Low-Temperature Signaling Pathways and Their Signaling Factors in Plant

Low temperature as abiotic stress adversely impacts plant growth and development, and limits the ecological distribution of plants as well. Throughout their long evolutionary history, plants have developed a range of complicated and precise molecular regulatory mechanisms to deal with low-temperature stress, involving the activation of signal transduction pathways and the regulation of related genes. In this review, we provide a systematic summary of the most recent research findings regarding three hypotheses of cellular perception of low-temperature signals and two major intracellular low-temperature signaling pathways, including CBF-dependent signaling pathways and CBF-independent signaling pathways. Focus is placed on the functions of each component of the ICE-CBF-COR signaling cascade as well as their interrelationships. This review concludes that although some progress has been made in the identification, function, and mechanism of low-temperature response genes, their roles in the low-temperature regulatory network and molecular mechanisms still need to be studied in detail, which will be of great significance for improving the low-temperature tolerance of plants and adapting to climate change.

Identification of rice (Oryza sativa L.) signal factors capable of inducing Agrobacterium vir gene expression

Two kinds of signal factors capable of inducing Agrobaorerium vir gene expression were purified and identified from leaf extracts of panicle-differentiating to flowering stage of rice (Oryza saliva L. cv. IR 72) detected by Agrobacterium vir(?) lacZ. fusion genes. The induction was similar to that observed with 5 μm actosyringone (AS). Based on the comprehensive analysis of the data by UV, IR, NMR, MS, HMQC and HMBC, the structures of these two signal factors are identified as 5, 7, 4’-trihydroxy-3’, 5’-dimethoxy-flavone (named tricin) and 5, 4’ -dihydroxy-3’, 5’ -dimethoxy-7- (β-D-glucosyloxy) -flavone, respectively. These results demonstrate that monocotyledonous plants do contain highly efficient vir gene inducing factors of Agrobacterium, and the reason why monocotyledonous plants are difficult to transform by Ayrobacterium is not due to absence of vir gene inducing factors, but due to the signal factors only produced in specific stage and tissue of monocotyledonous

Late SV40 factor:A key mediator of Notch signaling in human hepatocarcinogenesis

AIM:To investigate the relationship between late SV40 factor(LSF)and Notch signaling in the development and progress of hepatocellular carcinoma(HCC).METHODS:Liver cancer tissue specimens from 25 patients were analyzed for Notch-1 and LSF expression by immunohistochemistry.The correlation between expression and the biological effects of Notch-1 and LSF were analyzed using genetic and pharmacological strategies in HCC cell lines and human normal cell lines,including hepatic stellate cells(HSC)and human embryonic kidney epithelial cells(HEK).RESULTS:Immunohistochemistry showed that both Notch-1 and LSF were significantly upregulated in HCC samples(76%,19/25,P<0.0001 and 84%,21/25,P<0.0001,respectively)compared with non-cancer samples.Activation of Notch-1 by exogenous transfection of Notch1 intracellular domain increased LSF expression in HSC and HEK cells to levels similar to those seen in HepG2 cells.Furthermore,blocking Notch-1 activation with aγ-secretase inhibitor,DAPT,downregulated LSF expression in HepG2 cells.Additionally,a biological behavior assay showed that forced overexpression of LSF promoted HepG2 cell proliferation and invasion.CONCLUSION:LSF is a key mediator of the Notch signaling pathway,suggesting that it might be a novel therapeutic target for the treatment of HCC.

