Effects of invigorating-spleen and anticancer prescription on extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway in colon cancer mice model

BACKGROUND Colon cancer(CC)is one of the most common malignant tumors in the gastrointestinal system.Overall,CC had the third highest incidence but the second highest mortality rate globally in 2020.Nowadays,CC is mainly treated with capecitabine chemotherapy regimen,supplemented by radiotherapy,immunotherapy and targeted therapy,but there are still limitations,so Chinese medicine plays an important role.AIM To investigate the effects of invigorating-spleen and anticancer prescription(ISAP)on body weight,tumor inhibition rate and expression levels of proteins in extracellular-signal-regulated kinase(ERK)/mitogen-activated protein kinase(MAPK)signaling pathway in CC mice model.METHODS The CC mice model were established and the mice were randomly divided into 5 groups,including the control group,capecitabine group,the low-dose,mediumdose and high-dose groups of ISAP,with 8 mice in each group,respectively.After 2 weeks of intervention,the body weight and tumor inhibition rate of mice were observed,and the expression of RAS,ERK,phosphorylated ERK(p-ERK),C-MYC and matrix metalloproteinase 2(MMP2)proteins in the tissues of tumors were detected.RESULTS Compared with the control group,the differences of body weight before and after treatment was much smaller in the groups of ISAP,with the smallest difference in the high-dose group of ISAP,while the capecitabine group had the greatest difference,indicating ISAP had a significant inhibiting effect on the growth of transplanted tumor in mice.The expression of RAS protein was decreased in the low-and medium-dose groups of ISAP,and the change of p-ERK was significant in the medium-and high-dose groups of ISAP.MMP2 protein expression was significantly decreased in both the low-dose and medium-dose groups of ISAP.There were no significant changes in ERK in the ISAP group compared to the capecitabine group,while RAS,MMP2,and C-MYC protein expression were reduced in the ISAP group.The expression level of C-MYC protein decreased after treated with ISAP,and the decrease was the most significant in the medium-dose group of ISAP.CONCLUSION ISAP has a potential inhibiting effect on transplanted tumor in mice,and could maintain the general conditions,physical strength and body weight of mice.The expression levels of RAS,p-ERK,MMP2 and c-myc were also decreased to a certain extent.By inhibiting the expression of upstream proteins,the expression levels of downstream proteins in ERK/MAPK signaling pathway were significantly decreased.Therefore,it can be concluded that ISAP may exert an anti-tumor effect by blocking the ERK/MAPK signaling pathway and inhibiting the expression of MMP2 and c-myc proteins.

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.

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.

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.

Insulin-like growth factor binding protein-5 influences pancreatic cancer cell growth

AIM： To investigate the functional significance of insulin-like growth factor binding protein-5 （IGFBP-5） overexpression in pancreatic cancer （PaC）.METHODS： The effects of IGFBP-5 on cell growth were assessed by stable transfection of BxPC-3 and PANC-1 cell lines and measuring cell number and DNA synthesis. Alterations in the cell cycle were assessed by flow cytometry and immunoblot analyses. Changes in cell survival and signal transduction were evaluated after mitogen and phosphatidylinositol activated protein kinase 3-kinase （PI3K） inhibitor treatment.RESULTS： After serum deprivation, IGFBP-5 expression increased both cell number and DNA synthesis in BxPC-3 cells, but reduced cell number in PANC-1 cells. Consistent with this observation, cell cycle analysis of IGFBP-5-expressing cells revealed accelerated cell cycle progression in BxPC-3 and G2/M arrest of PANC-1 cells. Signal transduction analysis revealed that Akt activation was increased in BxPC-3, but reduced in PANC-1 cells that express IGFBP-5. Inhibition of PI3K with LY294002 suppressed extracellular signal-regulated kinase-1 and -2 （ERK1/2） activation in BxPC-3, but enhanced ERK1/2 activation in PANC-1 cells that express IGFBP-5. When MEK1/2 was blocked, Akt activation remained elevated in IGFBP-5 expressing PaC cells; however, inhibition of PI3K or MEK1/2 abrogated IGFBP-5-mediated cell survival.CONCLUSION： These results indicate that IGFBP-5 expression affects the cell cycle and survival signal pathways and thus it may be an important mediator of PaC cell growth.

Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injury

Cold-inducible RNA-binding protein（CIRP）, a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5℃ on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP m RNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury.

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.

Cross-Talk between Extracellular S1P/S1P2 and P42/44 MAPK in bcr/abl Positive Chronic Myeloid Leukemia Cells

Objective： BCR/ABL oncoprotein-expression is associated with uncontrolled cell growth. Sphingosine kinase 1 （SPK1） regulates the production of sphingosine 1-phosphate （S1P）, a key lipid signal molecular in cell proliferation and survival. The objective of this study was to elucidate the roles of S1P and its receptors in bcr/abl positive chronic myeloid leukemia （CML） cells. Methods： The expressions of SIP receptors： S1P1, S1P2 and S1P3 in CML cells were detected by RT-PCR. SPK1 expression, activity and extracellular S1P were determined in ECV304 and HL-60 cells which were transfected with bcr/abl gene. To elucidate the relationship between the BCR/ABL, ERK/MAPK （extracellular signal-regulated kinase/mitogen-activated protein kinase）, SPK/S 1P and S 1P/S 1 P2 signal pathways, bcr/abl positive CML cell line K562 was treated with STI571, PD98059, N,N-dimethyl sphingosine （DMS） and JTE-013. Results： Retrovirus-mediated overexpression of bcr/abl gene in ECV304 and HL-60 cells resulted in upregulation of the expression, activity of SPK1 and increase of the secretion of SIP, whereas treatment of STI571 and PD98059 decreased the BCR/ABL-induced S1P secretion. Treatment of DMS reduced S1P secretion and P42/44MAPK phosphorylation. S1P2-selective antagonist JTE-013 could also decrease P42/44MAPK phosphorylation. Conclusion： These results suggest that BCR/ABL up-regulates extracellular sphingosine 1-phosphate through sphingosine kinase 1 and there is cross-talk between SPK1/S1P/S1P2 and P42/44MAPK in bcr/abl positive CML cells.

不同级别胶质瘤患者MEK、ERK、CHKα蛋白的阳性表达情况及临床意义

目的探讨不同级别胶质瘤患者胆碱激酶α(CHKα)、细胞外信号调节激酶(ERK)及丝裂原激活蛋白激酶(MEK)蛋白的阳性表达情况及临床意义。方法回顾性分析2019年1月至2022年12月长沙市第四医院神经外科收治的124例胶质瘤患者的临床资料。检测、统计不同级别胶质瘤患者MEK、ERK、CHKα蛋白的阳性表达情况。结果不同肿瘤最大径、年龄、WHO分级及预后胶质瘤患者MEK、ERE、CHKα蛋白的高、低表达情况比较,差异均有统计学意义(P<0.05)。Ⅰ、Ⅱ、Ⅲ、Ⅳ级胶质瘤患者MEK蛋白阳性表达率分别为9.62%、7.69%、94.08%和92.04%,ERK蛋白阳性表达率分别为9.57%、7.49%、94.52%和92.18%,CHKα蛋白阳性表达率分别为9.60%、7.53%、94.71%和92.23%,高、低级别胶质瘤患者MEK、ERK、CHKα蛋白阳性表达率比较,差异均有统计学意义(P<0.05)。随访结束后结果显示,124例患者中死亡52例,失访12例,获得随访112例,随访率为90.32%。结论不同级别胶质瘤患者MEK、ERK、CHKα蛋白的阳性表达情况有明显区别,并且与患者的疾病预后明显相关,对疾病的诊断和预后判断具有重要价值。

