The present study was undertaken to identify whether prostaglandin E2 receptor is the potential receptor/binding site for Ginkgolide A, Ginkgolide B, Ginkgolide K, and Bilobalide, the four main ingredients of the Ginkgo biloba L., leaves. Using functional assays, we identified EP4, coupled with Gs protein, as a target of Ginkgolide B. In human neuroblastoma SH-SY5Y cells suffered from oxygen-glucose deprivation/reperfusion, Ginkgolide B-activated PKA, Akt, and ERK1/2 as well as Src-mediated transactivation of epidermal growth factor receptor. These resulted in downstream signaling pathways, which enhanced cell survival and inhibited apoptosis. Knockdown of EP4 prevented Ginkgolide B-mediated Src, epidermal growth factor receptor (EGFR), Akt, and ERK1/2 phosphorylation and neuroprotective effects. Moreover, Src inhibitor prevented Ginkgolide B-mediated EGFR transactivation and the downstream Akt and ERK1/2 activation, while the phosphorylation of PKA induced by Ginkgolide B was not affected, indicating Ginkgolide B might transactivate EGFR in a ligand-independent manner. EP4 knockdown in a rat middle cerebral artery occlusion (MCAO) model prevented Ginkgolide B-mediated infarct size reduction and neurological assessment improvement. At the same time, the increased expressions of p-Akt, p-ERK1/2, p-PKA, p-Src, and p-EGFR and the deceased expression of cleaved capases-3 induced by Ginkgolide B in cerebral cortex were blocked due to EP4 knockdown. In conclusion, Ginkgolide B exerts neuroprotective effects in rat MCAO model through the activation of EP4 and the downstream transactivation of EGFR.

Sepsis, a systemic inflammatory response, caused by pathogenic factors including microorganisms, has high mortality and limited therapeutic approaches. Herein, a new soluble epoxide hydrolase (sEH) inhibitor series comprising a phenyl ring connected to a memantyl moiety via a urea or amide linkage has been designed. A preferential urea pharmacophore that improved the binding properties of the compounds was identified for those series via biochemical assay in vitro and in vivo studies. Molecular docking displayed that 3,5-dimethyl on the adamantyl group in B401 could make van der Waals interactions with residues at a hydrophobic pocket of sEH active site, which might indirectly explain the subnanomolar level activities of memantyl urea derivatives in vitro better than AR-9281. Among them, compound B401 significantly improved the inhibition potency with human and murine sEH IC50 values as 0.4 nM and 0.5 nM, respectively. Although the median survival time of C57BL/6 mice in LPS-induced sepsis model was slightly increased, the survival rate did not reach significant efficacy. Based on safety profile, metabolic stability, pharmacokinetic and in vivo efficacy, B401 demonstrated the proof of potential for this class of memantyl urea-based sEH inhibitors as therapeutic agents in sepsis.

目的 确定银杏内酯K(ginkgolide K,GK)对缺血性脑卒中诱导的神经血管单元损伤的保护作用及调节缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)通路机制.方法 采用小鼠小胶质细胞BV-2及人脐静脉细胞融合细胞EA.hy926氧糖剥离再灌注(oxygen-glucose deprivation and reperfusion,OGD/R)模型模拟脑缺血引起的神经血管单元损伤.细胞OGD 4 h后再灌注并给予GK干预.给药24 h时检测BV-2细胞上清中炎症因子水平及EA.hy926细胞损伤情况;Western blot检测BV-2细胞给药1 h后p-Akt、p-Erk和6 h后HIF-1α水平,并应用LY294002验证,同时检测EA.hy926细胞给药1 h后p-Akt及6 h后HIF-1α等蛋白变化.结果 GK明显抑制BV-2细胞上清TNF-α、IL-6、IL-1β分泌,激活p-Akt、抑制p-Erk并下调HIF-1α,且LY294002共同干预后,GK调节作用下降;同时,增加EA.hy926细胞活力和抑制LDH释放,上调p-Akt、HIF-1α、HO-1、VEGF并下调cleaved caspase-3/9水平.结论 GK可多效应减少缺血性脑卒中诱导的神经血管单元损伤,其机制可能与针对不同细胞HIF-1α调节作用不同有关.

