Neuroprotective mechanisms of rutin for spinal cord injury through anti-oxidation and anti-inflammation and inhibition of p38 mitogen activated protein kinase pathway

Rutin has anti-inflammatory, antioxidant, anti-viral, anti-tumor and immune regulatory effects. However, the neuroprotective effects of rutin in spinal cord injury are unknown. The p38 mitogen activated protein kinase （p38 MAPK） pathway is the most important member of the MAPK family that controls inflammation. We assumed that the mechanism of rutin in the repair of spinal cord injury is associated with the inhibition of p38 MAPK pathway. Allen’s method was used to establish a rat model of spinal cord injury. The rat model was intraperitoneally injected with rutin （30 mg/kg） for 3 days. After treatment with rutin, Basso, Beattie and Bresnahan locomotor function scores increased. Water content, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 levels, p38 MAPK protein expression and caspase-3 and -9 activities in T8–9 spinal cord decreased. Oxidative stress related markers superoxide dismutase and glutathione peroxidase levels increased in peripheral blood. Rutin exerts neuroprotective effect through anti-oxidation, anti-inflammation, anti-apoptosis and inhibition of p38 MAPK pathway.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer

AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters.METHODS: Westem blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3,p38 and mitogen or ERK activated protein kinaseMEK-1proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients.Immunohistochemistry was employed for their localization.RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526±65 760 vs122 807±65 515, P = 0.001), ERK-2 (168 471±95 051 vs120 469±72 874, P＜0.001), ERK-3 (118 651±71 513 vs70 934±68 058, P＜0.001), P38 (104 776±51 650 vs82 930±40 392, P= 0.048) and MEK-1(116 486±45 725 vs101 434±49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging [average ratio of integral optic density (IOD)tumor:IODnormal in TNM Ⅰ, Ⅱ, Ⅲ, Ⅳ tumors was 1.43±0.34,5.08±3.74, 4.99±1.08, 1.44±1.02, n = 42, P = 0.023] and serosa invasion (4.31±4.34 vs 2.00±2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage Ⅲ and Ⅳ tumors were higher than those in stage Ⅰ and Ⅱ tumors (2.64±3.01 vs 1.01±0.33,P = 0.022; 2.05±1.54 vs 1.24±0.40, P = 0.030). Gastric cancer tissues with either lymph node involvement (2.49±2.91vs 1.03±0.36, P= 0.023; 1.98±1.49 vs 1.24±0.44, P= 0.036)or serosa invasion (2.39±2.82 vs 1.01±0.35, P = 0.022;1.95±1.44 vs 1.14±0.36, P = 0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann Ⅱ tumors,expression of ERK-2 and ERK-3 was increased compared with Borrmann Ⅲ tumors (2.57±1.86 vs 1.23±0.60, P = 0.022;5.50±5.05 vs 1.83±1.21, P = 0.014). Borrmann Ⅳ tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P＞0.05).Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site.CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers.Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MAPK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.

Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Abstract Objective:To explore the effects of γ-irradiation on mitogen-activatedprotein kinases(MAPKs) and role intracellular calcium in this event in intestinal epithelial cell line 6(IEC-6 cells).Methods:After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca^2+ chelator were exposed to γ-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry.Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting.Results:In response to γ-irradiation,phosphorylation of ERK was not significantly observed ,while the levels of phos-phorylated c-Jun NH2-terminal kinase(JNK) and p38 MAPK were increased in 30 min and reached the peak 2h after exposure to 6Gy γ-irradiation,though the cell viability was significantly lowered 12h.On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK.Chelation of in-tracellular Ca^2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca^2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 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.

Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma

Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK 1/2 in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK 1/2 or ERK 1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK 1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK 1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

下调PDCD4抑制MAP2K3和p38 MAPK的表达减轻LPS诱导的肾小管上皮细胞炎症和凋亡

目的研究程序性细胞死亡蛋白4(programmed cell death protein 4,PDCD4)在脓毒症诱导的急性肾损伤(acute kidney injury,AKI)中的作用机制,以及调控PDCD4表达通过丝裂原活化蛋白激酶3(mitogen-activated protein kinase 3,MAP2K3)和p38蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)对脓毒症AKI起到潜在治疗作用。方法用脂多糖(lipopolysaccharide,LPS)刺激人肾小管上皮细胞(HK-2)构建脓毒症AKI细胞模型。进一步用腺病毒介导siRNA和过表达载体抑制和上调AKI细胞模型中PDCD4的表达;CCK-8法检测细胞增殖;用DCFH-DA及激光共聚焦显微镜检测细胞中ROS水平,用总SOD活性检测试剂和MDA检测试剂盒检测细胞中SOD和MDA水平;免疫共沉淀验证PDCD4和MAP2K3之间的蛋白相互作用;TUNEL染色法检测细胞凋亡;RT-qPCR和Western blot检测PDCD4及相关基因的mRNA和蛋白表达水平;ELISA法检测患者血清中炎症相关因子水平。结果LPS诱导可以促进HK-2细胞中PDCD4表达,下调PDCD4可抑制LPS诱导的HK-2细胞的炎症、氧化应激及细胞凋亡。数据库预测及免疫共沉淀证实PDCD4可以与MAP2K3相互作用,且在LPS诱导的HK-2细胞中,MAP2K3表达水平显著增强。MAP2K3过表达和p38 MAPK激动剂可以减轻PDCD4下调对LPS诱导的细胞炎症和氧化应激的影响并抑制细胞凋亡。结论下调PDCD4可以通过抑制MAP2K3和p38 MAPK从而抑制LPS诱导的肾小管上皮细胞的炎症和凋亡。

甲状腺癌组织中DCBLD2和MAP4K3的表达及与临床病理特征及预后的关系

目的 探讨甲状腺癌组织中盘状蛋白含CUB和LCCL结构域2(discoidin CUB and LCCL domain containing 2,DCBLD2)、丝裂原活化蛋白激酶激酶激酶激酶3(mitogen-activated protein kinase kinase kinase kinase 3,MAP4K3)的表达及与临床病理特征及预后的关系。方法 选取2016年1月~2020年6月在扬州大学附属兴化市人民医院诊治的92例甲状腺癌患者。采用免疫组织化学(immunohitochemistry,IHC)检测癌组织及癌旁组织中DCBLD2和MAP4K3的表达。比较不同临床病理特征甲状腺癌患者DCBLD2和MAP4K3表达差异。Kaplan-Meier曲线分析不同DCBLD2和MAP4K3表达患者无进展生存预后的差异。多因素COX分析影响甲状腺癌无进展生存预后的危险因素。结果 甲状腺癌组织中DCBLD2(67.39%),MAP4K3(65.22%)阳性率高于癌旁组织(5.43%,6.52%),差异具有统计学意义(χ^(2)=76.262,68.894,均P<0.05)。癌组织中DCBLD2与MAP4K3的表达呈显著正相关(r=0.742,P<0.05)。TNM分期Ⅲ~Ⅳ期、并发淋巴结转移癌组织中DCBLD2的阳性率(87.18%,93.75%)和MAP4K3的阳性率(84.62%,90.63%)分别高于Ⅰ~Ⅱ期(52.83%,50.94%)、无淋巴结转移癌组织(53.33%,51.67%),差异具有统计学意义(χ^(2)=11.230~15.513,均P<0.05)。DCBLD2阳性和阴性组患者三年无进展生存率分别为74.19%(46/62),93.33%(28/30)。MAP4K3阳性和阴性组患者三年无进展生存率分别为75.00%(45/60),90.63%(29/32)。DCBLD2阳性组,MAP4K3阳性组患者三年累积无进展生存率低于DCBLD2阴性组、MAP4K3阴性组,差异具有统计学意义(χ^(2)=4.533,4.138,P=0.033,0.046)。DCBLD2阳性(OR=1.659,P=0.001)、MAP4K3阳性(OR=1.606,P=0.001)、肿瘤TNM分期Ⅲ~Ⅳ期(OR=1.766,P=0.001)和并发淋巴结转移(OR=1.868,P=0.001)是影响甲状腺癌患者无进展生存预后的独立危险因素。结论 甲状腺癌组织中DCBLD2和MAP4K3表达升高,两者均参与甲状腺癌的发生发展,有助于评估甲状腺癌患者的无进展生存预后。

