Helicobacter pylori induces epithelial-mesenchymal transition in gastric carcinogenesis via the AKT/GSK3β signaling pathway

Helicobacter pylori (H. pylori) is a main risk factor for gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is involved in the development and progression of H. pylori-associated GC. However, the exact molecular mechanism of this process remains unclear. The AKT/GSK3β signaling pathway has been demonstrated to promote EMT in several types of cancer. The present study investigated whether H. pylori infection induced EMT, and promoted the development and metastasis of cancer in the normal gastric mucosa, and whether this process was dependent on AKT activation. The expression levels of the EMT-associated proteins, including E-cadherin and N-cadherin, were determined in 165 gastric mucosal samples of different disease stages by immunohistochemical analysis. The expression levels of E-cadherin, N-cadherin, AKT, phosphorylated (p-)AKT (Ser473), GSK3β and p-GSK3β (Ser9) were further determined in H. pylori-infected Mongolian gerbil gastric tissues and cells co-cultured with H. pylori by immunohistochemical analysis and western blotting. The results indicated that the expression levels of the epithelial marker E-cadherin were decreased, whereas the expression levels of the mesenchymal marker N-cadherin were increased during gastric carcinogenesis. Their expression levels were associated with H. pylori infection. Furthermore, H. pylori infection resulted in downregulation of E-cadherin expression and upregulation of N-cadherin expression in Mongolian gerbils and GES-1 cells. In addition, an investigation of the associated mechanism of action revealed that p-AKT (Ser473) and p-GSK3β (Ser9) were activated in GES-1 cells following co-culture with H. pylori. Furthermore, following pretreatment of the cells with the AKT inhibitor VIII, the expression levels of E-cadherin, N-cadherin, p-AKT and p-GSK3β did not show significant differences between GES-1 cells that were co-cultured with or without H. pylori. The levels of p-AKT and p-GSK3β were increased in H. pylori-infected Mongolian gerbils. In conclusion, the present study demonstrated that H. pylori infection activated AKT and resulted in the phosphorylation and inactivation of GSK3β, which in turn promoted early stage EMT. These effects were AKT-dependent. This mechanism may serve as a prerequisite for GC development.     

Absence of FGFR3–TACC3 rearrangement in hematological malignancies with numerical chromosomal alteration

FGFR–TACC, found in different tumor types, is characterized by the fusion of a member of fibroblast grown factor receptor (FGFR) tyrosine kinase (TK) family to a member of the transforming acidic coiled-coil (TACC) proteins. Because chromosome numerical alterations, hallmarks of FGFR–TACC fusions are present in many hematological disorders and there are no data on the prevalence, we studied a series of patients with acute myeloid leukemia and myelodysplastic syndrome who presented numerical alterations using cytogenetic traditional analysis. None of the analyzed samples showed FGFR3–TACC3 gene fusion, so screening for this mutation at diagnosis is not recommended.     

Characterization of membrane occupation and recognition nexus repeat containing 3, meiosis expressed gene 1 binding partner,in mouse male germ cells

Mammalian spermatogenesis is a well-organized process of cell development and differentiation. Meiosis expressed gene 1 (MEIG1) plays an essential role in the regulation of spermiogenesis. To explore potential mechanisms of MEIG1''s action, a yeast two-hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 (MORN3). MORN3 mRNA is only abundant in mouse testis. In the testis, Morn3 mRNA is highly expressed in the spermiogenesis stage. Specific anti-MORN3 polyclonal antibody was generated against N-terminus of the full-length MORN3 protein, and MORN3 expression and localization was examined in vitro and in vivo. In transfected Chinese hamster ovary cells, the antibody specifically crossed-reacted the full-length MORN3 protein, and immunofluorescence staining revealed that MORN3 was localized throughout the cytoplasm. Among multiple mouse tissues, about 25 kDa protein, was identified only in the testis. The protein was highly expressed after day 20 of birth. Immunofluorescence staining on mixed testicular cells isolated from adult wild-type mice demonstrated that MORN3 was expressed in the acrosome in germ cells throughout spermiogenesis. The protein was also present in the manchette of elongating spermatids. The total MORN3 expression and acrosome localization were not changed in the Meig 1-deficient mice. However, its expression in manchette was dramatically reduced in the mutant mice. Our studies suggest that MORN3 is another regulator for spermatogenesis, probably together with MEIG1.     

