Effect of ginsenoside Rg1 on hematopoietic stem cells in treating aplastic anemia in mice via MAPK pathway

BACKGROUND Aplastic anemia(AA)presents a significant clinical challenge as a life-threatening condition due to failure to produce essential blood cells,with the current the-rapeutic options being notably limited.AIM To assess the therapeutic potential of ginsenoside Rg1 on AA,specifically its protective effects,while elucidating the mechanism at play.METHODS We employed a model of myelosuppression induced by cyclophosphamide(CTX)in C57 mice,followed by administration of ginsenoside Rg1 over 13 d.The invest-igation included examining the bone marrow,thymus and spleen for pathological changes via hematoxylin-eosin staining.Moreover,orbital blood of mice was collected for blood routine examinations.Flow cytometry was employed to identify the impact of ginsenoside Rg1 on cell apoptosis and cycle in the bone marrow of AA mice.Additionally,the study further evaluated cytokine levels with enzyme-linked immunosorbent assay and analyzed the expression of key proteins in the MAPK signaling pathway via western blot.RESULTS Administration of CTX led to significant damage to the bone marrow’s structural integrity and a reduction in hematopoietic cells,establishing a model of AA.Ginsenoside Rg1 successfully reversed hematopoietic dysfunction in AA mice.In comparison to the AA group,ginsenoside Rg1 provided relief by reducing the induction of cell apoptosis and inflammation factors caused by CTX.Furthermore,it helped alleviate the blockade in the cell cycle.Treatment with ginsenoside Rg1 significantly alleviated myelosuppression in mice by inhibiting the MAPK signaling pathway.CONCLUSION This study suggested that ginsenoside Rg1 addresses AA by alleviating myelosuppression,primarily through modulating the MAPK signaling pathway,which paves the way for a novel therapeutic strategy in treating AA,highlighting the potential of ginsenoside Rg1 as a beneficial intervention.

Effect of the Tiaobufeishen decoction on Caveolin-1-p38 MAPK signaling pathway and mechanism of improving the tracheobronchomalacia in chronic obstructive pulmonary disease

Objective: Reliving the rela ti onship of the Caveolin-1-p38 MAPK signaling pathway and COPD tr acheob ronchomalacia, and resea rch the mechanism of Tiaobufeishen decoc tion imp rove the regression of the weasand cartilage cells. Methods: Flow cytometry was used to analyze the apoptosis rate to determine the optimal concentration of Tiaobufeishen decoction and CSE, CCK8 assay was used to dete rmine the op ti mal concent ration of P38-MAPK specific inhibitor. The COPD cell model was created by tracheal chondrocyte which dispose by optimal concent ration CSE, then add the IL-1P set up the chond rocyte degene ration model, use the method of toluidine blue staining and immunohistochemical authenticate degeneration of cartilage. This research included control group, model group, model-Tiaobufeishen group, model-blocker group. When the model was set up succeed, add the Tiaobufeishen decoction and P38-MAPK blocke r in the model-Tiaobufeishen and model-blocke r gr oups, r espectively. Weste rn Blot was used to detect the exp ression of caveolin-1 and p-p38 in the chond rocyte. RT-PCR was used to detect the expression of MMP3 and caveolin-1 in the matrix. Results: The cell activity was not influence by the concentration of Tiaobufeishen decoction and blocker, the concentration of the CSE model was moderation. Compared with control group, the level of caveolin-1, p38MAPK, MMP3 in the model group was significant increase, moreover, the result of toluidine blue staining and immunohistochemical methods show that the chond rocyte has obvious reg ression. The exp ression of caveolin-1, p38MAPK, and MMP3 have significant decrease than the control group, and the reduction of chondrocyte degeneration. Conclusion: The caveolin-1-p38MAPK signaling pathway play an important role in the morbidity of the tracheobronchomalacia. Tiaobufeishen decoction could decrease the exp ression of the caveolin-1, p-p38, MMP3, inhibit the activa tion of the caveolin-1-p38MAPK signaling pathway, therefore, it can improve the tracheobronchomalacia.

