A rapid and sensitive liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed and validated for the simultaneous determination of H002 and its phosphorylated metabolite, H002-P and hydroxylated...A rapid and sensitive liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed and validated for the simultaneous determination of H002 and its phosphorylated metabolite, H002-P and hydroxylated metabolite H002-M, in rat blood. H001, an analogue of H002, was used as the internal standard.Blood samples were prepared by simple protein precipitation. The analytes and internal standard were separated on a Zorbax SB-C18 column with a gradient mobile phase consisting of methanol and water containing 0.1% formic acid at a flow rate of 0.2 mL /min with an operating temperature of 20 1C. The detection was performed on a triple quadrupole tandem mass spectrometer with positive electrospray ionization in multiple-reaction monitoring mode.Linear detection responses were obtained from 0.2–100 ng/mL for H002 and H002-M, while 0.5–100 ng/mL for H002-P. The intra- and inter-day precision(RSD%) was within 11.76%, with the accuracy(RE%) ranging from –9.84% to 9.12%. The analytes were shown to be stable during sample storage, preparation and analytic procedures.The method was applied to determine the pharmacokinetics of H002 in rats, and a preliminary study showed that the pharmacokinetics of H002 correlated with its biological effect on peripheral blood lymphocytes.展开更多
The liver is the central organ involved in lipid metabolism and the gastrointestinal(GI)tract is responsible for nutrient absorption and partitioning.Obesity,dyslipidemia and metabolic disorders are of increasing publ...The liver is the central organ involved in lipid metabolism and the gastrointestinal(GI)tract is responsible for nutrient absorption and partitioning.Obesity,dyslipidemia and metabolic disorders are of increasing public health concern worldwide,and novel therapeutics that target both the liver and the GI tract(gut-liver axis)are much needed.In addition to aiding fat digestion,bile acids act as important signaling molecules that regulate lipid,glucose and energy metabolism via activating nuclear receptor,G protein-coupled receptors(GPCRs),Takeda G protein receptor 5(TGR5)and sphingosine-1-phosphate receptor 2(S1PR2).Sphingosine-1-phosphate(S1P)is synthesized by two sphingosine kinase isoforms and is a potent signaling molecule that plays a critical role in various diseases such as fatty liver,in-flammatory bowel disease(IBD)and colorectal cancer.In this review,we will focus on recent findings related to the role of S1P-mediated signaling pathways in the gut-liver axis.展开更多
目的:探讨开玄解毒方对于咪喹莫特诱导银屑病样小鼠模型的症状、体征改善及其对1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)水平的调节作用。方法:选用BALB/c小鼠25只,随机均分为正常组、模型组、甲氨蝶呤组、开玄解毒中剂量组和高剂量...目的:探讨开玄解毒方对于咪喹莫特诱导银屑病样小鼠模型的症状、体征改善及其对1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)水平的调节作用。方法:选用BALB/c小鼠25只,随机均分为正常组、模型组、甲氨蝶呤组、开玄解毒中剂量组和高剂量组,除正常组外,其余组采用外涂咪喹莫特法建立银屑病样皮损模型,连续干预7 d后取材。期间对皮损拍照并计算银屑病面积和严重程度指数(psoriasis area and severity index,PASI)评分;HE染色观察皮损病理改变及测量表皮厚度;对脾脏及胸腺拍照并称重;免疫组化法检测表皮Ki67、S1P表达;蛋白质印迹法检测皮损KRT17、Claudin、Occludin表达;qRT-PCR检测IL-17、IL-23、S1PRs mRNA表达水平。结果:与模型组比较,开玄解毒方可缓解咪喹莫特诱导的小鼠银屑病样皮损,降低PASI评分,减轻表皮增生程度(P均<0.01),改善胸腺、脾脏指数及血管增生情况(P<0.01或P<0.05),降低炎症因子IL-17、IL-23 mRNA表达(P<0.01),修复皮肤屏障(P<0.05)。同时,开玄解毒方可下调表皮中S1P及S1PRs mRNA表达(P<0.05)。结论:开玄解毒方能够减轻银屑病局部皮损症状,其药效机制可能与调节S1P水平有关。展开更多
