Exosomes derived from microglia overexpressing miR-124-3p alleviate neuronal endoplasmic reticulum stress damage after repetitive mild traumatic brain injury

We previously reported that miR-124-3p is markedly upregulated in microglia-derived exosomes following repetitive mild traumatic brain injury.However,its impact on neuronal endoplasmic reticulum stress following repetitive mild traumatic brain injury remains unclear.In this study,we first used an HT22 scratch injury model to mimic traumatic brain injury,then co-cultured the HT22 cells with BV2 microglia expressing high levels of miR-124-3p.We found that exosomes containing high levels of miR-124-3p attenuated apoptosis and endoplasmic reticulum stress.Furthermore,luciferase reporter assay analysis confirmed that miR-124-3p bound specifically to the endoplasmic reticulum stress-related protein IRE1α,while an IRE1αfunctional salvage experiment confirmed that miR-124-3p targeted IRE1αand reduced its expression,thereby inhibiting endoplasmic reticulum stress in injured neurons.Finally,we delivered microglia-derived exosomes containing miR-124-3p intranasally to a mouse model of repetitive mild traumatic brain injury and found that endoplasmic reticulum stress and apoptosis levels in hippocampal neurons were significantly reduced.These findings suggest that,after repetitive mild traumatic brain injury,miR-124-3 can be transferred from microglia-derived exosomes to injured neurons,where it exerts a neuroprotective effect by inhibiting endoplasmic reticulum stress.Therefore,microglia-derived exosomes containing miR-124-3p may represent a novel therapeutic strategy for repetitive mild traumatic brain injury.

MicroRNA-363-3p inhibits colorectal cancer progression by targeting interferon-induced transmembrane protein 1

BACKGROUND The molecular mechanisms of colorectal cancer development and progression are far from being elucidated.AIM To investigate the role of microRNA-363-3p(miR-363-3p)in the progression of colorectal cancer.METHODS Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues.PITA 6 was utilized to predict the targets of miR-363-3p.Dual-luciferase reporter system was used to validate the target of miR-363-3p.Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells’clonogenic survival ability and migration ability,respectively.Cell proliferation was examined by cell counting kit-8 assay.Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1(IFITM1)in colorectal cancer tissues and adjacent tissues.The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients.A colorectal cancer cell line with a deficiency of IFITM1 was constructed,and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified.RESULTS MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues.IFITM1 was characterized as a direct target of miR-363-3p.Overexpression of miR-363-3p led to decreased clonogenic survival,proliferation,and migration of colorectal cancer cells,which could be reversed by forced IFITM1 expression.CONCLUSION MiR-363-3p can constrain clonogenic survival,proliferation,and migration of colorectal cancer cells via targeting IFITM1.

Genetic Diversity of Chinese Soybean mosaic virus Strains and Their Relationships with Other Plant Potyviruses Based on P3 Gene Sequences

Soybean mosaic virus （SMV）, a member of the genus Potyvirus, is a major pathogen of soybean plants in China, and 16 SMV strains have been identified nationwide based on a former detailed SMV classification system. As the P3 gene is thought to be involved in viral replication, systemic infection, pathogenicity, and overcoming resistance, knowledge of the P3 gene sequences of SMV and other potyviruses would be useful in efforts to know the genetic relationships among them and control the disease. P3 gene sequences were obtained from representative isolates of the above-mentioned 16 SMV strains and were compared with other SMV strains and 16 Potyvirus species from the National Center for Biotechnology GenBank database. The P3 genes from the 16 SMV isolates are composed of 1041 nucleotides, encoding 347 amino acids, and share 90.7-100% nucleotide （NT） sequence identities and 95.1-100% amino acid （AA） sequence identities. The P3 coding regions of the 16 SMV isolates share high identities （92.4-98.9% NT and 96.0-100% AA） with the reported Korean isolates, followed by the USA isolates （88.5-97.9% NT and 91.4-98.6% AA）, and share low identities （80.5-85.2% NT and 82.1-84.7% AA） with the reported HZ 1 and P isolates from Pinellia ternata. The sequence identities of the P3 genes between SMV and the 16 potyviruses varied from 44.4 to 81.9% in the NT sequences and from 21.4 to 85.3% in the AA sequences, respectively. Among them, SMV was closely related to Watermelon mosaic virus （WMV）, with 76.0-81.9% NT and 77.5-85.3% AA identities. In addition, the SMV isolates and potyvirus species were clustered into six distinct groups. All the SMV strains isolated from soybean were clustered in Group I, and the remaining species were clustered in other groups. A multiple sequence alignment analysis of the C-terminal regions indicated that the P3 genes within a species were highly conserved, whereas those among species were relatively variable.

