Gut bacterial β-glucuronidases (GUS) play an important role in deconjugation of various O-glucuronides, which are tightly linked with the drug-induced intestinal toxicity. Increasing evidence has indicated that inhibition of bacterial GUS could alleviate GUS-associated intestinal toxicity, but the potent and broad-spectrum inhibitors against multiple bacterial GUS have been rarely reported. This study aimed to find potent and broad-spectrum GUS inhibitors from Ginkgo biloba. It was found that amentoflavone displayed relatively strong inhibition on three GUS including CpGUS, SpasGUS and EcGUS. Further investigations demonstrated that amentoflavone could inhibit GUS-mediated PNPG hydrolysis in a dose-dependent manner with IC50 values of 2.36 μM, 2.88 μM and 3.43 μM for CpGUS, SpasGUS and EcGUS, respectively. Inhibition kinetic studies showed that amentoflavone functioned as a non-competitive inhibitor against all tested GUS with Ki values of less than 2 μM. Docking simulations indicated that amentoflavone could tightly bind on allosteric sites of three GUS mainly via hydrogen bonding interactions, and the number of hydroxyl groups of amentoflavone played crucial roles in these interactions. Collectively, our findings suggested that amentoflavone was a potent broad-spectrum inhibitor against bacterial GUS, which can be used as a promising lead compound for developing novel agents to alleviate GUS-associated intestinal toxicity.

生物制药发展势头迅猛，越来越多的生物药物进入临床应用，生物制药人才需求日益增加。为了适应我国高校的“新工科”建设要求，解决当前课堂教学内容定式化、学生思维方式死板等问题，以培养学生的工学思维能力为导向，对《生物制药工艺学》课程的教学模式及方法的革新途径进行了探讨，建立了以“工艺单元操作、流程设计及工艺建模”为主的教学模式，并对实际教学效果进行了评价。通过本文的探索，可为生物制药专业的“新工科”改革提供借鉴。

Objectives High-fat diet has been considered a risk factor for the development of pancreatic cancer. It is also shown to significantly impact composition and dysbiosis of gut microbiota in both humans and animals. However, there is little information on the effect of high-fat diet on the development of pancreatic cancer or upon the gut microbiota of patients with pancreatic cancer in humans or animal models. Methods In this study, the effect of high-fat diet on cancer pathology and the gut microbiota was investigated by a carcinogen-induced pancreatic cancer mouse model. Results Compared with carcinogen alone, mice with high-fat diet and carcinogen showed more obvious pathological changes in pancreatic tissue; increased levels of proinflammatory cytokine tumor necrosis factor-α, interleukin-6, interleukin-10, and carbohydrate antigen 242; and increased expression of cancer-associated biomarkers mucin-4 and claudin-4 in pancreatic tissue. Moreover, there is a significant change in the gut microbiota between the carcinogen group and the carcinogen with high-fat diet group. We identified that Johnsonella ignava especially existed in the carcinogen with high-fat diet group, which may contribute to pancreatic cancer development. Conclusions Our results revealed that high-fat diet changed the composition of the gut microbiota and was involved in carcinogen-induced pancreatic cancer progression.

Pancreatic lipase (PL), a crucial enzyme in the digestive system of mammals, has been proven as a therapeutic target to prevent and treat obesity. The purpose of this study is to evaluate and characterize the PL inhibition activities of the major constituents from Fructus Psoraleae (FP), one of the most frequently used Chinese herbs with lipid-lowering activity. To this end, a total of eleven major constituents isolated from Fructus Psoraleae have been obtained and their inhibition potentials against PL have been assayed by a fluorescence-based assay. Among all tested compounds, isobavachalcone, bavachalcone and corylifol A displayed strong inhibition on PL (IC50 < 10 μmol·L−1). Inhibition kinetic analyses demonstrated that isobavachalcone, bavachalcone and corylifol A acted as mixed inhibitors against PL-mediated 4-methylumbelliferyl oleate (4-MUO) hydrolysis, with the Ki values of 1.61, 3.77 and 10.16 μmol·L−1, respectively. Furthermore, docking simulations indicated that two chalcones (isobavachalcone and bavachalcone) could interact with the key residues located in the catalytic cavity of PL via hydrogen binding and hydrophobic interactions. Collectively, these finding provided solid evidence to support that Fructus Psoraleae contained bioactive compounds with lipid-lowering effects via targeting PL, and also suggested that the chalcones in Fructus Psoraleae could be used as ideal leading compounds to develop novel PL inhibitors.

