Malignant ascites effusion (MAE) is a common complication of advanced malignant tumors with limited treatments. Euphorbia lathyris (EL) has a long history of application in patients with edema and ascites. Herein, we reported for the first time a mode in which EL and EL Pulveratum (PEL) spontaneously formed natural microemulsions (ELM and PELM) without the addition of any carriers and excipients, and found that the protein and phospholipid contained in them encapsulated fatty oil and diterpenoid esters through non-covalent interactions. The denaturation and degradation of protein in PELM resulted in stronger binding of diterpenoid esters to the hydrophobic region of protein, which facilitated the sustained and slow release of diterpenoid esters and improved their bioavailability in vivo, thereby retaining the efficacy of preventing MAE while alleviating the irritation of intestinal mucosa. The mechanism by which PELM retained efficacy might be related to increased feces moisture and urine volume, and decreased expression of AVPR2, cAMP, PKA and AQP3 in MAE mice. And its mechanism of reducing intestinal mucosal irritation was related to decreased cell apoptosis, amelioration of oxidative stress, elevation of mitochondrial membrane potential, and up-regulation of Occludin and Claudin-1 expression in IEC-6 cells. This nano-adjuvant-free natural microemulsions may be a promising therapeutic strategy in the field of phytochemistry for promoting the application of natural and efficient nano-aggregates spontaneously formed by medicinal plants in MAE, and provide a new perspective for advancing the development of the fusion of Chinese herbal medicine and nanomedicine and its clinical translation.

Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1β and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L1, L2, and L3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1β expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L1, L2, and L3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine.

目的 探讨延胡索总碱贴片的安全性及其对神经病理性疼痛大鼠的镇痛作用及其机制.方法 ①安全性实验:采用单次、多次给药的皮肤刺激性实验及皮肤过敏性实验,评价延胡索总碱贴片的安全性.②镇痛实验:取48 只雄性SD大鼠,随机分为正常组、假手术组、模型组、布洛芬组、双氯芬酸钠贴片组及延胡索总碱贴片高、中、低剂量组,每组 6 只.除正常组和假手术组外,其余组大鼠采用坐骨神经慢性压迫损伤方法诱导神经病理性疼痛模型,术后第8 天开始,布洛芬组灌胃给药,双氯芬酸钠贴片组及延胡索总碱贴片高、中、低剂量组分别给予双氯芬酸钠贴片及延胡索总碱贴片腹部贴敷,其余组腹部贴敷空白贴片,均每天1 次,连续14 d.分别于术前及干预后1,3,7,10,14 d测定各组大鼠热缩足反射潜伏期;采用Western blot法检测各组大鼠干预结束后脊髓中Toll样受体4(TLR4)、核因子κB(NF-κB)、p38 丝裂原活化蛋白激酶(p38MAPK)、信号传导和转录激活因子3(STAT3)蛋白表达情况.结果 ①给予延胡索总碱贴片后,皮肤刺激反应评分均小于0.5 分,致敏率为0,组织结构与正常皮肤无差异.②镇痛实验中,延胡索总碱贴片中、高剂量组干预后3 d和延胡索总碱贴片各剂量组干预后7,10,14 d患侧的热缩足反射潜伏期均明显长于同期模型组(P均<0.05);干预结束后,延胡索总碱贴片各剂量组大鼠脊髓中 TLR4、NF-κB、p38MAPK蛋白相对表达量均明显低于模型组(P均<0.05),STAT3 蛋白相对表达量均明显高于模型组(P均<0.05).结论 延胡索总碱贴片安全性良好,可抑制慢性坐骨神经挤压损伤神经病理性疼痛大鼠痛觉敏感,其机制可能与抑制脊髓中TLR4、NF-κB、p38MAPK表达并上调STAT3 的表达有关.

Objective Using the stable isotope ratio method for the authenticity identification and variety identification of refined honey. Methods In this paper, a total of 17 samples of different varieties of refined honey were used to obtain refined honey proteins by precipitation with sodium tungstate solution and sulphuric acid solution. The isotope mass spectrometer was used to simultaneously detect the δ 13 C values of refined honey proteins and refined honey as well as the δ 18 O and δ 2 H values of refined honey, processed of the results obtained, analysed the authenticity of the samples and conduct a variety identification study. Results Tested of the resulting honey samples, the results showed that four batches of refined honey did not up to standard, two batches of C-4 vegetable syrup were detected as adulterated, and two batches of protein were not detected. The δ 18 O and δ 2 H values of refined honey were also found to be effective in distinguishing the varietal origin of refined honey to a certain extent. Conclusions The stable isotope ratio method is useful in the authenticity identification of refined honey, and provides new ideas to further promote the authenticity of refined honey and variety identification research.

