mediated apoptosis pathway, did not display obvious change in the expression. CONCLUSION: It is suggested that mitochondria mediated apoptosis, but not death receptor mediated apoptosis got involved in the JNK3 gene deficiency induced RGCs protection. Our study provides a novel insight into the isoform-specific role of JNKs in neurotrauma and indicates some cues for its therapeutics.

Saponins from Panax japonicus attenuate age-related neuroinflammation via regulation of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways

Neuroinflammation is recognized as an important pathogenic factor for aging and related cognitive disorders. Mitogen-activated protein kinase and nuclear factor kappa B signaling pathways may mediate neuroinflammation. Saponins from Panax japonicus are the most abundant and bioactive members in rhizomes of Panax japonicus, and show anti-inflammatory activity. However, it is not known whether saponin from Panax japonicus has an anti-inflammatory effect in the aging brain, and likewise its underlying mechanisms. Sprague-Dawley rats were divided into control groups(3-, 9-, 15-, and 24-month-old groups) and saponins from Panax japonicus-treated groups. Saponins from Panax japonicus-treated groups were orally administrated saponins from Panax japonicus at three doses of 10, 30, and 60 mg/kg once daily for 6 months until the rats were 24 months old. Immunohistochemical staining and western blot assay results demonstrated that many microglia were activated in 24-month-old rats compared with 3-and 9-month-old rats. Expression of interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2, and inducible nitric oxide synthase increased. Each dose of saponins from Panax japonicus visibly suppressed microglial activation in the aging rat brain, and inhibited expression levels of the above factors. Each dose of saponins from Panax japonicus markedly diminished levels of nuclear factor kappa B, IκBα, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. These results confirm that saponins from Panax japonicus can mitigate neuroinflammation in the aging rat brain by inhibition of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways.

Stress-activated kinases as therapeutic targets in pancreatic cancer

Pancreatic cancer is a dismal disease with high incidence and poor survival rates.With the aim to improve overall survival of pancreatic cancer patients,new therapeutic approaches are urgently needed.Protein kinases are key regulatory players in basically all stages of development,maintaining physiologic functions but also being involved in pathogenic processes.c-Jun N-terminal kinases(JNK)and p38 kinases,representatives of the mitogen-activated protein kinases,as well as the casein kinase 1(CK1)family of protein kinases are important mediators of adequate response to cellular stress following inflammatory and metabolic stressors,DNA damage,and others.In their physiologic roles,they are responsible for the regulation of cell cycle progression,cell proliferation and differentiation,and apoptosis.Dysregulation of the underlying pathways consequently has been identified in various cancer types,including pancreatic cancer.Pharmacological targeting of those pathways has been the field of interest for several years.While success in earlier studies was limited due to lacking specificity and off-target effects,more recent improvements in small molecule inhibitor design against stress-activated protein kinases and their use in combination therapies have shown promising in vitro results.Consequently,targeting of JNK,p38,and CK1 protein kinase family members may actually be of particular interest in the field of precision medicine in patients with highly deregulated kinase pathways related to these kinases.However,further studies are warranted,especially involving in vivo investigation and clinical trials,in order to advance inhibition of stress-activated kinases to the field of translational medicine.

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

Signaling interactions among neurons impact cell fitness and death in Alzheimer's disease

The pathology of Alzheimer’s disease involves a long preclinical period,where the characteristic clinical symptoms of the changes in the brain are undetectable.During the preclinical period,homeostatic mechanisms may help prevent widespread cell death.Evidence has pointed towards selective cell death of diseased neurons playing a potentially protective role.As the disease progresses,dysregulation of signaling pathways that govern cell death contributes to neurodegeneration.Aberrant activation of the c-Jun N-terminal kinase pathway has been established in human and animal models of Alzheimer’s disease caused by amyloid-beta 42-or tau-mediated neurodegeneration.Clonal mosaic studies in Drosophila that examine amyloid-beta 42 in a subset of neurons suggest complex interplay between amyloid-beta 42-expressing and wild-type cells.This review examines the role of c-Jun N-terminal kinase signaling in the context of cell competition and short-range signaling interactions between amyloid-beta 42-expressing and wild-type neurons.Cell competition is a conserved phenomenon regulating tissue integrity by assessing the fitness of cells relative to their neighbors and eliminating suboptimal cells.Somatic clones of amyloid-beta 42 that juxtapose genetically distinct neuronal cell populations show promise for studying neurodegeneration.Generating genetic mosaics with labeled clones of amyloid-beta 42-or tau-expressing and wild-type neurons will allow us to understand how short-range signaling alterations trigger cell death in neurons and thereby contribute to the progression of Alzheimer’s disease.These approaches have the potential to uncover biomarkers for early Alzheimer’s disease detection and new therapeutic targets for intervention.