CX3CR1对创伤性骨髓炎大鼠骨骼肌微纤维、ERK/MAPK信号通路及炎症反应的影响

目的探索CX3CR1对创伤性骨髓炎大鼠骨骼肌微纤维、ERK/MAPK信号通路及炎症反应的影响。方法选取30只SPF级SD雄性大鼠,依据随机数字表法分为健康组、模型组、CX3CR1抑制组,每组10只。除健康组外,其余各组均建立创伤性骨髓炎模型。其中健康组、模型组大鼠均每日常规腹腔注射生理盐水,CX3CR1干预组向残腔内注射CX3CR1中和抗体进行处理。采用ELISA法检测血清中IL-6、IL-10、IL-1β、TGF-β水平,应用改良X线Norden评分检测骨骼肌微纤维,HE染色观察病理变化,免疫印迹及PCR检测股骨组织中细胞外信号调节蛋白激酶(Extracellular regulated protein kinase,ERK1/2)、丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)蛋白及mRNA表达。结果与健康组比较,模型组TGF-β、IL-1β、IL-10、IL-6等炎症因子含量均升高(P<0.05);与模型组比较,CX3CR1抑制组炎症因子含量降低(P<0.05)。与健康组比较,模型组随时间推移X线Norden评分升高(P<0.05);与模型组比较,CX3CR1抑制组X线Norden评分降低(P<0.05)。HE染色显示,健康组骨质完好;模型组可见大量炎性细胞浸润、灶性脓肿及坏死灶;CX3CR1抑制组大鼠的骨质明显改善,炎症反应降低。与健康组比较,模型组ERK1/2、MAPK蛋白及mRNA表达升高(P<0.05);与模型组比较,CX3CR1抑制组ERK1/2、MAPK蛋白及mRNA表达降低(P<0.05)。结论抑制CX3CR1可改善创伤性骨髓炎大鼠的疾病反应,可能与降低炎症反应、ERK/MAPK信号通路以及改善骨骼肌微纤维相关。

PBX3 promotes migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway

AIM:To investigate the role of pre-B-cell leukemia homeobox(PBX)3 in migration and invasion of colorectal cancer(CRC)cells.METHODS:We detected PBX3 expression in five cell lines and surgical specimens from 111 patients with CRC using real-time reverse transcription-polymerase chain reaction.We forced expression of PBX3 in low metastatic HT-29 and SW480 cells and knocked down expression of PBX3 in highly metastatic LOVO and HCT-8 cells.Wound healing and Boyden chamber assays were used to detect cell migration and invasionafter altered expression of PBX3.Western blot was performed to detect the change of signaling molecule ERK1/2 following PBX3 overexpression.RESULTS:High level of PBX3 expression was correlated with the invasive potential of CRC cells,and significantly associated with lymph node invasion(P=0.02),distant metastasis(P=0.04),advanced TNM stage(P=0.03)and poor overall survival of patients(P<0.05).Ectopic expression of PBX3 in low metastatic cells was shown to promote migration and invasion,while inhibited PBX3 expression in highly metastatic cells suppressed migration and invasion.Furthermore,upregulation of phosphorylated extracellular signal-regulated kinase(ERK)1/2 was found to be one of the targeted molecules responsible for PBX3-induced CRC cell migration and invasion.CONCLUSION:PBX3 induces invasion and metastasis of CRC cells partially through activation of the MAPK/ERK signaling pathway.

Synthetic lethal short hairpin RNA screening reveals that ring finger protein 183 confers resistance to trametinib in colorectal cancer cells

Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells,using a synthetic lethal short hairpin RNA(shRNA) screening approach.Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183(RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF 183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA(siRNA) was used to knock down RNF183 transiently.Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay(ELISA) were used to evaluate the protein abundance. MTT assay,colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8(IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.

