Experimental study on the apoptosis of cervical cancer Hela cells induced by juglone through c-Jun N-terminal kinase/c-Jun pathway

Objective: To study the regulatory effect and molecular mechanism of juglone on apoptosis of cervical cancer Hela cells. Methods: Cervical cancer Hela cells were cultured and treated with different dosages of juglone (10, 20, and 40 pmol/L, respectively) and c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10, 20, and 40 mu mol/L. respectively). Then cellular proliferative activity and the expression of JNK/c-Jun pathway molecule and apoptotic molecule in the cells were detected. Results: After 6, 12. 18 and 24 h of treatment, the value for proliferative activity of cells treated with juglone was significantly lower than that of control group (p<0.05), and the anti-proliferative effect was more significant as the treatment period and juglone dosage increased (P<0.05). The protein expressions of Box, CytC, Fas, FasL, Caspase-3, and p-c-Jun in cells treated with juglone were significantly higher than those of control group (P<0.05), and the protein expressions of Bax, CytC, Fas. FasL, Caspase-3, p-JNK and p-c-Jun increased more remarkably as the juglone dosage increased (P<0.05). In cells treated with 40 pmol/L juglone and SP600125, the protein expressions of Bax, CytC, Fas. Fast.. and Caspase-3 were significantly lower than those of cells treated with 40 pmol/L juglone (J<0.05), and the protein expressions of Bax, CytC, Fas, FasL and Caspase-3 reduced more remarkably as the SP600125 dosage increased (P<0.05). Conclusion: Juglone can increase the expression of apoptotic molecules in mitochondrial pathway and death receptor pathway by activating JNK/c-Jun pathway, thus inducing apoptosis of cervical cancer cells.

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.

S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

AIM： To determine the role of c-Jun N-terminal kinase （JNK） activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosylmethionine （AdoMet）. METHODS： Primary hepatocyte cultures were pretreated with 100 IJmol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays. JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by EIIman＇s method and reactive oxygen species （ROS） generation by cell cytometry. RESULTS： The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decrease in ethanol-induced apoptosis （P〈 0.05）. AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity, and prevented cytochrome c release and pro-caspase 3 cleavage.CONCLUSION： The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease （ALD） treatment.

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.

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.

Effects of Different Therapeutic Methods and Typical Recipes of Chinese Medicine on Activation of c-Jun N-terminal Kinase in Kupffer Cells of Rats with Fatty Liver Disease

Objective： To observe the effects of different therapeutic methods and the recipes of Chinese medicine （CM） on the activation of c-Jun N-terminal kinase （JNK） in Kupffer cells of rats with fatty liver disease and to explore the mechanisms of these therapeutic methods. Methods： By using a random number table, 98 rats were randomly divided into 7 groups： control group, model group, and 5 treatment groups, including soothing Liver （Gan） recipe group, invigorating Spleen （Pi） recipe group, dispelling dampness recipe group, promoting blood recipe group, and complex recipe group. Rats in the control group were fed with normal food and distilled water by gastric perfusion, while rats in the model group were fed with high-fat food and distilled spirits by gastric perfusion. Rats in the 5 treatment groups were fed with high-fat food and corresponding recipes by gastric perfusion. Twelve weeks later, all rats were sacrificed and liver tissues were stained for pathohistological observation. Kupffer cells were isolated from livers of rats to evaluate JNK and phospho-JNK expressions by Western blotting. Results： The grade of hepatic steatosis was higher in the model group than the control group （P〈0.05）. Compared with the model group, the grade of fatty degeneration in soothing Liver recipe group and invigorating Spleen recipe group were significantly ameliorated （P〈0.05）. Expressions of JNK and phospho-JNK in Kupffer cells were significantly higher in the model group than those in the control group （P〈0.05, P〈0.01）. Compared with the model group, expressions of JNK in all treatment groups decreased, especially in invigorating Spleen recipe group and promoting blood recipe group （P〈0.05）. Compared with the model group, expressions of phospho-JNK in all treatment groups declined significantly （P〈0.01）, especially in soothing Live recipe group and invigorating Spleen recipe group. Conclusions： The high expressions of JNK and phospho-JNK in Kupffer cells might play an important role in the pathogenesis of fatty liver disease in rats. The recipes of CM, especially invigorating Spleen recipe and soothing Liver recipe, might protect liver against injury by reducing the total JNK protein content and inhibiting the activation of JNK protein in Kupffer cells of fatty liver model rats, which showed beneficial effects on fatty liver disease.

