没食子酸对过敏性鼻炎小鼠的抗过敏作用研究

目的 探究没食子酸对过敏性鼻炎(AR)小鼠的抗过敏作用.方法 将100只小鼠随机分为空白组、模型组,低剂量实验组、中剂量实验组和高剂量实验组,每组20只.空白组小鼠不做处理,其他组小鼠通过卵清蛋白(OVA)诱导构建AR模型,低、中、高剂量实验组中分别采用20,40和80 mg·kg-1没食子酸进行治疗.用酶联免疫吸附法...     

葛根素下调Atg5、LC3Ⅰ/Ⅱ蛋白对缺血再灌注脑损伤小鼠的神经保护作用

目的 观察葛根素通过下调自噬相关基因5(Atg5)、微管相关蛋白轻链Ⅰ/Ⅱ(LC3Ⅰ/Ⅱ)蛋白表达发挥抗缺血再灌注脑损伤小鼠的神经保护作用。方法 将ICR小鼠48只随机分为4组，分别为假手术组、模型组和低剂量、高剂量实验组，每组12只。实验时间共10 d,于第6天夹闭小鼠双侧颈总动脉缺血30 min后进行再灌注，建立缺血性脑损伤小鼠模型，低、高剂量实验组小鼠造模前后5天均腹腔注射葛根素(50,100 mg·kg^-1·d^-1)。用平衡木实验评价小鼠的平衡能力，用自主活动实验评价小鼠的日常活动能力，用蛋白质印迹法检测各组小鼠大脑皮层自噬关键蛋白Atg5、LC3Ⅰ/Ⅱ的相对表达。结果 在术后1 d,假手术组、模型组和低、高剂量实验组小鼠穿越平衡木的时间分别为(5.71±4.23),(21.23±9.49),(10.48±6.50)和(11.33±8.91)s;在术后3 d各组小鼠穿越平衡木的时间分别为(6.09±2.73),(16.83±7.51),(9.48±5.94)和(8.09±5.17)s;在术后5 d各组小鼠穿越平衡木的时间分别为(5.3...     

新疆阿魏乙酸乙酯部位对S-180腹水瘤模型小鼠的影响

目的 探讨新疆阿魏乙酸乙酯提取物对S-180腹水瘤模型小鼠的影响.方法 将120只KM小鼠随机分为正常对照组(等体积溶剂),模型组(等体积溶剂),顺铂组(5 mg/kg)及阿魏乙酸乙酯部位低、中、高剂量组(实验Ⅰ组、Ⅱ组、Ⅲ组,给药剂量以生药计1.0,2.0,3.0 g/kg),各20只.将含2×106 mL-1 S-...     

癸源煎配方颗粒对卵巢储备功能下降模型小鼠的改善作用及机制研究

目的:研究癸源煎配方颗粒(GDFG)对卵巢储备功能下降(DOR)模型小鼠的改善作用及机制。方法:将动情周期正常的42只雌性ICR小鼠随机分为对照组、模型组、戊酸雌二醇组(阳性对照,0.15 mg/kg)和GDFG低、中、高剂量组(0.75、1.49、2.98 g/kg),每组7只。除对照组外,其余各组小鼠均腹腔注射顺铂(3 mg/kg)复制DOR模型。造模成功后,给药组小鼠灌胃相应药物,模型组和对照组小鼠灌胃生理盐水,每天1次,连续4周。末次给药后,采用酶联免疫吸附试验测定小鼠血清中抗米勒管激素(AMH)和促卵泡生成素(FSH)水平;采用苏木精-伊红(HE)染色法观察小鼠卵巢组织病理学形态;采用免疫组化法观察小鼠卵巢组织中AMHR受体Ⅱ(AMHRⅡ)、Smad4蛋白的分布情况;采用Western blot法检测小鼠卵巢组织中AMHRⅡ、Smad4蛋白的表达水平。结果:与对照组比较,模型组小鼠血清中AMH水平和卵巢组织中AMHRⅡ、Smad4蛋白表达水平均显著降低(P<0.01),血清中FSH水平显著升高(P<0.01);卵泡组织中可见卵泡皱缩、卵泡核丢失、卵巢间质纤维化、黄体疏松;AMHRⅡ、Smad4蛋白主要分布在卵泡膜上和卵巢间质中。与模型组比较,GDFG各剂量组小鼠血清中AMH水平和卵巢组织中AMHRⅡ、Smad4蛋白表达水平均显著升高(P<0.01),血清中FSH水平均显著降低(P<0.05或P<0.01);卵巢组织中可见各级卵泡、卵泡形态改善,未见明显核丢失和卵丘形成;AMHRⅡ、Smad4蛋白主要分布在卵泡核(GDFG高剂量组除外)及卵巢颗粒细胞膜上(GDFG中剂量组以分布在窦卵泡为主),成熟卵泡核周围或黄体中有少许分布。结论:GDFG可改善DOR模型小鼠的卵巢功能,其机制可能与升高血清中AMH水平和卵巢组织中AMHRⅡ、Smad4蛋白表达水平,改善AMHRⅡ、Smad4蛋白在卵巢颗粒细胞膜和卵泡核内的分布以及降低血清中FSH水平有关。     