Total flavone of Abelmoschus manihot suppresses epithelial-mesenchymal transition via interfering transforming growth factor-β1 signaling in Crohn's disease intestinal fibrosis

AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells

Yamanaka 因素(Oct3/4， Sox2， Klf4， c-Myc ) 高度在胚胎的茎(ES ) 被表示房间，和他们的在表示上能在老鼠和人的体的房间导致 pluripotency，显示这些因素调整为 ES 房间 pluripotency 必要的发展发信号网络。然而，发信号的小径的全身的分析由 Yamanaka 因素调整了充分还没被描述了。在这研究，我们鉴别一个整个染色体上的内长的 Yamanaka 因素的目标倡导者在 E14.1 鼠标 ES 房间用薄片(染色质 immunoprecipitation ) 放大 -on-chip，并且我们发现这四个因素共同占据 58 个倡导者。有趣地，当 Oct4 和 Sox2 作为核心因素被分析时， Klf4 工作了为发展规定提高核心因素，而 c-Myc 似乎在调整新陈代谢起一个不同作用。小径分析表明 Yamanaka 因素一起调整由 16 条发展发信号小径，其九条在 ES 房间代表更早未知的小径组成的一个发展发信号网络，包括 apoptosis 和房间周期小径。我们进一步从在老鼠 ES 房间检验 Yamanaka 因素的最近的研究分析了数据。有趣地，这分析也揭示了 16 条发展发信号小径， 14 条小径重叠，由这研究揭示了，尽管有目标基因和发信号的小径由每个单个 Yamanaka 因素调整了在这二数据集之间显著地不同。我们建议 Yamanaka 因素极其调整由约十二条关键发展发信号小径组成也维持 ES 房间并且可能的 pluripotency 导致 pluripotent 干细胞的一个发展发信号网络。

Inhibition of epidermal growth factor receptor signaling protects human malignant glioma cells from hypoxia - induced cell death

Epidermal growth factor receptor(EGFR)signaling has become an importanttarget for drug development becauseEGFR signaling enhances tumor cell proliferation,migration,and invasion and inhibits apoptosis.However,theresults of clinical trials using EGFR inhibitors in patients with solid tumors have been disappointing.Here,wereport a protective effect of the EGFR inhibitors AG1478 and PD153035 against cell death induced by acute hy-poxia,which contrasts with their proapoptotic effects under normoxia.Under hypoxic conditions,both agents re-

Role of the Arabidopsis thafiana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses

Jasmonic 酸(JA ) 是对草食动物攻击，病原体感染并且机械伤害调整植物防卫回答的重要植物激素。在这份报告，我们提供了生物化学、基因的证据证明 Arabidopsis thaliana NAC 家庭蛋白质 ANAC019 和 ANAC055 可能作为抄写使活跃之物工作调整辩护基因的导致 JA 的表示。在 JA 发信号的二 NAC 基因的角色与 anac019 anac055 被检验两倍异种并且与转基因的植物 overexpressing ANAC019 或 ANAC055。两倍变异的植物显示出的 anac019 anac055 稀释了导致 JA 的植物的存储 PROTEIN1 (VSP1 ) 和 LIPOXYGENASE2 (LOX2 ) 表示，而二 NAC 基因显示出的转基因的植物 overexpressing 提高了导致 JA 的 VSP1 和 LOX2 表示。二 NAC 基因的 导致JA 的表示取决于 COI1 和 AtMYC2 的功能和发现 ANAC019 的那 overexpression 部分救了 atmyc2-2 异种的JA相关的显型，带了我们到二 NAC 蛋白质 AtMYC2 下游地扮演调整 发信号JA 的防卫回答的一个假设。证实这个想法的进一步的证据来自观察到 necrotrophic 真菌的两倍异种高显示出的 anac019 anac055 的反应类似到 atmyc2-2 异种的。

Three important components in the regeneration of the cavernous nerve： brain-derived neurotrophic factor, vascular endothelial growth factor and the JAK/STAT signaling pathway

Retroperitoneal operations, such as radical prostatectomy, often damage the cavernous nerve, resulting in a high incidence of erectile dysfunction. Although improved nerve-sparing techniques have reduced the incidence of nerve injury, and the administration of phosphodiesterase type 5 inhibitors has revolutionized the treatment of erectile dysfunction, this problem remains a considerable challenge. In recent years, scientists have focused on brain-derived neurotrophic factor and vascular endothelial growth factor in the treatment of cavernous nerve injury in rat models. Results showed that both compounds were capable of enhancing the regeneration of the cavernous nerve and that activation of the Janus kinase （JAK）/signal transducer and activator of transcription （STAT） pathway played a major role in the process.

