Design and oral hypoglycemic activity of novel oral hypoglycemic peptide ODA / 中国药科大学学报

@#Incretin promotes insulin secretion through a glucose-dependent mechanism, involving glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). Therefore, their correspondingly specific receptors GLP-1R and GIPR are suitable targets for the treatment of type 2 diabetes. Based on the oral hypoglycemic peptide OHP2 designed by our team, we further designed a new oral hypoglycemic peptide, ODA to reduce glucose. Compared with OHP2, ODA exhibited better lipophilicity as well as the enhanced endocytosis and transcytosis in Caco-2 cells. In addition, ODA remained the ability to activate GLP-1R and enhanced the binding ability to GIPR. The hypoglycemic efficacy of the low-dose ODA (0.53 mg/kg) is comparable to that of OHP2 (1.06 mg/kg). These results indicated that ODA could be a new oral drug with potential for the treatment of type 2 diabetes.

Design and antitumor activity of programmed cell death ligand 1 epitope peptide vaccine / 中国药科大学学报

@#Several programmed cell death protein 1 (PD-1) or its ligand (PD-L1) immune checkpoint blocking antibodies are available for clinical treatment, but only some patients show clinical response, so an alternative strategy for tumor immunotherapy is needed.A therapeutic tumor vaccine targeting PD-L1 is a meaningful attempt.In this study, we designed an epitope peptide vaccine targeting PD-L1, and then screened the immunogenic PD-L1 epitope peptide based on the humanized immune system (HIS) mouse model and further investigated its anti-tumor activity.The results show that the designed and screened PD-L1-B1 epitope peptide vaccine not only successfully induced PD-L1-specific humoral and cellular immunity, but also exhibit anti-tumor activity.In addition, immunotherapy increased T-lymphocyte infiltration of tumors and reshaped the tumor immunosuppressive microenvironment.In conclusion, PD-L1-B1 epitope peptide vaccine exhibits potent anti-tumor activity and may be an effective alternative immunotherapeutic strategy for patients insensitive to PD-1/PD-L1 blockade.

Effects and mechanisms of SNP-9 on Parkinson''s disease cell model induced by rotenone / 中国药科大学学报

@#In this article, the effects and mechanisms of SNP-9 on Parkinson''s disease (PD) cell model were investigated.SH-SY5Y cells were treated with rotenone to establish PD cell model; the effects of rotenone and SNP-9 on cell viability were detected by MTT assay; Hoechst/PI double staining assay was used to detect the effects of rotenone and SNP-9 on cell apoptosis; DCFH-DA probe was used to detect the effects of rotenone and SNP-9 on cellular reactive oxygen species (ROS) levels; and Western blot was used to detect the effects of rotenone and SNP-9 on protein levels of tyrosine hydroxylase (TH), α-synuclein (α-syn), Bcl-2 and Bax.The results showed that SNP-9 could alleviate abnormalities in cell viability, levels of TH and α-syn, apoptosis, ROS and apoptotic relative protein Bax/Bcl-2 induced by rotenone.Our findings suggest that SNP-9 may alleviate rotenone-induced injury in neuronal cells by regulating cell apoptosis related pathway.

Effect of homologous overexpression of nucleoside diphosphate kinase on the yield and molecular weight of hyaluronic acid / 中国药科大学学报

@#To investigate the effect of nucleoside diphosphate kinase (NDK) on the synthesis of hyaluronic acid, nucleoside diphosphate kinase gene (ndk) was overexpressed along with the hyaluronic acid-producing genes in recombinant B.subtilis. Two engineered strains named Hp8tg and Pn8tg were constructed.Uniform hyaluronic acid (HA) could be obtained from both engineered strains.HA produced by both recombinant strains was confirmed by monosaccharide composition analysis, Fourier transform infrared spectometry and nuclear magnetic resonance spectroscopy.Inducing conditions of HA fermentation were optimized by response surface methodology.Overexpression of ndk could increase the production and molecular weight of HA by 1.3-fold and 1.1-fold, respectively. This study revealed for the first time that overexpression of ndk could relieve the inhibition effect of uridine diphosphate (UDP) on Class II HA synthase and increase the production and molecular weight of HA, which proves to be an efficient strategy for the production of HA, and the preparation of other polysaccharides.