槲皮素调控p38 MAPK/NF-κB/NLRP3信号通路对大鼠分泌性中耳炎的作用机制

目的 探究槲皮素(quercetin, Que)对大鼠分泌性中耳炎(secretory otitis media, SOM)的作用及机制。方法 选取雄性SD大鼠48只,随机性分为对照组(Control)、模型组(Model)、Que组(100 mg·kg^(-1))、Que+p38 MAPK激动剂组(100 mg·kg^(-1)Que+2 mg·kg^(-1)Anisomycin),每组各12只。除Control组外,其余各组大鼠均采取腹腔注射卵清蛋白复制分泌性中耳炎大鼠模型,造模成功后,各组大鼠进行相应干预21 d。给药干预结束后,苏木精-伊红(HE)染色观察各组大鼠中耳组织形态学变化;酶联免疫吸附法(ELISA)测定血清TNF-α、IL-6、IL-1β、IL-18及中耳灌洗液中IFN-γ、IL-4水平;实时定量PCR (RT-qPCR)和Western blot检测中耳黏膜组织p38 MAPK/NF-κB/NLRP3信号通路相关mRNA及蛋白表达变化。结果 与Control组相比,Model组大鼠表现中耳内黏膜层增厚,伴有大量炎性细胞浸润,血清炎性因子TNF-α、IL-6、IL-1β、IL-18及中耳灌洗液中IL-4增加,IFN-γ降低(P <0.01),中耳黏膜组织p-p38MAPK/MAPK、p-NF-κB/NF-κB、NLRP3、ASC、pro-caspase-1、cleaved-caspase-1、IL-1β、IL-18蛋白及mRNA表达水平均明显升高(P <0.01)。与model组相比,Que能够明显改善中耳内黏膜增厚,抑制炎性细胞浸润,降低TNF-α、IL-6、IL-1β、IL-18、IL-4及中耳黏膜组织p38 MAPK/NF-κB/NLRP3通路mRNA和蛋白表达,升高IFN-γ(P <0.05或P <0.01)。与Que组相比,Anisomycin能够逆转Que对SOM大鼠的保护作用。结论 Que能够有效改善SOM发生、发展,其机制与抑制p38 MAPK/NF-κB/NLRP3信号通路介导的炎性反应相关。

基于ERK/P38MAPK信号通路研究苗药金乌健骨方防治奥沙利铂诱导的神经病理性疼痛的作用机制

目的基于细胞外调节蛋白激酶/丝裂原活化蛋白激酶p38(extracellular regulated kinase/p38 mitogen-activated kinaseERK/p38MAPK)信号通路探究苗药金乌健骨方防治奥沙利铂(oxaliplatin,OXA)诱导的神经病理性疼痛(chemotherapy-induced neuropathic pain,CINP)的作用机制。方法SPF级雌性SD大鼠40只,随机分为对照组、模型组、西药组、金乌健骨方组,每组10只。除对照组外,其余大鼠腹腔注射OXA 2 mg·kg^(-1)·d^(-1),连续给药5 d,建模慢性CINP模型。建模的同时,金乌健骨方组灌胃给药金乌健骨方24 g·kg^(-1)·d^(-1),西药组灌胃给药度洛西汀6.45 mg·kg^(-1)·d^(-1),对照组与模型组给予等量的生理盐水,连续给药15 d。分别于给药0、3、6、9、12、15 d检测大鼠机械痛阈值、热痛阈值,治疗结束后检测血清肿瘤坏死因子α(tumor necrosis factor-α,TNF-α),白细胞介素^(-1)β(interleukin^(-1)β,IL^(-1)β),白细胞介素-6(interleukin-6,IL-6)水平,并对脊髓L4-L5膨大神经组织进行病理检测,比较大鼠脊髓L4-L5背根神经节组织p-ERK1/2、p38MAPK、c-Jun氨基末端激酶(c-Jun N-terminal ki-nase,JNK)、细胞癌基因fos(Cellular oncogene fos,c-Fos)、环磷酸腺苷反应原件结合蛋白(cAMP response element binding protein,CREB)、核蛋白因子-κB(nucleoprotein factor-κB,Nf-κB)蛋白表达情况。结果对照组0~15 d机械痛阈值变化差异无统计学意义(P>0.05),第3天后,模型组械痛阈值开始下降,第9天后,模型组与金乌健骨方组、西药组机械痛阈值差异有统计学意义,金乌健骨方组、西药组机械痛阈值高于模型组,西药组高于金乌健骨方组(P<0.05);对照组0~15 d热痛阈值变化差异无统计学意义(P>0.05),模型组热痛阈值下降,第0天后,模型组与金乌健骨方组、西药组热痛阈值差异有统计学意义,金乌健骨方组、西药组热痛阈值高于模型组,西药组高于金乌健骨方组(P<0.05)。与对照组比较,模型组血清IL^(-1)β,IL-6及TNF-α水平升高,p-ERK1/2、p38MAPK、JNK、c-Fos、CREB、Nf-κB蛋白表达上调(P<0.05),与模型组比较,金乌健骨方组、西药组血清IL^(-1)β,IL-6及TNF-α水平降低,p-ERK1/2、p38MAPK、JNK、c-Fos、CREB、Nf-κB蛋白表达下调(P<0.05),与金乌健骨方组比较,西药组血清IL^(-1)β,IL-6及TNF-α水平降低,p-ERK1/2、p38MAPK、JNK、c-Fos、CREB、Nf-κB蛋白表达下调(P<0.05)。结论苗药金乌健骨方能够改善OXA诱导的神经病理性疼痛,其机制可能与调节ERK/p38MAPK通路有关。

