Gga-miRNA-181-5p family facilitates chicken myogenesis via targeting TGFBR1 to block TGF-βsignaling

MicroRNAs(miRNAs)have been demonstrated to control chicken skeletal muscle growth,however,the potential function of the miR-181-5p family in chicken myogenesis remains largely unknown.Here,our study identified the two chicken(Gallus gallus;Gga)miR-181-5p family members widely expressed in various tissues,specifically miR-181a-5p and miR-181b-5p.Besides,the breast muscles of fast-growing broilers expressed higher levels of miR-181a-5p and miR-181b-5p than those of slow-growing layers.Functionally,miR-181a-5p and miR-181b-5p both promote the expression level of myogenic factors including myogenin(MyoG),myogenic differentiation 1(MyoD1),and myosin heavy chain(MyHC),meanwhile accelerating the myotube formation of skeletal muscle satellite cells(SMSCs).Mechanistically,miR-181a-5p and miR-181b-5p directly bind to the 3′untranslated region(UTR)of the transforming growth factor beta receptor 1(TGFBR1)mRNA,further reducing the expression of TGFBR1.TGFBR1 is a key Transforming growth factor beta(TGF-β)signaling transduction receptor and had a negative function in muscle cell differentiation.Furthermore,knockdown of TGFBR1 facilitated the expression of chicken myogenic factors,boosted myotube formation,and decreased the SMAD family member 2/3(SMAD2/3)phosphorylation in chicken SMSCs.SMAD2/3 are downstream of TGF-βsignaling,and miR-181a-5p and miR-181b-5p could reduce the expression of TGFBR1 to further diminish the SMAD2/3 phosphorylation.Our findings revealed that the miR-181-5p family targets TGFBR1 to break the TGF-βsignaling transduction,which resulted in promoting chicken skeletal muscle development.

Electroacupuncture improves myocardial fibrosis in heart failure rats by attenuating ECM collagen deposition through modulation of TGF-β1/Smads signaling pathway

Background: To explore the effects of electroacupuncture on cardiac function and myocardial fibrosis in rat models of heart failure, and to elucidate the underlying mechanism of electroacupuncture in heart failure treatment. Methods: Healthy male Sprague-Dawley rats were allocated into three groups: Sham group, Model group, and electroacupuncture (Model + EA) group, with each group comprising 8 rats. The model underwent a procedure involving the ligation of the left anterior descending coronary artery to induce a model of heart failure. The Model + EA group was used for 7 consecutive days for electroacupuncture of bilateral Shenmen (HT7) and Tongli (HT5), once a day for 30 min each time. Left ventricular parameters in rats were assessed using a small-animal ultrasound machine to analyze changes in left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular ejection fraction, and left ventricular fractional shortening. Serum interleukin-1β (IL-1β), cardiac troponin (cTn), and N-terminal brain natriuretic peptide precursor levels were measured using ELISA. Histopathological changes in rat myocardium were observed through HE staining, while collagen deposition in rat myocardial tissue was assessed using the Masson staining method. Picro sirius red staining, immunohistochemical staining, and RT-qPCR were utilized to distinguish between the various types of collagen deposition. The expression level of TGF-β1 and SMAD2/3/4/7 mRNA in rat myocardial tissues was determined using RT-qPCR. Additionally, western blot analysis was conducted to assess the protein expression levels of TGF-β1, SMAD3/7, and p-SMAD3 in rat myocardial tissues. Results: Compared with the Sham group, the left ventricular ejection fraction and left ventricular fractional shortening values of the Model group were significantly decreased (P < 0.01);the left ventricular end-diastolic volume and left ventricular end-systolic volume values were remarkably increased (P < 0.01);serum N-terminal brain natriuretic peptide precursor content was increased (P < 0.01);serum IL-1β and cTn levels were increased (P < 0.01);myocardial collagen volume fraction were increased (P < 0.01);and those of the expression of TGF-β1 and SMAD2/3/4 mRNA was increased (P < 0.01);the expression of SMAD7 mRNA was decreased (P < 0.01);the protein expression levels of TGF-β1, SMAD3, and p-Smad3 were increased (P < 0.01);the protein expression level of SMAD7 was decreased (P < 0.01) in the Model group. Compared to the Model group, the expression levels of the proteins TGF-β1, SMAD3, and p-Smad3 in myocardial tissue were found to be decreased (P < 0.01), and the expression level of the protein SMAD7 was found to be increased (P < 0.01) in the Model + EA group;the collagen volume fraction and deposition of type Ⅰ /Ⅲ collagen were decreased (P < 0.01) in the Model + EA group. Conclusion: Electroacupuncture alleviates myocardial fibrosis in rats with heart failure, and this effect is likely due to attributed to the modulation of the TGF-β1/Smads signaling pathway, which helps reduce collagen deposition in the extracellular matrix.