Effects and mechanisms of SNP-9 on Aβ25-35-induced damage in bEnd.3 cells / 中国药科大学学报

@#In order to investigate the effects of neuroprotective peptide SNP-9 which is derived from silk fibroin hydrolysate on the injury of the blood-brain barrier in Alzheimer′s disease (AD), Aβ25-35 was used to damage brain microvascular endothelial cells bEnd.3 to establish AD injury model and drug intervention was performed.MTT assay was used to detect the effects of SNP-9 and Aβ25-35 on cell viability.RT-qPCR was used to determine the effects of SNP-9 and Aβ25-35 on the mRNA levels of tight junctions (TJs)-related ZO-1, occludin and claudin-5.Western blot was used to detect the effects of SNP-9 and Aβ25-35 on the protein levels of TNF-α, phosphorylated NF-κB, NF-κB, IκBα and RAGE.The results showed that SNP-9 reduced bEnd.3 cell damage induced by Aβ25-35, and improved the abnormal mRNA levels of ZO-1, occludin and claudin-5 in model cells.It alleviated the abnormal protein levels of TNF-α, phosphorylated NF-κB, IκBα and RAGE induced by Aβ25-35. These results suggest that SNP-9 may regulate the levels of TNF-α in model cells by influencing RAGE/NF-κB pathway, and then ameliorate TJs-related abnormalities and alleviate bEnd.3 cell injury induced by Aβ25-35.

Advanced structure analysis and structure-activity relationship of polysaccharide SGP-2 from Sarcandra glabra / 中国药科大学学报

@#SGP-2 was an acidic polysaccharide with good hypoglycemic activity isolated from Sarcandra glabra (Thunb.) Nakai in the previous study. This study used the laser particle size analysis, transmission electron microscopy (TEM) and atomic force microscopy(AFM) analysis techniques to analyze the advanced structure of SGP-2 in the deionized water and Na2SO4 solution and discuss the structure-activity relationship between the advanced structural characterizations and the α-glucosidase inhibition activities of SGP-2 and its derivative in vitro.Results showed that SGP-2 presented aggregates and spheres in the deionized water.AFM analysis showed that the diameter of SGP-2 was 33 nm and the height was 1.84 nm, whereas compact spherical conformations with high degrees of branching were observed in 0.05 mol/L Na2SO4 solution and SGP-2 had smaller particle size in saline solution compared with that in water.SGP-2 treated by 0.5 mol/L urea and dialysis at the concentration of 1 000 μg/mL showed 98.8% inhibition activity of that from untreated SGP-2. The inhibition rate of short rod conformation with branches reached 83.3% when the temperature rose up to 140 °C, and the α-glucosidase inhibition activity was even higher than that of untreated SGP-2 under the same condition; while SGP-2 with the tangled glycan chains under the condition of carboxyl group reduced had much lower inhibition activity.Therefore, the spherical structure or the short rod conformation with branches played an important role in the activity of SGP-2. This research provides a theoretical basis for further study of structure-function relationship between the advanced structure and activity of polysaccharides.

Effects and mechanisms of microRNA-23b on neuronal apoptosis induced by Aβ25-35 / 中国药科大学学报

@#In this study, the effects and mechanisms of miR-23b in the AD cell model were explored. Aβ25-35 was used to induce neuronal injury model, and cell viabilities were detected by MTT assay. The effect of miR-23b on the apoptotic levels of Aβ25-35-induced SH-SY5Y cells was analyzed using Annexin V-FITC/PI detection kit. The effect of miR-23b on the mitochondrial membrane potential of Aβ25-35-induced SH-SY5Y cells was examined using JC-1 fluorescent probe. The levels of cell apoptosis-related proteins and autophagy-related proteins were detected by Western blot. The results showed that miR-23b could alleviate the apoptosis and the abnormal mitochondrial membrane potential in SH-SY5Y cells induced by Aβ25-35.This study suggested that miR-23b may attenuate Aβ25-35-induced neuronal apoptosis by regulating apoptosis and autophagy-related pathway.