Ethnopharmacological relevance: Ginkgo biloba L. is a kind of traditional Chinese medicinal material with a long history. Its main active ingredients, ginkgolides, can be used for the treatment of stroke and other cardio-cerebrovascular diseases. Ginkgo Diterpene Lactone Meglumine Injection (GDLI), a modernized TCM, has attracted much attention because of its neuroprotective and anti-inflammatory properties. Aim of the study: To uncover the effects of GDLI on ischemic stroke patients, as well as the underlying biomarkers involved in sub-acute stroke. Materials and methods: We used a state-of-the-art targeted proteomics chip to investigate the association between numerous serum proteins (1101 proteins) and the sub-acute phase post-ischemic stroke. Then, the relative proteins of anti-apoptosis, anticoagulant, and neuroprotection of GDLI were verified in animal models. Results: Compared with the serum from healthy volunteers, we identified 15 up-regulated proteins and 26 downregulated proteins (FC >= 1.5) involved in inflammatory response, immune response, and nervous system development in the sub-acute ischemic stroke. The pro-inflammatory proteins, such as IL17, MSP-R, G-CSF-R, TLR3, MIP-3 beta, TNFRSF19, and TNFRSF12, were significantly increased in serum, illustrating that the chronic inflammatory state was evident in the sub-acute stage of ischemic stroke. However, the common pro-inflammatory proteins, such as IL-1 beta, IL-6, IL-8, TNF-alpha, IFN-gamma, and IL-10, known to be up-regulated in acute stroke, had close or lightly lower levels than healthy humans (FC >= 1.5, P > 0.05). And some cytokines (IL3, CCL13, TNFRSF3, IL10 R beta, HLA-A, IL-1 F8/FIL1 eta, TNFRSF8, CCL18) were also markedly down-regulated in the sub-acute phase of stroke. These proteins are highly associated with the onset of stroke-induced immunosuppression and post-stroke infection. Moreover, we noticed that Ginkgo Diterpene Lactone Meglumine Injection (GDLI) treatment for 14 days was helpful to the recovery of patients in the subacute period. After the treatment of GDLI, it was observed that several inflammatory cytokines (i.e. IL-17 and IL-28A), chemokine (i.e. CCL14), and Coagulation Factor III were reduced. Meanwhile, the anti-inflammatory cytokines (IL-10 R alpha, GREMLIN, and Activin C) and neurotrophic factors (Neurturin and IGFBP2) were found to be up-regulated in stroke patients through self-control observation. Finally, we identified the IGFBP2 as a novel marker in the animal models. Conclusions: In summary, the potential markers in sub-acute stroke patients were highly different from known protein markers in the acute phase of ischemic stroke. The serum protein IGFBP2 could be novel biomarkers for the treatment of GDLI in sub-acute stroke patients. Our present findings provide an innovative insight into the novel treatment of GDLI in ischemic stroke therapy.

Ginkgolide terpenoid lactones, including ginkgolides and bilobalide, are two crucial bioactive constituents of extract of Ginkgo biloba (EGb) which was used in the treatment of cardiovascular and cerebrovascular diseases. The aims of this study were to investigate the antioxidant effects and mechanism of ginkgolides (ginkgolide A (GA), ginkgolide B (GB), ginkgolide K (GK)) and bilobalide (BB) against oxidative stress induced by transient focal cerebral ischemia. In vitro, SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD) for 4 h followed by reoxygenation with ginkgolides and BB treatments for 6 h, and then cell viability, superoxide dismutase (SOD), and ROS were respectively detected using kit. Western blot was used to confirm the protein levels of hemeoxygenase-1 (HO-1), quinone oxidoreductase l (Nqo1), Akt, phosphorylated Akt (p-Akt), nuclear factor-E2-related factor2 (Nrf2), and phosphorylated Nrf2 (p-Nrf2). GB combined with different concentrations of LY294002 (PI3K inhibitor) were administrated to SH-SY5Y cells for 1 h after OGD, and then p-Akt and p-Nrf2 levels were detected by western blot. In vivo, 2 h of middle cerebral artery occlusion (MCAO) model was established, followed with reperfusion and GB treatments for 24 and 72 h. The infarct volume ratios were confirmed by TTC staining. The protein levels of HO-1, Nqo1, SOD1, Akt, p-Akt, Nrf2, and p-Nrf2 were detected using western blot and immunohistochemistry (IHC). Experimental data in vitro confirm that GA, GB, GK, and BB resulted in significant decrease of ROS and increase of SOD activities and protein levels of HO-1 and Nqo1; however, GB group had a significant advantage in comparison with the GA and GK groups. Moreover, after ginkgolides and BB treatments, p-Akt and p-Nrf2 were significantly upregulated, which could be inhibited by LY294002 in a dose-dependent manner, meanwhile, GB exhibited more effective than GA and GK. In vivo, TTC staining indicated that the infarct volume ratios in MCAO rats were dramatically decreased by GB in a dose-dependent manner. Furthermore, GB significantly upregulated the protein levels of HO-1, Nqo1, SOD, p-Akt, p-Nrf2, and Nrf2. In conclusion, GA, GB, GK, and BB significantly inhibited oxidative stress damage caused by cerebral ischemia reperfusion. Compared with GA, GK, and BB, GB exerts the strongest antioxidant stress effects against ischemic stroke. Moreover, ginkgolides and BB upregulated the levels of antioxidant proteins through mediating the Akt/Nrf2 signaling pathway to protect neurons from oxidative stress injury.