TGF-β1/ERK信号通路与糖尿病肾病肾纤维化的发病机制研究进展

肾纤维化是糖尿病肾病(diabetic nephropathy, DN)发生发展并导致终末期肾脏疾病(end-stage renal disease, ESRD)的主要病理变化,转化生长因子-β1(TGF-β1)是已知最重要的致纤维化因子。越来越多研究表明TGF-β1/ERK信号通路是介导肾纤维化的重要非Smad依赖途径通路,在肾纤维化中发挥重要作用。本文对肾纤维化发病机制及TGF-β1/ERK信号通路在DN肾纤维化中的作用进展进行综述。

Acupuncture at Back-Shu point improves insomnia by reducing inflammation and inhibiting the ERK/NF-κB signaling pathway

BACKGROUND Insomnia is a disease where individuals cannot maintain a steady and stable sleep state or fail to fall asleep.Western medicine mainly uses sedatives and hypnotic drugs to treat insomnia,and long-term use is prone to drug resistance and other adverse reactions.Acupuncture has a good curative effect and unique advantages in the treatment of insomnia.AIM To explore the molecular mechanism of acupuncture at Back-Shu point for the treatment of insomnia.METHODS We first prepared a rat model of insomnia,and then carried out acupuncture for 7 consecutive days.After treatment,the sleep time and general behavior of the rats were determined.The Morris water maze test was used to assess the learning ability and spatial memory ability of the rats.The expression levels of inflammatory cytokines in serum and the hippocampus were detected by ELISA.qRTPCR was used to detect the mRNA expression changes in the ERK/NF-κB signaling pathway.Western blot and immunohistochemistry were carried out to evaluate the protein expression levels of RAF-1,MEK-2,ERK1/2 and NF-κB.RESULTS Acupuncture can prolong sleep duration,and improve mental state,activity,diet volume,learning ability and spatial memory.In addition,acupuncture increased the release of 1L-1β,1L-6 and TNF-αin serum and the hippocampus and inhibited the mRNA and protein expression of the ERK/NF-κB signaling pathway.CONCLUSION These findings suggest that acupuncture at Back-Shu point can inhibit the ERK/NF-κB signaling pathway and treat insomnia by increasing the release of inflammatory cytokines in the hippocampus.

Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway

The current study aimed to assess the effect of timosaponin AⅢ(T-AⅢ)on drug-metabolizing enzymes during anticancer therapy.The in vivo experiments were conducted on nude and ICR mice.Following a 24-day administration of T-AⅢ,the nude mice exhibited an induction of CYP2B10,MDR1,and CYP3A11 expression in the liver tissues.In the ICR mice,the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration.The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6,MDR1,and CYP3A4,along with constitutive androstane receptor(CAR)activation.Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression.Furthermore,other CAR target genes also showed a significant increase in the expression.The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice.Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation,with this effect being partially reversed by the ERK activator t-BHQ.Inhibition of the ERK1/2 signaling pathway was also observed in vivo.Additionally,T-AⅢ inhibited the phosphorylation of EGFR at Tyr1173 and Tyr845,and suppressed EGF-induced phosphorylation of EGFR,ERK,and CAR.In the nude mice,T-AⅢ also inhibited EGFR phosphorylation.These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