Design and antitumor activity of immune checkpoint B7-H3 epitope vaccine / 中国药科大学学报

@#B7-H3 is an immune checkpoint molecule overexpressed on the surface of a variety of tumors, and is is an ideal target for tumor immunotherapy. In this study, nitrolated T cell epitope designed in the early stage of the laboratory was used to construct an epitope vaccine that can target immune checkpoint B7-H3. The vaccine can significantly inhibit tumor growth in the CT26 colon cancer model, and has a significant synergistic effect with the PD-L1 protein vaccine. B7-H3 vaccine can increase the proportion of CD4+ T cells in splenic T lymphocytes and the proportion of CD8+ T cells in tumor-infiltrating T lymphocytes, while reducing the proportion of suppressor Treg cells in tumor-infiltrating CD4+ T lymphocytes, which effectively improves tumor immunosuppressive microenvironment. Research results suggest that the B7-H3 epitope vaccine can be used as an effective tumor vaccine candidate molecule.

Effects of VHL inhibitor on rotenone-induced Caenorhabditis elegans model of Parkinson′s disease / 中国药科大学学报

@#To investigate the effects of VHL (von Hippel-Lindau) inhibitor on Caenorhabditis elegans (C.elegans) model of Parkinson''s disease (PD),C.elegans were exposed to rotenone and treated with VHL inhibitor VH298.The death,dopaminergic neurodegeneration and mitochondrial unfolded protein response (mito-UPR) of transgenic strains with the markers zcIs9 and otIs181 exposed to different concentrations of rotenone were investigated. The death,dopaminergic neurodegeneration,and changes of behaviors including head thrashes,body bends and foraging behavior of C.elegans model of PD treated with different concentrations of VH298 were explored.The results showed that different concentrations of rotenone can lead to the death,dopaminergic neurodegeneration and abnormal mito-UPR of transgenic nematodes with zcIs9; otIs181,while the VHL inhibitor can decrease the death rate and alleviate dopaminergic neurodegeneration of rotenone-induced C.elegans model of PD.The VHL inhibitor can also attenuate the behavioral abnormalities of head thrashes,body bends and foraging behavior of C.elegans model.These results suggest that rotenone may cause mitochondrial damage in the transgenic nematodes with zcIs9; otIs181, and then destroy mitochondrial homeostasis,thereby resulting in dopaminergic neurodegeneration and death of the nematodes. The VHL inhibitor VH298 may promote the survival of rotenone-induced C.elegans model of PD,and alleviate dopaminergic neurodegeneration,thereby improving the behavioral abnormalities of C.elegans model of PD.

Effects and mechanisms of FGF21 on neuronal damage induced by rotenone / 中国药科大学学报

@#In this study,the SH-SY5Y cell model with Parkinson"s disease-like lesions was established by using rotenone,and the effect and mechanism of fibroblast growth factor 21 (FGF21) on the cell model were explored. Different concentrations of FGF21 were used to treat neuronal injury model induced by rotenone,and cell viabilities were detected by MTT assay. Effects of FGF21 and rotenone on the apoptotic levels of SH-SY5Y cells were analyzed by using Annexin V-FITC detection kit. Western blot was used to assess the effects of FGF21 and rotenone on the protein levels of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in SH-SY5Y cells. Effects of FGF21 and rotenone on reactive oxygen species (ROS) levels in SH-SY5Y cells were tested using DCFH-DA fluorescent probe. The results showed that FGF21 could reduce the damage in SH-SY5Y cells induced by rotenone,inhibit cell apoptosis,alleviate the abnormalities of TH and α-syn in SH-SY5Y cells induced by rotenone and down-regulate the abnormal ROS levels in SH-SY5Y cells. The results suggested that FGF21 may attenuate rotenone-induced neuronal damage through regulation of oxidative stress.