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。

车叶草苷调节RAS/RAF/MEK/ERK信号通路对肝细胞癌细胞活性及裸鼠移植瘤生长的影响

目的探讨车叶草苷调节Ras蛋白(Ras protein,RAS)/Raf蛋白激酶(Raf protein kinase,RAF)/丝裂原激活蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号通路对肝细胞癌(hepatocellular carcinoma,HCC)细胞活性及裸鼠移植瘤生长的影响。方法体外培养人HCC-LM3以细胞计数试剂盒(cell counting kit 8,CCK-8)法筛选车叶草苷体外最佳作用浓度。将HCC-LM3细胞随机分为对照组(细胞正常培养,不作其他干预)、车叶草苷组(3 mmol/L车叶草苷进行干预)、ML-098组(0.5μmol/L RAS激活剂ML-098进行干预)、车叶草苷+ML-098组(车叶草苷3 mmol/L和RAS激活剂ML-098终浓度0.5μmol/L联合干预)。以CCK-8法、流式细胞术分别检测各组HCC-LM3细胞活性、凋亡率;Western blot检测各组HCC-LM3细胞增殖、凋亡相关蛋白表达及RAS/RAF/MEK/ERK信号通路相关蛋白表达。制备HCC-LM3裸鼠原位癌模型并随机分为对照组(5 ml/kg生理盐水)、车叶草苷低剂量组(25 mg/ml车叶草苷)、车叶草苷中剂量组(50 mg/ml车叶草苷)、车叶草苷高剂量组(100 mg/ml车叶草苷)。检测各组裸鼠肝体比、肿瘤长径。以Ki67免疫组织化学染色法检测各组裸鼠肿瘤细胞增殖指数;Western blot检测各组裸鼠肿瘤RAS/RAF/MEK/ERK信号通路相关蛋白表达。结果与对照组相比,车叶草苷组HCC-LM3细胞凋亡率、裂解凋亡蛋白酶3(Cleaved caspase-3)与B细胞淋巴瘤2相关X蛋白(Bcl-2 associated X protein,Bax)、含半胱氨酸的天冬氨酸蛋白水解酶9(cysteinyl aspartate specific proteinase 9,caspase-9)蛋白表达均升高(P均<0.05),细胞活性、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)与RAS蛋白表达、p-RAF/RAF、p-MEK/MEK、p-ERK/ERK均降低(P均<0.05);ML-098对HCC-LM3细胞各指标的作用与车叶草苷相反(P<0.05)。与车叶草苷组相比,车叶草苷+ML-098组HCC-LM3细胞凋亡率、Cleaved caspase-3与Bax、caspase-9蛋白表达均降低(P均<0.05),细胞活性、PCNA与RAS蛋白表达、p-RAF/RAF、p-MEK/MEK、p-ERK/ERK均升高(P均<0.05)。与对照组相比,车叶草苷低、中、高剂量组裸鼠肝体比、肿瘤长径、肿瘤细胞增殖指数、RAS蛋白表达、p-RAF/RAF、p-MEK/MEK、p-ERK/ERK均降低(P均<0.05),并呈一定剂量依赖性(P均<0.05)。结论车叶草苷可抑制RAS/RAF/MEK/ERK信号通路激活,降低HCC细胞体外细胞活性并促使其凋亡,延缓其裸鼠移植瘤生长。

