Anti-inflammatory mechanism of annexin A1 mimetic peptide Ac2-26 on lung injury induced by cardiopulmonary bypass in rats / 中华胸心血管外科杂志

Objective:To observe the effect of annexin A1 peptidomimetic Ac2-26 on lung injury in rats with cardiopulmonary bypass, and analysis its anti-inflammatory mechanism.Methods:Eighteen male SD rats were divided into 3 groups(n=6) according to the random number table: sham operation group(S group), ischemia reperfusion group(IR group), Ac2-26 group(A group). The sham operation group received femoral arteriovenous puncture and catheterization, only the left lung was opened and the cardiopulmonary bypass tube was not connected. The other two groups were established with cardiopulmonary bypass left lung ischemia-reperfusion injury model. The IR group was given the same volume 10 minutes before cardiopulmonary bypass with the normal saline solution. Group A was injected with Ac2-26(1 mg/kg) through the tail vein 10 minutes before cardiopulmonary bypass. The arterial blood of the three groups of rats was taken for blood gas analysis before CPB(T1), immediately after opening the left lung hilum(T2), and at the end of the experiment(T3) to calculate OI and RI. At T1 and T3, the femoral vein blood was taken to measure the number of PMN. At T3, pulmonary artery blood was taken to measure the number of PMN and VCAM-1 content, left lung tissue was taken to measure NE and MPO content by ELISA, lung tissue neutrophil apoptosis was measured by Tunel method, and lung tissue ICAM-1 and AnxA1 protein expression was measured by Western-Blot.Results:At T3, compared with group S, the number of PMN in peripheral blood and pulmonary artery blood and the content of pulmonary artery VCAM-1 in IR group were significantly higher, the content of MPO and NE in lung tissue increased, the lung tissue pathological damage score increased, OI decreased and RI increased, all P<0.05. Compared with IR group, the lung tissue pathological damage score of rats in group A was significantly reduced, the number of PMN in peripheral blood and pulmonary artery blood and the content of VCAM-1 in pulmonary artery blood were significantly reduced, the expression of AnxA1 and ICAM-1 in the lung tissue of rats decreased; the content of MPO and NE in lung tissue decreased, and the apoptosis rate of PMN increased, all P<0.05. Conclusion:Ac2-26 annexin A1 peptidomimetics can inhibit the aggregation of neutrophils in the lung, weaken the adhesion of neutrophils to the pulmonary vascular endothelium, promote the apoptosis of neutrophils in the lungs, and reduce the content of NE and MPO. It has a protective effect on lung injury after cardiopulmonary bypass in rats.

Development of a novel stability indicating RP-HPLC method for quantification of Connexin43 mimetic peptide and determination of its degradation kinetics in biological fluids / 药物分析学报

Connexin43 mimetic peptide (Cx43MP) has been intensively investigated for its therapeutic effect in the management of inflammatory eye conditions, spinal cord injury, wound healing and ischemia-induced brain damage. Here, we report on a validated stability–indicating reversed-phase high performance liquid chromatography(RP-HPLC)method for the quantification of Cx43MP under stress conditions.These included exposure to acid/base, light, oxidation and high temperature. In addition, the degradation kinetics of the peptide were evaluated in bovine vitreous and drug-free human plasma at 37 ℃. Detection of Cx43MP was carried out at 214 nm with a retention time of 7.5 min. The method showed excellent linearity over the concentration range of 0.9–250μg/mL(R2≥0.998),and the limits of detection(LOD)and quantification(LOQ) were found to be 0.90 and 2.98 μg/mL, respectively. The accuracy of the method determined by the mean percentage recovery at 7.8, 62.5 and 250μg/mL was 96.79%, 98.25% and 99.06% with a RSD of<2.2%. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions and completely degraded within 24 h at 37 ℃(0% recovery)and within 12 h at 80 ℃(0.34% recovery).Cx43MP was found to be more stable in bovine vitreous(t1/2slow=171.8 min)compared to human plasma(t1/2slow=39.3 min)at 37 ℃ according to the two phase degradation kinetic model. These findings are important for further pre-clinical development of Cx43MP.

A Sporadic Case of Epstein Syndrome: A Rare Cause of Refractory Thrombocytopenia / 대한내과학회지

A 37-year-old female presented to our hospital with a history of bleeding episodes (excessive bleeding after tooth extraction, gum bleeding, easy bruising, and excessive menstruation) and severe thrombocytopenia (2,000/µL). She had no family history of bleeding tendency or thrombocytopenia. No peripheral lymphadenopathy or splenomegaly was noted. The patient's white blood cell count was normal; hemoglobin was 9.7 g/dL. A peripheral blood smear showed markedly decreased platelets, with occasional giant or large platelets. Bone marrow examination found increased megakaryocytes. The patient also complained of hearing difficulty; a hearing test indicated sensory-neural hearing impairment. Her thrombocytopenia was refractory to treatment with glucocorticosteroids, intravenous gamma-globulin, and danazol. In the 13 years following her initial presentation, the patient required anti-hypertensive treatment, a hearing-aid for progressive hearing loss, and started maintenance kidney dialysis. Her clinical history of refractory thrombocytopenia, progressive hearing impairment, and renal failure suggested myosin heavy chain 9 gene-related congenital syndrome (Epstein syndrome), which was confirmed by the presence of a heterozygous deletion mutation, c.221_223del, (p.Lys74del) in peripheral leukocyte deoxyribonucleic acid.