Activation of c-Jun N-terminal kinase 1/2 regulated by nitric oxide is associated with neuronal survival in hippocampal neurons in a rat model of ischemia

Background C-Jun N-terminal kinase （JNK） signaling pathway plays a critical role in cerebral ischemia. Although the mechanistic basis for this activation of JNK1/2 is uncertain, oxidative stress may play a role. The purpose of this study was to investigate whether the activation of JNK1/2 is associated with the production of endogenous nitric oxide （NO）. Methods Ischemia and reperfusion （I/R） was induced by cerebral four-vessel occlusion. Sprague-Dawley （SD） rats were divided into 6 groups： sham group, I/R group, neuronal nitric oxide synthase （nNOS） inhibitor （7-nitroindazole, 7-NI） given group, inducible nitric oxide synthase （iNOS） inhibitor （2-amino-5,6-dihydro-methylthiazine, AMT） given group, sodium chloride control group, and 1% dimethyl sulfoxide （DMSO） control group. The levels of protein expression and phospho-JNK1/2 were detected by Western blotting and the survival hippocampus neurons in CA1 zone were observed by cresyl violet staining. Results The study illustrated two peaks of JNK1/2 activation occurred at 30 minutes and 3 days during reperfusion. 7-NI inhibited JNK1/2 activation during the early reperfusion, whereas AMT preferably attenuated JNK1/2 activation during the later reperfusion. Administration of 7-NI and AMT can decrease I/R-induced neuronal loss in hippocampal CA1 region. Conclusion JNK1/2 activation is associated with endogenous NO in response to ischemic insult.

Inhibition of c-Jun N-terminal Kinase Signaling Pathway Alleviates Lipopolysaccharide-induced Acute Respiratory Distress Syndrome in Rats

Background： An acute respiratory distress syndrome （ARDS） is still one of the major challenges in critically ill patients. This study aimed to investigate the effect of inhibiting c-Jun N-terminal kinase （JNK） on ARDS in a lipopolysaccharide （LPS）-induced ARDS rat model. Methods： Thirty-six rats were randomized into three groups： control, LPS, and LPS ＋ JNK inhibitor Rats were sacrificed 8 h alter LPS treatment. The lung edema was observed by measuring the wet-to-dry weight （W/D） ratio of the lung. The severity of ptdmonary inflammation was observed by measuring myeloperoxidase （MPO） activity of lung tissue. Moreover, the neutrophils in bronchoalveolar lavage fluid （BALF） were cotinted to observe the airway inflammation. In addition, lung collagen accumulation was quantified by Sircol Collagen Assay. At the same time, the pulmonary histologic examination was perlbrmed, and lung injury score was achieved in all three groups. Results： MPO activity in lung tissue was found increased in rats treated with LPS comparing with that in control （I.26 ＋ 0.15 U in LPS vs. 0.77 ± 0.27 U in control, P 〈 0.05）. Inhibiting ,INK attenuated LPS-induced MPO activity upregulation （0.52 ± 0. 12 U in LPS ＋ JNK inhibitor vs. 1.26 ± 0.15 U in LPS, P 〈 0.05）. Neutrophils in BALF were also found to be increased with LPS treatment, and inhibiting ,INK attenuated LPS-induced neutrophils increase in BALF （255.0 ± 164.4 in LPS vs. 53 （44.5-103） in control vs. 127.0 ± 44.3 in LPS JNK inhibitor, P 〈 0.05）. At the same time, the lung injury score showed a reduction in LPS ＋ JNK inhibitor group comparing with that in LPS group （ 13.42 ± 4.82 vs. 7.00 ± 1.83, P 0.001 ）. However, the lung W/D ratio and the collagen in BALF did not show any difl＇erences between LPS and LPS ＋ JNK inhibitor group.Conclusions： Inhibiting JNK alleviated LPS-induced acute lung inflammation and had no effects on pulmonary edema and fibrosis..INK inhibitor might be a potential therapeutic medication in ARDS, in the context of reducing lung inflammatory.