丹参多糖经MAPK/ERK信号轴抑制对肺癌细胞A549增殖、迁移和凋亡的影响

目的 探讨丹参多糖经丝裂原激活蛋白激酶(MAPK)/细胞外信号调节激酶(ERK)信号轴抑制对肺癌细胞A549增殖、迁移和凋亡的影响。方法 体外培养A549细胞,用不同质量浓度(0,1,2,4,8,16,32,64 mg/mL)丹参多糖干预48 h后,确定丹参多糖的半数抑制浓度(IC50);将A549细胞分为对照组、高剂量组、中剂量组和低剂量组(分别以0,8,4,2 mg/mL丹参多糖干预),以及抑制剂组(40μmol/L PD 98059)。采用划痕愈合试验检测A549细胞的迁移水平,采用流式细胞术检测A549细胞的凋亡情况,同时采用实时荧光定量聚合酶链反应法和免疫印迹法分别检测A549细胞中MAPK、ERK、基质金属蛋白酶-9(MMP-9)、B细胞淋巴瘤2基因(Bcl-2)、胱天蛋白酶3(caspase-3)mRNA和蛋白表达水平。结果 随着丹参多糖质量浓度的增加,A549细胞增殖率显著降低(P <0.05);丹参多糖对A549细胞的IC50为7.82 mg/mL,高、中、低剂量组干预剂量分别为8,4,2 mg/mL。与对照组比较,抑制剂组,高、中、低剂量组细胞的迁移距离及MMP-9,MAPK,ERK,Bcl-2 mRNA和蛋白表达水平均显著降低(P <0.05),细胞凋亡率、caspase-3 mRNA和蛋白表达水平均显著升高(P <0.05);与抑制剂组比较,高、中、低剂量组细胞的迁移距离及MMP-9,MAPK,ERK,Bcl-2 mRNA和蛋白表达水平均显著升高(P <0.05),细胞凋亡率、caspase-3 mRNA和蛋白表达水平均显著降低(P <0.05),且随丹参多糖剂量的增加呈量效依赖关系(P <0.05)。结论 丹参多糖可能通过MAPK/ERK信号轴诱导A549细胞凋亡,抑制A549细胞增殖和迁移。

mTOR和ERK/MAPK信号通路调控自噬在孤独症发病中的作用

孤独症是一种以重复刻板样行为和社交缺陷为主要特征的神经发育障碍性疾病,发病率高的特点使其逐渐成为研究的热点。中国与西方国家孤独症的发病率相似,约为1%,位于儿童精神疾病的前列^([1])。目前认为孤独症由环境和遗传因素共同决定,病因复杂,具体机制尚不明确。随着研究的深入,自噬在孤独症发病机制中的作用受到广泛关注。

欧前胡素调节ERK/MAPK信号通路对肺结核大鼠炎症反应的影响

目的 探讨欧前胡素(Imp)调节细胞外调节蛋白激酶(ERK)/有丝分裂原激活蛋白激酶(MAPK)信号通路对肺结核大鼠炎症反应的影响。方法 64只大鼠随机分为对照组12只及造模组52只,造模组大鼠通过尾部注射结核杆菌方法建立肺结核大鼠模型,之后随机分为模型组、Imp低(Imp-L,25 mg/kg)、中(Imp-M,50 mg/kg)、高剂量(Imp-H,100 mg/kg)组、Imp-H+ERK特异性激活剂(EGF,100 mg/kg Imp+25 mg/kg EGF)组。干预结束后,主动脉采血,ELISA检测各组大鼠血清中白细胞介素-6(IL-6)、γ干扰素(IFN-γ)、环氧化酶-2(COX-2)水平;分离肺组织,HE、Tunel分别检测大鼠肺组织病理学变化及细胞凋亡情况;统计肺组织中结核杆菌菌落数;Western blot检测p-ERK1/2/ERK1/2、p38 MAPK表达水平。结果 与对照组相比,模型组肺组织严重病变,IL-6、IFN-γ、COX-2、细胞凋亡率、结核杆菌菌落数、p-ERK1/2/ERK1/2、p38 MAPK表达均显著增加(P<0.05);与模型组相比,Imp-L组、Imp-M组、Imp-H组病理损伤得到缓解,IL-6、IFN-γ、COX-2、细胞凋亡率、结核杆菌菌落数、p-ERK1/2/ERK1/2、p38 MAPK表达显著降低,以Imp-H组变化最为显著(P<0.05);与Imp-H组相比,Imp-H+EGF组病理损伤进一步加重,IL-6、IFN-γ、COX-2、细胞凋亡率、结核杆菌菌落数、p-ERK1/2/ERK1/2、p38 MAPK表达显著增加(P<0.05)。结论 Imp可降低肺结核炎症反应,与抑制ERK/MAPK信号通路的激活有关。

Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.