3-甲基嘌呤对脂多糖诱导小鼠急性肺损伤的保护作用及其机制研究

目的:研究自噬抑制剂3-甲基嘌呤(3-MA)对脂多糖(LPS)诱导小鼠急性肺损伤的保护作用及其机制。方法:取小鼠随机分为正常对照组、模型(LPS 15 mg/kg)组、药物对照(3-MA 20 mg/kg)组和低、高剂量治疗(LPS 15 mg/kg+3-MA 20、40 mg/kg)组,每组10只。除正常对照组和药物对照组外,其余各组小鼠ip LPS复制急性肺损伤模型,药物对照组和低、高剂量治疗组小鼠分别于建模前1 h ip相应剂量的3-MA。建模6 h后分别测量各组小鼠的肺湿/干质量比(W/D),HE染色观察肺组织病理变化;Western blot法检测肺组织肿瘤坏死因子α(TNF-α)、NF-κB p65、LC3BⅡ/Ⅰ、激活型半胱氨酸氨基蛋白酶3(Cleaved-caspase-3蛋白的表达水平。结果:与正常对照组比较,模型组小鼠的W/D值和TNF-α、NF-κB p65、LC3BⅡ/Ⅰ、Cleaved-caspase-3蛋白表达均增强(P<0.01);与模型组比较,低剂量治疗组小鼠的W/D值和TNF-α、NF-κB p65、LC3BⅡ/Ⅰ、Cleaved-caspase-3蛋白表达均减弱(P<0.05),高剂量治疗组小鼠仅LC3BⅡ/Ⅰ蛋白表达减弱(P<0.01)。结论:在LPS诱导的急性肺损伤小鼠模型中,过度自噬可能通过激活NF-κB通路参与炎症反应并诱导细胞凋亡;3-MA适度抑制自噬可减轻炎症反应并起到保护肺组织的作用。     

磷酸肌酸对小鼠移植性S180肉瘤血管生成的作用及机制研究

目的 探讨磷酸肌酸(creatine phosphate,Cp)对小鼠移植性S180肉瘤血管生成的作用及其相关机制.方法 建立昆明小鼠S180肉瘤模型,60只小鼠随机分为4组:0.9%氯化钠溶液对照组(对照组)、Cp低剂量组(200 mg/kg)、Cp中剂量组(400 mg/kg)、Cp高剂量组(800 mg/kg),观察4组小鼠移植瘤重量;流式细胞术测定基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)及金属蛋白酶组织抑制因子-2(tissue inhibitors of metalloproteinases,TIMP-2)蛋白的表达;免疫组化检测肿瘤组织微血管密度(microvasvular density,MVD);RT-PCR检测移植瘤组织中血管内皮生长因子(vascular endothlial growth factor,VEGF)及碱性成纤维细胞生长因子(base fibroblast growth,bFGF)mR-NA的表达水平.结果 对照组、Cp低剂量组和中剂量组移植瘤重量、VEGF、bFGFmRNA、MMP-2、TIMP-2水平比较,差异均无统计学意义(P>0.05);Cp高剂量组与对照组、Cp低、中剂量组比较,小鼠移植性肉瘤质量明显减小,MVD数显著减少,VEGF、bFGFmRNA和MMP-2蛋白表达明显减少,TIMP-2蛋白表达明显增加,差异均有统计学意义(P<0.01).结论 Cp低、中剂量对肿瘤血管生成无明显影响;Cp高剂量对肿瘤血管生成具有明显的抑制作用,其机制可能与通过下调MMP-2、VEGF和bFGF、上调TIMP-2的表达水平有关.     