Specific effects of c-Jun NH2-terminal kinaseinteracting protein 1 in neuronal axons

c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.

c-Jun氨基端激酶信号通路调控急性呼吸窘迫综合征中重要炎症介质表达机制的研究进展

急性呼吸窘迫综合征(ARDS)是一种临床常见的危重症,其核心问题在于过度的炎症反应。c-Jun氨基端激酶(JNK)是促分裂原活化的蛋白质激酶(MAPK)家族最重要的成员之一,在调控细胞增殖、分化、凋亡、自噬及炎症等多个重要功能中发挥作用。JNK信号通路被过度激活时,会导致炎症反应的持续激活和炎症因子的过度生成,从而对机体造成严重损害。因此,明确JNK通过哪些信号通路参与炎症反应,对于控制ARDS的炎症进程和调节机体的免疫反应平衡具有重要意义。

恩格列净通过c-Jun氨基末端激酶信号通路改善2型糖尿病性骨质疏松症的机制研究

目的探讨恩格列净通过c-Jun氨基末端激酶(JNK)信号通路改善2型糖尿病性骨质疏松症(T2DOP)的机制。方法将大鼠随机分为假手术组(sham组)、T2DOP组、EM组(予恩格列净干预)和EM+Anisomycin组(予EM+JNK激活剂Anisomycin干预),每组各10只。收集4组大鼠体重、血糖、血酸水平、骨代谢指标及股骨生物力学特征。采用Micro-CT测定股骨显微结构;HE染色检测股骨组织病理学变化;蛋白质印迹法(Western blot)检测骨组织中氨基末端激酶1(JNK1)、磷酸化JNK1(p-JNK1)、c-Jun、磷酸化c-Jun(p-c-Jun)蛋白表达水平并分组进行比较。结果T2DOP组大鼠体重明显低于sham组,空腹血糖(FPG)、甘油三酯(TG)、总胆固醇(TC)及低密度脂蛋白胆固醇(LDL-C)水平均明显高于sham组;EM组大鼠体重明显高于T2DOP组,FPG、TG、TC及LDL-C水平均明显低于T2DOP组,TG、TC及LDL-C水平均明显高于sham组;EM+Anisomycin组大鼠体重明显低于EM组,FPG、TG、TC及LDL-C水平均明显高于EM组(P<0.05)。T2DOP组大鼠血清骨特异性转录因子(CBF-α1)、Ⅰ型前胶原氨基端原肽(PⅠNP)、骨钙素(OC)及Ⅰ型胶原交联羧基端肽(CTXⅠ)水平、股骨最大负荷、断裂挠度、弹性模量、骨小梁骨密度(BMD)、骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)均明显低于sham组,骨小梁间隔(Tb.Sp)高于sham组;EM组大鼠血清CBF-α1、PⅠNP、OC及CTX-1水平、股骨最大负荷、断裂挠度、弹性模量、BMD、BV/TV、Tb.N、Tb.Th均明显高于T2DOP组,Tb.Sp低于T2DOP组;EM组大鼠血清CBF-α1、PⅠNP及OC水平、股骨最大负荷、断裂挠度、弹性模量、BMD、BV/TV、Tb.N、Tb.Th均明显低于sham组,CTX-1水平及Tb.Sp均高于sham组;EM+Anisomycin组大鼠血清CBF-α1、PⅠNP、OC及CTX-1水平、股骨最大负荷、断裂挠度、弹性模量、BMD、BV/TV、Tb.N、Tb.Th均明显低于EM组,Tb.Sp高于EM组(P<0.05)。T2DOP组大鼠股骨组织中JNK1、p-JNK1、c-Jun、p-c-Jun蛋白表达水平均明显高于sham组;EM组大鼠股骨组织中JNK1、p-JNK1、c-Jun、p-c-Jun蛋白表达水平均明显低于T2DOP组;EM+Anisomycin组大鼠股骨组织中JNK1、p-JNK1、c-Jun、p-c-Jun蛋白表达水平均明显高于EM组(P<0.05)。结论恩格列净可改善T2DOP大鼠骨代谢量失衡和骨微结构,其作用机制可能和抑制JNK/c-Jun信号通路激活有关。

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.

