Berberine Induces Cell Apoptosis through Cytochrome C/Apoptotic Protease-Activating Factor 1/Caspase-3 and Apoptosis Inducing Factor Pathway in Mouse Insulinoma Cells

Objective: To investigate apoptotic effects of berberine, a significant alkaloids component existing in Rhizoma coptidis, and its possible acting mechanism in insulinoma cells. Methods: Different concentrations of berberine were used to treat mouse insulinoma(MIN6) cells for various period of time. The viability and apoptosis of the cells were analyzed using methylthiazolyldiphenvl-tetrazolium bromide assay, flow cytometry and enzyme-linked immuno sorbent assay. Changes in the relating pro-and anti-apoptosis proteins were detected by western-blotting. Results: The half-maximal inhibitory concentration(IC50) of berberine was 5.7 μmol/L on MIN6 cells viability for 16 h. Berberine caused a 20% reduction(P0.05) in cell number after only 4-h incubation; which reached 50% after 24 h(P0.01). Berberine treatment for 16 h significantly increased the level of DNA fragmentation. The flow cytometry showed the apoptotic rate increased 2.9-and 4.6-fold after treating with berberine(5 μmol/L) for 8 and 16 h, while 3-and 8.7-fold after 10 μmol/L treatment for 8 and 16 h(P0.01). Berberine treatment dramatically elevated the expression ratio of Bax to Bcl-2. Meanwhile, berberine notably increased the apoptosis-inducing factors and cytochrome C transforming from the mitochondria to the cytoplasm. Apoptotic protease-activating factor 1(Apaf-1) was subsequently activated after cytochrome C release. Furthermore, caspase-3 and poly adenosine diphosphate-ribose polymerase were also activated to trigger apoptosis cascade. Conclusion: High concentration(5 and 10 μmol/L) of berberine could induce the apoptosis of MIN6 cells through cytochrome C/Apaf-1/caspase-3 and apoptosis inducing factor(AIF) pathway.     

Liver-specific knockout of histone methyltransferase G9a impairs liver maturation and dysregulates inflammatory,cytoprotective, and drug-processing genes

1. Methyltransferase G9a is essential for a key gene silencing mark, histone H3 dimethylation at lysine-9 (H3K9me2). Hepatic G9a expression is down-regulated by xenobiotics and diabetes. However, little is known about the role of G9a in liver. Thus, we generated mice with liver-specific knockout (Liv-KO) of G9a.

2. Adult G9a Liv-KO mice had marked loss of H3K9me2 proteins in liver, without overt liver injury or infiltration of inflammatory cells. However, G9a-null livers had ectopic induction of certain genes normally expressed in neural and immune systems. Additionally, G9a-null livers had moderate down-regulation of cytoprotective genes, markedly altered expression of certain important drug-processing genes, elevated endogenous reactive oxygen species, induction of ER stress marker Chop, but decreased glutathione and nuclear Nrf2. microRNA-383, a negative regulator of the PI3K/Akt pathway, was strongly induced in G9a Liv-KO mice. After LPS treatment, G9a Liv-KO mice had aggravated lipid peroxidation and proinflammatory response.

3. Taken together, the present study demonstrates that G9a regulates liver maturation by silencing neural and proinflammatory genes but maintaining/activating cytoprotective and drug-processing genes, in which the G9a/miR-383/PI3K/Akt/Nrf2?(Chop) pathways may play important roles. G9a deficiency due to genetic polymorphism and/or environmental exposure may alter xenobiotic metabolism and aggravate inflammation and liver dysfunction.

     

miR-34c-3p与M2型肿瘤相关巨噬细胞在三阴性乳腺癌中的表达及意义

目的:检测miR-34c-3p与M2型肿瘤相关巨噬细胞（tumor-associated macrophage,TAM)在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达，并探讨其在乳腺癌发病中的意义。方法:选择2017年2月至2018年1月于西京医院就诊的68例TNBC患者作为实验组(A组)，以同期就诊的70例乳腺纤维腺瘤患者作为对照组(B组)。采用Real-time RT-PCR 检测组织标本中miR-34c-3p的表达；流式细胞检测M2型TAM的表达；ELISA法检测血清中IL-10的表达。并进一步分析miR-34c-3p的表达与乳腺癌M2型TAM及血清IL-10水平的相关性。结果:A、B组miR-34c-3p的相对表达量分别为0.84±0.08、1.29±0.71，A组明显低于B组，差异有统计学意义(P<0.05)。与B组比较，A组M2型TAM(CD68+CD163+)细胞比例显著升高(P<0.05)。A组血清IL-10的表达［(19.69±4.93) pg/ml］较B组［(17.26±3.51) pg/ml］显著升高，差异有统计学意义(P<0.05)。TNBC组织中miR-34c-3p的表达与M2型TAM比例及血清IL-10表达均呈显著负相关(r=-0.508，r=-0.656，P<0.05)。结论:TNBC组织中miR-34c-3p的表达下调，M2型TAM比例及血清IL-10表达均显著升高，促进TNBC进展。     