Sphingolipids are ubiquitous components of cell membranes. Their metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and sur...Sphingolipids are ubiquitous components of cell membranes. Their metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. S1P is generated by phosphorylation of sphingosine catalyzed by sphingosine kinase-1 (SPHK1). The purpose of this study is to explore the roles of S1P, S1P receptors, and sphingosine kinases in malignant musculoskeletal tumors. Twenty-one tumor samples (7 liposarcomas, 3 chondrosarcomas, 6 osteosarcomas, 5 MFH) obtained at open biopsy, and four human MFH cell lines (Nara H, Nara F, TNMY1, GBS-1) were used. We examined the mRNA expression of S1P receptors by RT-PCR, and the expression levels of SPHK by Real-time PCR. We used 4 MFH cell lines to analyze SPHK1 proteins by Western blotting. SPHK1 siRNA was transfected into MFH cell lines by lipofection method. Cell proliferation (control and transfected with siRNA) was assayed using WST-8 (Cell Counting Kit-8) assay. All high grade malignant tumors expressed S1P1, S1P2, S1P3 receptors, whereas the expression of S1P1 receptor was detected in 50% of low-grade malignant tumors, S1P2 receptor in 30%, and S1P3 in 50%. No statistically significant difference was found in the expression level of SPHK1 between high-grade and low-grade malignant tumors by Real-time PCR. By results of Western blotting, proteins of SPHK1 were expressed in all MFH cell lines. In MFH cell lines, transfection with SPHK1 siRNA oligonucleotides resulted in approximately 50 to 80% suppression of SPHK1 mRNA expression as determined by real-time PCR. Down-regulation of SPHK1 with small interfering RNA significantly reduced SPHK1 protein levels by Western blotting. Knock down of SPHK1 expression significantly decreased cell proliferation of all MFH cells. These results suggest that the expression of S1P receptors may play an important role for cell proliferation and may correlate with histologic grade in malignant bone and soft tissue tumors, and that SPHK1 may be one of essential mo展开更多
基金supported by the National Science and Technology Major Project of China(Nos.2012ZX09301002-001-007 and2012ZX09301002-006)National Natural Science Foundation of China(NSFC,Nos.81202545,81302847 and 81473096)
文摘A rapid and sensitive liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed and validated for the simultaneous determination of H002 and its phosphorylated metabolite, H002-P and hydroxylated metabolite H002-M, in rat blood. H001, an analogue of H002, was used as the internal standard.Blood samples were prepared by simple protein precipitation. The analytes and internal standard were separated on a Zorbax SB-C18 column with a gradient mobile phase consisting of methanol and water containing 0.1% formic acid at a flow rate of 0.2 mL /min with an operating temperature of 20 1C. The detection was performed on a triple quadrupole tandem mass spectrometer with positive electrospray ionization in multiple-reaction monitoring mode.Linear detection responses were obtained from 0.2–100 ng/mL for H002 and H002-M, while 0.5–100 ng/mL for H002-P. The intra- and inter-day precision(RSD%) was within 11.76%, with the accuracy(RE%) ranging from –9.84% to 9.12%. The analytes were shown to be stable during sample storage, preparation and analytic procedures.The method was applied to determine the pharmacokinetics of H002 in rats, and a preliminary study showed that the pharmacokinetics of H002 correlated with its biological effect on peripheral blood lymphocytes.
基金This work was supported by the USA National Institutes of Health(NIH)grants R01 DK104893 and R01DK-057543VA Merit Award I01BX004033 and 1I01BX001390Research Career Scientist Award(IK6BX004477)from the Department of Veterans Affairs.