葫芦巴碱调节PI3K/Akt/NF-κB信号通路对变应性接触性皮炎大鼠免疫反应的影响

目的探讨葫芦巴碱对变应性接触性皮炎(ACD)大鼠免疫、炎性反应及PI3K/Akt/NF-κB信号通路的影响。方法60只SD大鼠随机分为正常组、ACD组、葫芦巴碱(低、中、高)剂量组(20、40、80 mg/kg)和PI3K抑制剂(LY294002)组(40 mg/kg),每组10只。除正常组外,其余组大鼠采用2,4二硝基氟苯(DNFB)诱导ACD模型。给药结束后,通过录像观察大鼠挠痒行为;HE染色检测大鼠耳皮肤组织病理学变化;ELISA检测大鼠血清IgE及Th1、Th2、Th17型细胞因子(IFN-γ、IL-4、IL-17)水平;Western blot检测大鼠耳皮肤组织中炎性因子(IL-1β、IL-6)蛋白及PI3K/Akt/NF-κB信号通路相关蛋白表达。结果与正常组比较,ACD组大鼠挠痒次数增加,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值升高(P<0.05),耳皮肤组织角化过度且可见大量炎性细胞浸润;与ACD组比较,葫芦巴碱(低、中、高)剂量组和LY294002组大鼠挠痒次数减少,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值降低(P<0.05),耳皮肤组织病理损伤均有所改善,且葫芦巴碱各给药组呈剂量依赖效应;葫芦巴碱高剂量组和LY294002组大鼠上述指标比较,差异无统计学意义(P>0.05)。结论葫芦巴碱可抑制ACD大鼠皮肤组织中PI3K/Akt/NF-κB信号通路激活,调控Th1、Th2、Th17型免疫应答并减轻皮肤局部和全身炎性反应。

S100P、GPC3在胃癌中的表达及其与病理特征的关系

目的研究S100钙结合蛋白(S100P)、磷脂酰肌醇蛋白多糖3(GPC3)在胃癌中的表达水平及其与病理特征的关系。方法选取2020年1月至2022年12月于河南科技大学第一附属医院行手术治疗的95例胃癌患者作为胃癌组,同期选取60例于医院体检的健康人群作为对照组。采用酶联免疫吸附法检测两组受检者的血清S100P、GPC3水平;取胃癌患者术中胃癌组织及癌旁组织,采用试剂盒检测两组织中增殖与侵袭基因[蛋白酪氨酸磷酸酶(PTEN)、存活蛋白(survivin)、细胞周期蛋白依赖性激酶抑制剂1(p21)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)]含量;采用Pearson法分析S100P、GPC3水平与胃癌增殖与侵袭基因表达的相关性,分析S100P、GPC3水平与患者临床病理特征关系,采用Logistic回归模型分析胃癌病理分级的危险因素。结果胃癌组患者的血清S100P水平为(11.87±3.49)μg/L,明显低于对照组的(17.53±4.11)μg/L,GPC3水平为(5.29±1.71)ng/mL,明显高于对照组的(3.31±0.54)ng/mL,差异均有统计学意义(P<0.05);胃癌组织中的Survivin蛋白含量为(77.56±10.35)ng/mg,明显高于癌旁组织的(45.28±7.14)ng/mg,PTEN、p21、Caspase-3蛋白含量分别为(24.17±4.53)ng/mg、(30.48±4.57)ng/mg、(11.36±1.62)ng/mg,明显低于癌旁组织的(38.45±6.19)ng/mg、(47.82±7.20)ng/mg、(18.75±2.46)ng/mg,差异均有统计学意义(P<0.05);Pearson法分析结果显示,Survivin蛋白表达与S100P呈负相关(r=-0.693,P<0.05),与GPC3呈正相关(r=0.686,P<0.05),PTEN、p21、Caspase-3蛋白表达与S100P呈正相关(r=0.704、0.667、0.652,P<0.05),与GPC3呈负相关(r=-0.745、-0.722、-0.693,P<0.05);S100P、GPC3表达水平与患者TNM分期、病情严重程度有关(P<0.05);Logistic回归分析结果显示,S100P、GPC3表达水平为影响胃癌病理分级的危险因素(P<0.05)。结论胃癌患者血清S100P水平下降,GPC3水平上升,两者与病理分级、增殖侵袭基因表达密切相关,可作为评估胃癌早期的有效生物标志物。