The inhibitory effect of ginger flavine on S180 sarcoma in mice

目的 探讨姜黄素对小鼠S180肉瘤的影响.方法 40只小鼠随机分为四组:对照组每只每天灌胃蒸馏水10ml;低、中、高剂量组每天分别灌胃姜黄素50、100、200mg/kg;连续10d.观察瘤重、体重及测定肝组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性.结果 低、中、高剂量组的姜黄素对S180肉瘤生长抑制率分别为12.5％、37.5％、47.3％;与对照组比较,姜黄素各剂量组肝组织SOD活力均有所升高,且中剂量组SOD活力明显高于对照组(P＜0.05);中、高剂组的小鼠肝组织GSH-Px活力明显高于对照组(P＜0.05).结论 姜黄素对小鼠S180肉瘤有抑制作用.     

不同生境人参茎叶总皂苷对心律失常小鼠的作用比较

目的 探讨园参茎叶总皂苷(GSLS)和林下山参茎叶总皂苷(MSLS)对心律失常小鼠的改善作用.方法 将80只SPF级BALB/c小鼠随机分为正常组、模型组、园参茎叶总皂苷低(GSLS-L)、中(GSLS-M)、高(GSLS-H)剂量组、林下山参茎叶总皂苷低(MSLS-L)、中(MSLS-M)、高(MSLS-H)剂量组....     

PCSK9单抗的应用对高脂模型小鼠血脂影响的实验研究

目的 观察前蛋白转化枯草杆菌/丝氨酸蛋白酶9 (PCSK9)单抗在高脂模型小鼠降血脂方面的作用.方法 高脂饲料喂养建立小鼠高脂模型,并采用不同剂量(0.25、0.5、1 mg/kg)的PCSK9单抗进行静脉注射.全自动生化分析仪检测小鼠血清TC、TG、HDL-C、LDL-C水平,Western blot检测小鼠肝脏LDL受体(LDL-R)蛋白水平.结果 模型组、PCSK9低、中、高剂量组TC、TG、LDL-C水平都明显高于空白组(P＜0.05),HDL-C水平都明显低于空白组(P＜0.05);PCSK9低、中、高剂量组TC、TG、LDL-C水平都明显低于模型组(P＜0.05),HDL-C水平都明显高于模型组(P＜0.05),且存在明显的剂量依赖性.另外,模型组、PCSK9低、中剂量组小鼠肝组织中LDL-R的表达水平明显低于空白组(P＜0.05);PCSK9低、中、高剂量组小鼠肝组织中LDL-R的表达水平明显高于模型组(P＜0.05).结论 PCSK9单抗具有较好的降血脂作用,有望成为治疗心血管疾病的新药.     