基于EGFR/MAPK/ERK信号通路探讨鳖甲煎丸对MHCC-97H肝癌细胞皮下瘤的抑瘤作用

目的基于表皮生长因子受体(epidermal growth factior receptor,EGFR)/丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号通路探究鳖甲煎丸对MHCC-97H肝癌细胞皮下瘤的抑瘤作用及作用机制。方法选取30只雄性BLAB/c裸鼠,建立MHCC-97H肝癌细胞皮下瘤模型。造模成功后随机分为模型组,鳖甲煎丸低、中、高剂量组(0.55、1.1、2.2 g/kg),西药组(乐伐替尼4 mg/kg+吉非替尼80 mg/kg),每组6只。鳖甲煎丸低、中、高剂量组灌胃2次/d,西药组每周灌胃5 d,模型组予以等量生理盐水2次/d灌胃,每组连续干预2周。观察大鼠一般情况;计算各组大鼠抑瘤率;HE染色观察病理形态学变化;RT-qPCR检测瘤体组织中EGFR、丝裂原活化蛋白质激酶激酶(mitogen-activated protein kinase kinase,MEK)、ERK1、ERK2 mRNA表达水平;Western blot检测EGFR、磷酸化的EGFR(p-EGFR)、MEK、磷酸化的MEK(p-MEK)、ERK1、ERK2、磷酸化的ERK1/2(p-ERK1/2)、基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)、细胞周期蛋白D1(cell cycle protein D1,Cyclin D1)、神经型钙黏附蛋白(N-cadherin)、上皮型钙黏附蛋白(E-cadherin)相对表达水平。结果与模型组比较,鳖甲煎丸低、中、高剂量组及西药组精神、反应、进食饮水等情况均明显改善。与第0天比较,各组第14天体质量明显降低(P<0.01)。与模型组、鳖甲煎丸低剂量组比较,鳖甲煎丸中、高剂量组和西药组瘤体质量减轻(P<0.05,P<0.01)。鳖甲煎丸低、中、高剂量组和西药组抑瘤率分别为20%、47.73%、55.91%、75.45%。与模型组比较,鳖甲煎丸低、中、高剂量组及西药组肿瘤细胞排列疏松,边界模糊,细胞核固缩、破裂,其中西药组最明显。与模型组比较,鳖甲煎丸低、中、高剂量组和西药组EGFR、MEK、ERK1、ERK2 mRNA表达水平明显下降(P<0.01);与鳖甲煎丸低剂量组比较,鳖甲煎丸高剂量组和西药组EGFR、MEK、ERK1、ERK2 mRNA表达水平显著降低(P<0.01),鳖甲煎丸中剂量组ERK1 mRNA表达水平显著降低(P<0.01);与鳖甲煎丸中剂量组比较,西药组EGFR、ERK2 mRNA表达水平降低(P<0.05,P<0.01)。与模型组比较,鳖甲煎丸中、高剂量组和西药组p-EGFR/EGFR、p-MEK/MEK、p-ERK1/ERK1、p-ERK2/ERK2、MMP-1、Cyclin D1、N-cadherin蛋白相对表达水平下降(P<0.05,P<0.01),E-cadherin蛋白相对表达水平明显升高(P<0.01)。与鳖甲煎丸高剂量组比较,西药组p-EGFR/EGFR、p-ERK1/ERK1、MMP-1、Cyclin D1、N-cadherin蛋白相对表达水平明显下降(P<0.01),E-cadherin蛋白相对表达水平升高(P<0.05)。结论鳖甲煎丸可能通过抑制EGFR/MAPK/ERK信号通路激活,从而下调MMP-1、Cyclin D1、N-cadherin蛋白,上调E-cadherin蛋白表达,进而对MHCC-97H肝癌细胞皮下瘤产生显著的抑制作用。

澳洲茄碱对肺癌A549细胞生物活性、瘤体抑制及MAPK/ERK1/2的影响

目的澳洲茄碱对肺癌A549细胞生物活性、瘤体抑制及MAPK/ERK1/2信号的影响。方法将人肺癌细胞株A549分为空白组、低剂量澳洲茄碱组、中剂量澳洲茄碱组、高剂量澳洲茄碱组及顺铂组,分别加入浓度为0、15、20、25μmol/L的澳洲茄碱及2μg/mL顺铂培养。MTT检测活性;流式细胞仪检测凋亡率;Transwell法检测侵袭;划痕试验检测划痕愈合率。25只裸鼠分为空白组、低剂量澳洲茄碱组、中剂量澳洲茄碱组、高剂量澳洲茄碱组及顺铂组,皮下注射0.3 mL的0、15、20、25μmol/L的澳洲茄碱及2μg/mL顺铂共培养A549细胞,观察瘤体质量及抑瘤率。结果澳洲茄碱组降低A549细胞活性,凋亡率升高,侵袭、划痕愈合率、MAPK、p-MAPK、ERK1/2及p-ERK1/2蛋白水平降低(P<0.05)。结论澳洲茄碱可抑制肺癌细胞增殖、侵袭及转移,并加快凋亡,作用呈现剂量依赖性且高剂量效果最佳,研究机制可能与抑制MAPK/ERK1/2信号表达相关。