Ghrelin通过JNK信号通路抑制LPS诱导的肺泡巨噬细胞凋亡

【目的】观察体外条件下加入外源性ghrelin对内毒素(LPS)诱导大鼠肺泡巨噬细胞NR8383凋亡以及吞噬能力的影响,并探讨JNK(c-Jun N-terminal kinase)信号通路在其中所起的作用。【方法】用CCK-8(Cell Counting Kit-8)法检测加入LPS或者ghrelin与LPS共孵育后NR8383的细胞毒性;流式细胞技术、原位末端标记法(TUNEL)检测细胞凋亡率;Western blot检测JNK、phospho-JNK信号通路蛋白以及cleaved caspase-3、Bax、Bcl-2凋亡相关分子蛋白的表达;激光共聚焦技术检测巨噬细胞的吞噬能力;使用ghrelin受体拮抗剂[D-Lys-3]-GHRP-6、JNK特异性抑制剂SP600125分别抑制ghrelin受体及JNK激酶的激活。【结果】CCK-8检测结果显示LPS可显著抑制NR8383增殖,而ghrelin可呈浓度依赖性地阻断这种抑制作用;流式和TUNEL检测进一步证实ghrelin可显著降低LPS所致NR8383凋亡作用(P<0.05),而应用ghrelin受体拮抗剂[D-Lys-3]-GHRP-6可以减弱ghrelin的抗凋亡效果(P<0.05);LPS可以激活JNK激酶活性,并改变其下游凋亡相关蛋白的表达,其中促凋亡蛋白Bax以及cleaved caspase-3表达上调,抗凋亡蛋白Bcl-2表达下调,应用ghrelin可以逆转LPS对JNK激酶的激活,继而下调Bax以及cleaved caspase-3的表达,上调Bcl-2的表达,差异均具有统计学意义(P<0.05);ghrelin还可以维持LPS持续刺激下NR8383的吞噬能力(P<0.05)。【结论】ghrelin通过下调JNK信号通路的激活抑制LPS诱导的NR8383的凋亡,并维持其吞噬能力。

黄芪注射液拮抗大鼠脑缺血再灌注损伤的保护作用和机制

目的探讨黄芪注射液对大鼠脑缺血再灌注损伤的神经保护作用和机制。方法成年健康雄性Wistar大鼠70只,应用线栓法经左侧颈外-颈内动脉插线建立大脑中动脉阻塞再灌注模型,经腹腔注射1g/L黄芪注射液(3ml/kg)干预治疗。Longa法评价大鼠神经功能缺损程度,氯化三苯基四氮唑(TTC)染色观察脑梗死体积,苏木素-伊红染色观察顶叶皮质区神经元形态结构变化,透射电子显微镜观察神经细胞的超微结构,流式细胞术检测细胞凋亡,Western blotting检测c-Jun氨基末端激酶3(JNK3)蛋白表达,RT-PCR检测JNK3 mRNA表达。结果经黄芪注射液治疗后,大鼠顶叶皮质区神经元JNK3 mRNA和JNK3蛋白表达较对照组显著减低,凋亡细胞数量显著减少,脑梗死体积显著缩小,神经元形态和超微结构以及动物神经行为功能显著改善。结论黄芪注射液可能通过下调神经元JNK3基因表达而抑制细胞凋亡,缩小脑梗死体积,改善动物的神经行为功能。