胶质瘤组织中miR-218的表达及其对血管生成的作用

目的 明确miR-218在胶质瘤组织中的表达，探讨其对胶质瘤血管生成的作用及机制。方法 采用荧光实时定量PCR法和免疫印迹法检测胶质瘤组织和细胞中miR-218、P70核糖体S6激酶1（p70s6k1）的表达情况，采用基质胶塞实验和小管形成实验分别在体内外检测miR-218对新血管生成的影响。结果 21例胶质瘤组织标本中miR-218表达较7例癌旁组织标本明显下调，且表达量与WHO分级有关。过表达miR-218能显著抑制血管生成。MiR-218直接靶向p70s6k1。过表达p70s6k1能部分逆转miR-218对血管新生的抑制作用。结论 MiR-218能通过靶向p70s6k1的表达调控胶质瘤血管生成，进而影响胶质瘤的进展。     

藿朴夏苓汤治疗早期新型冠状病毒肺炎（COVID-19）湿邪郁肺证物质基础及功效的网络分析

目的筛选藿朴夏苓汤治疗早期新型冠状病毒肺炎(COVID-19)湿邪郁肺证的物质基础,预测其作用机制。方法查阅文献及临床报道分析藿朴夏苓汤与早期COVID-19湿邪郁肺证的方证关系。运用TCMSP数据库筛选藿朴夏苓汤中潜在活性成分,将活性成分与新型冠状病毒(SARS-CoV-2)3CL水解酶、血管紧张素转化酶Ⅱ(ACE2)进行分子对接,根据结合能筛选与两者均有较好结合作用的核心成分。借助Cytoscape软件构建关键成分靶点蛋白互作网络,筛选出核心靶点;通过STRING数据库进行核心靶点的GO分析,Cytoscape软件Clue GO插件进行Pathway、KEGG富集分析。结果方证关系分析藿朴夏苓汤用于治疗早期COVID-19湿邪郁肺证,筛选出藿朴夏苓汤中潜在作用成分12个,核心靶点67个。其中通草中的通脱木皂苷元Ⅰ,茯苓、猪苓中的过氧麦角甾醇,半夏中的黄芩苷与SARS-CoV-2 3CL水解酶、ACE2均具有较好的结合活性。GO、Pathway、KEGG富集分析结果显示藿朴夏苓汤中12个潜在作用成分参与调节刺激反应、信号转导、细胞死亡等生物过程以及白介素信号通路、癌症EGFR信号通路、酪氨酸激酶信号转导途径、编程性细胞死亡途径、MAPK信号通路等。结论藿朴夏苓汤以化湿解毒、宣肺透邪治疗早期COVID-19湿邪郁肺证患者,菲酮、黄芩苷、酸枣仁皂苷、啤酒甾醇、常春藤皂苷元、过氧麦角甾醇、柠檬二烯醇、麦角甾-7,22-二烯-3-酮、通脱木皂苷元Ⅰ、泽泻醇B-23-醋酸酯、泽泻醇B、新橙皮苷可能为其主要的物质基础,通过阻断SARS-CoV-2病毒蛋白合成,阻止病毒进入宿主细胞,通过调控白介素信号通路、MAPK信号通路、PI3K-Akt信号通路、T细胞受体信号通路、C型凝集素受体信号通路,抑制相关炎症因子的表达发挥作用。     

基于UPLC-Q/TOF-MS分析淫羊藿炮制前后黄酮组分的变化规律

目的基于UPLC-Q/TOF-MS技术建立淫羊藿炮制前后指纹图谱,对其全成分进行分析并找出标志性化学成分,以明确淫羊藿炮制前后黄酮组分的变化规律。方法采用UPLC-Q/TOF-MS技术,在正离子模式下采集淫羊藿生品及炮制品样品数据,并在此基础上根据正交-偏最小二乘判别分析(OPLS-DA)整体探究9个不同产地、批次的淫羊藿炮制前后化学成分的差异。结果从淫羊藿生品及炮制品中寻找并鉴定出9个标志性化学成分,即8-乙烯-山柰酚、淫羊藿素、淫羊藿次苷I、淫羊藿素-3-O-葡萄糖苷、异戊醇基箭藿苷B、1,3-异戊二烯基朝藿定C、1,3-异戊二烯基-箭藿苷B-7-O-葡萄糖醛酸、3-O-(4-乙酰氧基)鼠李糖-2-O-(间二乙酰氧基)葡萄糖-淫羊藿苷及其同分异构体。结论淫羊藿炮制后黄酮组分结构发生变化,次级糖苷增加,多级糖苷减少,淫羊藿黄酮组分总体向低糖苷组分转化,进一步阐明了淫羊藿加热炮制后黄酮组分的变化规律。