姜黄素通过p38 MAPK/NLRP3抑制肠道病毒71型诱导的细胞焦亡

肠道病毒-A71型(EV-A71)重症感染患儿多表现为过激的炎症反应,病毒感染引起的细胞焦亡可能是机体炎症发生的重要原因之一。本文旨在探究姜黄素对EV-A71病毒感染引起的细胞焦亡与细胞损伤的保护作用及可能的机制。首先,观察了姜黄素对EV-A71引起的细胞毒性的影响。CCK8检测结果显示,EV-A71感染降低细胞的增殖活力;LDH测定表明,病毒增加细胞培养上清中LDH的释放,造成了细胞的损伤;DAPI核染色及Dil细胞膜染色后观察到,EV-A71感染引起了细胞的形态变化和数量减少。姜黄素可以逆转病毒引起的上述变化,提示姜黄素对病毒感染的细胞毒性具有保护作用。细胞焦亡发生时,可促进炎症因子IL-1β的成熟、产生和释放。我们观察了EV-A71及姜黄素干预对细胞IL-1β产生的影响。Western印迹结果显示,病毒感染细胞内IL-1β的活化增加。ELISA检测结果显示,EV-A71病毒感染引起细胞上清中IL-1β的分泌水平增加;qPCR测定结果显示,EV-A71病毒感染细胞中IL-1β的转录水平上调;而姜黄素干预可抑制染毒细胞IL-1β的活化和分泌。Western印迹检测细胞内焦亡相关分子的变化。结果显示,EV-A71感染可诱导细胞发生焦亡,并呈时间和剂量依赖性,分子NOD样受体热蛋白结构域相关蛋白3(NLRP3)、GSDMD、胱天蛋白酶1(caspase-1)参与EV-A71诱导细胞发生的焦亡;姜黄素干预可以有效抑制EV-A71诱导的细胞细胞焦亡。另外,Western印迹检测显示,姜黄素可以减少细胞内EV-A71结构蛋白VP1的水平,通过检测细胞上清中病毒的CCID50发现,姜黄素可以降低细胞上清中病毒的滴度。最后,对姜黄素抗焦亡机制进行了探索,发现EV-A71感染诱导了细胞自噬并激活了p38/NLRP3通路,姜黄素能抑制自噬标志蛋白LC3及自噬底物p62的降解,并抑制p38/NLRP3的激活。总之,本研究明确了姜黄素通过对自噬溶酶体阶段及p38/NLRP3通路的抑制,有效减轻了EV-A71诱导的细胞焦亡,为姜黄素在抗EV-A71感染的临床应用提供参考。