Apoptosis and non-alcoholic fatty liver diseases

The number of patients with nonalcoholic fatty liver diseases(NAFLD) including nonalcoholic steatohepatitis(NASH), has been increasing. NASH causes cirrhosis and hepatocellular carcinoma(HCC) and is one of the most serious health problems in the world. The mechanism through which NASH progresses is still largely unknown. Activation of caspases, Bcl-2 family proteins, and c-Jun N-terminal kinase-induced hepatocyte apoptosis plays a role in the activation of NAFLD/NASH. Apoptotic hepatocytes stimulate immune cells and hepatic stellate cells toward the progression of fibrosis in the liver through the production of inflammasomes and cytokines. Abnormalities in glucose and lipid metabolism as well as microbiota accelerate these processes. The production of reactive oxygen species, oxidative stress, and endoplasmic reticulum stress is also involved. Cell death, including apoptosis, seems very important in the progression of NAFLD and NASH. Recently, inhibitors of apoptosis have been developed as drugs for the treatment of NASH and may prevent cirrhosis and HCC. Increased hepatocyte apoptosis may distinguish NASH from NAFLD, and the improvement of apoptosis could play a role in controlling the development of NASH. In this review, the association between apoptosis and NAFLD/NASH are discussed. This review could provide their knowledge, which plays a role in seeing the patients with NAFLD/NASH in daily clinical practice.

Tumor necrosis factor-α mediates JNK activation response to intestinal ischemia-reperfusion injury

AIM:To investigate whether tumor necrosis factor-α(TNF-α)mediates ischemia-reperfusion(I/R)-induced intestinal mucosal injury through c-Jun N-terminal kinase(JNK)activation.METHODS:In this study,intestinal I/R was induced by 60-min occlusion of the superior mesenteric artery in rats followed by 60-min reperfusion,and the rats were pretreated with a TNF-α inhibitor,pentoxifylline,or the TNF-α antibody infliximab.After surgery,part of the intestine was collected for histological analysis.The mucosal layer was harvested for RNA and protein extraction,which were used for further real-time polymerase chain reaction,enzyme-linked immunosorbent assay and Western blotting analyses.The TNF-α expression,intestinal mucosal injury,cell apoptosis,activation of apoptotic protein and JNK signaling pathway were analyzed.RESULTS:I/R significantly enhanced expression of mucosal TNF-α at both the mRNA and protein levels,induced severe mucosal injury and cell apoptosis,activated caspase-9/caspase-3,and activated the JNK signaling pathway.Pretreatment with pentoxifylline markedly downregulated TNF-α at both the mRNA and protein levels,whereas infliximab pretreatment did not affect the expression of TNF-α induced by I/R.However,pretreatment with pentoxifylline or infliximab dramatically suppressed I/R-induced mucosal injury and cell apoptosis and significantly inhibited the activation of caspase-9/3 and JNK signaling.CONCLUSION:The results indicate there was a TNFα-mediated JNK activation response to intestinal I/R injury.

Neuroprotective effects of Activin A on endoplasmic reticulum stress-mediated apoptotic and autophagic PC12 cell death

Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum(ER) stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein(CHOP) and cleaved-caspase-3] and biomarkers of autophagy(Beclin-1 and light chain 3), and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1(IRE1), tumor necrosis factor receptor-associated factor 2(TRAF2), apoptosis signal-regulating kinase 1(ASK1), c-Jun N-terminal kinase(JNK) and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

CSN1 inhibits c-Jun phosphorylation and down-regulates ectopic expression of JNK1

CSN1 is a component of the COP9 signalosome(CSN),a conserved protein complex with pleiotropic functions in many organs and cell types.CSN regulates ubiquitinproteasome dependent protein degradation via the deneddylation and the associated deubiquitination activities.In addition,CSN associates with protein kinases and modulates cell signaling,particularly the activator protein 1(AP-1)pathway.We have shown previously that CSN1 suppresses AP-1 transcription activity and inhibits ultraviolet(UV)and serum activation of c-fos expression.Here we show that CSN1 can inhibit phosphorylation of proto-oncogene c-Jun product and repress c-Jun dependent transcription.Further,CSN1 dramatically downregulates ectopic expression of c-Jun N-terminal kinase 1(JNK1)in cultured cells.The decline in JNK1 is not caused by excessive proteolysis or by 3′UTR-dependent mRNA instability,but by CSN1-dependent repression of one or multiple steps in transcriptional and posttranscriptional mechanisms.Thus,in contrast to CSN5/Jab1,which promotes AP-1 activity,CSN1 displays a negative effect on the AP-1 pathway.Finally,we discuss about the dynamic equilibrium of the CSN complexes in regulation of the AP-1 pathway.