BcSDR1 is involved in regulation of glucose transport and cAMP and MAPK signaling pathways in Botrytis cinerea

Botrytis cinerea is a typical necrotrophic pathogenic fungus that causes severe diseases in a wide range of plant species, leading to significant economic losses. Our previous study showed that BcSDR1 positively regulates growth,development, and pathogenicity of B. cinerea. However, the regulation mechanism of BcSDR1 and the relationship between BcSDR1 and cAMP and MAPK signaling pathways are not well understood. In this study, transcriptome data showed that BcSDR1 is involved in glucose transmembrane transport, signal transduction, secondary metabolism, and other biological processes. BcSDR1 mutant(BCt41) showed remarkably weak sensitivity to cAMP and MAPK signaling pathways specific inhibitors, SQ22536 and U0126, and significantly decreased cAMP content. The key genes of cAMP and MAPK signaling pathways, BcGB1, BcBTP1, BcBOS1, BcRAS1, and BcBMP3 were significantly upregulated,whereas BcPLC1, BcBCG1, BcCDC4, BcSAK1, BcATF1, and BcBAP1 were significantly downregulated(P<0.05).BcSDR1 was obviously upregulated in BcBCG2, BcBCG3, BcPKA1, and BcPKAR RNA interference(RNAi) mutants, but significantly downregulated in BcPKA2, BcBMP1, and BcBMP3 RNAi mutants. Thus, BcBCG2, BcBCG3, BcPKA1, and BcPKAR negatively regulate BcSDR1 expression, whereas BcPKA2, BcBMP1, and BcBMP3 positively regulate BcSDR1expression.

虫草素调节MAPK/AP-1信号通路对慢性阻塞性肺疾病大鼠肺组织损伤的影响

目的 基于丝裂原活化蛋白激酶(MAPK)/激活蛋白-1(AP-1)信号通路探讨虫草素(Cor)对慢性阻塞性肺疾病大鼠肺组织损伤的改善作用。方法 利用烟熏联合脂多糖滴入气管的方法建立慢性阻塞性肺疾病大鼠模型,将造模大鼠随机分为模型组(COPD)、虫草素低、中、高剂量组(Cor-L、M、H,10、20、50 mg/kg)、阳性对照组(NAC,0.5 g/kg),另设置正常培养的大鼠为对照组(NC)。使用肺功能仪检测大鼠肺功能指标呼气峰流速(PEF)、用力肺活量(FVC);HE染色观察大鼠肺组织病理学变化;对肺泡灌洗液白细胞进行计数分离;ELISA法检测肺泡灌洗液中TNF-α、IL-6和LTB4水平;微量法试剂盒检测肺组织中MDA、SOD、GSH-Px水平;Western Blot检测MAPK/AP-1信号通路蛋白表达。结果 与NC组比较,COPD组大鼠肺组织出现肺泡肿大,炎性细胞浸润等病理学变化,肺功能(PEF、FVC)、巨噬细胞比率、淋巴细胞比率、SOD、GSH-Px活性显著下降,白细胞数目、中性粒细胞比率、TNF-α、IL-6和LTB4水平、MDA含量、p-ERK/ERK、p-JNK/JNK、p-p38/p38、C-jun、C-fos蛋白表达显著上升(P<0.05);与COPD组比较,Cor各剂量组、NAC组大鼠肺组织病理学变化明显好转,肺功能(PEF、FVC)、巨噬细胞比率、淋巴细胞比率、SOD、GSH-Px活性显著上升,白细胞数目、中性粒细胞比率、TNF-α、IL-6和LTB4水平、MDA含量、p-ERK/ERK、p-JNK/JNK、p-p38/p38、C-jun、C-fos蛋白表达显著下降(P<0.05);NAC组与Cor-H组上述指标无显著差异(P>0.05)。结论 虫草素可以抑制MAPK/AP-1信号通路,减轻炎症反应和氧化应激,改善慢性阻塞性肺疾病大鼠肺组织病理损伤。

