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.

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.

Role of JNK signaling pathway in sensitivity to radiotherapy of nasopharyngeal carcinoma

Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods:Human nasopharyngeal carcinoma CNE multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Western blot was employed to analyze the activity of JNK signaling pathway in MCS after X-ray irradiation,and the expression of caspase-3 protein before and after using SP600125(a special inhibitor of JNK).X-ray induced cell apoptosis in MCS before and after treated with SP600125 were detected by TUNEL.Results:The level of JNK phosphorylation in MCS was a dynamic course after radiation,and there was a phosphorylation peaks at 2 h later,the apoptotic rate of MCS(P < 0.05) and the expression of caspase-3 protein(P < 0.05) were significantly increased after treated with SP600125.Conclusion:The transient activation of JNK played a important role in sensitivity to radiotherapy of CNE MCS via mediating survival signals,blocking this pathway accelerate cell apoptosis,which may be related to the increased expression of caspase-3.

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。

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.

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.

TRAF2-MLK3 interaction is essential for TNF-α-induced MLK3 activation

Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulation. The mecha- nism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors （TRAFs） are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half （amino acids 511-847）. Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.

紫杉醇通过MLK_3/JNK_3信号通路对海马神经元损伤的保护作用

目的研究紫杉醇对脑缺血/再灌注后海马神经元损伤的保护作用的分子机制。方法采用SD大鼠四动脉结扎脑缺血模型(4-VO),在缺血前30 min脑室注射紫杉醇(4、8、12、20μg/kg),应用免疫沉淀及免疫印迹方法分析JNK3/MLK3/Fas-L的蛋白表达量和激活情况;焦油紫染色,观察紫杉醇对大鼠海马神经元的作用。结果紫杉醇显著抑制了脑缺血/再灌注后JNK3/MLK3/Fas-L的激活;焦油紫染色观察,紫杉醇对大鼠海马神经元的凋亡有明显的抑制作用。结论紫杉醇对脑缺血/再灌注诱导的海马神经元的损伤具有保护作用,这种保护作用和JNK3介导的信号通路密切相关,为临床治疗脑中风提供理论依据。

Combination of Total Astragalus Extract and Total Panax Notoginseng Saponins Strengthened the Protective Effects on Brain Damage through Improving Energy Metabolism and Inhibiting Apoptosis after Cerebral Ischemia-Reperfusion in Mice

Objective： To explore the effects and molecular mechanisms of the combination between total Astragalus extract （TAE） and total Panax notoginseng saponins （TPNS） against cerebral ischemia- reperfusion injury. Methods： C57BL/6 mice were randomly divided into sham-operated group, model group, TAE （110 mg/kg） group, TPNS （115 mg/kg） group, TAE-TPNS combination group and Edaravone （4 mg/kg） group, treated for 4 days, then, cerebral ischemia-repeffusion injury was established by bilateral common carotid artery （CCA） ligation for 20 min followed by reperfusion for 1 and 24 h. Results： TPNS could increase adenosine triphosphate （ATP） level, TAE and TAE-TPNS combination increased ATP, adenosine diphosphate （ADP） contents and Na＋-K＋-ATPase activity, and the effects of TAE-TPNS combination were stronger than those of TAE or TPNS alone after reperfusion for 1 h. After reperfusion for 24 h, TAE, TPNS and TAE-TPNS combination significantly increased neurocyte survival rate and decreased the apoptosis rate as well as down-regulated the expression of phosphorylated c-June N-terminal kinasel/2 （p-JNK1/2）, cytochrome C （Cyt C）, cysteine aspartic acid-specific protease （Caspase）-9 and Caspase-3. Furthermore, the effects in TAE-TPNS combination were better than those in TAE or TPNS alone. Conclusion： The combination of TAE 110 mg/kg and TPNS 115 mg/kg could strengthen protective effects on cerebral ischemia injury, the mechanism underlying might be related to improving jointly the early energy metabolism, and relieving the delayed apoptosis via inhibiting the mitochondrial apoptosis pathway of JNK signal transduction.