N2L,a novel lipoic acid-niacin dimer,attenu⁃ates ferroptosis and decreases lipid peroxida⁃tion in HT22 cells

OBJECTIVE N2L is a novel lipoic acid-niacin dimer regulating lipid metabolism with multifunction,including antioxidant effect.We investigated the protective effect of N2L and the underlying mechanisms under the ferroptosis inducer RAS-selective lethality 3(RSL3)treat⁃ment in HT22 cells.METHODS HT22 cells were pretreated with N2L and then were treated with RSL3 to establish a ferroptosis cell model.MTT assay was used to detect the cell survival rate.Free radical probe(dihydroethidium,DHE)and ferrous probe FerroOrange were used to detect the contents of free radicals and ferrous ions in cells.The ultrastructure of mitochondria of treat⁃ed cells was observed by transmission electron microscope.The expression of ferroptosis-relat⁃ed proteins acyl-CoA synthetase long-chain family member 4(ACSL4),glutathione peroxidase 4(GPX4),cyclooxygenase-2(COX-2),ferritin Heavy Chain 1(FTH1),nuclear factor E2-related factor 2/heme oxygenase-1,and phosphoryla⁃tion levels of the c-Jun N-terminal kinase(JNK)/extracellular regulated protein kinases(ERK)pathway were detected by Western blotting.RE⁃SULTS RSL3 decreased the cell viability and induced excessive accumulation of(reactive oxy⁃gen species)ROS in HT22 cells.N2L pretreat⁃ment effectively protected HT22 cells against lipid peroxidation.What′s more,N2L recovered GPX4 protein expression and blocked the increase of COX-2 and ACSL4 expressions.Moreover,N2L also significantly prevented FTH1 from downregulation and maintained iron homeo⁃stasis.Finally,N2L pretreatment could decrease JNK/ERK activation induced by RSL3.CON⁃CLUSION N2L is an excellent ferroptosis inhibi⁃tor,and its anti-ferroptosis mechanism may be related to the reduction of lipid peroxidation and the regulation of iron homeostasis.

Exosomes from PM^(2.5)-treated Human Bronchial Epithelial Cells Increase Lung Cancer Metastatic Potential

Objective Fine particulate matter(PM^(2.5))is an air pollutant that has become of great concern in recent years.Numerous studies have found that PM^(2.5)may contribute to lung cancer,but the pathogenesis has not yet been fully elucidated.In this study,we explored the roles of exosomes from bronchial epithelial cells in PM^(2.5)-promoted lung cancer metastasis.Methods Exosomes were isolated from cell supernatants.An animal model of lung metastasis(established by tail vein injection of A549-luc)and in vitro studies with lung cancer cell lines were used to investigate the effects of exosomes derived from PM^(2.5)-treated human bronchial epithelial cells(PHBE-exo).Results The animal experiments revealed that PHBE-exo-treated mice showed stronger luciferase activity and a larger relative metastatic region in the lungs,thus indicating that PHBE-exo promoted the metastatic potential of lung cancer.Additionally,PHBE-exo promoted the migration,invasion and epithelial-to-mesenchymal transition of lung cancer cells,in a manner mediated by activation of c-Jun Nterminal kinase.Conclusion These results implied that PM^(2.5)may promote the development of lung cancer through exosomes derived from bronchial epithelial cells,thus providing a potential interventional target for lung cancer.These findings broadened our understanding of cancer-promoting mechanisms of environmental pollutants.