中药痛消饮治疗寒凝血瘀证原发性痛经效果及对MAPK/ERK信号通路的影响

目的探讨中药痛消饮治疗寒凝血瘀证原发性痛经的效果及对丝裂原活化蛋白激酶/细胞外调节蛋白激酶(mitogen activated protein kinase/extracellular regulated protein kinase,MAPK/ERK)信号通路的影响。方法选取衡水市中医医院106例寒凝血瘀证原发性痛经患者,按照随机数字表法分为中药组和常规组,各53例。常规组给予布洛芬颗粒治疗,中药组给予中药痛消饮治疗,2组均于月经预来潮前7 d开始服药至月经来3 d后停服,共服用3个月经周期。比较2组疗效、治疗前后COX痛经症状量表(Cox dysmenorrhea symptom scale,CMSS)评分、疼痛视觉模拟评分(visual analog score,VAS)、血清疼痛介质[β-内啡肽(β-endorphins,β-EP)、前列腺素F2α(prostaglandin F2α,PGF2α)、前列腺素E 2(prostaglandin E 2,PGE 2)]水平、外周血单个核细胞MAPK/ERK信号通路相关因子[丝裂原活化细胞外调节激酶(mitogen activated extracellular regulated kinase,MEK)1、MEK2、ERK1、ERK2 mRNA]水平、子宫微循环状态[子宫动脉血流阻力指数(resistance index,RI)、搏动指数(pulsatility index,PI)、收缩期峰值/舒张期峰值(systolic peak/diastolic peak,S/D)]及不良反应发生率。结果中药组总有效率高于常规组(P<0.05)。2组治疗后VAS评分、CMSS量表持续时间和严重程度评分低于治疗前,且中药组低于常规组(P<0.05);2组治疗后血清β-EP、PGE 2水平高于治疗前,且中药组高于常规组(P<0.05);2组治疗后PGF2α水平,外周血单个核细胞MEK1、MEK2、ERK1、ERK2 mRNA相对表达量,子宫动脉PI、RI、S/D低于治疗前,且中药组低于常规组(P<0.05)。2组不良反应发生率比较差异无统计学意义(P>0.05)。结论中药痛消饮治疗寒凝血瘀证原发性痛经疗效显著,能通过抑制MAPK/ERK信号通路相关因子和疼痛介质表达显著减轻痛经症状,还可改善子宫微循环状态,且安全性良好。

