c-Jun N-terminal kinase is required for thermotherapyinduced apoptosis in human gastric cancer cells

AIM:To investigate the role of c-Jun N-terminal kinase(JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro.Following thermotherapy at 43 ℃ for 0,0.5,1,2 or 3 h,the cells were cultured for a further 24 h with or without the JNK specific inhibitor,SP600125 for 2 h.Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)] and flow cytometry(Annexin vs propidium iodide).Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.The production of p-JNK,Bcl-2,Bax and caspase-3 proteins was evaluated by Western blotting.The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction.RESULTS:The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy,and was 32.7%,30.6%,43.8% and 52.9% at 0.5,1,2 and 3 h post-thermotherapy,respectively.Flow cytometry analysis revealed an increased population of SGC790l cells in G0/G1 phase,but a reduced population in S phase following thermotherapy for 1 or 2 h,compared to untreated cells(P < 0.05).The increased number of SGC-790l cells in G0/G1 phase was consistent with induced apoptosis(flow cytometry) following thermotherapy for 0.5,1,2 or 3 h,compared to the untreated group(46.5% ± 0.23%,39.9% ± 0.53%,56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%,P < 0.01),respectively.This was supported by the TUNEL assay(48.2% ± 0.4%,40.1% ± 0.2%,61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%,P < 0.01) respectively.More importantly,the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment(P < 0.01),and peaked at 2 h.A similar pattern was detected for Bax and caspase-3 proteins.Bcl-2 increased at 0.5 h,peaked at 1 h,and then decreased.Furthermore,the JNK specific inhibitor,SP600125,suppressed p-JNK,Bax and caspase-3 at the protein level in SGC790l cells following thermotherapy,compared to mock-inhibitor treatment,which was in line with the decreased rate of apoptosis.The expression of Bcl-2 was consistent with thermotherapy alone.CONCLUSION:Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels,and up-regulated the expression of Bax and caspase-3 proteins.Bcl-2 may play a protective role during thermotherapy.Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.

Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling

Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway.

Mitogen-activated protein kinase phosphatase 1 protects PC12 cells from amyloid beta-induced neurotoxicity

The mitogen-activated protein kinase（MAPK） signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosphatase 1（MKP1） has an inhibitory effect on the p38 MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 sh RNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42（Aβ42）. The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha（TNF-α） and interleukin-1β（IL-1β） m RNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase（JNK） expression levels were assessed using western blot assay. Reactive oxygen species（ROS） levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase d UTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.

Serine-threonine protein kinase activation may be an effective target for reducing neuronal apoptosis after spinal cord injury

The signaling mechanisms underlying ischemia-induced nerve cell apoptosis are poorly understood. We investigated the effects of apoptosis-related signal transduction pathways following ischemic spinal cord injury, including extracellular signal-regulated kinase（ERK）, serine-threonine protein kinase（Akt） and c-Jun N-terminal kinase（JNK） signaling pathways. We established a rat model of acute spinal cord injury by inserting a catheter balloon in the left subclavian artery for 25 minutes. Rat models exhibited notable hindlimb dysfunction. Apoptotic cells were abundant in the anterior horn and central canal of the spinal cord. The number of apoptotic neurons was highest 48 hours post injury. The expression of phosphorylated Akt（pAkt） and phosphorylated ERK（p-ERK） increased immediately after reperfusion, peaked at 4 hours（p-Akt） or 2 hours（p-ERK）, decreased at 12 hours, and then increased at 24 hours. Phosphorylated JNK expression reduced after reperfusion, increased at 12 hours to near normal levels, and then showed a downward trend at 24 hours. Pearson linear correlation analysis also demonstrated that the number of apoptotic cells negatively correlated with p-Akt expression. These findings suggest that activation of Akt may be a key contributing factor in the delay of neuronal apoptosis after spinal cord ischemia, particularly at the stage of reperfusion, and thus may be a target for neuronal protection and reduction of neuronal apoptosis after spinal cord injury.