The purpose of the present study was to explore the protective effect and functional mechanism of ginkgolide K (GK: C20H22O9) on cerebral ischemia. SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD) to simulate an ischemic model in vitro. Cell viability, reactive oxygen species (ROS), nuclear staining with Hoechst 33258 and mitochondrial membrane potential were detected following 4 h of exposure to OGD. Subsequently, the expression levels of the apoptosis-related proteins, caspase-9, caspase-3, Bcl-2, Bax, p53 and c-Jun, as well as the mitogen-activated protein kinases (MAPKs) signaling molecules were detected by western blot analysis. GK significantly elevated the cell viability and decreased the generation of ROS and the number of apoptotic cells in a dose-dependent manner. Furthermore, GK markedly decreased the protein expression levels of p-p38, p-JNK, p-p53, p-c-Jun and the expression levels of Bcl-2, Bax, cleaved caspase-9 and caspase-3. In conclusion, GK demonstrated a neuroprotective effect on the simulated cerebral ischemia in vitro, and this effect was mediated through the inhibition of the mitochondria-mediated apoptosis pathway triggered by ROS-evoked p38 and JNK activation.

In this study, we aimed to investigate the effects of diterpene ginkgolides meglumine injection (DGMI) on paraquat (PQ)-induced lung injury and pulmonary fibrosis in rats. Male SD rats were challenged by PQ (20?mg/kg, i.p.) with or without either DGMI (1.25, 2.5, 5?mg/kg, i.p.) or Edaravone (EDA, 6?mg/kg, i.p.) posttreatment 2?h after PQ administration. Lung tissues were removed for biochemical analyses and pathological examinations on day 1, day 3, day 7, day 14 and day 21. Results showed that the administration of DGMI significantly increased the survival of PQ-challenged rats. At the same time, DGMI reversed the increase of Malondialdehyde (MDA) level and the decrease of Super Oxide Dismutase (SOD) level in lung tissues. Moreover, lung to body weight ratio, Interleukin-1beta (IL-1?), Interleukin-6 (IL-6) and Tumor necrosis factor-alpha (TNF-?) levels in lung tissues were reduced compared with the model group. H&E and Masson staining revealed that DGMI (5?mg/kg) alleviated histological injury and pulmonary fibrosis, and EDA (6?mg/kg) exerted approximate effects. Immunohistochemistry staining presented that the benefit effects of DGMI were associated with its ability to activate Akt-Nrf-2 pathway. In conclusion, these results suggest that DGMI possesses potential role in future therapies for PQ-induced lung injury and pulmonary fibrosis.

OBJECTIVE To investigate the protective effects and mechanism of diterpene ginkgolides meglumine injection (DGMI) against oxidative stress induced by oxygen-glucose deprivation (OGD) in SH-SY5Y cells. METHODS SH-SY5Y cells were divided into five groups: normal control, model control (OGD group) and drug(25 mg · L- 1) administration groups including DGMI group, extract of ginkgo biloba leaves injection group (EGBLI) and lactones ginkgo biloba injection group (LGBI). The cells suffered from oxygen-glucose deprivation (OGD) for 4 h, followed by reoxygenation with drugs for 6 h. Then, cell viabilities were detect using CCK-8 assays, reactive oxygen species (ROS) levels using fluorescence probe DCFH-DA and superoxide dismutase (SOD) activities using WST-1 test. Western blotting was used to detected protein levels of hemeoxygenase-1(HO-1), NAD(P)H, quinone oxidore?ductase l (Nqo1), protein kinase B (Akt), phosphorylated Akt (p-Akt), nuclear factor-E2-related factor2 (Nrf2) and phosphorylated Nrf2 (p-Nrf2). The cells were induced by OGD for 4 h, followed by reoxygen?ation and DGMI for 1 h, combined with different concentrations of PI3K inhibitor (LY294002) (at the final concentration of 12.5, 25 and 50 μmol · L-1) before the protein levels of AKT, p-AKT, Nrf2 and p-Nrf2 were detected by Western blotting. RESULTS SH-SY5Y cells induced by OGD for 4 h resulted in an increase in ROS(P<0.01), but a decrease in cell viabilities(P<0.01), SOD activities(P<0.01), and antioxidant protein levels ( Akt, p-Akt, Nrf2, p-Nrf2, HO-1 and Nqo1) (P<0.01). Compared with OGD group, treatment with reoxygenation and drugs (DGMI,EGBLI and LGBI respectively) for 6 h resulted in a decrease in ROS (P<0.01), but an increase in cell viabilities, SOD activities and antioxidant protein levels of p-Nrf2, HO-1, Nqo1 and p-Akt(P<0.05,P<0.01). DGMI group showed the best efficiently. Moreover, after OGD for 4 h, compared with DGMI group, combining reoxygenation and DGMI with LY294002 for 1 h resulted in a concentration-dependent inhibition of the protein levels of p-AKT and p-Nrf2(P<0.01). CONCLUSION DGMI 25 mg · L-1 can inhibit oxidative stress in SH-SY5Y cells induced by OGD by increasing the activity and expression of Nrf2 through PI3K/Akt pathway, which may be one of the mechanisms by which DGMI protects neurons from stroke.