Objective To study the mechanism of extracts from Qianjinzi(Euphorbiae Semen)and Qianjinzishuang(Euphorbiae Semen Pulveratum)mediating Toll-like receptor 4(TLR4)signaling pathway in Caco-2 cells via regulating lipid rafts.Methods Caco-2 cells were divided into Euphorbiae Semen low-,medium-and high-dose(50,200,800 μg/mL)groups,Euphorbiae Semen Pulveratum low-,medium-and high-dose(50,200,800 μg/mL)groups and control group.Flow cytometry was used to detect the lipid rafts abundance of Caco-2 cells membrane.Confocallaser scanning microscope was used for studying the co-localization of TLR4 and lipid rafts in Caco-2 cells.Western blotting was applied to detect TLR4,nuclear factor-κB p65(NF-κB p65),p-NF-κB p65 and NOD-like receptor family pyrin domain containing 3(NLRP3)protein expressions in Caco-2 cells.Furthermore,ELISA was applied to determine the levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the supernatant of Caco-2 cells.And the levels of IL-1β and TNF-α were detected in Caco-2 cells after transfection with TLR4.Results Compared with control group,Euphorbiae Semen extracts could increase the content of lipid rafts(P＜0.01),promote the recruitment of TLR4 to lipid rafts(P＜0.01),increase the protein expressions of TLR4,NF-κB p65,p-NF-κB p65 and NLRP3,as well as induce the release of IL-1β and TNF-α in Caco-2 cells(P＜0.05,0.01,0.001).However,the effects of Euphorbiae Semen Pulveratum extracts on the abundance of lipid rafts,the recruitment of TLR4 to lipid rafts,the conduction of TLR4-mediated signaling pathways and the secretion of pro-inflammatory factors were significantly weaker than those of Euphorbiae Semen extracts at the same dose(P＜0.05,0.01,0.001).In addition,the results of siTLR4 transfection in vitro further confirmed that the content of IL-1β and TNF-α were significantly decreased after transfection of TLR4 in Caco-2 cells(P＜0.05,0.01,0.001).Conclusion The attenuation mechanism of processing for Euphorbiae Semen may be closely related to interfering the recruitment of TLR4 to lipid rafts by regulating lipid rafts abundance,and ultimately affecting the cascade of TLR4 signaling protein contained in lipid rafts.

目的 研究延胡索总碱贴片中延胡索总碱在不同条件下的稳定性.方法 以延胡索总碱贴片中的延胡索甲素、延胡索乙素以及延胡索丑素的含量,24h体外累积释放量及黏附力为指标,进行了强光照射、高温、高湿的影响因素试验、加速试验及长期留样试验.结果 制剂对光照、高温、高湿稳定,加速试验、长期留样试验稳定性较好,延胡索总碱贴片中的延胡索甲素、延胡索乙素和延胡索丑素的含量、24h体外累计释放量及延胡索总碱贴片黏附力无显著性变化.结论 延胡索总碱贴片质量可控,稳定性良好.