Toxicities of amyloid-beta and tau protein are reciprocally enhanced in the Drosophila model

Extracellular aggregation of amyloid-beta(Aβ)and intracellular tau tangles are two major pathogenic hallmarks and critical factors of Alzheimer’s disease.A linear interaction between Aβand tau protein has been characterized in several models.Aβinduces tau hyperphosphorylation through a complex mechanism;however,the master regulators involved in this linear process are still unclear.In our study with Drosophila melanogaster,we found that Aβregulated tau hyperphosphorylation and toxicity by activating c-Jun N-terminal kinase.Importantly,Aβtoxicity was dependent on tau hyperphosphorylation,and flies with hypophosphorylated tau were insulated against Aβ-induced toxicity.Strikingly,tau accumulation reciprocally interfered with Aβdegradation and correlated with the reduction in mRNA expression of genes encoding Aβ-degrading enzymes,including dNep1,dNep3,dMmp2,dNep4,and dIDE.Our results indicate that Aβand tau protein work synergistically to further accelerate Alzheimer’s disease progression and may be considered as a combined target for future development of Alzheimer’s disease therapeutics.

Pomegranate extract inhibits EMT in clear cell renal cell carcinoma in a NF-κB and JNK dependent manner

Objective:Clear cell renal cell carcinoma(ccRCC)is the most common subtype of renal cell carcinoma(RCC)and is characterized by biallelic inactivation of the von Hippel-Lindau(VHL)tumor suppressor gene.One effect of VHL inactivation is hypoxia inducible factor alpha(HIFa)-independent constitutive activation of nuclear factor kappa B(NF-κB)and c-jun N-terminal kinase(JNK).Both NF-κB and JNK drive ccRCC growth and epithelial to mesenchymal transition(EMT).The purpose of this study was to determine the biochemical effects of pomegranate juice extracts(PE)on RCC cell lines.Methods:The pre-clinical effects of PE on NF-κB,JNK,and the EMT phenotype were assayed,including its effect on proliferation,anchorage-independent growth,and invasion of pVHLdeficient RCCs.Results:PE inhibits the NF-κB and JNK pathways and consequently inhibits the EMT phenotype of pVHL-deficient ccRCCs.The effects of PE are concentration-dependent and affect not only biochemical markers of EMT(i.e.,cadherin expression)but also functional manifestations of EMT,such as invasion.These effects are manifested within days of exposure to PE when diluted 2000-fold.Highly dilute concentrations of PE(106 dilution),which do not impact these pathways in the short term,were found to have NF-κB and JNK inhibitory effects and ability to reverse the EMT phenotype following prolonged exposure.Conclusion:These findings suggest that PE may mediate inhibition growth of pVHL-deficient ccRCCs and raises the possibility of its use as a dietary adjunct to managing patients with active surveillance for small,localized,incidentally identified renal tumors so as to avoid more invasive procedures such as nephrectomy.

Potential roles of vitamin D binding protein in attenuating liver injury in sepsis

Background:In sepsis,vitamin D binding protein(VDBP)has been shown to be low-expressed.The current study examined the relationship between serum VDBP level and liver injury in sepsis patients,as well as in a mouse model for sepsis and in cultured liver epithelial cell line exposed to lipopolysaccharide(LPS).Methods:The human study included 78 sepsis patients and 50 healthy volunteers.Sepsis patients were categorized into sepsis survivor group(n=43)and sepsis non-survivor group(n=35)based on 28-day mortality for data analysis.Adult male C57BL/6 mice were subjected to cecal ligation and puncture(CLP).Serum samples were collected on day 1,3,5 and 7 to determine the levels of VDBP,25-hydroxyvitamin D[25(OH)D_(3)],1,25-dihydroxyvitamin D[1,25(OH)_(2)D_(3)],interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-α).Potential protective effects of VDBP overexpression against LPS-induced liver damage were examined in cultured THLE2 cells.Results:Serum levels of VDBP,25(OH)D_(3),and 1,25(OH)_(2)D_(3)were significantly lower in sepsis patients vs.the healthy control(P<0.001),as well as in the sepsis non-survivor group vs.the sepsis survivor group(P<0.001,P=0.0338,or P=0.0013,respectively).Lower serum VDBP level was associated with higher Acute Physiology and Chronic Health Evaluation(APACHE)II score(r=−0.2565,P=0.0234)and Sequential Organ Failure Assessment score(r=−0.3522,P=0.0016),but lower serum albumin(ALB,r=0.4628,P<0.001)and total protein(TP,r=0.263,P=0.02).In CLP mice,there was a 5-day period of serum VDBP reduction,followed by return towards the baseline on day 7.VDBP was also decreased in LPS-treated THLE2 cells(P<0.001).VDBP overexpression reduced LPS-induced THLE2 damage.Reduced damage was associated with decreased oxidative stress and inactivation of the c-Jun N-terminal kinase signaling pathway.Conclusion:VDBP may be protective against sepsis-induced liver injury.