目的:探讨海藻类功能食品对肠道微生态失调小鼠的菌群调整及对非特异性免疫的调节作用,以进一步开发利用海洋藻类资源.方法:将SPF级昆明种小鼠随机分为正常对照组和干预组,干预组首先采用大剂量混合抗生素灌胃建立小鼠肠道微生态失调模型,再根据后期干预方式的不同分为:模型组、自然恢复组(正常喂养)和治疗组(海藻糖类配方鼠粮喂养);收集各组小鼠新鲜粪便,利用PCR-DGGE方法检测肠道菌群结构变化,获得细菌群落指纹图谱并进行分析,同时切取差异显著条带进行序列分析;酶联免疫吸附实验检测血清中IL-2、TNF-α生成水平,进而初步评价海藻类食品对小鼠非特异性免疫的影响.结果:抗生素灌胃后导致小鼠肠道菌群失调,相关免疫细胞因子水平下降,模型组小鼠体内Barnesiella intestinihominis等抑制机体免疫力的菌群量明显高于正常及治疗组,治疗组小鼠乳酸杆菌等有益菌及肠道菌群多样性恢复较好且小鼠血清细胞因子浓度明显升高.结论:海藻类功能食品能够帮助恢复肠道微生态平衡,增强实验小鼠免疫功能.

目的 探究海参多糖对抗生素所致小鼠肠道菌群失衡的调整作用.方法 超声浸提法提取海参多糖,硫酸-苯酚法测定多糖含量;通过抗生素连续灌胃1周,构建小鼠肠道紊乱模型;将18只昆明种小鼠随机分为正常对照、自然恢复和海参多糖干预组(每组6只),定期留取粪便,采用PCR-DGGE技术获得肠道菌群分子指纹图谱,进行相似性、多样性及主要差异条带序列的分析.结果 提取海参多糖的浓度为1.92 mg/mL;正常对照组小鼠肠道菌群以有益菌,即乳杆菌属和梭菌属占主导地位;而在抗生素干预后,这两种菌的含量明显下降,并伴随检测出致病性卟啉单胞菌属;经海参多糖处理后,有益菌含量有所恢复,有害菌含量降低.结论 海参多糖对抗生素所致实验小鼠肠道菌群紊乱有一定的恢复作用.

Soluble (pro)renin receptor [s(P)RR], which is generated from cleavage of (P)RR, can be detected in plasma and urine. s(P)RR levels can reflect the severity of some diseases, such as renal lesions, gestational diabetes mellitus or hypertension, and obstructive sleep apnea syndrome. However, the relationship between s(P)RR levels and the severity of chronic heart failure remains undetermined. We studied s(P)RR levels in 118 patients with chronic heart failure with reduced ejection fraction (HFrEF), including 86 without renal dysfunction (HF) and 32 with renal dysfunction (HF + RF), and 28 healthy subjects (HS) to reveal the relationship between s(P)RR levels and other HFrEF parameters. Plasma s(P)RR levels were 22.2 +/- 4.1 ng/mL (HS), 26.4 +/- 5.3 ng/mL (HF) and 30.0 +/- 5.3 ng/mL (HF + RF). Plasma s(P)RR levels were significantly higher in the HF group than in the HS group (P < 0.001) and even more increased in the HF + RF group (P < 0.001 vs. the HS group and P < 0.05 vs. the HF group). Multivariate regression analysis revealed that the left ventricular mass index (LVMI) and estimated glomerular filtration rate (eGFR) were independently related to s(P)RR levels in HFrEF patients. In conclusion, high plasma s(P)RR levels are associated with left ventricular remodeling and, especially, with renal dysfunction. Therefore, s(P)RR is a promising evaluative indicator for the severity of HFrEF patients.