滋水清肝饮对慢性束缚应激抑郁小鼠海马ERK、GSK3β、CREB、BDNF蛋白表达的影响

目的基于ERK/GSK-3β/CREB/BDNF信号通路探究滋水清肝饮对慢性束缚应激(CRS)抑郁小鼠的作用。方法除空白组外,其余小鼠建立CRS抑郁模型,造模成功后分为模型组、盐酸氟西汀组(10 mg/kg)和滋水清肝饮低、中、高剂量组(8.835、17.670、35.340 g/kg),给予相应剂量药物干预,同时继续进行CRS处理。给药7、14 d进行糖水偏好实验及行为学实验。给药14 d后,HE染色观察小鼠海马组织形态学改变,采用试剂盒检测血清超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平,ELISA法检测血清5-羟色胺(5-HT)、肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)水平,RT-qPCR法检测海马组织BDNF、TNF-α、IL-1βmRNA表达,Western blot法检测海马组织ERK1/2、p-ERK1/2、GSK3β、p-GSK3β、CREB、BDNF蛋白表达。结果与模型组比较,给药14 d后,盐酸氟西汀组和滋水清肝饮中剂量组小鼠糖水偏好率升高(P<0.01),悬尾不动时间和强迫游泳不动时间缩短(P<0.01),海马组织神经细胞损伤有所改善,血清MDA、TNF-α、IL-1β水平降低(P<0.05,P<0.01),SOD活性和5-HT水平升高(P<0.05,P<0.01),海马组织TNF-α、IL-1βmRNA表达降低(P<0.01),BDNF mRNA和p-ERK1/2、p-GSK3β、CREB、BDNF蛋白表达升高(P<0.05,P<0.01)。结论滋水清肝饮可改善慢性束缚应激小鼠抑郁样行为,其机制可能与调节小鼠海马ERK/GSK3β/CREB/BDNF信号通路有关。

锌指蛋白-36缺陷抑制小鼠的成骨细胞分化:基于激活ERK/ MAPK通路

目的探究锌指蛋白-36(ZFP36)对成骨细胞分化的调控作用及机制。方法通过提取小鼠原代骨髓间充质干细胞,结合小鼠成骨细胞前体细胞系MC3T3-E1,在体外成骨分化诱导状态下观察Zfp36(编码ZFP36)的表达变化。通过干扰RNA技术构建Zfp36缺陷的细胞,观察成骨分化作用的改变。通过第二代转录组测序技术探究Zfp36缺陷细胞成骨分化过程中的转录组水平改变。通过ERK/MAPK信号抑制分子U0126验证Zfp36缺陷对成骨分化作用的调控机制。结果小鼠原代骨髓间充质细胞以及MC3T3-E2细胞中Zfp36表达在成骨分化0~14d过程中呈逐渐升高趋势,在第7天到达峰值时较第0天升高3.85倍(P<0.0001)。抑制上述细胞Zfp36的表达后,成骨分化过程中碱性磷酸酶染色及茜素红染色弱于对照组,成骨分化标志基因Alpl(P<0.01)、Sp7(P<0.001)、Bglap(P<0.01)、Ibsp(P<0.0001)表达显著减弱。转录本测序结果提示Zfp36缺陷细胞的下调基因富集到骨矿化相关基因集中,且与ERK信号相关。蛋白表达检测显示Zfp36缺陷细胞的磷酸化ERK蛋白比例较对照组升高2.1倍(P=0.0274)。通过分子化合物U0126抑制Zfp36缺陷细胞中被激活的ERK/MAPK信号,可观察到表型挽救现象,并且呈剂量依赖。Zfp36缺陷细胞在10μmol/L度U0126作用下碱性磷酸酶染色增强,成骨细胞分化标志基因Runx2(P<0.05)及Bglap(P<0.05)表达显著增高。结论ZFP36参与了小鼠成骨细胞的分化调控过程,Zfp36缺陷会引起ERK/MAPK信号通路的激活,进而抑制成骨细胞向骨细胞的分化。