Hepato-protective effect of thymoquinone against acetaminophen induced liver injury is associated with regulation of JNK and AMPK signaling pathway

OBJECTIVE To investigate the hepato-protective mechanism of thymoquinone(TQ) on the development of acetaminophen(APAP)-induced liver injury.METHODS In vivo,male kunming mice were injected with a single dose of 300 mg·kg^(-1) APAP.Some mice were pretreated with TQ(5 or 20 mg·kg^(-1))and N-acetylcysteine(NAC,300 mg·kg^(-1))2 h before APAP injection.Mice were euthanized at 2 h,6 h,12 h after APAP treatment.In vitro,human Chang liver cells were incubated with 3.125,6.25 or 12.5μmol·L^(-1) TQ,10μmol·L^(-1) SP600125 and 500μmol·L^(-1) AICAR in the presence of APAP for 24 h.Cell viability were analyzed by MTT assay,protein expressions were assessed by Western blot.RESULTS TQ pretreatment significantly reduced serum aminotransferase and increased hepatic glutathione(GSH)and glutathione peroxidase(GSH-PX)activities,while significantly inhibited interleukin-1β(IL^(-1)β)levels.TQ significantly inhibited c-Jun N-terminal kinase(JNK),extracellular signal regulated kinase(ERK)and P38 phosphorylation induced by APAP.Moreover,TQ inhibited phosphatidylinositol 3-kinase(PI3K)/mammalian target of rapamycin(m TOR)signaling activation and activated AMPK phosphorylation induced by APAP.In addition,TQ inhibited signal transducer and activator of transcription 3(STAT3)phosphorylation on APAP-induced liver injury.In vitro,APAP enhanced JNK phosphorylation and attenuated AMPK phosphorylation in Chang liver cel s,and these effects were blocked by pretreatment with TQ,SP600125(JNK inhibitor)and AICAR(AMPK activator).CONCLUSION Our findings suggest that TQ may actively prevent APAP-induced liver injury,and this effect may be mediated by JNK and AMPK signaling pathways.

Arrestin-mediated signaling: Is there a controversy?

The activation of the mitogen-activated protein(MAP) kinases extracellular signal-regulated kinase(ERK)1/2 was traditionally used as a readout of signaling of G protein-coupled receptors(GPCRs) via arrestins, as opposed to conventional GPCR signaling via G proteins. Several recent studies using HEK293 cells where all G proteins were genetically ablated or inactivated, or both non-visual arrestins were knocked out, demonstrated that ERK1/2 phosphorylation requires G protein activity, but does not necessarily require the presence of non-visual arrestins. This appears to contradict the prevailing paradigm. Here we discuss these results along with the recent data on gene edited cells and arrestinmediated signaling. We suggest that there is no real controversy. G proteins might be involved in the activation of the upstream-most MAP3Ks, although in vivo most MAP3K activation is independent of heterotrimeric G proteins, being initiated by receptor tyrosine kinases and/or integrins. As far as MAP kinases are concerned, the best-established role of arrestins is scaffolding of the three-tiered cascades(MAP3K-MAP2 K-MAPK). Thus, it seems likely that arrestins, GPCRbound and free, facilitate the propagation of signals in these cascades, whereas signal initiation via MAP3K activation may be independent of arrestins. Different MAP3Ks are activated by various inputs, some of which are mediated by G proteins, particularly in cell culture, where we artificially prevent signaling by receptor tyrosine kinases and integrins, thereby favoring GPCR-induced signaling. Thus, there is no reason to change the paradigm: Arrestins and G proteins play distinct non-overlapping roles in cell signaling.