针刺对脑心综合征大鼠脑组织细胞凋亡、炎症因子及丝裂原活化蛋白激酶/核因子-κB/激活蛋白-1信号通路的影响

目的探讨针刺对脑心综合征大鼠脑组织细胞凋亡、炎症因子及丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)/核因子-κB(nuclear factor-κB,NF-κB)/激活蛋白-1(activator protein-1,AP-1)信号通路的影响。方法将36只SD大鼠随机分为针刺组、模型组和对照组,各12只。采用胶原酶加肝素联合注射大鼠尾状核制备脑心综合征大鼠模型,对照组给予等剂量生理盐水向大鼠尾状核注入,手术操作过程同模型组。模型组和假手术组均不予针刺;针刺组选心俞穴、内关穴、风府穴和水沟穴,每日针刺1次,连续3天。采用原位末端凋亡(TdT-mediated dUTP-biotin nick end labeling,TUNEL)法测定脑组织细胞凋亡情况;神经功能采用Zea-Longa法评估;运用酶联免疫法测定白细胞介素(interleukin,IL)-1β、IL-6和肿瘤坏死因子(tumor necrosis factor,TNF)-α水平;采用Western Blot法测定MAPK、NF-κB、AP-1蛋白表达。结果模型组和针刺组大鼠脑组织细胞凋亡指数(apoptosis index,AI)高于假手术组(P<0.05);针刺组大鼠脑组织AI低于模型组(P<0.05)。模型组大鼠神经行为评分高于假手术组(P<0.05);针刺组大鼠神经行为评分低于模型组(P<0.05)。模型组大鼠IL-1β、IL-6和TNF-α水平高于假手术组(P<0.05);针刺组大鼠IL-1β、IL-6和TNF-α水平低于模型组(P<0.05)。模型组大鼠MAPK蛋白灰度值、NF-κB蛋白灰度值和AP-1蛋白灰度值高于假手术组(P<0.05);针刺组大鼠MAPK蛋白灰度值、NF-κB蛋白灰度值和AP-1蛋白灰度值低于模型组(P<0.05)。结论脑心综合征大鼠采用针刺可减轻大鼠脑组织细胞凋亡,减轻大鼠炎症因子,且可下调MAPK/NF-κB/AP-1信号通路表达。

Nucleolin Mediates LPS-induced Expression of Inflammatory Mediators and Activation of Signaling Pathways

Summary:In this study,we investigated the effects of nucleolin on lipopolysaccharide(LPS)-induced activation of MAPK and NF-KappaB(NF-kB)signaling pathways and secretion of TNF-a,IL-1βand HMGB1 in THP-1 monocytes.Immunofluorescence assay and Western blotting were used to identify the nucleolin expression in cell membrane,cytoplasm and nucleus of THP-1 monocytes.Inactivation of nucleolin was induced by neutralizing antibody against nucleolin.THP-1 monocytes were pretreated with anti-nucleolin antibody for 1 h prior to LPS challenge.The irrelevant IgG group was used as control.Secretion of inflammatory mediators(TNF-a,IL-1β and HMGB1)and activation of MAPK and NF-kB/I-kB signaling pathways were examined to assess the effects of nucleolin on LPS-mediated inflammatory response.Nucleolin existed in cell membrane,cytoplasm and nucleus of THP-1 monocytes.Pretreatment of anti-nucleolin antibody significantly inhibited the LPS-induced secretion of TNF-a,IL-1β and HMGB1.P38,JNK,ERK and NF-κB subunit p65 inhibitors could significantly inhibit the secretion of IL-1β,TNF-a and HMGB1 induced by LPS.Moreover,the phosphorylation of p38,JNK,ERK and p65(or nuclear translocation of p65)was significantly increased after LPS challenge.In contrast,pretreatment of anti-nucleolin antibody could significantly inhibit the LPS-induced phosphorylation of p38,JNK,ERK and p65(or nuclear translocation of p65).However,the irrelevant IgG,as a negative control,had no effect on LPS-induced secretion of TNF-a and IL-Iβ and phosphorylation of p38,JNK,ERK and p65(or nuclear translocation of p65).We demonstrated that nucleolin mediated the LPS-induced activation of MAPK and NF-κB signaling pathways,and regulated the secretion of inflammatory mediators(TNF-a,IL-1β and HMGB1).

Hypoxia-inducible factor-1α–mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2

BACKGROUND As human placenta-derived mesenchymal stem cells(hP-MSCs)exist in a physiologically hypoxic microenvironment,various studies have focused on the influence of hypoxia.However,the underlying mechanisms remain to be further explored.AIM The aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs.METHODS A hypoxic cell incubator(2.5%O2)was used to mimic a hypoxic microenvironment.Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs.The cell cycle was profiled by flow cytometry.Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing.CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction.Small interfering RNA-mediated hypoxia-inducible factor 1α(HIF-1α)or CD99 knockdown of hP-MSCs,luciferase reporter assays,and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis.Protein expression was assayed by western blotting;immunofluorescence assays were conducted to evaluate changes in expression levels.RESULTS Hypoxia enhanced hP-MSC proliferation,increased the expression of cyclin E1,cyclin-dependent kinase 2,and cyclin A2,and decreased the expression of p21.Under hypoxia,CD99 expression was increased by HIF-1α.CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation.CONCLUSION Hypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