基于粪便代谢组学的麻黄细辛附子汤毒性作用机制研究

目的 研究麻黄细辛附子汤(MXF)对正常小鼠的毒性作用机制。方法 将48只SPF级BABL/C小鼠随机分为空白组和MXF低、中、高剂量组,每组12只。MXF低、中、高剂量组小鼠分别按照11.262、33.786、45.050 g/kg的剂量灌胃给药,空白组小鼠灌胃等体积生理盐水,每日1次,连续灌胃7 d,记录体质量、肛温、生存率,并检测脏器指数、血清生化因子等指标。末次给药后收集小鼠粪便样品,采用超高效液相-质谱联用(UHPLC-QE/MS)技术检测。结果 与空白组相比,MXF中剂量组小鼠在给药第3～5天、MXF高剂量组小鼠给药第2～7天体质量显著低于空白组(P<0.05);各给药组小鼠肛温差异无统计学意义;MXF中、高剂量组小鼠的生存率分别为58.33%、50.00%。与空白组相比,MXF低、中、高剂量组小鼠的脾、肺、胸腺、肾上腺指数和肌酸激酶,MXF低、高剂量组小鼠的睾丸指数,MXF低剂量组小鼠的肌酸激酶同工酶与肌酸激酶比值,MXF中剂量组α-羟丁酸脱氢酶、乳酸脱氢酶、碱性磷酸酶,MXF中、高剂量组小鼠的尿素和胱抑素C的差异均有统计学意义(P<0.05)。经粪便代谢组学...     

Formulating protein therapeutics into particulate forms

This review is aimed at providing critical comments on selected approaches to formulating protein drugs into particulate forms feasible as practical pharmaceutical dosage forms. From a practical point of view, the need to formulate protein therapeutics into particulate forms includes inhalation and sustained-release delivery proteins, stabilizing and incorporating proteins into tissue engineering scaffolds and medical devices, as well as protecting and targeting protein therapeutics in an in vivo environment. For either of the applications, a common challenge is that proteins are easily denatured during particle-forming processes in which water–oil or water–air interfaces, multivalent ions or polyelectrolytes, strong shear stress and/or reactive crosslinking agents are often involved. Moreover, methods to protect proteins during the particle-forming processes must not compromise their pharmaceutical objectives, such as encapsulation efficiency, burst-free controlled release and storage convenience. Although numerous methods have been reported to formulate proteins into particulate systems, few of them meet the criteria above. To stimulate critical and interactive readings of the vast and booming information, the authors also provide their analysis regarding the feasibility of the formulation strategies summarized in this review.     

Inhibitors of protein farnesylation 1998

Protein farnesyltransferase (PFTase) inhibitors are being studied as mechanistically novel antitumour agents. Whilst the antiproliferative effects of PFTase inhibitors are well-documented in cell culture and rodent animal models, clinical studies which began in 1997 should soon reveal their utility in human cancer patients. This review summarises the scientific and patent literature covering PFTase inhibition that has been published since the previous two updates [1,2]. New biology with a potential impact on the utility of PFTase inhibitors is reviewed first, followed by a discussion of new PFTase inhibitors. As in earlier updates, compounds are grouped according to their kinetic mechanism of PFTase inhibition.     

Biological functions and structural biology of Plasmodium falciparum autophagy-related proteins: The under-explored options for novel antimalarial drug design

Malaria remains a threat to global public health and the available antimalarial drugs are undermined by side effects and parasite resistance, suggesting an emphasis on new potential targets. Among the novel targets, Plasmodium falciparum autophagy-related proteins (PfAtg) remain a priority. In this paper, we reviewed the existing knowledge on the functions and structural biology of PfAtg including the compounds with inhibitory activity toward P. falciparum Atg8-Atg3 protein–protein interaction (PfAtg8-PfAtg3 PPI). A total of five PfAtg (PfAtg5, PfAtg8, PfAtg12, PfAtg18, and Rab7) were observed to have autophagic and/or non-autophagic roles. Available data showed that PfAtg8 has conserved hydrophobic pockets, which allows it to interact with PfAtg3 to form PfAtg8-PfAtg3 PPI. Additionally, 2-bromo-N-(4-pyridin-2-yl-1,3-thiazol-2-yl) benzamide was identified as the most powerful inhibitor of PfAtg8-PfAtg3 PPI. Due to the dearth of knowledge in this field, we hope that the article would open an avenue to further research on the remaining PfAtg as possible drug candidates.     

