Aim An HPLC-UV method for the analysis of Traditional Chinese Medicine （TCM） preparation, Sijunzi decoction, has been developed. Four flavonoid marker compounds, liquiritigenin, liquiritin, isoliquiritigenin, and is...Aim An HPLC-UV method for the analysis of Traditional Chinese Medicine （TCM） preparation, Sijunzi decoction, has been developed. Four flavonoid marker compounds, liquiritigenin, liquiritin, isoliquiritigenin, and isoliquiritin, from Radix Glycyrrhizae were quantitatively analyzed in this preparation. Methods The separation was performed on a reversed-phase C18 column by using a gradient elution with mobile phase of （A） water-formic acid （100∶0.04, V/V） （pH 3） and （B） acetonitrile. The mobile phase gradient was set from 0-5 min at 10% of B and 45 min to 90% of B. The assay was carried out at a flow rate of 0.2 mL·min-1 at room temperature with the UV detection wavelengths at 280 nm and 368 nm. Results The extract （25 mg·mL^-1） of Sijunzi decoction contains 1.47 μg·mL^-1 liquiritigenin, 16.40 μg·mL^-1 liquiritin, 0.66 μg·mL^-1 isoliquiritigenin, and 2.12 μg·mL^-1 isoliquiritin. The recoveries for the sample preparation of the markers were 102.2%, 97.7%, 100.3%, and 99.9%, respectively. Conclusion The method is simple and accurate. This study reports a routine quantitative method for the analysis of multiple components in Sijunzi decoction by HPLC.展开更多
AIM To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction(TPSJ) on the epithelial barriers in vitro. METHODS Caco-2 cell monolayers were treated with or without ...AIM To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction(TPSJ) on the epithelial barriers in vitro. METHODS Caco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance(TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respecti-vely used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. western blotting was also used to evaluate the cellular expression of myosin light chain(MLC), phosphorylated MLC(pM LC), MLC kinase(MLCK), and nuclear factor(NF)-κB p65. RESULTS TPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cyto-kines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor(TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65. CONCLUSION Our results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.展开更多
目的探讨四君子汤对抗PD-1免疫治疗小鼠三阴性乳腺癌(triple negative breast cancer,TNBC)的疗效及作用机制。方法建立TNBC移植瘤小鼠模型,将小鼠随机分为4T1组(模型组)、四君子汤组、抗PD-1治疗组、四君子汤联合抗PD-1治疗组,每组6只...目的探讨四君子汤对抗PD-1免疫治疗小鼠三阴性乳腺癌(triple negative breast cancer,TNBC)的疗效及作用机制。方法建立TNBC移植瘤小鼠模型,将小鼠随机分为4T1组(模型组)、四君子汤组、抗PD-1治疗组、四君子汤联合抗PD-1治疗组,每组6只。四君子汤组小鼠按实验动物与人体表面积比换算给予4.85 g/(kg·d)四君子汤灌胃(每周5次),抗PD-1治疗组给予80μg/只抗PD-1抗体腹腔注射(每周2次),四君子汤联合抗PD-1治疗组则分别给予上述治疗,4T1组则灌胃等体积的0.9%氯化钠溶液(每周5次);连续干预3周。比较各组肿瘤体积大小,通过生存分析比较各组小鼠生存时间。采用C11-BODIPY染色法进行脂质过氧化评估各组小鼠肿瘤细胞脂质过氧化水平,并通过测量丙二醛(malondialdehyde,MDA)含量确认;采用7-氨基放线菌素D(7-aminoactinomycin D,7-AAD)染色法检测细胞死亡比例;采用实时定量PCR检测铁死亡相关基因表达。并进一步通过体外实验分析四君子汤有效成分(人参皂苷Rb1)对4T1癌细胞脂质过氧化和铁死亡的影响。结果与抗PD-1治疗组相比,四君子汤联合抗PD-1治疗组小鼠肿瘤体积明显缩小(P<0.01),四君子汤联合抗PD-1治疗组中位生存期明显延长(34 d vs.25 d,P<0.05)。与抗PD-1治疗组相比,四君子汤联合抗PD-1治疗组脂质过氧化平均荧光强度明显升高(P<0.05),肿瘤细胞中的MDA含量、癌细胞死亡率均明显升高(P均<0.01)。四君子汤组(与4T1组相比)和四君子汤联合抗PD-1治疗组(与抗PD-1治疗组相比)铁死亡抑制基因NRF2、GPX4、SLC7A11表达水平显著降低(P均<0.05),但铁死亡促进基因TFRC组间比较差异无统计学意义(P>0.05)。细胞实验结果显示:与4T1细胞空白对照组相比,人参皂苷Rb1单体对乳腺癌细胞脂质过氧化平均荧光强度的作用差异无统计学意义(P>0.05)。人参皂苷Rb1单体联合IFN-γ对乳腺癌细胞脂质过氧化平均荧光强度的作用明显高于单用IFN-γ(P<0.05)。人参皂苷Rb1干预下4T1癌细胞铁死亡抑制基因NRF2、GPX4、SLC7A11表达水平显著降低(P均<0.05)。结论四君子汤单独应用未见对小鼠TNBC有明显治疗效果,但可增强抗PD-1抗体免疫治疗小鼠TNBC的效果,其机制可能与四君子汤降低癌细胞铁死亡抑制基因表达有关。展开更多