Fibroblast growth factor receptor signaling as therapeutic targets in gastric cancer

Fibroblast growth factor receptors(FGFRs) regulate a variety of cellular functions, from embryogenesis to adult tissue homeostasis. FGFR signaling also plays significant roles in the proliferation, invasion, and survival of several types of tumor cells. FGFR-induced alterations, including gene amplification, chromosomal translocation, and mutations, have been shown to be associated with the tumor initiation and progression of gastric cancer, especially in diffuse-type cancers. Therefore, the FGFR signaling pathway might be one of the therapeutic targets in gastric cancer. This review aims to provide an overview of the role of FGFR signaling in tumorigenesis, tumor progression, proliferation, and chemoresistance. We also discuss the accumulating evidence that demonstrates the effectiveness of using clinical therapeutic agents to inhibit FGFR signaling for the treatment of gastric cancer.

Nexus of signaling and endocytosis in oncogenesis driven by non-small cell lung cancer-associated epidermal growth factor receptor mutants

Epidermal growth factor receptor(EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non-small cell lung cancer(NSCLC) patients that harbor EGFR kinase domain mutations exhibit oncogene addiction to mutant EGFR, which confers high sensitivity to tyrosine kinase inhibitors(TKIs). As patients invariably develop resistance to TKIs, it is important to delineate the cell biological basis of mutant EGFR-induced cellular transformation since components of these pathways can serve as alternate therapeutic targets to preempt or overcome resistance. NSCLC-associated EGFR mutants are constitutively-active and induce ligandindependent transformation in nonmalignant cell lines. Emerging data suggest that a number of factors are critical for the mutant EGFR-dependent tumorigenicity, and bypassing the effects of TKIs on these pathways promotes drug resistance. For example, activation of downstream pathways such as Akt, Erk, STAT3 and Src is critical for mutant EGFR-mediated biological processes. It is now well-established that the potency and spatiotemporal features of cellular signaling by receptor tyrosine kinases such as EGFR, as well as the specific pathways activated, is determined by the nature of endocytic traffic pathways through which the active receptors traverse. Recent evidence indicates that NSCLCassociated mutant EGFRs exhibit altered endocytic trafficking and they exhibit reduced Cbl ubiquitin ligasemediated lysosomal downregulation. More recent work has shown that mutant EGFRs undergo ligand-independent traffic into the endocytic recycling compartment, a behavior that plays a key role in Src pathway activation and oncogenesis. These studies are beginning to delineate the close nexus between signaling and endocytic traffic of EGFR mutants as a key driver of oncogenicprocesses. Therefore, in this review, we will discuss the links between mutant EGFR signaling and endocytic properties, and introduce potential mechanisms by which altered endocytic properties of mutant EGFRs may alter signaling and vice versa as well as their implications for NSCLC therapy.

Effect of hepatocyte growth factor signaling pathway activation on Plasmodium berghei infection

Objective:To explore the effect of hepatocyte growth factor signaling pathway activation on Plasmodium berghei infection.Methods:In this study,hepatocyte growth factor was detected by ELISA and Western blotting assay.Hepatocyte injury was detected by FITC-dextran absorption assay,and hepatocyte growth factor expression was shown to be expressed in the same injury cells by immunofluorescence against hepatocyte growth factor.In addition,Activation of hepatocyte growth factor and its receptor signaling pathway was detected with immunoprecipitation and detection of phosphorylation status.Results:It was found that injury of hepatocytes by sporozoite migration induced the secretion of hepatocyte growth factor and it was hepatocyte growth factor that rendered hepatocytes susceptible to Plasmodium sporozoite infection.In addition,hepatocyte infections depended on activation of the hepatocyte growth factor and its receptor signaling pathway.Conclusions:Our results indicate that hepatocyte growth factor and its receptor may possibly be potential targets for new approaches to malaria treatment.