大黄素抑制背根神经节压迫小鼠模型STAT3、VEGFA、p-ERK蛋白表达及其镇痛作用研究

目的 研究大黄素(Emodin,ED)对背根节压迫小鼠模型的镇痛作用以及对STAT3/VEGFA/p-ERK信号通路的影响。方法 建立背根神经节慢性压迫(chronic compression damage,CCD)小鼠疼痛模型,随机分为空白组、模型组、普瑞巴林组及ED低、中、高剂量组。通过检测动物机械痛敏和热辐射痛敏阈值、醋酸扭体实验评价镇痛效果;ELISA检测小鼠L4-L5节段脊髓组织中白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子(tumor necrosis factor-α,TNF-α)和CD18等炎症因子含量;Western blot和免疫荧光检测脊髓组织信号转导子和转录激活子3(signal transducer and activator of transcription 3,STAT3)、血管内皮生长因子(vascular endothelial growth factor,VEGF)和磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinases 1/2,p-ERK1/2)蛋白表达。结果 与空白组比较,模型组的机械痛敏、热辐射痛敏阈值显著降低(P<0.05),脊髓背角炎症因子表达显著增高(P<0.05),脊髓中VEGFA、STAT3、p-ERK的表达显著上调(P<0.05)。与模型组比,ED低、中、高剂量组CCD小鼠模型的机械痛敏、热辐射痛敏阈值升高(P<0.05);脊髓背角炎症因子表达降低(P<0.05);脊髓背角小胶质细胞STAT3、VEGFA、p-ERK的表达降低(P<0.05),醋酸诱导的小鼠急性疼痛中扭体次数减少(P<0.05)。结论 ED对背根节压迫小鼠模型有良好的镇痛作用,其机制可能与抑制脊髓背角炎症因子表达和STAT3/VEGFA/p-ERK介导的脊髓小胶质细胞活化有关。

基于TGF-β1/ERK1/2信号通路分析TM4SF1与鼻咽癌细胞增殖、侵袭、凋亡和自噬的关系

目的探讨四跨膜蛋白家族成员1(TM4SF1)对鼻咽癌细胞增殖、侵袭、凋亡和自噬的影响及机制。方法将CNE-2细胞分为si-TM4SF1组、si-NC组、LY3200882组、TGF-β1组、Control组。采用MTT法检测细胞增殖能力;Transwell法检测细胞侵袭能力;流式细胞仪检测细胞凋亡能力;单丹磺酰尸胺(MDC)检测细胞自噬能力;蛋白质印迹检测细胞中TGF-β1、ERK1/2、p-ERK1/2、LC3Ⅱ、LC3Ι、p62、Beclin1蛋白表达。结果和Control组相比,si-TM4SF1组和LY3200882组细胞中TM4SF1 mRNA表达、增殖、侵袭能力降低,细胞凋亡率和自噬小体荧光强度增加(P<0.05);和si-TM4SF1组相比,TGF-β1组细胞中TM4SF1 mRNA表达、增殖、侵袭能力增加,细胞凋亡率和自噬小体荧光强度降低(P<0.05)。和Control组相比,si-TM4SF1组和LY3200882组细胞中TGF-β1、p62蛋白及p-ERK1/2/ERK1/2比值降低,Beclin1蛋白及LC3Ⅱ/LC3Ι比值增加(P<0.05);和si-TM4SF1组相比,TGF-β1组细胞中TGF-β1、p62蛋白及p-ERK1/2/ERK1/2比值增加,Beclin1蛋白及LC3Ⅱ/LC3Ι比值降低(P<0.05)。结论低表达TM4SF1可能通过调控TGF-β1/ERK1/2信号抑制鼻咽癌细胞增殖和侵袭,促进细胞凋亡和自噬。