Advances of DNA-based nanomaterials in tumor therapy / 中国药科大学学报

@#Antitumor drugs usually have deficiencies such as poor water solubility, low targeting, poor stability, and difficulty in being taken up by tumor cells. The development of an ideal drug delivery vehicle is still an urgent problem to be solved in the field of cancer therapy. Due to their excellent sequence programmability, biocompatibility and biodegradability, DNA-based nanomaterials have been widely used as drug delivery vehicles for cancer treatment. Numerous studies have shown that DNA nanomaterials can effectively load cancer therapeutic agents, and achieve tumor targeted delivery, efficient cellular internalization as well as stimuli-responsive drug release. Starting from the history and development of DNA nanotechnology, this review illustrates the application progress of DNA nanomaterial as drug delivery vehicle in chemotherapy, gene therapy, immunotherapy and photodynamic therapy, and the future development is prospected so as to provide some reference for other researchers in this field.

Construction of mutant NDUFS7 plasmid and its effects on neural cells / 中国药科大学学报

@#To investigate the effects of NDUFS7 gene mutation on neurons, the mutant plasmid of pcDNA3.1(+)-NDUFS7 Q208STOP was constructed and transfected into differentiated SH-SY5Y cells. The effect of transfecting mutant plasmid on the viability of dopaminergic neural cells was detected by MTT assay. The effect of transfection of mutant plasmid on apoptosis was detected by Annexin Ⅴ-FITC/PI staining followed by flow cytometry assay. The changes in the expression levels of apoptosis-related proteins Bax and Bcl-2 in cells after transfection of mutant plasmid were detected by Western blot. The effects of transfection of mutant plasmid on the mitochondrial membrane potential in differentiated SH-SY5Y cells and the intervention effect of antioxidant Trolox were examined using JC-1 fluorescent probe. The intervention effect of Trolox on the apoptosis of differentiated SH-SY5Y cells transfected with mutant plasmid was detected by PI/Hoechst staining. The results showed that the subunit mutation of mitochondrial complex I in dopaminergic neurons could lead to decreased neuronal viability and increased apoptosis, while antioxidants could alleviate the abnormal mitochondrial membrane potential and apoptosis caused by transfection of mutant plasmids, suggesting that transfection of mutant plasmid of NDUFS7 gene could lead to apoptosis by causing abnormal mitochondrial function in dopaminergic neurons.

Effect and mechanism of FGF21 on astrocyte damage induced by Aβ25-35 / 中国药科大学学报

@#To investigate the effects and mechanism of fibroblast growth factor 21(FGF21)on astrocytes in AD-like lesions, Aβ25-35 was used to induce astrocyte model damaged. Cell model was established by inducing C6 astrocyte cell line and primary astrocyte damage with Aβ25-35. Different concentrations of FGF21 were used to intervene cell injury model induced by Aβ25-35, and cell viabilities were detected by MTT assay. Effects of FGF21 and Aβ25-35 on reactive oxygen species(ROS)levels in C6 cells were tested using DCFH-DA probe and flow cytometry. Western blot was used to assess the effects of FGF21 and Aβ25-35 on the activities of mitogen-activated protein kinases(MAPKs)in C6 cells. The results showed that FGF21 could reduce the damage of C6 cells and primary astrocytes induced by Aβ25-35, down-regulate the abnormal ROS level in C6 cells, and alleviate the abnormal phosphorylation levels of MEK1/2, ERK1/2 and p38 in C6 cells induced by Aβ25-35, suggesting that FGF21 may attenuate Aβ25-35-induced astrocyte damage by regulating ROS pathway and MAPKs signaling pathway.