Expression Patterns of OsPIL11,a Phytochrome-Interacting Factor in Rice,and Preliminary Analysis of Its Roles in Light Signal Transduction

The expression patterns of OsPILll, one of six putative phytochrome-interacting factors, were analyzed in different organs of transgenic tobacco （Nicotiana tabacum）. The expression of OsPIL 11 was organ-specific and was regulated by leaf development, abscisic acid （ABA）, jasmonic acid （JA） and salicylic acid （SA）. To further explore the role of OsPIL 11 in plant light signal transduction, a plant expression vector of OsPILll was constructed and introduced into tobacco. When grown under continuous red light, OsPILll-overexpressed transgenic tobacco exhibited shorter hypocotyls and larger cotyledons and leaves compared to wild-type seedlings. When grown under continuous far-red light, however, transgenic and wild-type seedlings showed similar phenotypes. These results indicate that OsPILll is involved in red light induced de-etiolation, but not in far-red light induced de-etiolation in transgenic tobacco, which lays the foundation for dissecting the function of OsPIL11 in phytochrome-mediated light signal transduction in rice.

Regulating effect of glycyrrhetinic acid on bronchial asthma smooth muscle proliferation and apoptosis as well as inflammatory factor expression through ERK1/2 signaling pathway

Objective: To study the influence of glycyrrhetinic acid(GA) on bronchial asthma(BA)smooth muscle proliferation and apoptosis as well as inflammatory factor expression and its molecular mechanism.Methods: Male SD guinea pigs were selected and made into asthma models, bronchial asthma smooth muscle cells were cultured and divided into BA group, GA group and GA + LM group that were treated with serum-free RPMI1640 culture medium, serumfree RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid, serum-free RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid and 100 ng/mL LM22B-10 respectively; normal guinea pigs were collected and bronchial smooth muscle cells were cultured as control group. The cell proliferation activity as well as the expression of proliferation and apoptosis genes, inflammatory factors and p-ERK1/2 was determined.Results: Proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6,YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in BA group were significantly higher than those of control group while m RNA expression levels of Bax,caspase-9 as well as caspase-3 were significantly lower than that of control group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in GA group were significantly lower than those of BA group(P < 0.05) while the m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly higher than those of BA group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40 of airway smooth muscle cell in GA + LM group were significantly higher than those of GA group(P < 0.05) while m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly lower that of GA group(P < 0.05).Conclusion: GA can inhibit the proliferation of bronchial smooth muscle cells and reduce the expression of inflammatory factors by inhibiting the phosphorylation of ERK1/2.

Spatial signalling mediated by the transforming growth factor-β signalling pathway during tooth formation

Tooth development relies on sequential and reciprocal interactions between the epithelial and mesenchymal tissues, and it is continuously regulated by a variety of conserved and specific temporal-spatial signalling pathways. It is well known that suspensions of tooth germ cells can form tooth-like structures after losing the positional information provided by the epithelial and mesenchymal tissues. However, the particular stage in which the tooth germ cells start to form tooth-like structures after losing their positional information remains unclear. In this study, we investigated the reassociation of tooth germ cells suspension from different morphological stages during tooth development and the phosphorylation of Smad2/3 in this process. Four tooth morphological stages were designed in this study. The results showed that tooth germ cells formed odontogenic tissue at embryonic day （E） 14.5, which is referred to as the cap stage, and they formed tooth-like structures at E16.5, which is referred to as the early bell stage, and E18.5, which is referred to as the late bell stage. Moreover, the transforming growth factor-β signalling pathway might play a role in this process.

Signaling pathways involved in the inhibition of epidermal growth factor receptor by erlotinib in hepatocellular cancer