基于ERK/TGF-β1/Smad4信号通路探讨章氏接骨丹含药血清对大鼠骨髓间充质干细胞成骨分化调控的影响

目的基于ERK/TGF-β1/Smad4信号通路探讨章氏接骨丹含药血清对大鼠骨髓间充质干细胞成骨分化调控的影响。方法取3月龄SPF级雌雄各半SD大鼠40只,随机分为药物组与蒸馏水组各20只,药物组按2.7 mL/(kg·d)灌服生药量浓度为1.554 g/mL接骨丹药液,蒸馏水组以等剂量蒸馏水灌胃,2组每日早晚各灌胃1次,灌胃7 d后取大鼠腹主动脉血制备接骨丹含药血清和空白血清。取2月龄SD大鼠双侧股骨和胫骨的骨髓液培养传代至第4代制备骨髓间充质干细胞模型,随机分为空白组、模型组、接骨丹组、抑制剂组,选用细胞外调节蛋白激酶(ERK)抑制剂对ERK通路进行抑制。空白组采用10%空白血清+α-MEM培养液进行培养;模型组采用10%空白血清+α-MEM培养液+ERK抑制剂进行培养;接骨丹组采用10%接骨丹含药血清+α-MEM培养液进行培养;抑制剂组采用10%接骨丹含药血清+α-MEM培养液+ERK抑制剂进行培养。4组均培养9 d后分别检测4组碱性磷酸酶(ALP)浓度,采用免疫荧光法测定Ⅰ型胶原(COLI)表达水平,采用qPCR法检测COLI、转化生长因子-β1(TGF-β1)、Smad4蛋白(Smad4)、Runx-2基因(Runx2)mRNA相对表达水平,采用Western blot检测COLI、TGF-β1、Smad4、Runx2、磷酸化ERK(p-ERK)蛋白表达量。结果与空白组比较,模型组ALP浓度和COLI表达水平均降低(P均<0.05),COLI、TGF-β1、Smad4、Runx2 mRNA相对表达水平及COLI、TGF-β1、Smad4、Runx2、p-ERK蛋白表达量均降低(P均<0.05);与模型组比较,接骨丹组ALP浓度和COLI表达水平均增加(P均<0.05),COLI、TGF-β1、Smad4、Runx2 mRNA相对表达水平及COLI、TGF-β1、Smad4、Runx2、p-ERK蛋白表达量均增加(P均<0.05)。与接骨丹组比较,抑制剂组ALP浓度和COLI表达水平均降低(P均<0.05),COLI、TGF-β1、Smad4、Runx2 mRNA相对表达水平及COLI、TGF-β1、Smad4、Runx2、p-ERK蛋白表达量均降低(P均<0.05)。结论章氏接骨丹能促进骨髓间充质干细胞成骨分化,其调控机制可能与上调ERK/TGF-β1/Smad4信号通路相关。

铜离子激活MAPK-ERK通路调控鼻咽癌放射敏感性

目的探究铜离子(Cu^(2+))对鼻咽癌(nasopharyngeal carcinoma,NPC)细胞放射敏感性的影响,寻找提升NPC放疗效果的潜在靶标。方法细胞Cu^(2+)检测对比正常鼻咽上皮细胞系NP69及多种NPC细胞系内Cu^(2+)的含量;分别加入0、5、10、20、50、100、200μmol/L Cu^(2+)溶液及0、0.05、0.1、0.2、0.5、1.0、2.0 mmol/L TEPA溶液,利用CCK-8实验测定NPC细胞内Cu^(2+)含量对辐照前后细胞存活率的影响,并确定后续实验中Cu^(2+)组及TEPA组所使用的药物浓度;CCK-8实验及克隆形成实验检测药物处理后各组NPC细胞放射敏感性的变化;Western blot检测各组NPC细胞辐照处理前后的DNA损伤情况及MAPK-ERK通路相关蛋白表达。结果NPC细胞中Cu^(2+)含量显著高于正常鼻咽上皮细胞(P<0.05)。添加Cu^(2+)溶液浓度低于50μmol/L时促进CNE1细胞的放疗抵抗性,添加0.1~0.2 mmol/L TEPA能提高SUNE1细胞放射敏感性(P<0.05)。与对照组比较,Cu^(2+)组NPC细胞放疗抵抗性增强,TEPA组NPC细胞放疗敏感性增强(P<0.05)。较之对照组而言,Cu^(2+)组细胞MAPK-ERK通路活化程度上调(P<0.05)。使用MAPK-ERK通路抑制剂SCH772984能够有效逆转Cu^(2+)介导的NPC放疗抵抗(P<0.05)。结论NPC细胞中Cu^(2+)含量升高,其通过激活MAPK-ERK通路增强NPC细胞的放疗抵抗性。