Advances of synthetic lipoproteins as drug nanovectors / 中国药科大学学报

@#Synthetic lipoproteins are efficient nanovectors for targeting delivery of biological drugs, chemical drugs and imaging contrast agents and so on. They have ultra-small particle size, excellent biocompatibility, favorable circulation half-life and specific lipoprotein-receptors-binding capacity of lipoprotein receptors. Compared with traditional natural lipoproteins, synthetic lipoproteins possess both native biochemical properties and functions, and superior drug delivery potentials. This paper introduces the development of lipoproteins as drug nanovectors and reviews preparation methods of synthetic lipoproteins, in vitro and in vivo applications and the approaches employed to expand the limited applications of lipoproteins as drug nanovectors. Further development of synthetic lipoproteins is also proposed.

Collagen mimetic peptide-PEG hybrid hydrogel for 3D culture of rabbit bone marrow mesenchymal stem cells / 国际生物医学工程杂志

Objective To construct a extracellular matrix-like collagen mimetic peptide-PEG hybrid hydrogel and to study the usage of this hydrogel in 3D culture of rabbit bone marrow mesenchymal stem cells (rBMSCs).Methods The hybrid hydrogel was synthesised by conjugating the cysteine at the end of the collagen mimetic peptide with the maleimine-modified multi-arm PEG.The circular dichroism spectra were used to characterize the triple helix structure and thermal stability of the collagen mimetic peptides.The rheology test and scanning electron microscopy were used to study the gelation process,mechanical strength and internal structure of the hydrogel.The rBMSCs were embedded in the hybrid hydrogel for 3D culture.The cell compatibility of the hydrogel and its effect on differentiation of the cells was studied.Results Collagen mimetic peptides could promote spontaneous formation of triple helix structure in the natural collagen,and the thermal transition temperature was 49.4 ℃.The formation process of the collagen mimetic peptides-PEG hybrid hydrogel was rapid,in which the porous network-like fibrous structure was formed.After the encapsulation of rBMSCs within the hydrogel for 24 h,most of the cells remained viable.Gene expression analysis showed that the hybrid hydrogel could affect the differentiation of rBMSCs.Conclusions The collagen mimetic peptide-PEG hybrid hydrogel possesses the characteristics of mild preparation condition,good mechanical strength and good cell compatibility,and is favorable to chondrocyte differentiation of rBMSCs.

Research progress in erythropoietin mimetic peptides / 天津医药

Erythropoietin (EPO) is an active glycoprotein synthesized by kidney. The physiological function of regulat?ing the synthesis of erythrocytes by EPO makes it as a clinical drug for treatment of anemia resulted from chronic kidney fail?ure. However, its short biological half-life makes frequent administration, which limits its wide clinical utility since the tough burden and pain on patients. Therefore, the development of EPO derivatives with good efficacy, less adverse reaction and long duration has been a hot spot in the field during several decades. There are currently many different variants of EPO derivatives including erythropoiesis stimulating agents (ESAs) on the market. This article aims to summarize the recent re?search progress in the development of erythropoietin derivatives, specially focusing on EPO mimetic peptides (EMP).

Papel protector de las lipoproteínas de alta densidad en sepsis: aspectos básicos e implicancias clínicas / Protective role of high density lipoproteins in sepsis: basic issues and clinical implications

High density lipoproteins (HDL) are responsible of reverse cholesterol transport and play an important antiatherogenic role. In recent years, several studies suggest that HDL have additional functions, including a possible anti-inflammatory activity in infectious conditions. Furthermore, available evidence indicates that the presence of lipopolysaccharide (LPS) within the circulation during infectious states induced by gram-negative bacteria may be involved in the decrease in HDL cholesterol levels and changes in lipoprotein composition, which have been associated with a higher mortality due to sepsis in animal models and in humans. In this article, we review this subject and also discuss possible mechanisms that explain the positive impact achieved by native HDL, reconstituted HDL, or HDL apolipoprotein peptides on the inflammatory response and mortality in models of endotoxemia. In this regard, it has been proposed that one of the mechanisms by which HDL protect against sepsis may be mediated by its binding ability and/or neutralizing capacity on LPS, avoiding an excessive response of the immune system. Thus, increasing blood levels of HDL and/or parenteral HDL administration may represent a new anti-inflammatory tool for managing septic states in humans.