Objective To investigate the effects of the extracts from Semen Euphorbiae before and after frosting on the toll-like receptor 4 （TLR4）/nuclear factor kappa-B （NF-κB）/nod-like receptor family pyrin domain containing 3 （NLRP3） signaling pathway. Methods According to the body weight， 42 BALB/c mice were randomly divided into a blank control group， Semen Euphorbiae low-， medium-， and high-dose groups （10.49， 20.98， and 41.96 g/kg， respectively） and Semen Euphorbiae Pulveratum low-， medium-， and high-dose groups （10.49， 20.98， and 41.96 g/kg， respectively）， with six rats per group. Mice received continuous gavage for 7 d. The contents of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in serum of mice were determined by enzyme linked immune sorbent assay. The protein and the mRNA expression of TLR4， NF-κB p65， and NLRP3 in colon tissues of mice were detected by Western blotting and real-time PCR. According to the body weight， 30 BALB/c mice were randomly divided into a blank control group， Semen Euphorbiae group， Semen Euphorbiae+TAK-242 group， Semen Euphorbiae Pulveratum group， and Semen Euphorbiae Pulveratum+ TAK-242 group， with six rats per group. The administration groups were respectively given 20.96 g/kg of Semen Euphorbiae or Semen Euphorbiae Pulveratum， and the continuous gavage was 7 d. The Semen Euphorbiae+TAK-242 group and Semen Euphorbiae Pulveratum+ TAK-242 group were intraperitoneally injected with TAK242 （3 mg/kg） 30 min before and on days 5， 6， and 7 of administration. The protein expression of NLRP3 and NF-κB p65， and the mRNA expression of NLRP3 and IL-1β in colon tissues of mice were assessed by Western blotting and real-time PCR. Results Semen Euphorbiae not only significantly promoted the release of IL-1β and TNF-α， but it also significantly increased the protein and the mRNA expression of TLR4， NF-κB p65 and NLRP3 （P ＜ 0.05， P ＜ 0.01）. However， upregulation of IL-1β and TNF-α secretion， and TLR4， NF-κB p65， NLRP3 protein， and mRNA expression were decreased by Semen Euphorbiae Pulveratum （P ＜ 0.05， P ＜ 0.01）. NLRP3 and NF-κB p65 protein expression and NLRP3 and IL-1β mRNA expression were inhibited after the intervention with the TLR4 inhibitor TAK-242 （P ＜ 0.01）. Conclusion Semen Euphorbiae could induce an inflammatory response of the colon tissue by activating the TLR4-mediated NF-κB/NLRP3 pathway， thus promoting the release of TNF-α and IL-1β. However， upregulation of the TLR4/NF-κB/NLRP3 signaling pathway and pro-inflammatory factor secretion were significantly alleviated by Semen Euphorbiae Pulveratum， indicating that the detoxification mechanism of Semen Euphorbiae frosting may be related to the decreased ability to cause inflammation and the intervention of TLR4/NF-κB/NLRP3 signaling pathway.

目的 运用网络药理学和分子对接探讨千金子二萜醇酯类成分治疗白血病的潜在作用机制.方法 选取千金子中7种代表性二萜醇酯类成分为研究对象,综合运用SwissTargetPre-diction、SEA、OMIM、Gene Cards数据库筛选及预测千金子二萜醇酯类成分治疗白血病的潜在靶点.通过STRING数据库和Cytoscape 3.7.2软件来建立PPI网络.使用DAVID数据库对筛选出的靶点进行基因本体(GO)生物学过程分析和京都基因与基因组百科全书(KEGG)通路分析,并应用Cytoscape 3.7.2构建"成分-靶点-通路"网络,最后对千金子二萜醇酯类成分与关键靶蛋白进行分子对接验证.结果 7种千金子二萜醇酯类成分涉及252个作用靶点,筛选得到75个治疗白血病的潜在靶点,PPI核心网络表明治疗白血病的关键靶点为CDK1、CDK2、CCNA2,基因富集分析及"成分-靶点-通路"网络图显示千金子二萜醇酯类成分可能通过调节PI3 K-Akt、ErbB、p53等信号通路来发挥对白血病的治疗作用.结论 本研究表明千金子二萜醇酯类成分可能通过调节细胞凋亡、增殖分化、转录、细胞周期等多种途径来治疗白血病,诠释了中药通过多成分、多靶点、多途径治疗疾病的研究思路,为进一步深入研究千金子二萜醇酯类成分治疗白血病的作用机制提供了科学依据.

以千金子中3种主要的二萜醇酯类成分提取率的综合评分为评价指标,通过Box-Behnken响应面法结合熵权法对千金子二萜醇酯类成分的闪式提取工艺进行优化.确定最佳提取工艺为:料液比1:14(g:mL)、提取时间2.5 min、提取电压130 V,在此条件下,3种二萜醇酯类成分提取率的综合评分为0.370,与模拟预测值(0.396)接近.该闪式提取工艺设计合理,可为千金子主要二萜醇酯类成分的快速高效提取提供一定的参考.