GNA12通过激活ERK信号通路促进鼻咽癌细胞增殖和侵袭迁移

目的探讨鸟嘌呤核苷酸结合蛋白α亚基12(G protein subunitα12,GNA12)对鼻咽癌细胞生物学行为的影响。方法利用GEPIA2、Human Protein Altas数据库预测GNA12在头颈部肿瘤中的表达情况,利用Keplan-Meier plotter数据库分析GNA12表达与头颈部肿瘤预后的关系。采用qRT-PCR实验检测正常鼻咽黏膜上皮细胞系(NP69)和鼻咽癌细胞系(CNE-2、5-8F、HK-1)中GNA12的表达水平。用携带si-GNA12和NC的干扰序列分别转染鼻咽癌细胞CNE-2和5-8F细胞系,构建敲低的细胞株(si-GNA12组)和对照细胞株(NC组)。采用划痕实验和Transwell实验检测细胞迁移能力,CCK-8和集落克隆实验检测细胞的增殖能力。采用Western blot实验检测上皮-间质转化中N-cadherin、E-cadherin、Snail以及Vimentin蛋白和ERK信号通路中ERK及p-ERK蛋白表达情况。结果数据库结果显示,头颈部肿瘤组织中GNA12表达高于正常头颈部组织(P<0.05)。GNA12表达越高,预后越差(P<0.05)。与正常鼻咽黏膜上皮细胞相比,鼻咽癌细胞系中GNA12 mRNA表达水平较高(P<0.05)。与NC组相比,si-GNA12组GNA12 mRNA表达水平下降(P<0.05)。与NC组相比,si-GNA12组CNE-2和5-8F细胞的增殖、迁移和侵袭均受到抑制(P<0.05)。Western blot显示,与NC组相比,si-GNA12组N-cadherin、Vimentin、Snail蛋白表达下降(P<0.05),E-cadherin蛋白表达上升(P<0.05),ERK信号通路相关蛋白p-ERK蛋白表达下降(P<0.05)。结论GNA12可能通过调控ERK信号通路促进鼻咽癌细胞的增殖和侵袭迁移。

老年冠心病患者PCI术后心肌再灌注损伤与ERK/MAPK信号通路的相关性

目的探讨老年冠状动脉粥样硬化性心脏病(冠心病)患者经皮冠状动脉介入治疗(PCI)术后心肌再灌注损伤的影响因素及与细胞外调节蛋白激酶/丝裂原活化蛋白激酶(ERK/MAPK)信号通路活化的相关性。方法选择2020年3月至2022年12月于郑州大学附属郑州中心医院心血管内科92例行PCI的老年冠心病患者作为研究对象,采用实时荧光定量PCR检测患者术前ERK、p38-MAPK mRNA的表达,评估PCI术后心肌再灌注损伤情况,分析其影响因素及与ERK/MAPK信号通路指标的关系。结果92例患者中20例(21.74%)PCI术后出现心肌再灌注损伤(损伤组,n=20)。损伤组患者PCI术前血流分级≤2级、Killip分级≥2级比例显著高于非损伤组患者,术前血清ERK、p38-MAPK mRNA表达量高于非损伤组,差异有统计学意义(P<0.05)。多因素回归分析示,术前血流分级≤2级、Killip分级≥2级及血清ERK、p38-MAPK水平升高是老年冠心病患者PCI术后出现心肌再灌注损伤的影响因素(P<0.05)。结论除血流分级、Killip分级外,术前ERK/MAPK信号通路相关指标高表达可增加老年冠心病患者PCI术后心肌再灌注损伤风险,临床应予以密切监测,以尽早识别心肌再灌注损伤并采取针对性干预措施。

线粒体内膜蛋白17(MPV17)通过阻断ERK通路抑制铁过载小鼠脾脏CD3^(+)T细胞铁死亡

目的探索铁过载对小鼠脾脏损伤的影响及线粒体内膜蛋白17(MPV17)在铁过载小鼠脾脏CD3^(+)T细胞铁死亡中的作用。方法将小鼠随机分为正常饮食组、高铁饮食组、高铁饮食联合铁死亡抑制剂ferrostatin-1(Fer-1)处理组、高铁饮食联合MPV17腺病毒注射组,每组5只。喂食8周后,取脾脏组织并固定,通过组织切片和HE染色法观察脾脏结构,碘化丙啶(PI)染色检测脾脏CD3^(+)T细胞死亡,脂质过氧化荧光探针C11 BODIPY 581/591检测脂质氧化,实时定量PCR检测溶质载体家族7成员11(SLC7A11)及前列腺素内过氧化物合成酶2(PTGS2)mRNA水平,流式细胞术检测M1、M2巨噬细胞比例,ELISA实验检测肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)及IL-6的含量。同时增加高铁饮食联合细胞外信号调节激酶(ERK)抑制剂处理组、ERK激活剂处理组、β半乳糖苷(β-gal)酶联合ERK激活剂处理组、MPV17联合ERK激活剂处理组,Western blot法检测MPV17、谷胱甘肽过氧化物酶4(GPX4)、磷酸化的ERK(p-ERK)水平,JC-1结合流式细胞术检测线粒体膜电位。结果与正常饮食组相比,高铁饮食组小鼠脾脏红髓形状不规则,白髓结构消失,脾脏CD3^(+)T细胞死亡增多,脂质过氧化物增多,SLC7A11及PTGS2表达升高,血液中M1/M2巨噬细胞比例升高,炎症因子含量升高;经Fer-1处理或过表达MPV17,部分恢复脾脏结构,CD3^(+)T细胞数量及脂质过氧化物减少,SLC7A11及PTGS2表达被抑制,M1/M2巨噬细胞比例及炎症因子的含量降低。高铁饮食导致GPX4表达降低,p-ERK表达升高,抑制ERK部分恢复GPX4表达,激活ERK降低GPX4表达;MPV17抑制ERK部分恢复GPX4表达,MPV17部分恢复因ERK激活导致的线粒体膜电势降低。结论铁过载可诱导小鼠脾脏CD3^(+)T细胞发生铁死亡,MPV17通过阻断ERK信号抑制高铁饮食诱导的脾脏CD3^(+)T细胞铁死亡。

