为了研究黑龙江省大庆地区鹅源大肠杆菌流行株的毒力基因分布情况.从大庆周边6个地区鹅养殖场发病鹅体内分离大肠杆菌,采用PCR方法及K-B纸片法进行了毒力基因检测和药敏试验.结果表明:53株大肠杆菌分离鉴定出48株大肠杆菌含有毒力基因,以气杆菌素iucD(48/53,91％)、强毒力岛FyuA(27/53,51.3％)、黏附素I型菌毛FimH(38/53,71.1％)、大肠杆菌外膜蛋白ompA(47/53,89％)为主要毒力基因;抗生素敏感率最高为庆大霉素和呋喃妥因;耐药率最高为四环素、复方新诺明和环丙沙星.本研究为黑龙江地区鹅大肠杆菌病的预防和控制提供了数据资料.

[目的]采用分子对接以及体外试验确定青蒿素对牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)复制的抑制作用,为抗病毒药物和制剂开发提供新思路.[方法]以BVDV-NS5B蛋白为作用靶点,通过PDB数据库检索蛋白三维晶体结构,并对其结构进行适当修饰处理;检索TCMSP数据库中的青蒿素结构,应用Autodock软件将两者进行分子对接及结合能打分.随后采用CCK-8试剂盒测定青蒿素对MDBK细胞的最大安全浓度;将选定浓度的青蒿素进行梯度稀释,采用先加病毒后加药物、先加药物后加病毒、中药和病毒同时作用的3种不同加药方式进行药物抗病毒试验,确定对病毒的最佳药物抑制浓度、预防浓度和杀灭浓度.应用实时荧光定量PCR法检测3种药物作用方式下BVDV的拷贝数,进一步明确药物对BVDV复制的作用.[结果]分子对接数据表明青蒿素与BVDV-NS5B存在相互作用,结合自由能为一28.6748 kJ/mol.青蒿素在MDBK细胞上的最佳药物安全浓度为100 μmol/L.3种作用方式下青蒿素浓度为100 μmol/L时均可有效影响BVDV的复制,青蒿素对BVDV的抑制作用最为明显.[结论]青蒿素可与BVDV-NS5B蛋白靶点互作,并能在MDBK细胞上有效抑制BVDV的复制,本研究为抗BVDV中药筛选奠定了基础.

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens affecting the cattle industry worldwide. Phlorizin, a kind of flavonoids extracted from apple tree roots, leaves, and fruits, has a variety of biological functions and has been widely used as a herbal supplement and food additive. Here, BALB/c mouse and Madin-Darby bovine kidney (MDBK) cells were used to explore the effect and mechanism of phlorizin against BVDV infection. The results showed that phlorizin significantly inhibited CP BVDV replication and improved the histopathological changes of duodenum and spleen in mice. In vitro studies also confirmed the activity of phlorizin against CP BVDV. Exploration on its potential mechanism suggested that phlorizin inhibited CP BVDV-induced beclin-1 level and the conversion rate of LC3B-I to LC3B-II. Interestingly, although phlorizin also showed a protective effect on MDBK cells, which were treated with 3-methyladenine A (3-MA), the effect was significantly weakened. Furthermore, phlorizin suppressed the stage of BVDV replication but showed no effect on stages of attachment and internalization. Our data further indicated that phlorizin promoted IFN-α and IFN-β levels, decreased IL-1β and IL-6 expression, and regulated RIG-I, MDA5, TLR3, and NLRP3 levels. Similar to CP BVDV results, in vivo and in vitro, phlorizin inhibited NCP BVDV (NY-1 and YNJG2020 strains) infection. These results were the first to be discovered that phlorizin might be used as a new dietary strategy for controlling BVDV infection.