目的 比较千金子制霜前后提取物对LO2细胞毒性的影响并探究千金子制霜减毒的作用机制.方法 将人肝LO2细胞分为7组:千金子生品低、中、高3个剂量组、千金子霜品低、中、高3个剂量组和阴性对照组.将细胞培养48 h后,分别予以100,200,400μg·mL-1的千金子生品、千金子霜品供试品溶液和等体积的无血清细胞培养液,各组设置3个复孔.分别用PI染色法和Annexin V-FITC/PI双重染法来检测细胞周期和凋亡情况,试剂盒法检测乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)和Na+-K+-ATP酶的活力.结果 阴性对照组、千金子生品低、高2个剂量组、千金子霜品低、高2个剂量组的细胞G2/M期比例依次为(22.47±2.29)％,(13.63±0.88)％,(6.12±0.83)％,(18.03±0.73)％和(9.88±0.75)％;这5组总凋亡率依次为(3.87±0.12)％,(9.93±0.05)％,(12.80±0.28)％,(6.90±0.08)％和(9.73±0.09)％.阴性对照组、千金子生品低、中、高3个剂量组、千金子霜品低、中、高3个剂量组的LDH活力依次为(21.66±1.78),(257.77±13.09),(452.44±10.80),(798.65±8.72),(145.16±5.65),(238.10±4.85)和(495.56±5.16)mU·mL-1;这7组的SOD活力依次为(17.03±0.41),(8.28±0.35),(6.01±0.37),(2.89±0.27),(12.87±0.21),(10.58±0.24)和(7.71±0.44)U·mg-1;这7组的Na+-K+-ATP酶活力依次为(15.84±0.54),(8.47±0.66),(7.00±0.13),(3.64±0.17),(12.88±0.40),(10.79±0.22)和(8.45±0.13)U·mg-1.上述指标,千金子生品低、中、高3个剂量组与阴性对照组相比,差异均有统计学意义(均P<0.01);千金子霜品3个剂量组与千金子生品3个剂量组相比,差异均有统计学意义(均P<0.01).结论 千金子制霜后可能通过减轻细胞氧化损伤,降低细胞膜通透性,改善肝细胞功能损伤,减少细胞凋亡来降低体外肝毒性.

目的:研究甘草炮制前后对免疫低下小鼠免疫调节作用的影响.方法:小鼠腹腔注射环磷酰胺制备免疫低下模型,给予生甘草、清炒甘草、蜜炙甘草的提取液,检测免疫功能学评价指标,包括免疫器官指数、半数溶血值、溶血空斑数、脾淋巴细胞增值率、自然杀伤(NK)细胞杀伤率、耳肿胀度、碳廓清指数、腹腔巨噬细胞吞噬率及吞噬指数.结果:与对照组小鼠比较,模型组小鼠各检测指标明显降低(P<0.05,P<0.01);与模型组小鼠比较,各给药组(生甘草组、清炒甘草组、蜜炙甘草组)小鼠各检测指标均能增强.其中,仅蜜炙甘草组能明显增强各检测指标(P<0.05,P<0.01);与生甘草组小鼠相比,清炒甘草组能明显增强血清溶血素和抗体生成细胞、脾淋巴细胞增殖率和NK细胞活性(P<0.05,P<0.01),但对胸腺指数、脾脏指数、腹腔巨噬细胞吞噬率和吞噬指数、DTH、碳廓清功能无明显影响,蜜炙甘草组能明显增强除胸腺指数的各项评价指标(P<0.05,P<0.01),但对胸腺指数无明显影响.结论:甘草蜜炙前后对小鼠非特异性免疫、体液免疫及细胞免疫功能均有促进作用,且蜜炙后作用更强,为蜜炙甘草机制研究提供参考.

Previous pharmacological studies have indicated that diterpenoids are the primary effective chemical cluster in the seeds of Euphorbia lathyris L. The seed products are used in traditional Chinese medicine in the forms of Semen Euphorbiae (SE) and Semen Euphorbiae Pulveratum (SEP). However, the metabolism of the plant's diterpenoids has not been well elucidated, which means that the in vivo metabolite products have not been identified. The current study screened the physiological metabolites of six diterpenes [Euphorbia factor L1 (L1), L2 (L2), L3 (L3), L7a (L7a), L7b (L7b), and L8 (L8)] in feces and urine of rats after oral administration of SE and SEP using UHPLC-Q-Exactive MS. A total of 22 metabolites were detected in feces and 8 in urine, indicating that the major elimination route of diterpenoids is via the colon. Hydrolysis, methylation, and glucuronidation served as the primary metabolic pathways of these diterpenoids. In sum, this study contributed to the elucidation of new metabolites and metabolic pathways of SE and SEP, and the new chemical identities can be used to guide further pharmacokinetic studies.

