连翘酯苷对体外培养细胞的毒性研究

探讨了连翘酯苷对体外培养细胞的毒性,以期为人们更好地研究利用连翘酯苷提供实验依据。实验采用细胞病变法观察连翘酯苷对猪肾细胞（PK-15）、猴肾细胞（Mare-145）、鸡胚肾细胞（CEK）、鸡外周血淋巴细胞（PBL）等4种细胞的形态损伤;用MTT法检测连翘酯苷作用这4种细胞后的细胞活性和细胞数量的变化。根据这两种方法的结果计算出连翘酯苷对这4种细胞的半数致死量。实验结果显示高剂量的连翘酯苷可致细胞发生病变,而低剂量组的细胞形态优于对照组。经SPSS16．0统计,连翘酯苷对PK-15、Mare-145、CEK3种细胞的半数致死量分别为137．8、87．1、384．4μg／mL,对PBL应用到中华人民共和国兽药典（2000年版）规定药量的10倍剂量,结果未发现致死性。因此,适当合理的连翘酯苷用量对体外培养的细胞没有毒性,反而有一定的维持细胞形态的营养作用和刺激细胞增殖的作用。     

Effects of dietary Forsythia suspensa extract supplementation to lactating sows and nursery pigs on post-weaning performance,antioxidant capacity,nutrient digestibility,immunoglobulins, and intestinal health

The aim of this study was to determine the effect of dietary Forsythia suspensa extract (FSE) supplementation to lactating sows and nursery pigs on post-weaning performance, antioxidant capacity, immunoglobulins, and intestinal health. Based on backfat, body weight (BW), and parity, 24 gestating sows (Landrace × Yorkshire) with average parity of 3.38 ± 0.61 and BW of 234 ± 6.81 kg were allotted into two dietary treatments (control vs. 100 mg/kg FSE) with 12 sows per treatment from day 107 of gestation to day 21 of lactation. After weaning, based on the initial BW and source litter, 192 nursery pigs (Duroc × [Landrace × Yorkshire], average BW of 6.98 ± 0.32 kg, weaned at day 21) were allotted into four dietary treatments with eight replicate pens per treatment, six pigs per pen for a 4-wk study. The treatments included the following: 1) CC (sows and their piglets both fed control diet); 2) CF (sows fed control diet and their piglets fed FSE diet [containing 100 mg/kg FSE]); 3) FC (sows fed FSE diet and their piglets fed control diet); and 4) FF (sows and their piglets both fed FSE diet). The MIXED procedures of SAS for a split-plot arrangement with sow diet as the whole plot and nursery diet as split plot were used to analyze the data. After weaning, piglets from FSE-fed sows had improved (P < 0.05) average daily gain and feed efficiency, and lower (P < 0.05) diarrhea rate in overall (day 1 to 28) compared with those from sows fed control diet. Piglets from FSE-fed sows also had higher (P < 0.05) contents of immunoglobulin G (IgG), growth hormone, superoxide dismutase (SOD), total antioxidant capacity in serum, villus height in ileum, and villus height to crypt depth ratio in jejunum, as well as lower (P < 0.05) content of malondialdehyde (MDA) in serum and crypt depth in ileum compared with those from sows fed control diet. Piglets fed FSE during nursery had increased (P < 0.05) concentrations of IgG, SOD, and catalase, and decreased (P < 0.05) MDA and tumor nuclear factor-α levels in serum compared with those fed control diet during nursery. Piglets from FC group had increased (P < 0.05) protein expression of occludin in jejunal mucosa and relative abundance of Lactobacillus on genus level in colon compared with those from CC group. In conclusion, for the performance and intestinal health, diets supplemented with FSE during lactation phase seemed more efficient to alleviate weaning stress than the nursery phase. In terms of the antioxidant status and immunoglobulins, FSE supplemented in both phases were efficient for nursery pigs.     