Melatonin induces apoptosis of colorectal cancer cells through HDAC4 nuclear import mediated by CaMKII inactivation

Melatonin induces apoptosis in many different cancer cell lines, including colorectal cancer. However, the precise mechanisms involved remain largely unresolved. In this study, we provide evidence to reveal a new mechanism by which melatonin induces apoptosis of colorectal cancer LoVo cells. Melatonin at pharmacological concentrations significantly suppressed cell proliferation and induced apoptosis in a dose‐dependent manner. The observed apoptosis was accompanied by the melatonin‐induced dephosphorylation and nuclear import of histone deacetylase 4 (HDAC4). Pretreatment with a HDAC4‐specific siRNA effectively attenuated the melatonin‐induced apoptosis, indicating that nuclear localization of HDAC4 is required for melatonin‐induced apoptosis. Moreover, constitutively active Ca²⁺/calmodulin‐dependent protein kinase II alpha (CaMKIIα) abrogated the melatonin‐induced HDAC4 nuclear import and apoptosis of LoVo cells. Furthermore, melatonin decreased H3 acetylation on bcl‐2 promoter, leading to a reduction of bcl‐2 expression, whereas constitutively active CaMKIIα(T286D) or HDAC4‐specific siRNA abrogated the effect of melatonin. In conclusion, the present study provides evidence that melatonin‐induced apoptosis in colorectal cancer LoVo cells largely depends on the nuclear import of HDAC4 and subsequent H3 deacetylation via the inactivation of CaMKIIα.     

Dietary n-3 long chain PUFA supplementation promotes a pro-resolving oxylipin profile in the brain

The brain is highly enriched in long chain polyunsaturated fatty acids (LC-PUFAs) that display immunomodulatory properties in the brain. At the periphery, the modulation of inflammation by LC-PUFAs occurs through lipid mediators called oxylipins which have anti-inflammatory and pro-resolving activities when derived from n-3 LC-PUFAs and pro-inflammatory activities when derived from n-6 LC-PUFAs. However, whether a diet rich in LC-PUFAs modulates oxylipins and neuroinflammation in the brain has been poorly investigated. In this study, the effect of a dietary n-3 LC-PUFA supplementation on oxylipin profile and neuroinflammation in the brain was analyzed. Mice were given diets deficient or supplemented in n-3 LC-PUFAs for a 2-month period starting at post-natal day 21, followed by a peripheral administration of lipopolysaccharide (LPS) at adulthood. We first showed that dietary n-3 LC-PUFA supplementation induced n-3 LC-PUFA enrichment in the hippocampus and subsequently an increase in n-3 PUFA-derived oxylipins and a decrease in n-6 PUFA-derived oxylipins. In response to LPS, n-3 LC-PUFA deficient mice presented a pro-inflammatory oxylipin profile whereas n-3 LC-PUFA supplemented mice displayed an anti-inflammatory oxylipin profile in the hippocampus. Accordingly, the expression of cyclooxygenase-2 and 5-lipoxygenase, the enzymes implicated in pro- and anti-inflammatory oxylipin synthesis, was induced by LPS in both diets. In addition, LPS-induced pro-inflammatory cytokine increase was reduced by dietary n-3 LC-PUFA supplementation. These results indicate that brain n-3 LC-PUFAs increase by dietary means and promote the synthesis of anti-inflammatory derived bioactive oxylipins. As neuroinflammation plays a key role in all brain injuries and many neurodegenerative disorders, the present data suggest that dietary habits may be an important regulator of brain cytokine production in these contexts.     