Long non-coding RNA DPP10-AS1 represses the proliferation and invasiveness of glioblastoma by regulating miR-24-3p/CHD5 signaling pathway

This investigation aimed to unveil new prospective diagnosis-related biomarkers together with treatment targets against glioblastoma.Methods:The expression levels of long non-coding RNA(lncRNA)DPP10-AS1 were assessed using real-time quantitative polymerase chain reaction(RT-qPCR)within both the patient tissue specimens and glioblastoma cell lines.The relationship between lncRNA DPP10-AS1 expression in glioblastoma and patient prognosis was investigated.Cell Counting Kit-8(CCK-8),transwell,and clonogenic experiments were utilized to assess tumor cells’proliferation,invasiveness,and migratory potentials after lncRNA DPP10-AS1 expression was up or down-regulated.Using an online bioinformatics prediction tool,the intracellular localization of lncRNA DPP10-AS1 and its target miRNA were predicted,and RNA-FISH verified results.A dual-luciferase reporter experiment validated the relationship across miR-24-3p together with lncRNA DPP10-AS1.MiR-24-3p expression within glioblastoma was identified through RT-qPCR,and potential link across miR-24-3p and lncRNA DPP10-AS1 was assessed using Pearson correlation analysis.Moreover,influence from lncRNA DPP10-AS1/miR-24-3p axis upon glioblastoma cell progression was assessed in vivo via a subcutaneous xenograft tumor model.Results:The expression of lncRNA DPP10-AS1 was notably reduced in both surgical specimens of glioblastoma and the equivalent cell lines.Low level of lncRNA DPP10-AS1 in glioblastoma is following poor prognosis.The downregulation of lncRNA DPP10-AS1 in glioblastoma cells resulted in enhanced cellular proliferation,migration,and invasion capabilities,accompanied by downregulated E-cadherin and upregulated vimentin and N-cadherin.Additionally,the observed upregulation of lncRNA DPP10-AS1 demonstrated a substantial inhibitory function upon proliferation,invasion,and migratory capabilities of LN229 cells.Subcellular localization disclosed that lncRNA DPP10-AS1 had a binding site that interacted with miR-24-3p.Upregulated miR-24-3p was detected in glioblastomas,displaying an inverse correlation with lncRNA DPP10-AS1 expression.MiR-24-3p downstream target has been determined as chromodomain helicase DNA binding protein 5(CHD5).LncRNA DPP10-AS1 affected the invasion and proliferation of glioblastoma by controlling the miR-24-3p/CHD5 axis.Conclusion:The present study demonstrated that lncRNA DPP10-AS1 can inhibit the invasive,migratory,and proliferative properties of glioblastoma by regulating the miR-24-3p/CHD5 signaling pathway.Consequently,lncRNA DPP10-AS1 has potential as a tumor suppressor and might be utilized for accurate diagnosis and targeted treatments of glioblastomas.

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.