Las lipoproteínas de alta densidad (HDL) son responsables del transporte reverso de colesterol y ejercen un importante papel anti-aterogénico. En los últimos años, diversos estudios indican que las HDL también tendrían otras funciones críticas, incluyendo una posible actividad anti-inflamatoria durante estados infecciosos. Además, la evidencia disponible sugiere que la presencia de lipopolisacárido (LPS) en la circulación durante estados infecciosos inducidos por bacterias gramnegativas podría estar involucrado en la disminución del colesterol HDL y los cambios en composición de esta clase lipoproteínas, lo cual se asociaría con una mayor tasa de mortalidad por sepsis en modelos animales y en humanos. En este trabajo, se revisan los antecedentes mencionados y además se discuten posibles mecanismos que explican la disminución de la respuesta inflamatoria y de la mortalidad que se logran en modelos de endotoxemia tratados con HDL o preparaciones similares. En este sentido, se ha propuesto que uno de los mecanismos protectores de las HDL estaría mediado por su capacidad de unión y/o neutralización del LPS, evitando una respuesta exacerbada del sistema inmune. De esta manera, el aumento de los niveles sanguíneos de HDL y/o su administración parenteral podrían constituir nuevas herramientas anti-inflamatorias para el manejo de estados sépticos en humanos.

The effect and mechanism of an apolipoprotein E mimetic peptide ApoE23 on plasma lipopolysaccharide levels in the septic mice / 中华急诊医学杂志

Objective To observe the effect of apolipoprotein E mimetic peptide (ApoE23) on lipopolysaccharide (LPS) levels in plasma and the regulatory role of ApoE23 on low density lipoprotein receptor (LDLR) on liver cells in the septic mice.Methods An ApoE mimetic peptide was designed and referred terminologically as ApoE23 in abbreviation.ApoE23 was synthesized by using solid phase synthesis assay and were refined by using high performance liquid chromatography (HPLC).The peptide was identified and confirmed by using electron spray ionization mass spectrometry and amino acid composition analysis.The C57BL mice infected with Salmonella typhimurium group B were treated with apoE23 injected into tail vein.The plasma LPS levels were measured by using immunoturbidimetry.The LDLR expression and level on liver cells were measured by real time PCR and western blot respectively.Results The plasma LPS levels significantly increased and the liver LDLR expression decreased in the septic mice.ApoE23 treatment markedly reduced the plasma LPS levels and redressed the LDLR down-expressions on liver cells both in mRNA and protein levels compared to the septic mice without ApoE23 treatment.Conclusions The reduction of LPS level after ApoE23 treatment may be associated with the modulation role of ApoE23 in LDLR expression on liver cells,and ApoE23 may be a potential agent against bacterial sepsis as well.One of possible mechanisms was most likely associated with effect of ApoE23 on LDLR expression.

Inhibitory effect of apolipoprotein A-I mimetic peptide D-4 F on scavenger receptor A1 in macrophage-derived foam cells / 中国病理生理杂志

AIM:To investigate the inhibitory effect of apolipoprotein A-I mimetic peptide D-4F on the scaven-ger receptor A1 ( SR-A1 ) in macrophage-derived foam cells induced by oxidized low-density lipoprotein ( ox-LDL ) . METHODS:RAW264.7 cells were pretreated with different concentrations (12.5, 25 and 50 mg/L) of D-4F or 50 mg/L inactive control peptide scrambled D-4F (sD-4F) for 1 h or endoplasmic reticulum stress (ERS) inhibitor 4-phenylbutyr-ic acid (5 mmol/L) for 30 min, followed by the treatment with 100 mg/L ox-LDL for 12 h.In addition, the cells were pre-treated with 50 mg/L D-4F or sD-4F for 1 h, and then stimulated with 2 mg/L tunicamycin (TM;an ERS inducer), for 4 h.The viability of the cells was measured by MTT assay, and the content of intracellular total cholesterol ( TC) was meas-ured by a tissue/cell TC assay.The protein and mRNA levels of SR-A1 and glucose-regulated protein 78 (GRP78) were analyzed by Western blotting and quantitative real-time PCR, respectively.The fluorescence intensity of DiI-ox-LDL in the cells was detected by a multifunctional microplate reader.RESULTS:D-4F significantly reduced ox-LDL-induced macro-phage injury and intracellular cholesterol accumulation, and attenuated the ox-LDL-induced expression of SRA1 and GRP78 in a dose-dependent manner.Additionally, D-4F significantly inhibited the TM-induced protein expression of SR-A1 and GRP78, and attenuated the uptake of ox-LDL by macrophages.CONCLUSION: D-4F reduces ox-LDL-induced macro-phage cholesterol accumulation and injury by inhibiting SR-A1 expression.The mechanism may be related to the inhibition of ERS signaling pathway mediated by GRP78.