基于网络药理学研究连翘叶提取物通过AMPK抗恩诺沙星诱导肝损伤的作用

【目的】 运用网络药理学并通过体内试验研究连翘叶提取物（Forsythia suspense leaves extract，FSLE）治疗恩诺沙星（enrofloxacin，ENR）诱导的肝损伤（enrofloxacin induced liver injury，EILI）的作用机制。【方法】 采用乙醇-酶法提取连翘叶有效成分，通过液相-质谱法（LC-MS）鉴定FSLE主要的化学成分。利用PubChem、Swiss ADME和Swiss Target Prediction数据库分析预测FSLE有效活性成分及作用靶点，通过GeneCards疾病数据库搜索EILI的作用靶点。运用STRING在线网站及Cytoscape 3.8.0软件构建FSLE治疗EILI的核心靶点，借助Metascape网站进行GO功能及KEGG通路富集分析，并用Cytoscape 3.8.0软件绘制"成分-靶点-信号通路"可视化网络。通过体内试验验证FSLE抗EILI的药理作用，采用实时荧光定量PCR和Western blotting检测不同处理组腺苷酸激活蛋白激酶（AMP-activated protein kinase，AMPK）的表达，通过商业化试剂盒检测还原型谷胱甘肽（glutathione，GSH）、过氧化氢（hydrogen peroxide，H₂O₂）和丙二醛（malondialdehyde，MDA）含量及总超氧化物歧化酶（superoxide dismutase，SOD）的活性。【结果】 结合LC-MS与各数据库分析得出FSLE活性成分有20种，对应靶点338个；EILI疾病靶点913个；药物-疾病交集靶点124个，包含FSLE治疗EILI核心靶点12个，KEGG通路20条，其中AMPK通路与FSLE治疗EILI密切相关。动物试验结果显示，FSLE可显著降低EILI小鼠血清中谷丙转氨酶（alanine transferase，ALT）和谷草转氨酶（aspartate transferase，AST）活力（P<0.05）。FSLE可显著升高小鼠肝脏AMPK的基因和蛋白表达量水平（P<0.05）。与正常对照组相比，FSLE可显著降低小鼠肝脏组织中MDA和H₂O₂含量（P<0.05），显著升高GSH含量和SOD活力（P<0.05），抑制EILI诱导的肝脏氧化应激。【结论】 FSLE可通过上调AMPK的表达抑制肝脏氧化应激发挥抗EILI的作用，对肝损伤药物的开发具有重要的意义。     

真空冷冻干燥技术在连翘加工中的应用研究

通过试验优化连翘的真空冷冻干燥工艺参数,得到较佳的工艺参数。以干燥速率、生产效率及面积收缩率为干燥特性指标,以连翘活性成分得率为成分指标,采用单因素分析结合正交试验,考查预冻速率、真空压强和隔板温度对连翘干燥特性和活性成分得率的影响。结果表明,连翘的干燥特性与预冻速率、隔板温度及真空压强等冻干工艺参数呈现显著的规律性,连翘活性成分提取率则与工艺参数无显著规律性。在优化的工艺参数为预冻速率为7.5℃/min,真空度为40 Pa,隔板温度为50℃的条件下,连翘活性物质连翘酯苷A、连翘苷、连翘脂素、芦丁的甲醇提取得率分别为3.648%,0.560%,0.037%,0.170%,连翘活性成分提取得率的综合评分最高为1.103 9%,且干燥特性为优。     

连翘叶酶-醇提取物抗药物性肝损伤作用研究

【目的】研究连翘叶酶 醇提取物（FLEAE）抗药物性肝损伤（DILI）的作用及其机制。【方法】将48只昆明小鼠随机分为6组，每组8只，分别为正常对照组（NC）、FLEAE对照组（FC）、DILI模型组（DILI）及FLEAE高、中、低剂量治疗组（HFC、MFC、LFC），其中NC组和FC组小鼠腹腔注射生理盐水，其余各组小鼠腹腔注射300 mg/kg APAP注射液，每天2次，连续注射4 d，第5天开始NC组和DILI组小鼠灌胃生理盐水，FC、HFC、MFC和LFC组小鼠分别灌胃FLEAE 600，600，400和200 mg/kg，每天2次，连续灌胃3 d。最后1次灌胃12 h后，检测各组小鼠谷丙转氨酶（ALT）、谷草转氨酶（AST）水平及氧化应激指标谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、过氧化氢（H₂O₂）和丙二醛（MDA）水平，肝组织切片苏木精伊红（HE）染色观察肝损伤情况。用网络药理学方法构建FLEAE调控DILI的靶点集，GO和KEGG分析靶点的基因功能及通路，筛选FLEAE抗DILI的核心靶点。qRT-PCR和Western Blot检测核心靶点PIK3CA及其下游凋亡抑制分子B细胞淋巴瘤-2（Bcl-2）基因和蛋白的表达水平。【结果】FLEAE可显著降低DILI组小鼠血液中的ALT和AST水平，改善药物性肝损伤。FLEAE可显著降低DILI小鼠的GSH和SOD水平，提高H₂O₂和MDA水平。网络药理学分析表明，FLEAE抗DILI靶点与凋亡过程的负调控、氧化应激等有关，主要富集于PI3K/Akt信号通路，可能通过PIK3CA发挥作用；qRT-PCR和Western Blot结果显示，FLEAE可能通过提高PIK3CA基因和蛋白的表达发挥抗DILI及抑制细胞凋亡的作用。【结论】FLEAE通过上调DILI中PIK3CA的表达抑制细胞凋亡和改善氧化应激，发挥抗DILI的作用。