Aim To investigate the protective effects of Diterpene Ginkgolides Meglumine Injection(DGMI)on SY5 Y cells damaged by oxygen-glucose deprivation and its functional mechanisms.Methods After 4 h of OGD,the cells were treated with 25 mg·L-1 drugs for 1 h.Subsequently,cell viabilities were measured by cell counting kit-8(CCK-8 kit)and cell apoptosis was measured by flow cytometric analysis.Furthermore, the mitochondrial membrane potential was detected by rhodamine123 staining.The levels of phospho-p38, phospho-p53,Bcl-2,Bax and cleaved caspase-9/3 were evaluated by western blot.Results DGMI signif-icantly increased the cell viabilities of SY5 Y cells dam-aged by OGD,and reduced OGD-elicited dissipation of mitochondrial membrane potential and cell apoptosis. Furthermore,DGMI also reduced p-p38,p-p53,Bax/Bcl-2 ratio,cleaved caspase-9 and cleaved caspase-3. Conclusion DGMI shows good neuroprotective effects on SY5 Y cells after oxygen-glucose deprivation.The underlying mechanisms may be associated with the sup-pression of p38/p53/Bcl-2 /caspase-9/caspase-3 sig-naling pathway.

研究银杏二萜内酯葡胺注射液(diterpene ginkgolides meglumine injection,DGMI)对缺氧缺糖/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤的人神经母细胞瘤细胞SH-SY5Y凋亡的抑制作用及其机制.采用试剂盒、Fluo3 AM法、Western blot检测SH-SY5Y细胞氧糖剥离损伤4h后,与药物一起复氧1h细胞乳酸脱氢酶(LDH)的漏出量、caspase-3/7酶活力、细胞质中核小体含量、胞浆游离钙离子浓度以及calpain、cleaved caspase-12蛋白量的变化.结果显示DGMI能极显著降低OGD/R损伤的SH-SY5Y细胞LDH的漏出量,抑制caspase-3/7酶活性,减少细胞核核小体的释放量,下调细胞内游离钙离子浓度、calpain和cleaved caspase-12蛋白量,抑制calpain凋亡信号通路保护神经细胞.DGMI对OGD/R诱导的SH-SY5Y细胞凋亡具有显著的抑制作用,其作用机制可能与抑制细胞内Ca2+/calpain/caspase-12/capase-3信号通路有关.

Aim To investigate the protective effects of YXETNZ injection on SH-SY5 Y cells damaged by oxygen-glucose deprivation ( OGD ) , and explore its functional mechanisms. Methods After 4 h of OGD, the cells were treated with 25 mg·L-1 drugs for 1 h. Subsequently, cell viabilities were measured by cell counting kit-8 ( CCK-8 kit ) and cell apoptosis was measured by caspase-3/7 assay kit according to manu-facturer’ s instructions. Furthermore, cell death was also detected by ELISA. The levels of phospho-Akt, phospho-PKA,phospho-Bad were evaluated by western blot. Results Oxygen-glucose deprivation significant-ly decreased the cell viabilities of SH-SY5Y cells, while YXETNZ injection significantly increased cell vi-abilities, phospho-Akt, phospho-PKA and phospho-Bad. Furthermore, YXETNZ injection also reduced the activities of caspase-3/7 and cytoplasmic histone-asso-ciated-DNA-fragments contents. Conclusion Our re-searches demonstrat that YXETNZ injection shows good neuroprotective effects on SH-SY5 Y cells after oxygen-glucose deprivation. The underlying mechanisms may be associated with activation of PI3 K/Akt/Bad/caspase-3/7 , cAMP/PKA/Bad/caspase-3/7 signaling pathway.