补肾益气化瘀冲剂促进骨质疏松大鼠骨折术后骨痂血管形成的作用机制

背景:补肾益气化瘀冲剂具有益气化瘀、补肾通络的功效,常用于治疗骨质疏松,目前关于补肾益气化瘀冲剂对骨质疏松性骨折骨痂血管形成的影响研究较少。目的:探究补肾益气化瘀冲剂上调细胞外调节蛋白激酶/丝裂原活化蛋白激酶(extracellular signal-regulated kinase/mitogen-activated protein kinases,ERK/MAPK)信号通路改善骨质疏松大鼠骨折术后骨痂血管形成的具体机制。方法:①收集骨质疏松SD大鼠骨髓间充质干细胞、C57BL/6小鼠骨髓单核细胞,MTT法检测不同剂量补肾益气化瘀冲剂对骨髓间充质干细胞的毒性。分别使用添加0,1.5 mg/mL补肾益气化瘀冲剂的培养基培养骨髓间充质干细胞、骨髓单核细胞,进行体外成骨分化、破骨分化实验。②144只SD大鼠随机分为假手术组、模型组、冲剂组、冲剂+PD98059组,每组36只,后3组切除双侧卵巢建立骨质疏松模型,假手术组仅切除卵巢附近部分脂肪组织。8周后所有大鼠接受左侧胫骨横断截骨,冲剂+PD98059组给予5 g/kg补肾益气化瘀冲剂灌胃,尾静脉注射0.3 mg/kg PD98059(ERK1/2抑制剂);冲剂组给予5 g/kg补肾益气化瘀冲剂灌胃,尾静脉注射等体积生理盐水;假手术组、模型组给予等体积生理盐水,尾静脉注射等体积生理盐水;每日给药1次,持续8周。X射线、微型计算机断层扫描、微血管灌注造影观察骨折愈合与骨痂血管生成情况,检测骨痂骨密度与机械强度,苏木精-伊红、Masson染色观察胫骨组织形态,Western blotting、免疫组化检测ERK/MAPK信号通路相关分子表达情况。结果与结论:①与0 mg/mL补肾益气化瘀冲剂组相比,1.5 mg/mL补肾益气化瘀冲剂组成骨细胞碱性磷酸酶活性、矿化水平、p-ERK1/2、p-p38 MAPK表达水平升高(P<0.05),破骨细胞密度、体积减小(P<0.05);②与假手术组相比,冲剂组大鼠第4,8周骨折愈合程度相近,Lane-Sandhu评分、愈伤组织总体积、矿化愈伤组织体积、矿化愈伤组织体积/愈伤组织总体积、骨小梁厚度、血管数量、间距、厚度、血管体积/总体积、骨密度、骨痂极限荷载、刚度、p-ERK1/2、p-p38 MAPK、p-IκB-α、p-p65表达水平差异无显著性意义(P>0.05),模型组、冲剂+PD98059组大鼠骨折愈合缓慢,Lane-Sandhu评分、愈伤组织总体积、矿化愈伤组织体积、矿化愈伤组织体积/愈伤组织总体积、骨小梁厚度、血管数量、厚度、血管体积/总体积、骨密度、骨痂极限荷载、刚度、p-ERK1/2、p-p38 MAPK、p-IκB-α、p-p65表达水平降低(P<0.05),血管间距增加(P<0.05);③提示补肾益气化瘀冲剂能够通过增强ERK/MAPK信号通路相关分子表达改善骨质疏松大鼠骨折愈合情况,促进骨痂血管形成,减轻骨质疏松症状。

舒芬太尼调节Ras/Raf/MEK/ERK信号通路对骨关节炎大鼠炎症反应和软骨细胞凋亡的影响

目的:探讨舒芬太尼(Suf)调节Ras/Raf/丝裂原激活蛋白激酶激酶(MEK)/细胞外信号调节激酶(ERK)信号通路对骨关节炎(OA)大鼠炎症反应和软骨细胞凋亡的影响。方法:SD大鼠分为正常对照组(NC组)、空白对照组(Blank组)、低剂量Suf组(Suf-L组,1μg/kg)、高剂量Suf组(Suf-H组,5μg/kg)、双醋瑞因(DC组,54 mg/kg)、Suf-H+ML-099(Ras/Raf/MEK/ERK通路激活剂)组(5μg/kg Suf+11.7 mg/kg ML-099),每组12只。除NC组外,其他组大鼠均采用横断大鼠右膝关节内侧半月板胫骨韧带构建OA模型,成功建模后开始给药,而NC组、Blank组大鼠需腹腔注射,而NC组、Blank组大鼠腹腔注射且灌胃等量生理盐水,每天给药1次,持续给药3周。末次给药24 h后,监测大鼠压痛阈值、热痛阈值变化;ELISA检测血清中TNF-α、IL-1β、IL-6水平;番红O-固绿染色检测软骨组织病理变化;TUNEL染色检测软骨细胞凋亡;Western blot检测软骨组织中Ras/Raf/MEK/ERK信号通路蛋白及基质金属蛋白酶13(MMP-13)蛋白表达。结果:与NC组比较,Blank组大鼠压痛阈值、热痛阈值降低(P<0.05),软骨组织病理损伤严重,TNF-α、IL-1β、IL-6水平、Mankin评分、软骨细胞凋亡率、Ras、Raf、MEK、p-ERK1/2、MMP-13蛋白表达升高(P<0.05);与Blank组比较,Suf-L组、Suf-H组、DC组对应指标变化趋势与上述相反(P<0.05);ML-099减弱了高剂量Suf对OA大鼠的影响。结论:Suf可能通过下调Ras/Raf/MEK/ERK通路抑制OA大鼠炎症和软骨细胞凋亡。