人β防御素3和γ干扰素在MAPK信号通路中的相互诱导及联合抗甲型流感病毒的机制

目的 探讨人β防御素3(HBD3)和γ干扰素(IFN-γ)在丝裂源活化蛋白激酶(MAPK)信号通路中相互诱导作用及体外联合抗甲型流感病毒(IAV)H1N1的作用。方法 不同剂量IFN-γ(12.5、25.0、50.0、100.0μg/L)或HBD3(0.25、0.50、1.00、2.00 mg/L)分别作用人支气管上皮细胞BEAS-2B 4、12、24、48 h,另设立p38 MAPK、ERK和JNK通路抑制剂预处理后、用IFN-γ(25.0μg/L)或HBD3(1.00 mg/L)处理BEAS-2B细胞4 h, qRT-PCR和ELISA检测HBD3及IFN-γ基因和蛋白表达,Western blot检测p-p38 MAPK、p-ERK、p-JNK蛋白表达;25.0μg/L IFN-γ、1.00 mg/L HBD3、HBD3 siRNA转染及IFN-γ中和抗体(0.50 mg/L)单独或联合作用BEAS-2B细胞、用5×TCID50H1N1感染,qRT-PCR和Western blot检测IAV核蛋白(NP)基因和蛋白的表达情况。结果 在BEAS-2B细胞中,不同剂量的IFN-γ与HBD3可互相诱导表达(P<0.05或P<0.01),且均可诱导p-p38 MAPK、p-ERK和p-JNK的表达上调(P<0.01);p38 MAPK、ERK和JNK通路抑制剂预处理,均明显下调IFN-γ诱导的HBD3表达、及HBD3诱导的IFN-γ表达(P<0.01);IFN-γ和HBD3单独或联合使用均可明显抑制BEAS-2B细胞中H1N1 NP的表达(P<0.05或P<0.01),转染HBD3 siRNA减弱了IFN-γ抑制H1N1 NP表达的作用(P<0.01),IFN-γ中和抗体介入对HBD3抑制H1N1 NP表达的作用无显著影响(P>0.05)。结论 在BEAS-2B细胞中,HBD3和IFN-γ可通过MAPK通路相互诱导表达,且2者联合使用可增强抗IAV H1N1的作用。

Roles of extra-cellular signal-regulated protein kinase 5 signaling pathway in the development of spinal cord injury

Background:In consideration of characteristics and functions,extra-cellular signal-regulated protein kinase 5(ERK5)signaling pathway could be a new target for spinal cord injury(SCI)treatment.Our study aimed to evaluate the roles of ERK5 signaling pathway in secondary damage of SCI.Methods:We randomly divided 70 healthy Wistar rats into five groups:ten in the blank group,15 in the sham surgery+BIX02188(sham+B)group,15 in the sham surgery+dimethyl sulfoxide(DMSO;sham+D)group,15 in the SCI+BIX02188(SCI+B)group,and 15 in the SCI+DMSO(SCI+D)group.BIX02188 is a specific inhibitor of the ERK5 signaling pathway.SCI was induced by the application of vascular clips(with the force of 30 g)to the dura on T10 level,while rats in the sham surgery group underwent only T9-T11 laminectomy.BIX02188 or DMSO was intra-thecally injected at 1,6,and 12 h after surgery or SCI.Spinal cord samples were taken for testing at 24 h after surgery or SCI.Results:Expression of phosphorylated-ERK5(p-ERK5)significantly increased after SCI.Application of BIX02188 indeed inhibited ERK5 signaling pathway and reduced the degree of spinal cord tissue injury,neutrophil infiltration and proinflammatory cytokine expression,nuclear factor-kB(NF-kB)activation and apoptosis(measured by TdT-mediated 20-deoxyuridine 50-triphosphate nickend labeling,expression of Fas-ligand,BCL2-associated X[Bax],and B-cell lymphoma-2[Bcl-2]).Double immunofluorescence revealed activation of ERK5 in neurons and microglia after SCI.Conclusion:ERK5 signaling pathway was activated in spinal neurons and microglia,contributing to secondary injury of SCI.Moreover,inhibition of ERK5 signaling pathway could alleviate the degree of SCI,which might be related to its regulation of infiltration of inflammatory cells and release of inflammatory cytokines,expression of NF-kB and cell apoptosis.