Research advances of bispecific antibody drugs in tumor therapy / 中国药科大学学报

@#Tumor immunotherapy is currently the new direction for the treatment of cancer. Bispecific antibody can bind two different antigens, so the development prospect in the field of tumor treatment is very attractive. The most compelling trifunctional antibody and bispecific T-cell engager in bispecific antibodies have been marketed separately, with representative drugs as catumaxomab and blinatumomab, respectively. So far, nearly 100 antitumor bispecific antibody drugs are undergoing clinical trials and in-depth understanding of their mechanisms of action will provide more powerful solutions for cancer treatment. This review summarizes the progress of catumaxomab, blinatumomab and current highly promising bispecific antibody drugs, for the further development and application of tumor therapy.

lmmunogenicity of HER2 vaccine containing p-nitrophenylalanine / 中国药科大学学报

In order to delete the immune tolerance of self-protein HER2 and produce an efficient immune response,the genetic code expansion technique was employed to introduce the immunogenic amino acid p-nitro-phenylalanine into the position 5 ,26 and 79 of the HER2 fragment and HER2 mutants containing p-nitropheny-lalanine were obtained.Prototype and mutant HER2 molecules with a purity of more than 95% were obtained through a nickel affinity column.Immunogenicity analysis of the HER2 vaccine showed that pNO2Phe79HER2 was able to produce high titer cross-reactive antibodies in C57BL/6 mice and that the mouse antisera could recognize SKBR-3 cells with intact HER2 receptor extracellular domain and cleave HER2 highly expressed HER2 +B16F10 cells through ADCC effect.These findings suggested that pNO2Phe79HER2 which incorporates p-nitro-phenylalanine could be used as a tumor vaccine candidate targeting HER2.

Preparative optimization and antitumor activity of selenylaed polysaccharides from the extract residue of Sarcandra glabra（Thunb.）Nakai / 中国药科大学学报

Selenylation modification of polysaccharides from the extract residue of Sarcandra glabra(Thunb.) Nakai was operated with sodium selenite as modification agent and barium chloride as the catalyst.The synthesis conditions were optimized by orthogonal experiment with the yields and contents of selenium as indicators.Then, the purity,molecular weight,infrared spectroscopy,thermophysical properties and antitumor activity of the seleny-lated polysaccharides were determined.Results showed that the optimum conditions for preparing selenylated polysaccharides from the extract residue of S.glabra(Thunb.)Nakai were at 70 ℃ for 7 h with 1.0％ nitric acid using 1.5 g barium chloride as catalyst.Under the optimum condition,the physicochemical properties of the derivatives were measured and the results showed that the total carbohydrate content was(94.89 ± 0.83)％,the yield was(21.93 ± 0.85)％,the content of selenium was(944.54 ± 13.91)μg/g,the molecular weight was 1 339 kD. In vitro antitumor activity indicated that exposure of HepG2 cells to the increasing concentrations of the crude polysaccharide from the residue of S.glabra(Thunb.)Nakai and its selenylated products decreased cell viability in dose-dependent and time-dependent manner.The selenium polysaccharide demonstrated a better antitumor activity compared with the raw polysaccharide,which could be explored as a potential antitumor drug from abandoned extracted residue for the functional foods and pharmaceutical industries.