目的 考察延胡索总碱的溶解度、油水分配系数等理化性质,研究促透剂及穴位、非穴位贴敷对其体外经皮渗透特性的影响,为指导其经皮给药剂型设计和制备提供参考.方法 建立酸性染料比色法和高效液相色谱(high performance liquid chromatography,HPLC)法对延胡索总碱、延胡索甲素、延胡索乙素及延胡索丑素的含量进行测定,应用饱和溶液法和经典摇瓶法检测延胡索总碱的溶解性及油水分配情况;采用Franz扩散池法考察大鼠神阙穴与非穴位皮肤体外经皮渗透性能.结果 结果显示,延胡索总碱纯度大于50％,其在10％乙醇的生理盐水中溶解性较好,可作为扩散池接收介质.当pH在5.5～7.2时,延胡索总碱的logP值控制在-1～0之间,说明延胡索总碱为强亲水性组分,延胡索甲素、延胡索乙素、延胡索丑素的logP值均在0～3之间,体外经皮渗透性良好,神阙穴用药组的24小时体外累积透过量和滞留量优于非穴位(P<0.05).结论 延胡索总碱具有适宜经皮渗透的理化性质及较好的经皮渗透性能,适宜制成经皮给药制剂,且经神阙穴用药具有一定优势,以上结果可为经皮给药剂型设计提供实验基础.

目的:比较千金子制霜前后提取物对正常人胚肾细胞HEK293的影响,探究千金子制霜减毒的作用机制.方法:采用HEK293细胞为体外模型,随机分为对照组,千金子生品低、中、高质量浓度(100、200、400μg·mL–1)组和千金子霜品低、中、高质量浓度(100、200、400μg·mL–1)组.细胞体外培养后,分别给予等体积的无血清培养液和千金子生品、霜品提取物溶液,各组均设置3个复孔.采用CCK-8法评价细胞存活率,利用倒置显微镜观察细胞数量及形态变化;碘化丙啶(PI)染色法和Annexin V-FITC/PI双重染色法检测细胞周期和凋亡情况;试剂盒法检测细胞上清液中乳酸脱氢酶(LDH)、谷胱甘肽(GSH)、肌酐(Cr)和细胞裂解液中尿素氮(BUN)水平.结果:与对照组比较,千金子生品各质量浓度组HEK293细胞存活率降低,细胞形态异常,细胞数量减少,G0/G1、G1/G2期细胞比例明显降低,S期细胞比例明显增加,细胞凋亡率升高(P<0.01),LDH、BUN、Cr水平升高(P<0.05,P<0.01),GSH水平降低(P<0.01);与千金子生品组比较,千金子霜品相应质量浓度可显著增加HEK293细胞存活率(P<0.01),改善细胞形态,增加G0/G1期、G1/G2期细胞比例,降低S期细胞比例,同时能明显降低LDH、BUN、Cr水平(P<0.05,P<0.01),提高GSH水平(P<0.01).结论:千金子制霜后可能通过减轻细胞周期阻滞和细胞氧化损伤改善肾细胞功能、减少细胞凋亡,从而降低体外肾毒性.

Magnetic nanoparticles(NPs) are characterized by a rich variety of properties. Because of their excellent physical and chemical properties, they have come to the fore in biomedicine and other fields. The magnetic NPs were extensively studied in magnetic separation of cells, targeted drug delivery, tumor hyperthermia, chemo-photothermal therapy, magnetic resonance imaging (MRI) and other biomedical fields. Magnetic NPs are increasingly used in magnetic resonance imaging (MRI) based on their inherent magnetic targeting, superparamagnetic enzyme-like catalytic properties and nanoscale size. Poly(lactic-co-glycolic acid) (PLGA) is a promising biodegradable material approved by FDA and EU for drug delivery. Currently, PLGA-based magnetic nano-drug delivery systems have attracted the attention of researchers. Herein, we achieved the effective encapsulation of sized-controlled polyethylene glycol-3,4-dihydroxy benzyl-amine-coated superparamagnetic iron oxide nanoparticles (SPIO NPs) and euphorbiasteroid into PLGA nanospheres via a modified multiple emulsion solvent evaporation method (W1/O2/W2). NPs with narrow size distribution and acceptable magnetic properties were developed that are very useful for applications involving cancer therapy and MRI. Furthermore, SPIO-PLGA NPs enhanced the MRI T2 relaxation properties of tumor sites.The prepared SPIO NPs and magnetic PLGA nanospheres can be promising magnetic drug delivery systems for tumor theranostics. This study has successfully constructed a tumor-targeting and magnetic-targeting smart nanocarrier with enhanced permeability and retention, multimodal anti-cancer therapeutics and biodegradability, which could be a hopeful candidate for anti-tumor therapy in the future.