为分析小鼠感染牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)诱导的NLRP3炎症小体表达情况及其对病毒复制的影响,选用40只4～6周龄雌性SPF级BALB/c小鼠,随机分成8组,其中BVDV接种小鼠7组,对照1组,每组5只.应用105 TCID50的CP型BVDV腹腔感染小鼠后,分别在感染后0、6、12、24、48、96、168和240 h采集小鼠的肠道、脾脏和血液,通过qRT-PCR和 Western Blot方法对小鼠十二指肠中NLRP3、IL-1β 和Caspase-1分别进行基因及蛋白水平的检测.应用qRT-PCR方法确定不同时间点小鼠十二指肠和血液中的病毒载量,同时于感染后第168 h检查十二指肠和脾脏病理组织变化.结果表明:1)小鼠感染BVDV早期(6、12和24 h)组织中的NLRP3、IL-1β和Caspase-1基因转录水平逐步升高,48 h未见明显变化,同时感染24 h时,蛋白表达水平均显著高于对照组(P＜0.05);2)小鼠感染BVDV后期(96、168和240 h)NLRP3、IL-1β和Caspase-1的基因转录水平呈现下降趋势,168 h时转录和蛋白水平降低最为显著(P＜0.05);3)感染小鼠十二指肠、脾脏和血液的病毒载量均在168 h达到高峰;第168 h采集的小鼠十二指肠有大量炎性细胞浸润,脾脏淋巴细胞变性和坏死,证明BVDV感染模型成立.综上,NLRP3炎症小体在BVDV感染早期被激活,然而,随着病毒载量的增加,可能对其活性有一定的抑制作用.

Curcumin is the most important active component in turmeric extracts. Curcumin, a natural monomer from plants has received a considerable attention as a dietary supplement, exhibiting evident activity in a wide range of human pathological conditions. In general, curcumin is beneficial to human health, demonstrating pharmacological activities of anti-inflammation and antioxidation, as well as antitumor and immune regulation activities. Curcumin also presents therapeutic potential in neurodegenerative, cardiovascular and cerebrovascular diseases. In this review article, we summarize the advancements made in recent years with respect to curcumin as a biologically active agent in malignant tumors, Alzheimer’s disease (AD), hematological diseases and viral infectious diseases. We also focus on problems associated with curcumin from basic research to clinical translation, such as its low solubility, leading to poor bioavailability, as well as the controversy surrounding the association between curcumin purity and effect. Through a review and summary of the clinical research on curcumin and case reports of adverse effects, we found that the clinical transformation of curcumin is not successful, and excessive intake of curcumin may have adverse effects on the kidneys, heart, liver, blood and immune system, which leads us to warn that curcumin has a long way to go from basic research to application transformation.

BACKGROUND: Ulcerative colitis (UC) is a relapsing and chronic inflammatory disease of the gastrointestinal tract, which seriously threatens human health. Zingerone (ZO) has been proven to be effective for many diseases. The purpose of this study is to investigate the protective effects and potential mechanisms of ZO extracted from ginger on dextran sulfate sodium (DSS)-induced mouse ulcerative colitis (UC). RESULTS: The results showed that ZO alleviated the weight loss of UC model mice, reduced the disease activity index scores, and inhibited the shortening of colon length. ZO also improved DSS-induced pathological changes in colon tissue and inhibited the secretion of pro-inflammatory cytokines in colon and mesenteric lymph nodes. Further mechanism analysis found that ZO inhibited DSS-induced nuclear factor-kappa B pathway activation, and regulated peroxisome proliferator-activated receptor gamma (PPAR gamma) expression. To further explore whether PPAR gamma was involved in the anti-UC effect of ZO, PPAR gamma inhibitor GW9662 was used. Although ZO also showed a protective effect on GW9662-treated colitis mice, the protective role was significantly weakened. Importantly, the administration of GW9662 significantly aggravated UC compared with the ZO + DSS group. In addition, we preliminarily found that ZO had the effects of inhibiting DSS-induced oxidative stress, maintaining intestinal barrier, and inhibiting the content of LPS and the population of Escherichia coli. CONCLUSIONS: These results indicated that supplementation with ZO might be a new dietary strategy for the treatment of UC. (C) 2022 Society of Chemical Industry.

Disinfection is key for controlling microbial contamination and ensuring the safe production of milk and dairy products. In this study, we developed a new disinfection method using quaternary ammonium surfactant N-dodecyl-2-(pyridin-1-yl) acetamide chloride as the main component to form a bactericidal complex with either chlorhexidine acetate or glutaraldehyde, and we evaluated the bactericidal effects, safety, and clinical application value of the compound disinfectants. An in vivo acute oral toxicity assay in mice showed an LD50 > 5000 mg/kg body weight without abnormality in pathological tissue sections. Comparison with commercially available products also showed that they have outstanding bactericidal effects. Clinical trials proved that the compound disinfectants have excellent bactericidal effects on the air and ground of the dairy farm and on the skin of cattle, especially in a dairy farm environment. Our findings confirm that the new compound disinfectants have excellent bactericidal performance and are safe to use as disinfectants to prevent mastitis and contamination of the cattle farm environment.