Influence of retinoic acid on TBX1 expression in myocardial cells induced by Shh and Fgf8

The aim of this study was to explore the regulatory mechanism of retinoic acid (RA) on the TBX1 gene expression in myocardial cells. Ventricular cardiocytes were isolated from neonatal rats and cultured, and then treated with different concentrations of retinoic acid. The expression of Shh and Fgf8 at mRNA and protein levels in neonatal rat myocardial cells were measured by using RT-PCR and Western blot technique, respectively. There was basal expression of Shh and Fgf8 in the control group. When treated with 3 × 10⁻⁷ mol/L RA, we observed that the expression of Shh mRNA and protein in neonatal rat myocardial cells were up-regulated by 1.51 (P < 0.05) and 1.10 times (P < 0.05), respectively. In comparison with the control group, under the concentration of 5 × 10⁻⁷ mol/L RA, they were up-regulated by 2.21 (P < 0.05) and 2.38 times (P < 0.05) individually. Meanwhile, we could detect that the expression of Fgf8 mRNA and protein were up-regulated by 2.50 times (P < 0.05) and 80% (P < 0.05) separately compared with the control group after stimulation of 3 × 10⁻⁷ mol/L RA, and they were up-regulated by 3.48 (P < 0.05) and 2.04 times (P < 0.05) individually after stimulation of 5 × 10⁻⁷ mol/L RA. The results indicated that RA could induce the expression of Shh and Fgf8 in neonatal rat myocardial cells. At the same time, it has shown that Shh and Fgf8 were involved in the regulation process of RA on TBX1 expression.     

儿童非霍奇金淋巴瘤EB病毒LMP-1和P53、bcl-2表达的关系

目的探讨儿童NHL EB病毒LMP-1和P53、bcl-2蛋白的表达及关系.方法采用免疫组化Envision法检测64例儿童NHL中LMP-1和P53、bcl-2蛋白.结果 (1)P53蛋白阳性表达39例(60.9%),表达强度与淋巴瘤恶性程度呈正相关;阳性表达率在低恶组与中、高恶性组间有显著性意义,P＜0.01.bcl-2蛋白阳性表达37例(53.8%),bcl高于TCL,低恶性高于高恶性.(2)LMP-1蛋白阳性表达45例(70.3%),阳性表达率与肿瘤恶性程度和年龄有统计学意义,P＜0.01;而与淋巴瘤免疫表型、性别和发病部位无关.LMP-1表达与P53及bcl-2的表达呈正相关.结论 EBV感染是儿童NHL发生发展不可忽视的病毒致病因素,其致病作用可能是通过上调P53、bcl-2蛋白实现的.     

Stabilizing Peptide Fusion for Solving the Stability and Solubility Problems of Therapeutic Proteins

Purpose Protein aggregation is a major stability problem of therapeutic proteins. We investigated whether a novel stabilizing peptide [acidic tail of synuclein (ATS) peptide] could be generally used to make a more stable and soluble form of therapeutic proteins, particularly those having solubility or aggregation problems. Methods We produced ATS fusion proteins by fusing the stabilizing peptide to three representative therapeutic proteins, and then compared the stress-induced aggregation profiles, thermostability, and solubility of them. We also compared the in vivo stability of these ATS fusion proteins by studying their pharmacokinetics in rats. Results The human growth hormone–ATS (hGH–ATS) and granulocyte colony-stimulating factor–ATS (G-CSF–ATS) fusion proteins were fully functional as determined by cell proliferation assay, and the ATS fusion proteins seemed to be very resistant to agitation, freeze/thaw, and heat stresses. The introduction of the ATS peptide significantly increased the storage and thermal stabilities of hGH and G-CSF. The human leptin–ATS fusion protein also seemed to be very resistant to aggregation induced by agitation, freeze/thaw, and heat stresses. Furthermore, the ATS peptide greatly increased the solubility of the fusion proteins. Finally, pharmacokinetic studies in rats revealed that the ATS fusion proteins are also more stable in vivo. Conclusion Our data demonstrate that a more stable and soluble form of therapeutic proteins can be produced by fusing the ATS peptide. E. N. Lee and Y. M. Kim equally contributed to this work.     