文摘Aim An HPLC-UV method for the analysis of Traditional Chinese Medicine （TCM） preparation, Sijunzi decoction, has been developed. Four flavonoid marker compounds, liquiritigenin, liquiritin, isoliquiritigenin, and isoliquiritin, from Radix Glycyrrhizae were quantitatively analyzed in this preparation. Methods The separation was performed on a reversed-phase C18 column by using a gradient elution with mobile phase of （A） water-formic acid （100∶0.04, V/V） （pH 3） and （B） acetonitrile. The mobile phase gradient was set from 0-5 min at 10% of B and 45 min to 90% of B. The assay was carried out at a flow rate of 0.2 mL·min-1 at room temperature with the UV detection wavelengths at 280 nm and 368 nm. Results The extract （25 mg·mL^-1） of Sijunzi decoction contains 1.47 μg·mL^-1 liquiritigenin, 16.40 μg·mL^-1 liquiritin, 0.66 μg·mL^-1 isoliquiritigenin, and 2.12 μg·mL^-1 isoliquiritin. The recoveries for the sample preparation of the markers were 102.2%, 97.7%, 100.3%, and 99.9%, respectively. Conclusion The method is simple and accurate. This study reports a routine quantitative method for the analysis of multiple components in Sijunzi decoction by HPLC.
基金Supported by the National Natural Science Foundation of China,No.81202635the Guangdong Provincial Bureau of Chinese Medicine,No.20151244
文摘AIM To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction(TPSJ) on the epithelial barriers in vitro. METHODS Caco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance(TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respecti-vely used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. western blotting was also used to evaluate the cellular expression of myosin light chain(MLC), phosphorylated MLC(pM LC), MLC kinase(MLCK), and nuclear factor(NF)-κB p65. RESULTS TPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cyto-kines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor(TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65. CONCLUSION Our results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.
文摘目的探讨四君子汤对抗PD-1免疫治疗小鼠三阴性乳腺癌(triple negative breast cancer,TNBC)的疗效及作用机制。方法建立TNBC移植瘤小鼠模型,将小鼠随机分为4T1组(模型组)、四君子汤组、抗PD-1治疗组、四君子汤联合抗PD-1治疗组,每组6只。四君子汤组小鼠按实验动物与人体表面积比换算给予4.85 g/(kg·d)四君子汤灌胃(每周5次),抗PD-1治疗组给予80μg/只抗PD-1抗体腹腔注射(每周2次),四君子汤联合抗PD-1治疗组则分别给予上述治疗,4T1组则灌胃等体积的0.9%氯化钠溶液(每周5次);连续干预3周。比较各组肿瘤体积大小,通过生存分析比较各组小鼠生存时间。采用C11-BODIPY染色法进行脂质过氧化评估各组小鼠肿瘤细胞脂质过氧化水平,并通过测量丙二醛(malondialdehyde,MDA)含量确认;采用7-氨基放线菌素D(7-aminoactinomycin D,7-AAD)染色法检测细胞死亡比例;采用实时定量PCR检测铁死亡相关基因表达。并进一步通过体外实验分析四君子汤有效成分(人参皂苷Rb1)对4T1癌细胞脂质过氧化和铁死亡的影响。结果与抗PD-1治疗组相比,四君子汤联合抗PD-1治疗组小鼠肿瘤体积明显缩小(P<0.01),四君子汤联合抗PD-1治疗组中位生存期明显延长(34 d vs.25 d,P<0.05)。与抗PD-1治疗组相比,四君子汤联合抗PD-1治疗组脂质过氧化平均荧光强度明显升高(P<0.05),肿瘤细胞中的MDA含量、癌细胞死亡率均明显升高(P均<0.01)。四君子汤组(与4T1组相比)和四君子汤联合抗PD-1治疗组(与抗PD-1治疗组相比)铁死亡抑制基因NRF2、GPX4、SLC7A11表达水平显著降低(P均<0.05),但铁死亡促进基因TFRC组间比较差异无统计学意义(P>0.05)。细胞实验结果显示:与4T1细胞空白对照组相比,人参皂苷Rb1单体对乳腺癌细胞脂质过氧化平均荧光强度的作用差异无统计学意义(P>0.05)。人参皂苷Rb1单体联合IFN-γ对乳腺癌细胞脂质过氧化平均荧光强度的作用明显高于单用IFN-γ(P<0.05)。人参皂苷Rb1干预下4T1癌细胞铁死亡抑制基因NRF2、GPX4、SLC7A11表达水平显著降低(P均<0.05)。结论四君子汤单独应用未见对小鼠TNBC有明显治疗效果,但可增强抗PD-1抗体免疫治疗小鼠TNBC的效果,其机制可能与四君子汤降低癌细胞铁死亡抑制基因表达有关。