Construction, expression, purification and neuroprotective activity of TAT-FGF21 fusion protein / 中国药科大学学报

@#In order to improve the brain distribution of fibroblast growth factor 21(FGF21), TAT-FGF21 fusion protein was designed and its neuroprotective activity was investigated. The recombinant plasmid of pET28a-TAT-FGF21 was constructed and transformed into E. coli BL-21(DE3)sensitive bacteria. The TAT-FGF21 fusion protein was purified by Ni-NTA affinity chromatography column after IPTG induced expression. The SH-SY5Y cell damage model was induced by Aβ25-35, and the TAT-FGF21 fusion protein was used to intervene. The effects of Aβ25-35 and TAT-FGF21 induced on SH-SY5Y cell viability were determined using MTT method; DCFH-DA fluorescent probe was used to detect the intervention effect TAT-FGF21 on reactive oxygen species(ROS)generation induced by Aβ25-35 in SH-SY5Y cells; the effects of Aβ25-35 and TAT-FGF21 on mitochondrial membrane potential in SH-SY5Y cells were detected with JC-1 fluorescent probe. The results showed that TAT-FGF21 could improve the viability of SH-SY5Y cells, reduce the intracellular ROS production level of SH-SY5Y cells, and enhance the mitochondrial membrane potential of SH-SY5Y cells, which indicate that TAT-FGF21 could protect neurons on SH-SY5Y cell injury induced by Aβ25-35 through alleviating oxidative damage.

Advances of combined immunotherapy in tumor / 中国药科大学学报

@#Immune checkpoints are inhibitory signaling pathways in the immune system that maintain balance with co-stimulatory molecules, maintain tolerance to their own tissues, and avoid autoimmune responses. The development of tumors is accompanied by the upregulation of co-suppressor molecules and related ligands, causing the decline or exhaustion of T cell functions, which makes tumor cells excape immune surveillance. The development of monoclonal antibodies against inhibitory receptors and ligands to regulate T cell activity and improve antitumor immune responses have achieved promising clininal results. Combined treatment of immunomodulatory signaling pathways also showed a certain of synergy. This article summarizes the evaluation of combined tumor immunotherapy strategies and synergies, and outlooks the feasibility of combined immunotherapy and the selection of immunization strategies.

Optimization of in vitro human follicular helper T cell differentiation condition / 中国药科大学学报

In order to explore the regulation mechanisms of follicular helper T cell (Tfh Cell) differentiation,optimized conditions of in vitro induction from both peripheral blood mononuclear cells and MAC sorted Na(i)ve CD4 + T cells to human Tfh cells were developed.Induction efficiency difference of TCR signal anti-hCD3e stimulation between coated on solid phase and in soluble phase was also determined.Differentiation efficiency of CD4 + CXCR5 + ICOS+PD-1 + Tfh cell was determined by FACS while the expression level of IL-21 in cell supernatant was determined by ELISA tests.An ultimate induction condition that 5 μg/mL coated overnight anti-hCD3e stimulated na(i)ve CD4 + T cells to differentiate into Tfh at an up to 20.4％ percentage was finally determined.The optimization of in vitro induction protocol of human Tfh provided an effective examine platform for the studies on Tfh differentiation mechanisms and related pharmacology,toxicity and metabolic experiments.

Protective effect of fibroin peptides on Aβ25-35-induced injury in SH-SY5Y cells and its mechanism / 中国药科大学学报

This study aimed at exploring the molecular mechanism of fibroin peptide in preventing the Aβ25-35-induced neuronal damages in SH-SY5Y cells.MTT was used to detect the effect of fibroin peptide on the changes of the Aβ25-35-induced injuries in SH-SY5Y cells;Western blot was employed to detect the effect of fibroin peptide on the changes of the Aβ25-35-induced hyperphosphorylation of Tau in SH-SY5Y cells;DCFH-DA probe method was used to detect the effect of fibroin peptide on the Aβ-induced production of intracellular reactive oxygen species (ROS) in SH-SY5Y cells.The results indicated that fibroin peptide could improve the activity of the PP2A and inhibit the activity of GSK-3β to decrease the hyperphosphorylation of Tau induced by Aβ25-35.Fibroin peptide could significantly prevent the Aβ25-35-induced neuronal damages and multisite Tau hyperphosphorylation.In addition,fibroin peptide could also reduce oxidative damage to protect neurons by significantly decreasing the Aβ25-35-induced production of intracellular ROS.