Computational re-design of protein structures to improve solubility

ABSTRACT

Introduction: The rapid development of protein therapeutics is providing life-saving therapies for a wide range of human diseases. However, degradation reactions limit the quality and performance of these protein-based drugs. Among them, protein aggregation is the most common and one of the most challenging to prevent. Aggregation impacts biopharmaceutical development at every stage, from discovery to production and storage. In addition, regulators are highly concerned about the impact of protein aggregates on drug product safety.

Area covered: Herein, the authors review existing protein aggregation prediction approaches, with a special focus on four recently developed algorithms aimed to predict and improve solubility using three-dimensional protein coordinates: SAP, CamSol, Solubis and Aggrescan3D. Furthermore, they illustrate their potential to assist the design of solubility-improved proteins with a number of examples.

Expert opinion: Aggregation of protein-based drugs is, traditionally, addressed via wet lab experiments, using trial and error approaches that are expensive, difficult to perform and time-consuming. The structure-based in silico methods we describe here can predict accurately aggregation propensities, allowing researchers to work with pre-selected, well-behaved, protein candidates. These methods should contribute to the reduction of the time to the marketplace along with industrial costs and improve the safety of future therapeutic proteins.     

Stability of the Thrombolytic Protein Fibrolase: Effect of Temperature and pH on Activity and Conformation

The effect of temperature and pH on the activity and conformation of the thrombolytic protein fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (22°C). At 37°C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alphahelical structure was lost in a cooperative transition (T _m of 50°C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the T _m determined by NMR was 46°C at pD 8. The effect of pH on the proteolytic activity and structure of fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of fibrolase decreased to 43°C at pH 5. Protein concentrations determined over the pH range 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.     

Prediction of colloidal stability of high concentration protein formulations

Abstract

A major aspect determining the colloidal properties of proteins in solution is the interaction between them and with surrounding molecules. These interactions can be described by the concentration dependency of the protein diffusivity (k_D), as derived by dynamic light scattering and was determined for different solutions of monoclonal antibodies varying in pH, ionic strength and presence/absence of co-solute(s). Concerning colloidal stability, protein solutions of different k_D values are evaluated, based on their initial solution opalescence, to assess protein association. The current investigation shows that solution conditions with large k_D values, indicating high repulsive protein–protein interactions, show lower initial opalescence, compared to solution conditions with low k_D values. Upon applying stirring stress, to assess colloidal stability, the trend is such that, the higher k_D values are, the more stable the protein solutions are, as long as the thermodynamic and conformational stability is not impaired. Besides, k_D allows ranking of solution conditions for highly concentrated immunoglobulin solutions up to concentrations of ～200?mg?mL^?1 with regard to protein self-association and thus opalescent properties. The present study shows that the protein interaction parameter k_D can be used as a surrogate parameter for a qualitative prediction of protein association and, thus, colloidal protein stability.     

Isoform-specific activities of the regulatory subunits of phosphatidylinositol 3-kinases – potentially novel therapeutic targets

ABSTRACT

Introduction: The main regulatory subunits of Class IA phosphatidylinositol 3-kinase (PI3K), p85α and p85β, initiate diverse cellular activities independent of binding to the catalytic subunit p110. Several of these signaling processes directly or indirectly contribute to a regulation of PI3K and could become targets for therapeutic efforts.

Areas covered: This review will highlight two general areas of p85 activity: (1) direct interaction with regulatory proteins and with determinants of the cytoskeleton, and (2) a genetic analysis by deletion and domain switches identifying new functions for p85 domains.

Expert Opinion: Isoform-specific activities of regulatory subunits have long been at the periphery of the PI3K field. Our understanding of these unique functions of the regulatory subunits is fragmentary and raises many important questions. At this time, there is insufficient information to translate this knowledge into the clinic, but some tempting targets have emerged that could move the field forward with the help of novel technologies in drug design and identification.