哈蟆油酶解物激活MAPK/ERK信号凋亡途径修复皮质酮诱导HT-22细胞损伤作用

为研究哈蟆油(Oviductus Ranae,OR)对海马神经元细胞保护作用机制,该研究通过皮质酮(Corticosterone,CORT)诱导HT-22细胞损伤模型探究哈蟆油酶解物对小鼠海马神经元细胞(HT-22)细胞损伤模型的保护作用及其作用机制。采用CORT与HT-22细胞共同培养建立HT-22细胞损伤模型,给予哈蟆油酶解物再次孵育,将ERK信号通路抑制剂PD98059加入HT-22细胞中。采用MTT法、Hochest 33258染色、流式细胞术、Western blot技术检测HT-22细胞凋亡情况和相关蛋白表达以及MAPK/ERK信号通路蛋白表达。结果显示高水平CORT能够诱导HT-22细胞损伤,哈蟆油酶解物能够使CORT诱导的HT-22细胞活力提升62.5%~87.5%,凋亡比例降低50%~70%。哈蟆油酶解物能够上调BCL-2、P-ERK1/2蛋白表达,同时下调BAX、Caspase-3、Caspase-9的蛋白表达。上述结果表明,高水平CORT是通过抑制MAPK/ERK信号通路促进凋亡的发生从而诱导HT-22细胞损伤,哈蟆油酶解物能够通过激活MAPK/ERK信号通路调节BLC-2、BAX、Caspase-3、Caspase-9凋亡相关蛋白的表达进而抑制凋亡的发生减轻神经元损伤,该作用机制为哈蟆油发挥抗抑郁作用的关键途径之一。

赤凤迎源针刺法对面神经损伤模型大鼠Ras/MEK/ERK信号通路的影响

目的探讨赤凤迎源针刺法对面神经超微结构、丝裂原活化的细胞外信号调节激酶(mitogen-activated extracellular signal-regulated kinase,MEK)、细胞外信号调节蛋白激酶(extracellular signal-regulated kinase,ERK)及血清KRas蛋白表达的影响。方法将SD大鼠随机分为空白对照组、模型组、常规针刺组、赤凤迎源针刺组,每组8只。除空白对照组大鼠正常饲养外,其他各组大鼠用面神经颊支压榨法诱导面神经损伤模型。造模成功后,予以干预2周。干预结束后,行电镜检查观察大鼠的面神经超微结构;通过HE染色观察面神经组织形态组织学变化;ELISA法检测血清KRas的表达水平;Western blot检测面神经组织MEK、ERK蛋白的表达水平。结果造模后,与空白对照组比较,另外3组大鼠动物行为学评分均明显升高(P<0.01)。针刺2周后,常规针刺组及赤凤迎源针刺组大鼠评分明显低于模型组(P<0.01),且赤凤迎源针刺组评分低于常规针刺组(P<0.01)。针刺2周后,与模型组对比,常规针刺组及赤凤迎源针刺组面神经轴突纤维排列较紧密,内质网较多,KRas蛋白表达明显升高(P<0.05),面神经MEK蛋白、ERK蛋白表达升高(P<0.05),且赤凤迎源针刺组均高于常规针刺组(P<0.05)。结论赤凤迎源针刺法可改善面神经损伤模型大鼠动物行为学,上调KRas、MEK及ERK表达水平,可能与激活Ras/MEK/ERK信号通路有关,从而修复面神经损伤。