[目的]对牛甲基转移酶METTL3基因进行真核表达并对其蛋白进行生物信息学分析.[方法]以MDBK细胞为模板,RT-PCR扩增牛METTL3基因片段,构建pcDNA3.0-METTL3载体并转染至293T细胞后,进行Western Blotting验证,通过生物信息学在线工具分析METTL3蛋白.[结果]PCR、双酶切鉴定、Western Blotting和生物信息学分析表明牛METTL3基因编码区序列全长为1760 bp,编码了580个氨基酸,蛋白的分子质量64.4469 kDa,PI为5.98,属于不稳定亲水性蛋白,不存在跨膜结构与信号肽.二级结构主要是由α螺旋和无规则卷曲构成,三级结构与二级结构预测结果一致.[结论]成功构建了pcDNA3.0-HA-METTL3真核表达载体,生物信息学分析表明,牛METTL3蛋白为在细胞质中分泌合成的可溶蛋白,α螺旋和无规则卷曲为二级结构中最主要的结构元件.

Bovine viral diarrhea virus (BVDV) is an important member of the family Flaviviridae and often causes immunosuppression. Previous studies have suggested that BVDV envelope protein Erns and the nonstructural autoprotease Npro can inhibit host innate immune responses. Herein, we found that BVDV NS4B, as a nonstructural protein necessary for replication, is involved in antagonizing the main RNA virus sensing pathway. Overexpression of BVDV NS4B protein significantly inhibited Sendai virus (SeV)-induced interferon-β promoter activity, IFN-β mRNA and IFN regulatory factor 3 (IRF3) phosphorylation levels. We also discovered that BVDV NS4B protein significantly inhibited RIG-I like receptor (RLRs)-mediated interferon-β (IFN-β) promoter activity and endogenous MDA5 mRNA levels. In addition, the BVDV NS4B protein directly interacts with N-terminal CARDs of MDA5, and co-localized with MDA5 or MDA5-2CARD in the cytoplasm. In summary, the results of this study indicate that the BVDV NS4B protein acts as an interferon-β antagonist through inhibiting the MDA5-mediated signal transduction pathway. Our study provides an in-depth understanding of the molecular mechanisms of BVDV evading the host's natural immune response.

目的 研究牛病毒性腹泻病毒(bovine viral diarrhea viruses,BVDV)N-端自身蛋白酶(Npro)的生物信息学特征及表达特性.方法 克隆NADL株BVDV Npro基因,利用生物信息学软件分析其编码蛋白的理化性质、疏水性、跨膜区、信号肽、二级及三级结构,并进行原核表达、纯化及鉴定.将纯化的重组Npro蛋白免疫昆明小鼠,制备鼠抗Npro蛋白多克隆抗体,采用ELISA法、间接免疫荧光试验及Western blot法进行多克隆抗体效价、特异性及蛋白反应原性检测.结果 PCR扩增的Npro基因全长504 bp,编码168个氨基酸,Npro基因与BVDV V026株属于同一分支.Npro蛋白属于外膜蛋白,有亲水性,无信号肽,二级结构以β折叠和无规则卷曲为主.重组表达质粒pET-28a-Npro经PCR及酶切鉴定正确,测序结果与原序列一致;表达的重组Npro蛋白相对分子质量约25000,纯化后纯度达95％以上,且免疫原性良好;制备的鼠抗Npro蛋白多克隆抗体效价为1∶128000,特异性良好.结论 成功在大肠埃希菌中表达了BVDV Npro蛋白,表达的Npro蛋白免疫原性良好,为进一步研究BVDV Npro蛋白的功能奠定了基础.

Salmonella, an important zoonotic pathogen, causes significant morbidity and mortality in both humans and animals. Phloretin mainly isolated from strawberries and apples has the effects of treating inflammation and pathogenic bacteria, but its protective efficacy and mechanism of action against Salmonella spp. are less clear. In this study, we found that phloretin alleviated body weight loss, colon length shortening, and colonic pathological damage caused by S. Typhimurium. Phloretin also decreased S. Typhimurium translocation to the mesenteric lymph nodes (MLN) and spleen. Further mechanism studies showed that phloretin significantly inhibited inflammation and oxidative stress levels in the colonic tissue. Phloretin also prevented S. Typhimurium-mediated impairment in the colon epithelium barrier by the regulation ZO-1 and occludin levels. Interestingly, phloretin did not inhibit S. typhimurium growth in vitro, but reduced the internalization of S. Typhimurium into Caco-2 cells. Taken together, these findings indicated that phloretin may be a new dietary strategy to combat the disease.