目的 研究含有浒苔等植物的功能性食品对糖尿病小鼠血糖浓度的影响,并应用PCR-DGGE技术评价其对昆明小鼠肠道菌群稳态的影响.方法 采用高脂饲料喂养昆明小鼠加腹腔注射链脲佐菌素(STZ)的方法建立糖尿病小鼠模型;将实验动物随机分为正常对照组、自然恢复组和功能性食品喂养组,连续给药4周,尾静脉采血测量血糖水平.收集小鼠新鲜粪便,提取粪便细菌基因组DNA,通过PCR-DGGE获得细菌群落指纹图谱,并进行相关软件分析,同时切离差异显著条带进行序列分析.结果 本实验采用的功能性食品对糖尿病小鼠有降糖作用,并使血糖值稳定地维持在较低水平.4周后,功能性食品喂养的糖尿病小鼠血糖水平相对普通饲料喂养的糖尿病小鼠血糖发生显著性下降(t=4.19,P＜0.01);给2型糖尿病小鼠提供普通饲料时,其肠道菌群种类和数目相对较少;而提供功能性食品时,肠道菌群种类和数目相对增多,特别是双歧杆菌、Prevotella oryzae、Barnesiella intestinihominis、Culturomica和Parabacteroides distasonis明显增多,但是Muribaculum较少.结论 高脂饲料结合STZ诱导的2型糖尿病小鼠肠道菌群发生显著改变,组方食品通过扶持肠道菌群,降低血糖水平.

Bacterial β-glucuronidases play key roles in the deconjugation of a variety of endogenous and drug glucuronides, thus have been recognized as important targets to modulate the enterohepatic circulation of various glucuronides. In this study, more than 30 natural flavonoids were collected and their inhibitory effects against E. coli β-glucuronidase (EcGUS) were assayed. The results demonstrated that some flavonoids including scutellarein, luteolin, baicalein, quercetin and scutellarin displayed strong to moderate inhibitory effects against EcGUS, with the IC50 values ranging from 5.76 μM to 29.64 μM, while isoflavones and dihydroflavones displayed weak inhibitory effects against EcGUS. Further investigation on inhibition kinetics revealed that scutellarein and luteolin functioned as potent competitive inhibitors against EcGUS-mediated PNPG hydrolysis, with the Ki values less than 3.0 μM. Molecular docking simulations demonstrated that scutellarein and luteolin could be well-docked into the catalytic site of EcGUS, while the binding areas of these two natural inhibitors on EcGUS were highly overlapped with that of PNPG on EcGUS. Additionally, the structure-inhibition relationships of natural flavonoids against EcGUS are also summarized, which will be very helpful for the medicinal chemists to design and develop more potent flavonoid-type inhibitors against EcGUS.

气道黏液在先天性免疫应答中发挥重要作用.但是,当气道丧失正常功能时在其近端及远端位置便会出现高黏液分泌状态和分泌物增多的现象,同时将引发相关症状,如咳嗽、咳痰、肺功能下降,气道阻塞,也成为导致慢性阻塞性肺疾病(COPD)患者病死率增高的重要原因.黏液产生和分泌的细胞及分子机制已在体外人工培养的上皮细胞和动物模型中得到很好验证.烟雾和微生物产物可有效地触发黏液的产生和分泌过程.目前,只有少数药物被证实在降低COPD患者黏液高分泌状态中发挥确切疗效,药物对人体黏液高分泌的作用仍没有得到有效评估.

Objective To develop a novel and environmental-friendly method for bioconversion of baicalin to baicalein with recombinant β-glucuronidase.Methods The β-glucuronidase encoding gene (GUS) was cloned from Escherichia coli wild strain K12, and transformed into Escherichia coli BL21 (DE3) with pET-28a (+) as the vector.β-Glucuronidase was overexpressed and then purified by Ni affinity chromatography in one step.The biotransformation of baicalin to baicalein was performed by the recombinant β-glucuronidase, and the reaction conditions were optimization with orthogonal design.Results The protein was identified by SDS-PAGE with 70 kDa, and the maximum expression level could be achieved after induction for 12 h with 0.6 μmol/L IPTG, and 320 mg of purified enzyme, which could be obtained from one liter of bacterial culture.With the catalysis of the recombinant β-glucuronidase, the baicalin was transformed to baicalein with 72.5% conversion ratio under optimized conditions.Conclusion A novel and environmental-friendly scheme for bioconversion of baicalin to baicalein with recombinant β-glucuronidase was established.The method would hold great promise for further applications in both scientific research and biomedical industry.