文摘The liver is the central organ involved in lipid metabolism and the gastrointestinal(GI)tract is responsible for nutrient absorption and partitioning.Obesity,dyslipidemia and metabolic disorders are of increasing public health concern worldwide,and novel therapeutics that target both the liver and the GI tract(gut-liver axis)are much needed.In addition to aiding fat digestion,bile acids act as important signaling molecules that regulate lipid,glucose and energy metabolism via activating nuclear receptor,G protein-coupled receptors(GPCRs),Takeda G protein receptor 5(TGR5)and sphingosine-1-phosphate receptor 2(S1PR2).Sphingosine-1-phosphate(S1P)is synthesized by two sphingosine kinase isoforms and is a potent signaling molecule that plays a critical role in various diseases such as fatty liver,in-flammatory bowel disease(IBD)and colorectal cancer.In this review,we will focus on recent findings related to the role of S1P-mediated signaling pathways in the gut-liver axis.
文摘目的:探讨开玄解毒方对于咪喹莫特诱导银屑病样小鼠模型的症状、体征改善及其对1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)水平的调节作用。方法:选用BALB/c小鼠25只,随机均分为正常组、模型组、甲氨蝶呤组、开玄解毒中剂量组和高剂量组,除正常组外,其余组采用外涂咪喹莫特法建立银屑病样皮损模型,连续干预7 d后取材。期间对皮损拍照并计算银屑病面积和严重程度指数(psoriasis area and severity index,PASI)评分;HE染色观察皮损病理改变及测量表皮厚度;对脾脏及胸腺拍照并称重;免疫组化法检测表皮Ki67、S1P表达;蛋白质印迹法检测皮损KRT17、Claudin、Occludin表达;qRT-PCR检测IL-17、IL-23、S1PRs mRNA表达水平。结果:与模型组比较,开玄解毒方可缓解咪喹莫特诱导的小鼠银屑病样皮损,降低PASI评分,减轻表皮增生程度(P均<0.01),改善胸腺、脾脏指数及血管增生情况(P<0.01或P<0.05),降低炎症因子IL-17、IL-23 mRNA表达(P<0.01),修复皮肤屏障(P<0.05)。同时,开玄解毒方可下调表皮中S1P及S1PRs mRNA表达(P<0.05)。结论:开玄解毒方能够减轻银屑病局部皮损症状,其药效机制可能与调节S1P水平有关。
文摘Sphingolipids are ubiquitous components of cell membranes. Their metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. S1P is generated by phosphorylation of sphingosine catalyzed by sphingosine kinase-1 (SPHK1). The purpose of this study is to explore the roles of S1P, S1P receptors, and sphingosine kinases in malignant musculoskeletal tumors. Twenty-one tumor samples (7 liposarcomas, 3 chondrosarcomas, 6 osteosarcomas, 5 MFH) obtained at open biopsy, and four human MFH cell lines (Nara H, Nara F, TNMY1, GBS-1) were used. We examined the mRNA expression of S1P receptors by RT-PCR, and the expression levels of SPHK by Real-time PCR. We used 4 MFH cell lines to analyze SPHK1 proteins by Western blotting. SPHK1 siRNA was transfected into MFH cell lines by lipofection method. Cell proliferation (control and transfected with siRNA) was assayed using WST-8 (Cell Counting Kit-8) assay. All high grade malignant tumors expressed S1P1, S1P2, S1P3 receptors, whereas the expression of S1P1 receptor was detected in 50% of low-grade malignant tumors, S1P2 receptor in 30%, and S1P3 in 50%. No statistically significant difference was found in the expression level of SPHK1 between high-grade and low-grade malignant tumors by Real-time PCR. By results of Western blotting, proteins of SPHK1 were expressed in all MFH cell lines. In MFH cell lines, transfection with SPHK1 siRNA oligonucleotides resulted in approximately 50 to 80% suppression of SPHK1 mRNA expression as determined by real-time PCR. Down-regulation of SPHK1 with small interfering RNA significantly reduced SPHK1 protein levels by Western blotting. Knock down of SPHK1 expression significantly decreased cell proliferation of all MFH cells. These results suggest that the expression of S1P receptors may play an important role for cell proliferation and may correlate with histologic grade in malignant bone and soft tissue tumors, and that SPHK1 may be one of essential mo