Ceramide from sphingomyelin hydrolysis differentially mediates mitogen-activated protein kinases (MAPKs) activation following cerebral ischemia in rat hippocampal CA1 subregion

Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvessel occlusion （4-VO） method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular- signal regulated kinase （ERK） and c-Jun N-terminal protein kinase （JNK） using antibodies against ERK, JNK and diphosphorylated ERK and JNK. Results： At lh reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation （P 〈 0.05）. The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed （P 〈 0.05） by the sphingomyelinase inhibitor. Conclusion： The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.

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.

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.

Neuronal effects of SP600125 pretreatment in a rat model of cerebral ischemia/reperfusion injury Inhibited down-regulation of DNA repair protein

BACKGROUND： Recent studies have shown that the selective inhibitor of c-Jun N-terminal kinases （JNKs） signaling pathway, SP600125, exhibits neuronal protective effects in a rat model of brain ischemia/reperfusion. OBJECTIVE： To determine the mechanisms of neuroprotective effects of SP600125 in a rat model of brain ischemia/reperfusion, and determine the role of the JNK signaling pathway in SP600125-induced effects. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Animal Experiment Center, Medical School of Xi＇an Jiaotong University from June 2007 to September 2008. MATERIALS： SP600125 was provided by Biosource, USA; rabbit anti-phospho-JNK （Thr183/Tyr185） polyclonal antibody from Cell Signaling Technology, USA; rabbit anti-X-ray repair cross-complementing protein 1 （XRCC1） and anti-Ku70 polyclonal antibodies from Santa Cruz Biotechnology, USA; and TUNEL kit from Beijing Huamei Biology, China. METHODS： A total of 108 male, 4-month-old, Sprague Dawley rats were randomly assigned to three groups, with 36 rats per group. The sham operation group and ischemia/reperfusion group （I/R group） were intracerebroventricularly injected with 10 μL 1% DMSO. The SP600125-treated group （pre-SP group） was given 10 μL SP600125 （3 μg/μL）. Thirty minutes later, brain ischemia was induced in the I/R and pre-SP groups using the four-vessel occlusion method. Specifically, whole brain ischemia was induced for 6 minutes, and the clips were released to restore carotid artery blood flow. Rats from each group were observed at 2, 6, 12, 24, 48, and 72 hours, with 6 rats for each time point. The sham operation group was treated with the same surgical exposure procedures, with exception of occlusion of the carotid artery. MAIN OUTCOME MEASURES： Hematoxylin-eosin staining was used to observe neuronal survival in the hippocampal CA1 region, TUNEL was used to detect apoptosis in the hippocampal CA1 region, and immunohistochemistry was used to detect expression of phospho-JNK, XRCC1, and Ku70. RESULTS： Following brain ischemia/reperfusion, neuronal survival significantly decreased, and the number of apoptotic cells significantly increased （P 〈 0.01）. Compared with the I/R group, neuronal survival significantly increased in the pre-SP group, and the number of apoptotic cells significantly decreased （P 〈 0.01）. Expression of phospho-JNK increased, and XRCC1 and Ku70 significantly decreased （P 〈 0.05） following ischemia/reperfusion. Compared with the I/R group, expression of phospho-JNK decreased, and XRCC1 and Ku70 significantly increased in the pre-SP group （P 〈 0.05）. Correlation analysis revealed an inverse correlation between phospho-JNK gray value and XRCC1 and Ku70 gray values in the hippocampal CA1 region （r = -0.983, -0.953, P 〈 0.01）. CONCLUSION： SP600125 treatment decreased apoptosis induced by global brain ischemia/reperfusion in the rat hippocampal CA1 region. Results suggested that the neuroprotective effects were due to inhibited phosphorylation of JNK and reduced down-regulation of XRCC1 and Ku70.