蛋白酶体20S亚基β8调控丝裂原活化蛋白激酶激酶/细胞外信号调节激酶通路对肾透明细胞癌细胞增殖、迁移和侵袭的影响

目的探究蛋白酶体20S亚基β8(PSMB8)对肾透明细胞癌(ccRCC)细胞增殖、迁移和侵袭的影响,以及是否通过调控丝裂原活化蛋白激酶激酶(MEK)/细胞外信号调节激酶(ERK)信号通路发挥其作用。方法采用癌症基因组图谱数据库分析ccRCC与正常组织PSMB8 mRNA的表达水平,并通过实时荧光定量PCR、Western blot和免疫组织化学染色等方法进一步检测PSMB8在ccRCC组织和细胞中的表达情况。构建稳定过表达和敲减PSMB8的细胞株,分别采用CCK-8法和平板克隆实验检测细胞的增殖能力,划痕愈合实验和Transwell实验检测细胞迁移和侵袭的能力。对PSMB8共表达基因进行京都基因与基因组百科全书通路富集分析,Western blot检测MEK/ERK通路相关蛋白的磷酸化水平,并加用ERK激动剂C16-PAF处理进行细胞功能学挽救实验。结果与正常组织比较,PSMB8 mRNA和蛋白在ccRCC组织中呈高表达(P均<0.001),且与临床患者的TNM分期显著相关(P<0.001);与阴性对照组比较,过表达PSMB8可以促进786-O、ACHN细胞的增殖(P=0.021,P=0.039)、迁移和侵袭(P均<0.001),敲减PSMB8可以抑制786-O、ACHN细胞的增殖(P=0.022,P=0.005)、迁移和侵袭(P均<0.001);PSMB8共表达基因通路富集分析提示其可能与丝裂原活化蛋白激酶通路相关(P<0.001);敲减PSMB8后786-O和ACHN细胞MEK1/2(P=0.017,P=0.016)、ERK1/2(P=0.010,P=0.040)蛋白磷酸化水平及ERK下游因子c-Myc(P=0.043,P=0.038)、c-Fos(P=0.025,P=0.008)和CyclinD1(P=0.006,P=0.047)转录水平均下调;与ERK激动剂C16-PAF处理组比较,敲减PSMB8+C16-PAF组明显抑制786-O、ACHN细胞的增殖(P=0.003,P=0.002)、迁移和侵袭能力(P均<0.001)。结论PSMB8通过激活MEK/ERK信号通路从而促进ccRCC细胞的增殖、迁移及侵袭。

盐酸安罗替尼联合化疗对宫颈癌患者癌组织血管新生能力及MEK/ERK通路的影响

目的研究盐酸安罗替尼联合化疗对宫颈癌患者癌组织血管新生能力及MEK/ERK通路的影响。方法选取苏州大学附属第二医院2020年8月—2022年8月收治的68例宫颈癌患者为研究对象,采用随机数字表法分为试验组(n=34)和对照组(n=34)。对照组接受常规化疗治疗,试验组在此基础上联合安罗替尼抗血管生成治疗。对比两组患者临床疗效,治疗前后血管新生能力[血管内皮生长因子(VEGF)、肿瘤微血管密度(MVD)],治疗后MEK/ERK通路分子水平。结果与对照组总有效率58.82%(20/34)相比,试验组88.23%(30/34)更高(χ^(2)=7.555,P<0.05);治疗后,两组患者VEGF、MVD水平均降低,且相比于对照组,试验组更低[对照组分别为(63.98±5.14)ng·g^(-1)、(13.02±1.65)个·mm^(-2),试验组分别为(48.06±5.35)ng·g^(-1)、(6.68±1.62)个·mm^(-2)](t=12.512、15.987,均P<0.05);与对照组患者相比,试验组患者MEK1、MEK2以及ERK1/2水平更低[对照组分别为(2.06±0.19)ng·mL^(-1)、(2.28±0.14)ng·mL^(-1)、(1.48±0.12)ng·mL^(-1),试验组分别为(1.23±0.21)ng·mL^(-1)、(1.06±0.15)ng·mL^(-1)、(0.84±0.11)ng·mL^(-1)](t=17.089、34.670、22.924,均P<0.05)。结论盐酸安罗替尼联合化疗能够有效抑制宫颈癌患者癌组织血管新生能力以及MEK/ERK通路分子水平,临床疗效较高。