灯盏乙素介入IP3R-Ca2+途径抑制β淀粉样蛋白诱导的神经细胞凋亡

目的：观察灯盏乙素（Scu）对β淀粉样蛋白（Aβ）诱导的细胞模型中1，4，5-三磷酸肌醇受体（IP₃R）-Ca²⁺途径的影响，探讨其在阿尔茨海默病（AD）病程中可能发挥的积极作用。方法：选用神经母细胞瘤细胞分为对照组、Scu处理组、Aβ处理组、Aβ+Scu （高、中、低）处理组及Aβ+IP₃R拮抗剂组，用CCK-8法筛选药物浓度并检测各组细胞生存率；用酶联免疫吸附法检测各组细胞中1，4，5-三磷酸肌醇（IP₃）的含量；用蛋白印迹和实时荧光定量聚合酶链反应方法检测各组细胞IP₃R和凋亡相关因子Caspase-3、Bcl-2、Bax的蛋白及mRNA的表达水平；用激光共聚焦显微镜观察各组细胞内Ca²⁺浓度的变化；用AnnexinV/PI双染法测定各组细胞的凋亡率。结果：与对照组和Scu处理组相比，Aβ处理组细胞存活率下降，IP₃含量升高，IP₃R、Bax和Caspase-3的蛋白及mRNA表达上调，Bcl-2蛋白及mRNA的表达下调，细胞胞浆内Ca²⁺浓度及细胞凋亡率升高；Aβ+Scu处理组细胞中各检测指标的变化与Aβ处理组的结果正好相反，IP₃R通道下游指标的变化与Aβ+IP₃R拮抗剂组基本一致。结论：Scu能够下调通路蛋白IP₃、IP₃R的表达，抑制Aβ介导的Ca²⁺内流所致的细胞凋亡，可能通过对IP₃R-Ca²⁺途径的调控来影响AD病程。     

Investigation of non-linear Mate1-mediated efflux of trimethoprim in the mouse kidney as the mechanism underlying drug-drug interactions between trimethoprim and organic cations in the kidney

The purpose of this study was to elucidate the involvement of Mate1 in the tubular secretion of trimethoprim and saturation of Mate1-mediated efflux to address the mechanisms underlying the pharmacokinetic drug interactions with trimethoprim. Trimethoprim is a more potent inhibitor of MATE2-K than MATE1 with K_i values (μM) of 0.030–0.28 and 2.4–5.9, respectively. Trimethoprim is a substrate of human MATE1 and MATE2-K with K_m values of 2.3 ± 0.9 and 0.018 ± 0.004 μM, and mouse Mate1, but not human OCT2, mouse Oct1 and Oct2. Pyrimethamine significantly reduced the renal clearance (CL_R) of trimethoprim (mL/min/kg) from 40.0 ± 5.1 to 20.1 ± 3.7 (p < 0.05). Trimethoprim was given to mice at three infusion rates (150, 500, and 1500 nmol/min/kg). Together with an increase in the plasma concentrations of trimethoprim, the CL_R (mL/min/kg) of trimethoprim decreased to 25.9 ± 3.2, 13.5 ± 5.7, and 8.92 ± 1.50 at the respective rates. Trimethoprim decreased the CL_R of rhodamine 123 in an infusion rate-dependent manner: 11.5 ± 1.3 (control), 5.17 ± 1.55, 1.31 ± 0.50, and 0.532 ± 0.180. These results suggest that Mate1 mediates the tubular secretion of trimethoprim, and at therapeutic doses, MATEs-mediated efflux can be saturated, and thereby, cause drug interactions with other MATE substrates.     