连翘脂素调节Ras/Raf/MEK/ERK信号通路对结直肠癌细胞恶性生物学行为的影响

目的 探究连翘脂素(PG)对结直肠癌细胞增殖、迁移、侵袭、凋亡以及上皮间质转化的影响及机制。方法 培养HCT116结直肠癌细胞,采用10~200μmol/L的连翘脂素处理细胞,检测细胞存活率,筛选最佳实验浓度。将细胞分为对照组(Control组),连翘脂素低、中、高浓度组(PG-L组、PG-M组、PG-H组),连翘脂素高浓度+转染慢病毒阴性对照组(PG-H+NC组),连翘脂素高浓度+Ras慢病毒组(PG-H+Ras组)。平板克隆形成实验检测细胞增殖;划痕实验检测细胞迁移;Transwell小室法检测细胞侵袭;流式细胞术以及Hoechst33258染色法检测细胞凋亡;Western blot法检测上皮间质转化(EMT)标志蛋白E型钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)以及信号通路相关蛋白p-Raf、p-MEK、p-ERK表达;建立荷瘤小鼠模型评价连翘脂素对结直肠癌肿瘤生长的影响。结果 10~200μmol/L的连翘脂素处理HCT116结直肠癌细胞,可以显著抑制细胞存活率,经计算IC50值为(140.4±2.147)μmol/L,选择10、50、100μmol/L连翘脂素进行后续实验;与Control组比较,PG-L组、PG-M组、PG-H组HCT116细胞克隆形成率、划痕愈合率、细胞侵袭个数以及Vimentin、p-Raf、p-MEK、p-ERK表达显著降低,细胞凋亡率以及E-cadherin表达显著升高,且呈浓度依赖性(P<0.05);与PG-H+NC组比较,PG-H+Ras组HCT116细胞克隆形成率、划痕愈合率、细胞侵袭个数以及Vimentin、p-Raf、p-MEK、p-ERK表达显著升高,细胞凋亡率以及E-cadherin表达显著降低(P<0.05);连翘脂素可以显著抑制结直肠癌移植瘤的生长。结论 连翘脂素可以抑制结直肠癌细胞增殖、迁移、侵袭、凋亡以及上皮间质转化,其作用机制可能与抑制Ras/Raf/MEK/ERK信号通路激活有关。

基于MAPK/ERK/NF-κB信号通路探讨中西医结合治疗原发性痛经的研究进展

痛经(Dysmenorrhea)是临床常见妇科疾病,中医亦称“经行腹痛”,是指女性在行经前后或月经期,下腹部出现痉挛性疼痛,亦可放射至腰骶部,伴或不伴有恶心呕吐、腹泻及头晕乏力等全身症状。痛经可分为原发性和继发性两类,其中原发性痛经指生殖器官无器质性病变者;继发性痛经则是由于盆腔器质性疾病,如子宫内膜异位症、子宫腺肌症、子宫肌瘤、盆腔炎或宫颈狭窄等引起者。目前西医治疗原发性痛经多以解痉止痛的药物治疗,疗效虽显著而快速,但是副作用较大,停药后易复发;而中医在临床上四诊合参,辨证论治,不仅可以标本同治,也有助于预防调摄。通过梳理归纳近10年国内外原发性痛经的文献资料,基于MAPK/ERK/NF-κB信号通路探析原发性痛经的中西医研究进展,以期为中医药防治原发性痛经提供理论依据。现将原发性痛经的研究现状综述如下。