Role of microRNA-21 in uveal melanoma cell invasion and metastasis by regulating p53 and its downstream protein

AIM： To reveal the insight mechanism of liver metastasis in uveal melanoma, we investigated cell functions of microRNA-21 in three different uveal melanoma cell lines and analyze the relationship of target gene p53 and its downstream targets which been found significant expression in our previous study.METHODS： Quantitative real-time polymerase chain reaction （qRT-PCR） was used to detect microRNA-21 expression in normal uveal tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct OCM-1, MuM-2B and M619 cell line with stable overexpression and inhibition of microRNA-21. In vitro cell function tests such as cell proliferation, cell apoptosis, cell circle and abilities of migration and invasion were examined by MTT, BrdU assay, flow cytometry, transwell assay and Matrigel invasion assay respectively. The target gene was predicted by bioinformatics and confirmed by using a dual luciferase reporter assay. The expression of p53 and its suspected downstream targets LIM and SH3 protein 1 （LASP1） and Glutathione S Transferase pi （GST-Pi） were determined by qRT-PCR in mRNA level and western blotting analysis in protein level. Finally, the effect of microRNA-21 in a xenograft tumor model was assessed in four-week-old BALB/c nude mice. RESULTS： Compared to normal uveal melanoma, expressions of microRNA-21 were significantly higher in uveal melanoma cell lines. Overexpression of microRNA-21 promoted proliferation, migration, and invasion of OCM-1, M619 and MuM-2B cells, while inhibition of microRNA-21 reveal opposite effects. Wild type p53 was identified as a target gene of microRNA-21-3p, and proved by dual luciferase reporter assay. Up-regulated microRNA-21 inhibited the expression of wild type p53 gene, and the increased expression of LASP1 in mRNA level and protein level, while down-regulated microRNA-21 presented opposite way. However, GST-pi showed the potential pattern as expected, but relative mRNA level showed no statistically significant difference in OCM-1 cells. Furthermore, the mRNA expression of GST-pi was decreased in microRNA-21 overexpressing MuM-2B, and increased in M619 cells with inhibition of microRNA-21. In vivo, inhibition of microRNA-21 reduced tumor growth with statistically significant difference.CONCLUSION： These findings provide novel insight into molecular etiology of microRNA-21 in uveal melanoma cell lines, and suggest that microRNA-21 might be a potential candidate for the diagnosis and prognostic factor of human uveal melanoma in future.

活性维生素D3对UUO模型大鼠P38MAPK信号通路的影响

目的探讨活性维生素D 3对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的影响及其机制。方法54只雄性SD大鼠随机分为UUO模型组、假手术组、活性维生素D 3干预组,其中36只SD大鼠建立UUO模型,活性维生素D 3干预组在建模起给予活性维生素D 30.5μg/d灌胃,分别于术后3、7、14 d各处死6只大鼠,免疫组化观察肾小管间质损害程度;Western印迹法检测P38MAPK及磷酸化P38MAPK蛋白表达量。结果与假手术组相比,UUO模型组磷酸化P38MAPK蛋白表达量随时间延长显著增加(P<0.01);活性维生素D 3干预组7 d、14 d磷酸化P38MAPK蛋白表达量较UUO模型组表达显著减少(P<0.05);P38MAPK蛋白表达量各组差异无统计学意义。结论活性维生素D 3抑制UUO大鼠肾组织中P38MAPK信号通路的激活,延缓肾间质纤维化。

P53蛋白在子宫颈癌中的高表达

利用免疫组织化学方法，对３９例子宫颈癌（５例原位癌。３例早期浸润癌，３１例浸润癌）组织石腊包理标本进行了Ｐ５３表达的检测，结果发现３９例子宫颈癌标本中．１９例有Ｐ５３的高表达（４８．７％）（２例原位癌、且例早期浸润癌、１６例浸润癌）。还发现Ｐ５３高表达阳性率在分化较好的浸润癌相对较低（４２．９％），在分化较差的浸润癌相对较高（７５％）。这些结果表明Ｐ５３的高表达在子宫颈癌的发生中是一个比较常见的基因改变，Ｐ５３高表达的阳性率与子宫颈癌的分化程度有关。