肠道正常菌群参与机体的物质代谢，营养物质的吸收合成，并能够促进生长发育，维持人体正常生理活动，其对胃肠道消化和免疫作用的发挥与肠杆菌、肠球菌、类杆菌和乳杆菌的关系密不可分。肠道黏膜免疫反应主要依靠肠腔内黏膜表面的免疫球蛋白（ sIgA为主）和淋巴细胞为主体的免疫活性细胞，共同完成肠道局部免疫。抗生素的使用可诱导肠道菌群失调，脑－肠轴参与了肠道菌群失调，其不仅可引起全身免疫疾病，还能导致肥胖、2型糖尿病及肠癌。

目的 获得具有典型生化特征、教学用的模拟患者血清标本,并确定其贮存温度和保质时间.方法 制备1％的酵母提取物溶液作为基底液.向基底液中添加14.0 mmol/L的葡萄糖和10.0 mmol/L的三酰甘油(TG),制备模拟的糖尿病血清标本;向基底液中添加10.0 mmol/L的TG,制备模拟的高甘油三酯血症血清标本;向基底液中添加28.0 g/L的白蛋白和10.0 mmol/L的TG,制备模拟的肾病综合征血清标本;向基底液中添加28.0 g/L的白蛋白和60 U/L的谷草转氨酶,制备模拟的肝硬化患者血清标本.测定各标本在室温、4℃和-20C条件下,存放1、7、30 d后各生化指标的变化情况.结果 模拟的患者血清标本外观与临床血清标本基本一致.模拟的糖尿病血清标本具有葡萄糖、TG升高的特征;模拟的高甘油三酯血症血清标本具有TG升高的特征;模拟的肾病综合征血清标本具有TG升高、白蛋白降低的特征;模拟的肝硬化血清标本具有谷草转氨酶升高、白蛋白降低的特征.4种标本均可在-20C保存30 d,除了模拟肾病综合征的白蛋白以外,其余各指标的变化差异均无统计学意义(P＞0.05).结论 获得了4种可供实验教学使用的模拟患者血清标本,确定了它们的最适贮存温度和保质时间.

葡萄糖醛酸结合反应是机体内重要的一类代谢反应,其中O-葡萄糖醛酸结合是最常见的反应类型.O-葡萄糖醛酸苷的制备对于准确评估葡萄糖醛酸代谢产物的药理活性及安全性、药物代谢和药动学定量分析等研究至关重要.利用尿苷二磷酸葡萄糖醛酸转移酶(UGT)生物合成目标代谢物是制备葡萄糖醛酸苷的重要手段之一.综述了近年来利用生物转化技术制备O-葡萄糖醛酸苷的研究进展,包括利用植物、微生物、动物来源及人重组UGT酶等不同方法制备O-葡萄糖醛酸苷的最新进展,同时还综述了近年来国内外通过对生物反应体系和转化工艺的改进与优化,实现葡萄糖醛酸苷高效制备的相关技术和工艺特点.

目的:随着人参药用和保健价值的不断发掘,人参皂苷引起人们越来越多的关注,但有关人参皂苷与肠道菌群之间相互作用的研究仍为空白领域,本实验旨在探明人参皂苷对小鼠肠道菌群的影响,以期为人参皂苷的推广应用提供理论基础和实验依据.方法:有机溶剂法提取人参皂苷,将正常BALB/c小鼠按2 mg/0.1 kg人参皂苷进行连续灌胃饲养,分别在灌胃第10d和第13d无菌收集小鼠粪便,提取肠道细菌基因组总DNA,应用PCR-DGGE技术获得肠道菌群分子指纹图谱,进行菌群结构相似性、多样性分析,并将感兴趣的优势条带进行切胶、测序分析,对获得的序列在GeneBank数据库比对.结果:灌胃人参皂苷后小鼠肠道菌群结构发生改变,荧光假单胞菌和丁酸梭菌数量明显增加.结论:人参皂苷使小鼠肠道的菌群结构和数量发生明显改变,天然的人参皂苷口服很难被直接吸收利用,因此推测人参皂苷可能以肠道菌群作为发挥生物学作用的靶点,进而行使提高健康水平等保健功能.