扇贝多肽抑制紫外线A波诱导的HaCaT细胞凋亡依赖p38 MAPK通路和caspase-3

目的从p38促细胞分裂剂激活性蛋白激酶(p38MAPK)通路和半胱天冬酶-3(caspase-3)的角度,研究扇贝多肽(poly-peptidefromChlamysfarreri,PCF)抑制紫外线A波(UVA)引起的HaCaT细胞凋亡的分子机制。方法实验分为6组:对照组、UVA模型组、UVA+5.68mmol.L-1维生素C阳性对照组、UVA+5.69mmol.L-1PCF组、UVA+2.84mmol.L-1PCF组、UVA+1.42mmol.L-1PCF组。以正交实验设计确立UVA诱导HaCaT细胞凋亡模型;琼脂糖凝胶电泳分析PCF、p38MAPK抑制剂(SB203580)及caspase-3特异性抑制剂(Ac-DEVD-CHO)对细胞凋亡的影响;蛋白质印迹法检测p38MAPK及磷酸化p38MAPK表达;流式细胞术检测caspase-3的活性。结果PCF能明显抑制UVA引起的HaCaT细胞凋亡;SB203580和Ac-DEVD-CHO对UVA诱导的HaCaT细胞凋亡有抑制作用;1.425.69mmol.L-1内的PCF可剂量依赖性抑制UVA引起的p38MAPK磷酸化及caspase-3的活化。结论PCF可抑制UVA诱导的HaCaT细胞凋亡,其作用机制与抑制p38MAPK通路和caspase-3活性有关。

二甲双胍调节MEK/ERK通路及基质金属蛋白酶在恶性肿瘤中应用的研究进展

二甲双胍是目前治疗2型糖尿病的一线用药,其通过改善胰岛素抵抗、抑制肝脏糖异生等途径降低血糖。近年发现,二甲双胍还可通过促分裂原活化的蛋白激酶激酶/胞外信号调节激酶信号通路以及基质金属蛋白酶诱导细胞周期停滞、促进细胞凋亡、抑制细胞侵袭迁移,并可与各种抗肿瘤药物联用发挥协同作用以抑制肿瘤进展。但目前仍缺乏二甲双胍可以抑制癌症患者病情进展的研究证据,未来还需要对二甲双胍在恶性肿瘤治疗中的作用及其有效性、安全性进行进一步研究。

芪丹通脉片对急性缺血再灌注大鼠心肌信号转导系统PI3K/Akt和Erk1/2的影响

目的观察中药芪丹通脉片对缺血再灌注大鼠心肌梗死面积、细胞凋亡和细胞信号转导系统中磷脂酰肌醇-3激酶(PI3K)、丝(苏)氨酸激酶(Akt)和细胞外信号调节激酶(Erk1/2)的影响。方法雄性SD大鼠随机分为假手术组、缺血再灌注组(模型组)和芪丹通脉片预处理组。生理盐水或芪丹通脉片灌胃7 d后,大鼠麻醉开胸,结扎冠状动脉前降支40 min,再灌注4 h。伊文氏兰和TTC染色测定心肌梗死范围,计算梗死面积(IS)与缺血区面积(AAR)的比值(%,IS/AAR);TUNEL方法定性和定量检测心肌细胞凋亡指数;Westernblot测定信号蛋白的表达。结果芪丹通脉片组的心肌梗死范围明显小于模型组[(28.8±8.4)%vs.(49.1±10.3)%,P<0.01];其心肌细胞凋亡指数亦显著低于模型组[(11.6±2.3)%vs.(20.3±4.5)%,P<0.01];而其Akt和Erk1/2的磷酸化表达水平增加,与模型组比较分别增加了2.5倍和1.9倍(P<0.01,P<0.05)。结论芪丹通脉片能够抑制缺血再灌注所致的心肌细胞损伤,并影响生存信号通路PI3K/Akt和Erk1/2的活化水平。