基于“心受气于脾”探讨心痛泰调控p38 MAPK/AP-1对动脉粥样硬化兔的中医证候和VSMC胶原纤维的影响

目的:基于"心受气于脾"探讨心痛泰调控p38丝裂原活化蛋白激酶(p38 MAPK)/转录激活因子-1(AP-1)对动脉粥样硬化兔的中医证候积分和血管平滑肌细胞(VSMC)胶原纤维的影响。方法:120只清洁级兔随机分为假手术组,痰瘀互结模型组,心痛泰低剂量组、中剂量组、高剂量组和瑞舒伐他汀组。采用高脂喂养+球囊损伤法,造成痰瘀互结病证结合动脉粥样硬化兔模型,造模后予以相应药物灌胃8周(心痛泰低、中、高剂量组和瑞舒伐他汀组的给药量分别为2.3,4.6,9.2 g·kg^(-1)和0.55 mg·kg^(-1))。给药周期结束时取腹主动脉,苏木素-伊红(HE)染色观察易损斑块的情况;免疫组化法(IHC)测定基质金属蛋白酶-9(MMP-9),组织基质金属蛋白酶抑制剂-1(TIMP-1);马松(Masson)染色观察平滑肌细胞胶原纤维分解情况;蛋白免疫印迹法(Western blot)测定主动脉组织p38 MAPK,AP-1蛋白的表达;采用中医证候评分表评价痰瘀互结证中医证候积分。结果:与模型组比较,心痛泰各剂量组、瑞舒伐他汀组MMP-9含量显著降低,TIMP-1含量显著升高,p38 MAPK蛋白的表达,AP-1的核转位显著降低(P<0.01);与心痛泰低剂量组比较,心痛泰中、高剂量组,瑞舒伐他汀组MMP-9明显降低,TIMP-1明显升高,p38 MAPK蛋白的表达,AP-1的核转位明显降低(P<0.05,P<0.01)。与模型组比较,心痛泰各剂量组、瑞舒伐他汀组的中医证候积分均明显改善(P<0.05,P<0.01);与心痛泰低剂量组比较,心痛泰中、高剂量组,瑞舒伐他汀组的中医证候积分均显著改善(P<0.01)。Masson染色显示,模型组平滑肌纤维排列紊乱,胶原分解增多,纤维帽变薄,斑块易损性增加;与模型组比较,心痛泰各组和瑞舒伐他汀组平滑肌细胞排列较为整齐,胶原纤维分解减少,斑块稳定性增加。结论:心痛泰可下调p38 MAPK,MMP-9的表达,提高TIMP-1的水平,减少AP-1核转位,减少平滑肌细胞胶原纤维的分解,改善痰瘀互结证的中医证候评分。心痛泰可能通过调控p38 MAPK/AP-1信号通路及下游的细胞因子,稳定易损斑块,改善动脉粥样硬化之痰瘀互结证。

p38 MAPK/AP-1信号通路在1,2-二氯乙烷中毒性脑水肿形成中对iNOS表达的上调作用

目的探究p38 MAPK/AP-1信号通路在小鼠1?2-二氯乙烷(1?2-DCE)中毒性脑水肿形成过程中对诱导型一氧化氮合酶(iNOS)表达的作用及其对脑水肿的影响。方法选取40只雌性昆明种小鼠随机分为对照组、染毒组、低剂量以及高剂量p38抑制剂组。染毒小鼠于染毒柜中1.2 mg/L 1?2-二氯乙烷染毒3.5 h/d,连续染毒3 d,低、高剂量抑制剂组小鼠于染毒前1 h腹腔注射200μl 3.75、15 mg/kg的p38 MAPK抑制剂(SB202190)。染毒结束次日取材,测定各组小鼠脑含水量及HE病理观察脑水肿,Western Blot检测各组小鼠脑组织中磷酸化p38、激活蛋白1(AP-1)两个亚基c-fos、c-jun的磷酸化形式表达水平以及iNOS的蛋白表达,Real-Time RT-PCR检测iNOS mRNA表达水平。结果单纯染毒组的小鼠出现抱爪现象,SB202190干预能够明显改善1?2-DCE中毒小鼠的抱爪症状。染毒组小鼠脑组织含水量与对照组小鼠相比明显增加,SB202190干预能够有效缓解小鼠出现脑水肿。单纯染毒组小鼠脑组织中p-p38蛋白、磷酸化c-jun和c-fos表达水平明显上调,iNOS蛋白和mRNA表达水平也显著增加,而SB202190能够显著降低iNOS、磷酸化c-jun和c-fos的表达水平。结论小鼠脑组织iNOS mRNA和蛋白表达水平在1?2-DCE中毒性脑水肿形成过程中显著上调。p38 MAPK/AP-1信号通路参与iNOS表达增多的调控过程。