瞄准：在肝细胞癌(HCC ) 检验导致 erlotinib 的生长抑制的内在的机制。方法：在基因表示的导致 Erlotinib 的改变用 cDNA 数组技术被评估；在蛋白质表示或蛋白质激活的变化用西方的弄污由于象 IGF-1-induced EGFR transactivation 一样的 erlotinib 治疗被调查。结果：Erlotinib 治疗禁止了 mitogen 激活和抄写(STAT ) 的激活的蛋白质(地图)-kinase 小径和信号变换器调停了表明哪个导致了调整由 cDNA 数组技术示威了的基因的 apoptosis 和房间周期的一个改变的表达式。象与象 Bcl-2， Bcl-X (L) 或 jun D 一样的 antiapoptotic 因素的一条下面规定联系的 caspases 和 gadds 一样的 proapoptotic 因素的 Overexpression 说明了让 erlotinib 的力量导致 apoptosis。支持 G1/S-transition 的房间周期管理者并且在 cyclin 依赖的激酶禁止者和 gadds 的表示上的 Downregulation 响应 erlotinib 贡献了 G1/G0-arrest 的正式就职。而且，我们显示了由 IGF-1-receptor 的调停 EGFR 的发信号的 transactivation 并且在受体受体十字谈话显示出 erlotinib 的禁止的效果。结论：我们的学习在 HCC 房间和 thus 使 EGFR-TK-inhibition 的行动的机制的理解清楚些可能便于 additively 或 synergistically 行动的联合治疗的设计。而且，我们对 erlotinib 处理作出回应的小径上的数据能在以后预言肿瘤的应答的海角到 EGFR-TKIs 是有用的。

Complement C3a signaling mediates production of angiogenic factors in mesenchymal stem cells

A major portion of the beneficial effect of mesenchymal stem cells (MSC) is due to the production of trophic and angiogenic factors by these cells, and one of the efforts to improve the therapeutic efficacy of these cells lies in enhancing this capacity. Since there is complement activation in all areas of tissue injury, and both C3a and C5a activate MSC, it was asked whether stimulation with C3a or C5a would upregulate the production of trophic factors by MSC. C3a caused significant up-regulation of various angiogenic factors, including VEGF, CXCL8/IL-8 and IL-6. In contrast there was no detectable production of the pro-inflammatory cytokines TNF-α and IL-1β in spite of nuclear translocation of NFκB. Although C5a also caused moderate up-regulation of angiogenic factors, the effect was borderline significant. Furthermore the production of angiogenic factors induced by C3a was of physiological relevance: Supernatants of MSCs cultured under serum-free conditions induced minimal tube formation of HUVECs as an in vitro measure of angiogenesis;tube formation was considerably enhanced, when supernatants from C3a-stimulated MSC were used, while C3a itself had no direct angiogenic effect on HUVECs. The signaling cascade responsible for the production of angiogenic factors by C3a or C5a could be defined as activation of the rho cascade which was necessary for nuclear translocation of NFκB p65 and of phospho-ERK1/2. Although rho was only transiently activated, inhibition of the rho or “downstream of it” of the NFκB pathway, prevented C3a-and C5a-induced up-regulation of angiogenic factors.

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P＜0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P＜0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P＜0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P＜0.01;r = 0.938, P＜0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L)was confirmed by Western blotting as well.CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.

Effects of (-)-Epigallocatechin gallate on some protein factors involved in the epidermal growth factor receptor signaling pathway

（-）-Epigallocatechin gallate （EGCG）, a major polyphenolic constituent of green tea, can inhibit activity of specific receptor tyrosine kinases （RTKs） and related downstream signal transduction pathways, resulting in the control of unwanted cell proliferation. The epidermal growth factor receptor （EGFR） signaling pathway is one of the most important pathways that regulates growth, survival,proliferation and differentiation in mammalian cells. This review addresses the effects of EGCG on some protein factors involved in the EGFR signaling pathway in a direct or indirect manner. Based on our understanding of the interaction between EGCG and these factors, and based on their structures, EGCG could be used as a lead compound for designing and synthesizing novel drugs with significant biological activity.