Pgp1和FoxP3在结直肠癌中的表达及其与预后的关系

目的探讨P-糖蛋白1(Pgp1)和核转录因子蛋白3(FoxP3)在结直肠癌中的表达及其与预后的关系。方法选取2012年2月至2015年2月我院保存的结直肠癌标本106例,同时选取癌旁组织标本64例作为对照,采用免疫组化染色法检测Pgp1和FoxP3表达,同时根据Pgp1和FoxP3表达情况将患者分为Pgp1和FoxP3双阴性表达(A组),Pgp1单阳性表达(B组)、FoxP3单阳性表达(C组),Pgp1和FoxP3双阳性表达(D组)。结果结直肠癌组织Pgp1和FoxP3蛋白阳性表达率分别为63.21%和43.40%,明显高于癌旁组织(P<0.05);结肠癌Pgp1蛋白阳性表达率为78.95%,明显高于直肠癌(P<0.05);FoxP3表达与患者临床病理特征以及治疗方案无明显关系(P>0.05);B组中位总体生存时间为52个月(95%CI:40.01～63.99),明显高于A组、D组和C组(P<0.05);A组和D组中位总体生存时间为40个月(95%CI:31.20～48.80)和34个月(95%CI:30.74～37.26),明显高于C组的24个月(95%CI:18.56～29.44),比较差异具有统计学意义(P<0.05)。结论 Pgp1和FoxP3表达与结直肠癌临床病理特征关系较弱,其中仅Pgp1表达与肿瘤位置有一定相关性;Pgp1和FoxP3表达与结直肠癌患者预后有一定关系,其中Pgp1阳性表达,FoxP3阴性表达者预后较好。

Research progress of hsa-miR-495-3p and proteolipid protein 1 involvement in the pathogenesis of schizophrenia

Schizophrenia(SCZ)is a serious mental illness whose etiology and pathogenesis are not yet clear.The level of miRNA may be a crucial factor in the occurrence and development of SCZ.This study found that miR-495 may regulate the susceptibility gene of SCZ and that proteolipid protein 1(PLP1)as a risk gene for schizophrenia may be involved in its pathogenesis.In this article we review the research progress related to hsa-miR-495-3p(miR-495),PLP1,and schizophrenia.

补肾活血方对兔NPMSCs移植治疗退变椎间盘P 38 MAPK信号通路的影响

目的探讨补肾活血方对兔髓核间充质干细胞(nucleus pulposus-derived mesenchymal stem cells,NPMSCs)移植治疗退变椎间盘P 38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,P 38 MAPK)信号通路的影响。方法选取128只SPF级健康新西兰大白兔,其中2例用于NPMSCs的获取、纯化及培养,选取24只为假手术组(A组);兔退变椎间盘模型成功100只,模型组(B组,24只)、补肾活血方组(C组,24只)、NPMSCs移植组(D组,24只)、补肾活血方+NPMSCs移植组(E组,24只),死亡6只。每组在治疗前、治疗后1周、治疗后2周、治疗后3周4个时间点均处死6只兔,取相应椎间盘,采用酶联免疫测定每组大兔不同时间点椎间盘P 38 MAPK、基质金属蛋白酶3(matrix metalloproteinase 3,MMP-3)、基质金属蛋白酶7(matrix metalloproteinase 7,MMP-7)的表达。结果(1)各组变化趋势:A组、B组组内不同时间点P 38 MAPK、MMP-3、MMP-7蛋白相对表达差异无统计学意义(P>0.05);C组、D组、E组P 38 MAPK、MMP-3、MMP-7蛋白相对表达与治疗前、治疗后1周、治疗后2周、治疗后3周呈下降趋势,各组组内差异有统计学意义(P<0.01)。(2)与A组比较:B组、C组、D组、E组P38MAPK、MMP-3、MMP-7蛋白相对表达在治疗前、治疗后1周、治疗后2周、治疗后3周均升高,组间差异有统计学意义(P<0.01)。(3)与B组比较:C组、D组、E组P 38 MAPK、MMP-3、MMP-7蛋白相对表达在治疗前差异无统计学意义(P>0.05);在治疗后1周、治疗后2周、治疗后3周均降低,组间差异有统计学意义(P<0.01)。(4)与C组比较:D组P 38 MAPK、MMP-3、MMP-7蛋白相对表达在治疗前、治疗后1周、治疗后2周、治疗后3周时同期差异无统计学意义(P>0.05);E组P 38 MAPK、MMP-3、MMP-7蛋白相对表达在治疗后1周、治疗后2周、治疗后3周均降低,组间差异有统计学意义(P<0.01)。(5)与D组比较:E组P 38 MAPK、MMP-3、MMP-7蛋白相对表达在治疗后1周、治疗后2周、治疗后3周均降低,组间差异有统计学意义(P<0.01)。结论补肾活血方对兔NPMSCs移植治疗退变椎间盘具有协同作用;其机制可能与抑制P 38 MAPK信号通路,下调MMP-3、MMP-7蛋白相关。