转化生长因子-β对基质金属蛋白酶及其组织抑制因子调控的研究进展

基质金属蛋白酶 (MMPs)及其组织抑制因子 (TIMPs)参与调控胞外基质 (ECM)的降解与重建 ,二者的协同作用以及表达的动态平衡保证组织的生理 /病理形态结构建成 ,并完成生长、分化、维持、降解的往复周期 .转化生长因子 β (TGF β)通过对MMPs和TIMPs家族成员因细胞类型而异的基因表达调控作用 ,表现出调节ECM重建的生物学效应 .TGF β可通过激活Smad通路、促分裂原活化蛋白激酶 (MAPK)通路 ,以及刺激激活蛋白 1(AP 1)复合体的形成 ,完成对MMPs和TIMPs基因表达的调控功能 .

Comparative Analysis of Protein Expression Concomitant with DNA Methyltransferase 3A Depletion in a Melanoma Cell Line

DNA methyltransferase 3A (Dnmt3a), a de novo methyltransferase, has attracted a great deal of attention for its important role played in tumorigenesis. We have previously demonstrated that melanoma is unable to grow in-vivo in conditions of Dnmt3a depletion in a mouse model. In this study, we cultured the Dnmt3a depletion B16 melanoma (Dnmt3a-D) cell line to conduct a comparative analysis of protein expression con-comitant with Dnmt3a depletion in a melanoma cell line. After two-dimensional separation, by gel electro-phoresis and liquid chromatography, combined with mass spectrometry analysis (1DE-LC-MS/MS), the re-sults demonstrated that 467 proteins were up-regulated and 535 proteins were down-regulated in the Dnmt3a-D cell line compared to the negative control (NC) cell line. The Genome Ontology (GO) and KEGG pathway were used to further analyze the altered proteins. KEGG pathway analysis indicated that the MAPK signaling pathway exhibited a greater alteration in proteins, an interesting finding due to the close relation-ship with tumorigenesis. The results strongly suggested that Dnmt3a potentially controls the process of tu-morigenesis through the regulation of the proteins (JNK1, p38α, ERK1, ERK2, and BRAF) involved in tu-mor-related pathways, such as the MAPK signaling pathway and melanoma pathway.

The functional analysis of transiently upregulated miR-101 suggests a “braking” regulatory mechanism during myogenesis

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and microRNAs(miRNAs).A group of muscle-specific miRNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth.However,the functional role and regulatory mechanism of most non-muscle-specific miRNAs with stage-specific changes during differentiation are largely unclear.Here,we describe the functional characterization of miR-101a/b,a pair of non-muscle-specific miRNAs that show the largest change among a group of transiently upregulated miRNAs during myogenesis in C2C12 cells.The overexpression of miR-101a/b inhibits myoblast differentiation by suppressing the p38/MAPK,Interferon Gamma,and Wnt pathways and enhancing the C/EBP pathway.Mef2a,a key protein in the p38/MAPK pathway,was identified as a direct target of miR-101a/b.Interestingly,we found that the long non-coding RNA(lncRNA)Malat1,which promotes muscle differentiation,interacts with miR-101a/b,and this interaction competes with Mef2a mRNA to relieve the inhibition of the p38/MAPK pathway during myogenesis.These results uncovered a“braking”role in differentiation of transiently upregulated miRNAs and provided new insights into the competing endogenous RNA(ceRNA)regulatory mechanism in myoblast differentiation and myogenesis.

地榆治疗皮肤光老化机制的网络药理学分析及验证

目的 运用网络药理学、分子对接技术及动物实验研究地榆抗皮肤光老化的活性成分及作用机制。方法 通过TCMSP数据库筛选地榆的活性成分及靶点;检索Drugbank、GeneCards、OMIM等数据库获取光老化疾病的靶点;采用STRING分析及Cytoscape软件构建蛋白相互作用(PPI)网络;对交集靶点进行GO功能注释和KEGG富集分析;选取核心靶点与对应的活性成分进行分子对接验证;最后,通过动物实验验证关键靶点。结果 筛选得到的地榆活性成分中,与光老化对应的潜在靶点158个,核心靶点11个,其中,包括MAPK、FOS靶点,GO、KEGG富集分析主要涉及PI3K/Akt通路、NF-κB通路,分子对接结果显示:槲皮素与MAKP1的结合最为稳定。动物实验表明:地榆提取物能显著改善小鼠皮肤光老化,并显著降低p-ERK1/2、p-p38、p-JNK、p-c-fos蛋白表达的水平。结论 地榆在治疗皮肤光老化方面具有多靶点和多通路的作用特点。