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.

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.

毛兰素通过JNK/c-Jun信号通路对2型糖尿病大鼠肝脏损伤的保护作用机制研究

目的基于c-Jun氨基末端激酶(JNK)/c-Jun信号通路对氧化应激反应的调控作用,研究毛兰素对2型糖尿病大鼠肝脏损伤的保护作用。方法于2021年10月至2022年2月腹腔注射链脲佐菌素构建糖尿病大鼠模型,将造模成功大鼠分为模型组、毛兰素低剂量组(10 mg/kg)、毛兰素高剂量组(40 mg/kg),及罗格列酮组(1.25 mg/kg)、毛兰素(40 mg/kg)+JNK激活组(5 mg/kg),每组10只,另取正常饲养大鼠10只作为对照组。连续给药6周后,通过血糖仪和胰岛素放射免疫分析试剂盒检测空腹血糖(FBG)、空腹胰岛素(FINS)水平,计算胰岛素抵抗指数(HOMA-IR)及胰岛素敏感指数(ISI);天平称取大鼠体质量和肝质量,计算肝脏指数;试剂盒检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量;HE染色观察肝脏组织病理学变化;western blotting法检测肝脏组织中JNK/c-Jun信号通路相关蛋白表达。结果与对照组相比,模型组肝脏指数[(4.26±0.12)g/100 g比(2.24±0.09)g/100 g]、FBG[(21.49±1.78)mmol/L比(5.14±0.45)mmol/L]、FINS[(80.17±6.38)mmol/L比(22.35±2.04)mmol/L]、HOMA-IR[(76.46±6.56)比(5.11±1.12)]、ALT[(138.71±10.21)U/L比(70.29±5.54)U/L]和AST活性[(77.21±5.08)U/L比(40.38±3.27)U/L]、MDA含量[(13.45±1.34)nmol/mg prot比(3.72±0.87)nmol/mg prot]、JNK/c-Jun信号通路相关蛋白表达均明显升高(P<0.05),体质量、ISI[(−7.45±0.18)比(−4.74±0.11)]、SOD[(100.79±11.22)U/mg prot比(223.46±19.86)U/mg prot]和GSH-Px活性[(24.42±1.74)U/mg prot比(56.79±3.18)U/mg prot]明显降低(P<0.05),且肝脏组织病理损伤较为严重;与模型组相比,毛兰素低剂量组、毛兰素高剂量组和罗格列酮组肝脏指数、FBG、FINS、HOMA-IR、ALT和AST活性、MDA含量、JNK/c-Jun信号通路相关蛋白表达均明显降低(P<0.05),体质量、ISI、SOD和GSH-Px活性明显升高(P<0.05),减轻肝脏损伤程度;与毛兰素高剂量组相比,毛兰素低剂量组、毛兰素+JNK激活组肝脏指数、FBG、FINS、HOMA-IR、ALT和AST活性、MDA含量、JNK/c-Jun信号通路相关蛋白表达均明显升高(P<0.05),体质量、ISI、SOD和GSH-Px活性明显降低(P<0.05),肝脏损伤恢复缓慢。结论毛兰素可通过调控JNK/c-Jun信号通路,抑制JNK/c-Jun通路蛋白表达,从而缓解氧化应激反应,改善糖尿病大鼠肝脏损伤。

基于c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶信号通路探讨阿魏酸钠对偏头痛大鼠炎症反应的抑制作用