Caspase-3 GST Pgp在非小细胞肺癌中的表达及其临床意义

目的探讨非小细胞肺癌(non-small-cell lung cancer,NSCLC)中谷胱甘肽S-转移酶-π(glutathione transferase-πGST-π)、P-糖蛋白(P-glucose protein)与半胱氨酸蛋白酶(Caspase-3)之间的关系以及他们的表达与临床意义。方法采用免疫组织化学法检测117例NSCLC中GST-πPgp的表达水平,流式细胞技术检测Caspase-3的表达;根据肿瘤耐药机制的不同,选择一个凋亡抑制元素与两个耐药元素对肿瘤的耐药进行多方面的分析评估。结果GST-πPgp的阳性率明显高于对照组(P<0.01),PgP与肿瘤的分型无关(P>0.05),两者在分期分化表达中随其差别增大、差异性增加,而产生统计学意义(P<0.05),Pgp在淋巴转移中高表达,GST-π与肿瘤的分型、分期、转移均无关(P>0.05);在分化表达中与前者相同。Caspase-3定量分析中,癌组与对照组、鳞癌与腺癌组、高中低分化组、淋巴转移与无淋巴转移组均有不同程度的统计学意义。结论Pgp高表达时对肿瘤的原发耐药增强,结合Caspase-3定量分析,凋亡指数均有不同程度的衰减。通过生物学行为研究,了解凋亡抑制是肿瘤耐药的主要原因,这有助于临床正确制订初始化疗方案和判断预后。

特异性p^(38)MAPK抑制剂SB203580对乳鼠小脑颗粒神经元的保护作用

目的 研究 p38丝裂原激活蛋白激酶 (MAPK)选择性抑制剂SB2 0 35 80对乳鼠小脑颗粒神经元凋亡的保护作用。方法 SD乳鼠小脑颗粒神经元培养 ,琼脂糖凝胶电泳 ,SAPK/JNK分析试剂盒作激酶分析。结果 PI 3 K的特异性抑制剂LY2 940 0 2诱导小脑颗粒神经元凋亡 ,但SB2 0 35 80通过抑制细胞凋亡而促进小脑颗粒神经元的存活 ,且有浓度依赖性。LY2 940 0 2诱导凋亡的颗粒神经元中c Jun的表达量和磷酸化水平均升高 ,JNK被激活。但是 ,当小脑颗粒神经元生长在含SB2 0 35 80的高钾培养基中 ,c Jun的表达量、磷酸化水平和JNK的活性都明显的降低。结论 SB2 0 35 80通过抑制JNK的活性 ,降低c Jun的表达和磷酸化水平 。

异甘草素通过PI3K/Akt通路诱导人胃癌SGC7901细胞凋亡

目的:初步研究异甘草素(isoliquiritigenin ISL)对人胃癌SGC7901细胞凋亡诱导作用的影响,并探讨信号通路中蛋白质丝氨酸苏氨酸激酶(protein kinase B,Akt)、磷酸化Akt(PAkt)及下游凋亡蛋白Bax表达水平的变化.方法:取对数生长期人胃癌SGC7901细胞分为对照组、ISL实验组,培养24、48、72 h后采用MTT实验检测ISL对细胞生长的抑制情况,摸索后续实验药物浓度和作用时间;应用流式细胞仪检测各组细胞凋亡情况;应用Western blot法检测凋亡调节蛋白Bax、Ak和P-Akt的表达.结果:10mmol/L ISL不能抑制胃癌细胞株SGC7901的生长,而25、50、100mm o l/LISL可明显抑制其生长,且呈时间和浓度依赖性.与对照组凋亡率3.23%±0.45%相比,25mmol/L组(6.13%±0.61%)、50mmol/L组(11.70%±0.75%)、100mmol/L组(26.60%±1.51%)凋亡率逐渐增高(P<0.05)随ISL浓度的增加,P-Akt蛋白表达水平逐渐降低,凋亡蛋白Bax表达水平逐渐增加(均P<0.05),各组间A k t蛋白水平差异无统计学意义.结论:ISL能够诱导SGC7901细胞凋亡,其机制可能与下调PI3K/AKT信号转导通路蛋白的表达以及上调其下游的凋亡蛋白Bax有关.