chβ2m gene was amplified from normal chicken peripheral blood lymphocytes by RT-PCR. It was cloned into pET-32a(+) vector and induced by IPTG, then purified with HiTrap affinity column. The results showed that the sequence of chβ2m gene amplified from chickens by RT-PCR was the same as that previously reported (M84767, AY989898, Z48921) and consists of 360 bp encoding 120 amino acids (aa). Most of the soluble recombinant chβ2m proteins existed in the bacterial supernatant induced with 0.8 M IPTG. Only one band of molecular weight of 34 kDa was purified by ion-exchange chromatography. Western blotling assay demonstrated that anti-His-tag monoclonal antibody could specifically recognize the recombinant protein. This work would provid us the basis materials for further study of the structure and functions of chβ2m.

To investigate the biological function of UL14 protein during the course of MDV infection,UL14 gene from CEF cells infected with RB1B strain was amplified by PCR.UL14 gene was cloned into pET32a(+) vector and expressed in E.coli BL21(DE3).Recombinant His-UL14 protein expressed in E.coli was soluble.The BALB/c mice were immunized with recombinant UL14 protein and resulting antiserum was used in IFA.The specific inflorescence was visualized in CEF cells infected with MDV strain RB1B.

We developed a panel of monoclonal antibodies (MAb) against chicken β2-microglobulin (chβ2M) by fusions between SP2/0 myeloma cells and spleen cells from mice immunized with a synthesized peptide corresponding to positions 91-119 of the COOH domain of chβ2M. Two of them, 6E7 and 3D1, identified as IgG1/κ, could react with chβ2M protein from avian macrophage HD11 cells and human 293T cells transfected with pcDNA3.1-chβ2M in immunofluorescence assays. Only a 12 kDa protein band of chβ2M could be detected in the HD11 and 293T/chβ2M cell lysates by Western blot analysis. Chicken β2M in serum and plasma could be found in Western blot by MAb 3D1. Moreover, MAb 3D1 also recognized the chβ2M antigen on the cell membranes in flow cytometry. Immunohistochemical staining with these MAbs revealed that chβ2M was present in chicken thymus, spleen, and bursa. These MAbs will be good tools for analyzing the mechanism of the chicken immune system.

Marek’s disease virus (MDV) is a highly cell-associated herpesvirus that causes a disease in chickens characterized by tumor formation and immunosuppression. The changes of major histocompatibility complex (MHC) expression in different MDV-infected cells are not completely understood. In this study, we investigated the expression of the Class I MHC and β2-microglobulin (β2m) genes in response to MDV infection at different time points by real-time PCR. In both in vitro and in vivo, the expression levels of Class I MHC and β2m genes were upregulated during early MDV infections in comparison to control cells; We also found that the expression of Class I MHC gene was downregulated in BudR (5-bromo-2′-deoxyuridine)-treated MSB1 cells at 48 h and MDV-infected chicken embryo fibroblast cells (CEF) at 120 and 168 h post infection (hpi); Furthermore, compared to control groups, Class I MHC and β2m expression levels were downregulated in peripheral blood lymphocytes (PBLC) from MDV-infected chickens at 14 and 28 days post infection (dpi); Interestingly, both Class I MHC and β2m gene expression levels increased again in PBLC from MDV RB1B-infected chickens at 35 dpi, in which MDV was in the latent or transformed infection stages. In addition, Class I MHC expression was clearly decreased in MDV-infected CEF at 120 hpi although β2m expression was significantly increased. These changes in Class I MHC and β2m gene expression might provide more insights into host-virus interaction.

Two monoclonal antibodies(MAbs) against chicken β2-microglobulin(β2-M) were prepared by fusions the SP2/0 cells with spleen cells from BALB/c mice immunized with a synthesized peptide(TF-29).The ELISA titers of two antibodies 3D1 and 6E7,identified as IgG1/κ,were 1∶25 600 and 1∶204 800 respectively.Both MAbs specifically recognized not only the recombinant β2-M expressed in prokaryotic cells,but also the native chicken β2-M in avian macrophage HD11 cells by western blot and immunofluorescence assays.Moreover,both MAbs were able to react with the β2-M from chicken serum and plasma in western blot assay.These MAbs would facilitate for further study of chicken β2-M in the regulation of the immune system in chicken.