Atsttrin reduces lipopolysaccharide-induced neuroinflammation by inhibiting the nuclear factor kappa B signaling pathway

Progranulin is closely related to neuronal survival in a neuroinflammatory mouse model and attenuates inflammatory reactions. Atsttrin is an engineered protein composed of three progranulin fragments and has been shown to have an effect similar to that of progranulin. Atsttrin has anti-inflammatory actions in multiple arthritis mouse models, and it protects against further arthritis development. However, whether Atsttrin has a role in neuroinflammation remains to be elucidated. In this study, we produced a neuroinflammatory mouse model by intracerebroventricular injection of 1 μL lipopolysaccharide(10 μg/μL). Atsttrin(2.5 mg/kg) was administered via intraperitoneal injection every 3 days over a period of 7 days before intracerebroventricular injection of 1 μL lipopolysaccharide(10 μg/μL). In addition, astrocyte cultures were treated with 0, 100 or 300 ng/mL lipopolysaccharide, with 200 ng/mL Atsttrin simultaneously. Immunohistochemistry, enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction were performed to examine the protein and mRNA levels of inflammatory mediators and to assess activation of the nuclear factor kappa B signaling pathway. Progranulin expression in the brain of wild-type mice and in astrocyte cultures was increased after lipopolysaccharide administration. The protein and mRNA expression levels of tumor necrosis factor-α, interleukin-1β and inducible nitric oxide synthase were increased in the brain of progranulin knockout mice after lipopolysaccharide administration. Atsttrin treatment reduced the lipopolysaccharide-induced increase in the protein and mRNA levels of tumor necrosis factor-α, interleukin-1β, matrix metalloproteinase-3 and inducible nitric oxide synthase in the brain of progranulin knockout mice. Atsttrin also reduced the expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase 3 mRNA in lipopolysaccharide-treated astrocytes in vitro, and decreased the concentration of tumor necrosis factor α and interleukin-1β in the supernatant. Furthermore, Atsttrin significantly reduced the levels of phospho-nuclear factor kappa B inhibitor α in the brain of lipopolysaccharide-treated progranulin knockout mice and astrocytes, and it decreased the expression of nuclear factor kappa B2 in astrocytes. Collectively, our findings show that the anti-neuroinflammatory effect of Atsttrin involves inhibiton of the nuclear factor kappa B signaling pathway, and they suggest that Atsttrin may have clinical potential in neuroinflammatory therapy.

Dihydroergotamine ameliorates liver fibrosis by targeting transforming growth factor β type Ⅱ receptor

BACKGROUND The transforming growth factor β(TGFβ) signaling pathway plays a crucial role in the development of liver fibrosis by activating TGFβ type Ⅱ receptor(TGFβR2), followed by the recruitment of TGFβR1 finally triggering downstream signaling pathway.AIM To find drugs targeting TGFβR2 that inhibit TGFβR1/TGFβR2 complex formation, theoretically inhibit TGFβ signaling pathway, and thereby ameliorate liver fibrosis.METHODS Food and Drug Administration-approved drugs were screened for binding affinity with TGFβR2 by virtual molecular docking. We identified 6 candidates and further explored their potential by Cell Counting Kit-8(CCK-8) cell cytotoxic experiment to validate toxicity and titrated the best cellular working concentrations. Next, we further demonstrated the detailed molecular working mechanisms using mutagenesis analysis. Finally, we used a mouse model to investigate its potential anti-liver fibrosis effect.RESULTS We identified 6 drug candidates. Among these 6 drugs, dihydroergotamine(DHE) shows great ability in reducing fibrotic gene expressions such as collagen, p-SMAD3, and α-SMA in TGFβ induced cellular model of liver fibrosis in LX-2 cells. Furthermore, we demonstrated that DHE binds to TGFβR2. Moreover, mutation of Leu27, Phe30, Thr51, Ser52, Ile53, and Glu55 of TGFβR2 disrupted the binding of TGFβR2 with DHE. In addition, DHE significantly improved liver fibrosis, as evidenced by Masson’s trichrome staining of liver sections. This is further supported by the width and the velocity of the portal vein, and serum markers of liver function. In line with those observations, DHE also decreased macrophages infiltration and extracellular matrix deposition in the liver.CONCLUSION DHE alleviates liver fibrosis by binding to TGFβR2 thereby suppressing TGFβ signaling pathway. We show here that as far as drug repurposing, DHE has great potential to treat liver fibrosis.