circ-HIPK3通过调控miR-1207-5p促进结直肠癌细胞增殖和转移的作用机制

目的探究环状RNA同源域相互作用蛋白激酶3(circ-HIPK3)通过调控微小RNA-1207-5p(miR-1207-5p)促进结直肠癌(CRC)细胞增殖和转移的作用机制。方法体外培养人CRC细胞系HCT166细胞,分别以pcDNA3.1-NC(空白组)与pcDNA3.1-circ-HIPK3(研究组)转染HCT166细胞48 h,于倒置荧光显微镜下观察转染效率,以不做任何处理的HCT166细胞作为对照组;采用荧光定量PCR检测细胞circ-HIPK3及miR-1207-5p mRNA表达,采用MTT实验及划痕实验分别检测细胞增殖及迁移能力,双荧光素酶实验检测circ-HIPK3与miR-1207-5p的靶向作用,Western blot法检测Wnt/β-catenin通路蛋白表达情况。结果研究组细胞增殖促进率、划痕愈合率、细胞中circ-HIPK3、miR-1207-5p mRNA相对表达水平及Wnt、β-catenin蛋白表达水平均高于对照组和空白组,差异有统计学意义(P<0.05),而对照组与空白组上述各项指标比较差异无统计学意义(P>0.05);双荧光素酶实验结果显示circ-HIPK3上存在miR-1207-5p的结合位点。结论circ-HIPK3通过下调CRC中miR-1207-5p的相对表达,促进细胞增殖和转移,其作用机制可能与激活Wnt/β-catenin通路有关。

PI3K-Akt信号通路对人大肠癌hct-8/FU耐药细胞P-GP表达和耐药性的影响

目的:研究磷脂酰肌醇3-激酶/蛋白激酶B(PI3K-Akt)信号通路对人大肠癌hct-8/FU耐药细胞P-糖蛋白(P-GP)表达和耐药性的影响,探讨其逆转hct-8/FU多药耐药性的作用。方法:MTT法检测人大肠癌hct-8细胞、hct-8/FU耐药细胞、PI3K-Akt通路抑制剂LY294002处理的hct-8/FU耐药细胞对5-FU的半数生长抑制率和耐药指数;蛋白质印迹法检测hct-8、hct-8/FU细胞、LY294002处理的hct-8/FU耐药细胞PI3K、Akt、P-Akt、P-GP的表达变化。结果:大肠癌hct-8细胞对5-FU的半数生长抑制率为(43.2±1.4)mg.L-1,hct-8/FU耐药细胞为(516.00±20.03)mg.L-1,耐药指数为11.9;LY294002处理hct-8/FU耐药细胞后半数生长抑制率为(58.2±4.3)mg.L-1,耐药指数为1.37,较用药前对5-FU的敏感性提高,逆转指数为8.8(P<0.01);hct-8/FU耐药细胞PI3K、Akt、P-Akt、P-GP表达较hct-8细胞明显增加(均P<0.01),LY294002作用后其PI3K、Akt、P-Akt、P-GP表达较用药前明显下降(P<0.01)。结论:PI3K-AKT信号通路可能通过促进人大肠癌hct-8 P-GP的表达,增加其对5-FU的耐药性,降低肿瘤细胞对药物的敏感性。