Synthesis,in vivo and in silico evaluation of novel 2,3-dihydroquinazolin-4(1H)-one derivatives as potential anticonvulsant agents

In light of the pharmacophoric structural requirements for achieving anticonvulsant activity, a series of N-(1-methyl-4-oxo-2-un/substituted-1,2-dihydroquinazolin-3[4H]-yl)benzamide (4a-g) and N-(1-methyl-4-oxo-2-un/substituted-1,2-dihydroquinazolin-3[4H]-yl)-2-phenylacetamide (4h-n) derivatives were synthesized in two steps starting from the reaction of N-methyl isatoic anhydride with the appropriate hydrazide and followed by condensation with the appropriate aldehyde. The anticonvulsant activities of the synthesized compounds were evaluated according to the anticonvulsant drug development (ADD) programme protocol. Among the synthesized compounds, 4n showed promising activity in both the maximal electroshock (MES) and pentylenetetrazole (PTZ) tests with median effective dose (ED₅₀) values of 40.7 and 6 mg/kg, respectively. The six most promising derivatives, 4b , 4a , 4c , 4f , 4j , and 4i , showed very low ED₅₀ values in the PTZ test (3.1, 4.96, 8.68, 9.89, 12, and 13.53 mg/kg, respectively). All the tested compounds showed no to low neurotoxicity in the rotarod test with a wide therapeutic index. Docking studies of compound 4n suggested that GABA_A binding could be the mechanism of action of these derivatives. The in silico drug likeliness parameters indicated that none of the designed compounds violate Lipinski's rule of five and that they are able to cross the blood–brain barrier.

负载IL-4及BMP-2的氧化石墨烯-羧甲基壳聚糖凝胶对骨免疫和骨修复的早期影响研究

目的探讨负载 IL-4 和 BMP-2 的氧化石墨烯（graphene oxide，GO）-羧甲基壳聚糖（carboxymethyl chitosan，CMC）凝胶诱导巨噬细胞 M2 型分化及对 BMSCs 成骨分化的影响。方法取 CMC、GO 制备混合溶液后，分别添加 PBS、IL-4、BMP-2 或 IL-4+BMP-2，在交联剂作用下制备单纯或负载不同因子的 GO-CMC 凝胶支架；取单纯 GO-CMC 凝胶表征观测，包括大体、扫描电镜及傅里叶变换红外吸收光谱仪（Fourier transform infrared spectroscopy，FTIR）检测，以单纯 CMC 凝胶作为对照；取负载不同因子的 GO-CMC 凝胶行体外缓释实验。取 4～5 周龄 SPF 级 SD 雌性大鼠分离培养巨噬细胞，分别与单纯以及负载不同因子的 GO-CMC 凝胶培养，24 h 后行 CD206 免疫荧光检测巨噬细胞分化情况；取第 3 代大鼠 BMSCs 分别与单纯以及负载不同因子的 GO-CMC 凝胶成骨诱导培养，10 d 后行 ALP 染色观测早期成骨，21 d 行茜素红染色观测晚期成骨。结果大体观察 GO-CMC 凝胶呈棕色、半透明状；扫描电镜观察示，GO-CMC 凝胶孔径及孔壁厚度与单纯 CMC 凝胶相似，但内壁粗糙度增加；FTIR 检测显示 CMC 发生聚合形成凝胶。体外缓释实验示 3 种负载不同因子的 GO-CMC 凝胶缓释性能相似，均呈线性缓慢释放因子。CD206 免疫荧光检测示 GO-CMC 凝胶可诱导巨噬细胞 M2 型分化，ALP 及茜素红染色示 GO-CMC 凝胶可诱导 BMSCs 成骨分化；其中负载 IL-4+BMP-2 的 GO-CMC 凝胶作用最显著（P<0.05）。 结论负载 IL-4 和 BMP-2 的 GO-CMC 凝胶可诱导巨噬细胞 M2 型分化，增强 BMSCs 成骨分化能力，为后期骨缺损修复及骨免疫调节研究提供了新的策略。