目的 探讨阿魏酸钠(SF)通过调控JNK/p38 MAPK信号通路对偏头痛大鼠炎症反应的抑制作用。方法 腹腔注射硝酸甘油制备偏头痛大鼠模型,模型制作成功后随机分为模型组、SF低剂量(SF-L)组(50 mg/kg)、 SF高剂量(SF-H)组(100 mg/kg)、SF+JNK抑制剂(SF+SP600125)组(SF 100 mg/kg+SP600125 10 mg/kg)、 SF+JNK激活剂[SF+茴香霉素(AN)]组(SF 100 mg/kg+AN 5 mg/kg),每组12只,另取12只SD大鼠不做处理作为空白组。给药结束24 h后观察各组大鼠行为学变化,ELISA法检测血清中5-羟色胺(5-HT)、一氧化氮(NO)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)水平,TUNEL染色观察脑组织神经元凋亡情况,免疫组织化学法检测脑组织中TNF-α、 IL-6、降钙素基因相关肽(CGRP)表达,Western blotting法检测脑组织中JNK/p38 MAPK通路相关蛋白的表达。结果 与空白组比较,模型组大鼠挠头次数以及爬笼次数明显增加,神经元凋亡率显著升高;血清5-HT含量显著降低,NO、TNF-α和IL-6水平显著升高;脑组织中TNF-α、IL-6和CGRP表达以及磷酸化JNK(p-JNK)/JNK、磷酸化p38 MAPK(p-p38 MAPK)/p38 MAPK比值均显著升高(均P<0.05)。与模型组比较,SF-L组、SF-H组大鼠挠头次数及爬笼次数显著减少,神经元凋亡率显著降低;血清中5-HT含量显著升高,NO、TNF-α和IL-6水平显著降低;脑组织中TNF-α、IL-6和CGRP表达以及p-JNK/JNK、p-p38 MAPK/p38 MAPK比值均显著降低(均P<0.05)。与SF-H组比较,SF+SP600125组能够显著增强SF对偏头痛大鼠的保护作用;SF+AN组能够显著逆转SF对偏头痛大鼠的保护作用。结论 SF可能通过抑制JNK/p38 MAPK信号通路的表达,有效抑制偏头痛大鼠神经源性炎症反应,减少神经元凋亡,实现对偏头痛大鼠的保护作用。

c-Jun氨基末端激酶与首发精神分裂症患者认知功能及预后的相关性研究

目的探讨首发精神分裂症患者血清c-Jun氨基末端激酶(JNK)表达与认知功能及出院1年预后的相关性。方法对168例首发精神分裂症患者进行临床资料调查、认知功能评估和出院后随访。根据有无认知功能受损,168例患者被分为认知功能受损组65例和认知功能正常组103例。采用Pearson相关分析法分析患者血清JNK表达水平与认知功能[MATRICS共识认知成套测试(MCCB)得分]的相关性。采用Logistic回归分析探讨患者认知功能受损的影响因素,绘制受试者工作特征(ROC)曲线分析血清JNK表达水平等因素对患者认知功能受损的预测效能,采用Cox回归分析法对患者出院后1年的随访预后进行单因素和多因素分析。结果认知功能受损组血清JNK表达水平为(78.12±9.28)%,高于认知功能正常组的(59.38±6.17)%,差异有统计学意义(t=10.420,P<0.001)。首发精神分裂症患者血清JNK表达水平与MCCB得分呈显著负相关(r=-0.492,P<0.001)。血清JNK表达水平是首发精神分裂症患者认知功能受损的独立危险因素(OR=3.080,95%CI:1.864~12.157);血清JNK表达水平预测患者认知功能受损的曲线下面积(AUC)为0.756,最佳截断值为73.49,特异度为0.885,敏感度为0.913。出院后随访1年,高JNK水平患者的无病生存期短于低JNK水平患者,差异有统计学意义(P<0.05)。Cox回归分析显示,年龄、睡眠障碍、抑郁、焦虑、血清JNK表达水平均为首发精神分裂症患者出院后随访预后不良的独立影响因素(P<0.05)。结论首发精神分裂症患者血清JNK表达水平与认知功能、出院1年预后存在相关性,临床医务人员应密切监测患者血清JNK表达水平。