丁苯酞对阿尔茨海默病模型大鼠海马CA1区GSK-3β及p-Tau蛋白表达的影响

目的观察丁苯酞(NBP)软胶囊对阿尔茨海默病(AD)模型大鼠海马CA1区丝氨酸蛋白激酶-3β(GSK-3β)、p-Tau蛋白表达的影响,探讨NBP治疗AD的机制。方法将72只雄性SD大鼠随机分为假手术组(24只)、AD模型组(24只)、NBP治疗组(24只)。采用β-淀粉样蛋白1-42(Aβ1-42)制作AD模型,假手术组注射5μL生理盐水。造模成功后4周,NBP治疗组给予NBP与食用油混合后制成的NBP混合溶液灌胃;假手术组和AD模型组采用同等剂量的食用油进行灌胃。采用Morris水迷宫实验比较各组大鼠穿越平台次数。采用Western blot法分别于给药后1、2、4和8周检测海马CA1区GSK-3β、p-Tau蛋白的表达情况。结果与假手术组比较,AD模型组大鼠各时间点穿越平台次数显著减少(均P<0.05);与AD模型组比较,NBP治疗组各时间点大鼠穿越平台次数均增加(均P<0.05);与假手术组同期比较,AD模型组大鼠海马CA1区p-Tau、GSK-3β蛋白相对表达水平升高,差异有高度统计学意义(P<0.01);与AD模型组同期比较,NBP治疗组大鼠海马CA1区p-Tau、GSK-3β蛋白相对表达水平降低,差异有高度统计学意义(P<0.01);与假手术组同期比较,NBP治疗组大鼠海马CA1区p-Tau、GSK-3β蛋白相对表达水平升高,差异有高度统计学意义(P<0.01)。结论NBP可通过下调GSK-3β、p-Tau蛋白的表达,发挥对AD模型大鼠的脑保护作用。

miR-214-3p对缺糖缺氧再灌注诱导PC12细胞凋亡的作用及机制

目的探讨miR-214-3 p对缺糖缺氧再灌注诱导PC12细胞凋亡的作用及机制。方法利用数据库TargetScan 7.2预测miR-214-3 p和B淋巴细胞瘤因子2样蛋白2(BCL2L2)之间的结合位点;选取人胚肾293T细胞共转染分为BCL2L2 mRNA 3′UTR野生型荧光素酶报告载体+miR-214-3 p mimic(wt+mimic)组、BCL2L2 mRNA 3′UTR野生型荧光素酶报告载体+mimic negative control(wt+NC)组、BCL2L2 mRNA 3′UTR突变型荧光素酶报告载体+miR-214-3 p mimic(mut+mimic)组及BCL2L2 mRNA 3′UTR突变型荧光素酶报告载体+mimic NC(mut+NC组),检测4组293T细胞荧光素酶的发光活性;肾上腺嗜铬神经细胞瘤12(PC12)细胞转染miR-214-3 p mimic(mimic组)、miR-214-3 p mimic NC(mimic NC组)、miR-214-3 p inhibitor(inhibitor组),miR-214-3 p inhibitor NC(inhibitor NC组),采用蛋白免疫印迹法检测BCL2L2蛋白表达;PC12细胞作为对照(Control组),用缺氧缺糖(OGD/R)处理PC12细胞构建缺血再灌注细胞模型(OGD/R组),用miR-214-3 p mimic转染PC12细胞,然后用OGD/R处理PC12细胞(OGD/R+mimic组),采用实时荧光定量聚合酶链式反应检测Control组和OGD/R组miR-214-3 p及BCL2L2 mRNA表达,采用蛋白免疫印迹法检测Control组、OGD/R组及OGD/R+mimic组裂解胱天蛋白水解酶3(Cleaved-caspase3)、B淋巴细胞瘤-2相关X(Bax)及B淋巴细胞瘤因子2(Bcl2)的蛋白表达。结果数据库Targetscan 7.2预测结果表明,miR-214-3 p与BCL2L2的3′UTR区之间存在结合位点;荧光素酶报告基因实验结果表明,wt+mimic组荧光素酶活性低于wt+NC组,mut+mimic组和mut+NC中荧光素酶活性无明显差异;mimic NC组BCL2L2的蛋白表达高于mimic组,inhibitor NC组BCL2L2的蛋白表达低于inhibitor组(P<0.05);与Control组比较,OGD/R组PC12细胞中BCL2L2 mRNA和蛋白表达水平降低、miR-214-3 p mRNA表达升高,差异有统计学意义(P<0.05);OGD/R组Cleaved-caspase-3和Bax表达分别高于Control组、低于OGD/R+mimic组,但Bcl2表达分别低于Control组、高于OGD/R+mimic组(P<0.05)。结论过表达miR-214-3 p可靶向结合并下调BCL2L2的表达,从而加速PC12细胞的凋亡。