Expression and identification of recombinant fusion protein of thrombopoietin mimetic peptide and growth hormone in Pichia pastoris / 第三军医大学学报

Objective To fulfill high-efficient expression of rTMP-GH,a recombinant fusion protein of thrombopoietin mimetic peptide and human growth hormone,in Pichia pastoris.Methods cDNA fragment coding for rTMP-GH was amplified by PCR,and subsequently cloned into the expression vector pPIC9K.The linearlized plasmid pPIC9K-rTMP-GH was transformed into Pichia pastoris GS115 cells and screened in MD/MM plates under pressure of G418.The recombinant fusion protein rTMP-GH was identified by Western blotting,and its biological activity was analyzed by its ability on colony formation of megakaryoblasts.Results The pPIC9K-rTMP-GH expression plasmid was successfully constructed.GS115 cells with high expression of rTMP-GH was screened out,and the rough purified rTMP-GH showed promotive effect on megakaryoblast colony formation.Conclusion High-efficient expression of rTMP-GH in Pichia pastoris is achieved.

Effects of high density lipoprotein and apolipoprotein A-Ⅰ mimetic peptide on the expression of adiponectin in 3T3-L1 adipocytes / 中国药理学通报

Aim To explore the effect of high density lipoprotein and apolipoprotein A-I mimetic peptide on the secretion and the expression of adiponectin in fully differentiated 3T3-L1 adipocytes in inflammatory status and the possible mechanism.Methods Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentrations of high density lipoprotein(10~100 mg?L-1) and apolipoprotein A-I mimetic peptide(1~50 mg?L-1)with 100 ?g?L-1 lipopolysaccharide(n=3).Evaluate the levels of adiponectin in supernatant,the mRNA expression of adiponectin and PPAR?,and the activity of NF-?B.Results During LPS stimulation adiponectin concentrations in supernatant decreased from 0.25?0.03 ?g?L-1 to 0.14?0.02 ?g?L-1(P

Anti-HER2/neu Peptide Producing Vector System for Biologic Therapy - Is It Possible to Mass-produce the Peptide?

A humanized monoclonal antibody against HER2 has been using in a clinical setting and has been shown to possess therapeutic properties. A mimetic peptide against HER2 was also reported to bind to the HER2 receptor with some therapeutic potential. Based on a previous report and the sequence of Herceptin, we designed oligonucleotides of anti-HER2 mimetic peptides, named V2 and V3 peptides, in order to develop a peptide- producing vector system for biologic therapy against HER2- overexpressing cancers. We also adopted the sequence of a previously reported mimetic peptide, V1 (Park BW et al. Nat. Biotechnol, 2000, 18: 194-198), as a reference peptide. We examined the effects of the V2 and V3 peptides against the HER2-overexpressing cell lines, SK-BR-3 and T6-17. Transient transfection of the construct expressing V1 and V2 inhibited cell proliferation in HER2-overexpressing cell lines by 20 - 30%, but had no effect on the HER2-negative NIH3T3 cells. The proliferation inhibition shown by V2 was slightly better than that shown by V1. Recombinant peptides V2 and V3 were produced on a large scale in an E. coli system, and the V2 peptide showed anti-HER2-specific tumor cell proliferation inhibition of 10% to 30%. Current results suggest that anti-HER2 mimetic peptides, overexpressed by a constitutive promoter or produced in an E. coli system, could specifically inhibit the proliferation of HER2-expressing cancer cells. Further efforts to augment the biologic specificity and efficacy and to develop new technologies for the purification of the peptide from the E coli system are needed.

Effects of a new recombinant fusion protein rTMP-GH on proliferation of megakaryocytes in vitro / 第三军医大学学报

Objective To investigate the effects of rTMP-GH, a recombinant fusion protein of thrombopoietin mimetic peptide (TMP) and human growth hormone (GH), on the proliferation and thrombocytopoiesis of cultured megakaryocytes. Methods After being treated with 100 ng/ml of rTMP-GH for 7 d, Dami cells, a kind of megakaryocyte cell line, were analyzed by observing the numbers of colony forming unit. Meanwhile, Western blotting and RT-PCR were applied to detect the expression changes of globin transcription factor-1 (GATA-1), which is a main regulator of thrombocytopoiesis in megakaryocytes. Results The numbers of colony forming unit were markedly increased in cultured Dami cells incubated with rTMP-GH. Compared with normal control and GH treatment groups, both the mRNA and protein levels of GATA-1 were up-regulated significantly in Dami cells treated with rTMP-GH. Conclusion rTMP-GH has a strong ability to promote the proliferation and thrombocytopoiesis of megakaryocytes.