目的 通过PCR-DEEG方法比较长期使用农药的土地和正常的土地菌群结构和数量的变化,阐述农药对土壤微生态环境的影响.方法 收集长期使用农药的土壤和未使用农药的土壤,提取细菌基因组 DNA,采用针对16S rDNA V3区的PCR-DGGE技术获得土壤菌群指纹图谱,进行相似性、多样性分析.结果 长期使用农药的实验组土壤菌群结构与正常组比较差异有统计学意义(P＜0.05).长期使用农药的土壤中出现四种特异性菌种:Candidatus saccharimonas、Anabaena cylindrical、Caldilinea aerophila和Sphingobiumchlorophenolicum.Streptomyces niveus、Thermoanaerobacter菌种在长期使用农药的实验组含量增多,且差异均具有统计学意义(P＜0.01).结论 长期使用农药在一定程度上使土壤细菌种群的结构和数量发生变化,破坏了土壤微生态平衡.

Pinus massoniana bark proanthocyanidins (PMBPs), an active component isolated from Pinus massoniana bark, has been reported to possess a wide range of biochemical properties. Here, we investigated the anti-tumor effect of PMBPs on ovarian cancer. The results indicated that PMBPs significantly reduced the growth of ovarian cancer cells and induced dose-dependent apoptosis. The underlying mechanisms involved were elucidated to include the loss of mitochondrial membrane potential, down-regulation of the anti-apoptotic protein Bcl-2 and the activation of Caspase 3/9, suggesting that PMBPs triggered apoptosis through activation of mitochondria-associated apoptotic pathway. In addition, wound healing and transwell chamber assays revealed that PMBPs could suppress migration and invasion of ovarian cancer cells. PMBPs dramatically inhibited MMP-9 activity and expression, blocked the activity of NFκB and the activation of ERK1/2 and p38 MAPK. Our findings suggest that PMBPs has the potential to be developed as an anti-tumor drug for ovarian cancer treatment and/ or disease management.

微生物学是一门综合性课程，既包括理论基础，又包括实践技能。同时作为生命科学的重要学科之一，微生物学具有广度的多交叉融合性和深度的多层次进展性，因而起着生长点的作用，它的核心知识和技能被广泛应用于临床及生物技术生产实践中［1，2］。成为沟通临床医学和基础医学的桥梁学科。医学院校生物技术、生物制药专业如何合理巧妙地利用医学院校得天独厚的教学资源，对培养出具有一定医学背景、能够解决临床实际问题的创新、应用型人才具有重要意义，也将为学生的可持续发展奠定坚实的基础。因此，围绕微生物学知识体系的特点有效地调动学生学习积极性和主动性，切实提高教育教学质量，充实学生对这一学科的知识储备，使踏入生物学大门的学生对微生物学有一个较整体的认识和广泛的了解，能够掌握涉及微生物形态、结构、功能、生理代谢、遗传变异、生态分布、分类进化等方面的基本知识［3］，为后续课程学习和就业做出必要的准备，成为微生物学教学的关键。

目的 应用PCR-DGGE方法探究高脂高糖和高纤维膳食对实验小鼠肠道菌群的影响,解析不同饮食与肠道菌群之间的关系.方法 选用12只18～22 g清洁级实验小鼠,随机分为高脂高糖饲料组和高纤维饲料组,在第0、2、4、6周分别取小鼠粪便,提取基因组DNA后采用聚合酶链式反应—变性梯度凝胶电泳(PCR-DGGE)方法获得肠道菌群的分子指纹图谱,并进行肠道菌群多样性、相似性及优势条带分析.结果 DGGE图谱表明,随着高脂高糖饮食时间的延长,拟杆菌、厚壁菌和变形菌的数量发生了明显变化,而高纤维饮食中拟杆菌、厚壁菌的数量发生了改变,变形菌的数量无明显变化.结论 高脂高糖和高纤维饮食对肠道菌群的种类和数量均有一定影响.