Glycyrrhizic acid activates chicken macrophages and enhances their Salmonella-killing capacity in vitro

Objective： Salmonella enterica remains a major cause of food-borne disease in humans, and Salmonella Typhimurium （ST） contamination of poultry products is a worldwide problem. Since macrophages play an essential role in controlling Salmonella infection, the aim of this study was to evaluate the effect of glycyrrhizic acid （GA） on immune function of chicken HD11 macrophages. Methods： Chicken HD11 macrophages were treated with GA （0, 12.5 25, 50, 100, 200, 400, or 800 pg/ml） and lipopolysaccharide （LPS, 500 ng/ml） for 3, 6, 12, 24, or 48 h. Evaluated responses included phagocytosis, bacteria-killing, gene expression of cell surface molecules （cluster of differentiation 40 （CD40）, CD80, CD83, and CD197） and antimicrobial effectors （inducible nitric oxide synthase （iNOS）, NADPH oxidase-1 （NOX-1）, interferon-γ （IFN-γ）, LPS-induced tumor necrosis factor （TNF）-α factor （LITAF）, interleukin-6 （IL-6） and IL-IO）, and production of nitric oxide （NO） and hydrogen peroxide （H202）. Results： GA increased the internalization of both fiuorescein isothiocyanate （FITC）-dextran and ST by HD11 cells and markedly decreased the intracellular survival of ST. We found that the messenger RNA （mRNA） expression of cell surface molecules （CD40, CDSO, CD83, and CD197） and cytokines （IFN-γ, IL-6, and IL-10） of HD11 cells was up-regulated following GA exposure. The expression of iNOS and NOX-1 was induced by GA and thereby the productions of NO and H202 in HD11 cells were enhanced. Notably, it was verified that nuclear factor-κB （NF-κB） and c-Jun N-terminal kinase （JNK） pathways were responsible for GA-induced synthesis of NO and IFN-γ gene expression. Conclusions： Taken together, these results suggested that GA exhibits a potent immune regulatory effect to activate chicken macrophages and enhances Salmonella-killing capacity.

Hydroxychavicol,a polyphenol from Piper betle leaf extract,induces cell cycle arrest and apoptosis in TP53-resistant HT-29 colon cancer cells

This study aims to elucidate the antiproliferative mechanism of hydroxychavicol(HC).Its effects on cell cycle,apoptosis,and the expression of c-Jun N-terminal kinase(JNK)and P38 mitogen-activated protein kinase(MAPK)in HT-29 colon cancer cells were investigated.HC was isolated from Piper betle leaf(PBL)and verified by high-performance liquid chromatography(HPLC),nuclear magnetic resonance(NMR),and gas chromatography-mass spectrometry(GC-MS).The cytotoxic effects of the standard drug 5-fluorouracil(5-FU),PBL water extract,and HC on HT-29 cells were measured after 24,48,and 72 h of treatment.Cell cycle and apoptosis modulation by 5-FU and HC treatments were investigated up to 30 h.Changes in phosphorylated JNK(pJNK)and P38(pP38)MAPK expression were observed up to 18 h.The half maximal inhibitory concentration(IC_(50))values of HC(30μg/mL)and PBL water extract(380μg/mL)were achieved at 24 h,whereas the IC_(50)of 5-FU(50μmol/L)was obtained at 72 h.Cell cycle arrest at the G0/G1 phase in HC-treated cells was observed from12 h onwards.Higher apoptotic cell death in HC-treated cells compared to 5-FU-treated cells(P<0.05)was observed.High expression of pJNK and pP38 MAPK was observed at 12 h in HC-treated cells,but not in 5-FU-treated HT-29 cells(P<0.05).It is concluded that HC induces cell cycle arrest and apoptosis of HT-29 cells,with these actions possibly mediated by JNK and P38 MAPK.

Mitochondrial damage and biogenesis in acetaminophen-induced liver injury

Liver injury and acute liver failure caused by acetaminophen(APAP)overdose is the clinically most important drug toxicity in Western countries.Mechanistic investigations have revealed a central role of mitochondria in the pathophysiology.Excess formation of the reactive metabolite N-acetyl-p-benzoquinone imine(NAPQI)after an overdose leads to hepatic glutathione depletion,mitochondrial protein adducts formation and an initial oxidant stress,which triggers the activation of mitogen activated protein(MAP)kinase cascade ultimately leading to c-jun N-terminal kinase(JNK)phosphorylation.Phospho-JNK translocates to the mitochondria and amplifies the oxidative and nitrosative stress eventually causing the mitochondrial membrane permeability transition pore opening and cessation of adenosine triphosphate(ATP)synthesis.In addition,mitochondrial matrix swelling ruptures the outer membrane and releases endonucleases,which cause nuclear deoxyribonucleic acid(DNA)fragmentation.Together,the nuclear DNA damage and the extensive mitochondrial dysfunction result in necrotic cell death.However,the procell death signaling events are counteracted by adaptive responses such as autophagy and mitochondrial biogenesis.The improved mechanistic insight into the pathophysiology leads to better understanding of the mechanisms of action of the existing antidote N-acetylcysteine and justifies the clinical testing of novel therapeutics such as 4-methylpyrazole and calmangafodipir.