Let-7a gene knockdown protects against cerebral ischemia/reperfusion injury

The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies

AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

Anti-parkinsonian effects of octacosanol in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine-treated mice

Our previous research showed that octacosanol exerted its protective effects in 6-hydroxydopamine-induced Parkinsonian rats. The goal of this study was to investigate whether octacosanol would attenuate neurotoxicity in 1-methyl-4-phenyl-l,2,3,6 tetrahydropyridine （MPTP）-treated C57BL/6N mice and its potential mechanism. Behavioral tests, tyrosine hydroxylase immunohistochemistry and western blot were used to investigate the effects of octacosanol in a mouse model of Parkinson＇s disease. Oral administration of octacosanol （100 mg/kg） significantly improved behavioral impairments Jn mice treated by MPTP and markedly ameliorated morphological appearances of tyrosine hydroxylase-positive neuronal cells in the substantia nigra. Furthermore, octacosanol blocked MPTP-induced phosphorylation of p38MAPK and JNK, but not ERK1/2. These findings implicated that the protective effects afforded by octacosanol might be mediated by blocking the phosphorylation of p38MAPK and JNK on the signa transduction in vivo. Considering its excellent tolerability, octacosanol might be considered as a candidate agent for clinical application in treating Parkinson＇s disease.

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

AIM To investigated the relationships between HER2, c-Jun N-terminal kinase(JNK) and protein kinase B(AKT) with respect to metastatic potential of HER2-positive gastric cancer(GC) cells.METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcriptionpolymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility.CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC.

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.

Vasopressin decreases neuronal apoptosis during cardiopulmonary resuscitation

The American Heart Association and the European Resuscitation Council recently recommend- ed that vasopressin can be used for cardiopulmonary resuscitation, instead of epinephrine. However, the guidelines do not discuss the effects of vasopressin during cerebral resuscitation. In this study, we intraperitoneally injected epinephrine and/or vasopressin during cardiopul- monary resuscitation in a rat model of asphyxial cardiac arrest. The results demonstrated that, compared with epinephrine alone, the pathological damage to nerve cells was lessened, and the levels of c-Iun N-terminal kinase and p38 expression were significantly decreased in the hippo- campus after treatment with vasopressin alone or the vasopressin and epinephrine combination. No significant difference in resuscitation effects was detected between vasopressin alone and the vasopressin and epinephrine combination. These results suggest that vasopressin alone or the vasopressin and epinephrine combination suppress the activation of mitogen-activated protein kinase and c-Iun N-terminal kinase signaling pathways and reduce neuronal apoptosis during cardiopulmonary resuscitation.

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.

Increased expression of receptor for advanced glycation end-products worsens focal brain ischemia in diabetic rats

A rat model of diabetes mellitus was induced by a high fat diet, followed by focal brain ischemia induced using the thread method after 0.5 month. Immunohistochemistry showed that expression of receptor for advanced glycation end-products was higher in the ischemic cortex of diabetic rats compared with non-diabetic rats with brain ischemia. Western blot assay revealed increased phosphorylated c-Jun N-terminal kinase expression, and unchanged phosphorylated extracellular signal-regulated protein kinase protein expression in the ischemic cortex of diabetic rats compared with non-diabetic rats with brain ischemia. Additionally, phosphorylated p38 mitogen-activated protein kinase protein was not detected in any rats in the two groups. Severity of limb hemiplegia was worse in diabetic rats with brain ischemia compared with ischemia alone rats. The results suggest that increased expression of receptor for advanced glycation end-products can further activate the c-Jun N-terminal kinase pathway in mitogen-activated protein kinase, thereby worsening brain injury associated with focal brain ischemia in diabetic rats.

SIGNAL MECHANISM OF INHIBITION OF BIFIDOBACTERIA ON GROWTH OF COLON CANCER

Objective: To explore the antitumor mechanisms of bifidobacteria adolescence in vivo. Methods: The content of extracellular signal regulated proteins (ERK)1/2, C-Jun N-terminal kinase (JNK), p38, c-fos and c-jun in nude mouse transplanted large bowel carcinoma was detected by using laser confocal microscopy. The expression of NF-κB was determined by immunohistochemistry. Results: After the nude mouse transplanted tumor was treated with bifidobacteria, the average fluorescent strength of ERK1/2, JNK, c-fos and c-jun was significantly lower than that in tumor control group (P<0.01). The average fluorescent strength of p38 was not obvious difference in the two groups (P>0.05). The positive cell density of NF-κB in large bowel carcinoma transplantation tumors in Bifidobacterium injection group was markedly lower than that in tumor group(P<0.01). Conclusion: bifidobacteria adolescence could markedly decrease the activity of ERK1/2 and JNK, the expression c-fos and c-jun, and the activity of NF-κB.

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.