1.Click chemistry-based enrichment strategy for tracing cellular fatty acid metabolism by LC-MS/MS
Ru-Jie YANG ; Jian ZOU ; Jia-Yue LIU ; Jiang-Kun DAI ; Jian-Bo WAN
Journal of Pharmaceutical Analysis 2023;13(10):1221-1231
Fatty acids(FAs),which were initially recognized as energy sources and essential building blocks of biomembranes,serve as the precursors of important signaling molecules.Tracing FA metabolism is essential to understanding the biochemical activity and role of FAs in physiological and pathological events.Inspired by the advances in click chemistry for protein enrichment,we herein established a click chemistry-based enrichment(CCBE)strategy for tracing the cellular metabolism of eicosapentaenoic acid(EPA,20:5 n-3)in neural cells.Terminal alkyne-labeled EPA(EPAA)used as a surrogate was incubated with N2a,mouse neuroblastoma cells,and alkyne-labeled metabolites(ALMs)were selectively captured by an azide-modified resin via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction for enrichment.After removing unlabeled metabolites,ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatography mass spectrometry(LC-MS)analysis.The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from the complicated matrices.In addition,this method can overcome current detection limits by enhancing MS sensitivity of targets,improving the chromatographic separation of sn-position glycerophospholipid regioisomers,facilitating structural characterization of ALMs by a specific MS/MS fragmentation signature,and providing versatile fluorescence detection of ALMs for cellular distribution.This CCBE strategy might be expanded to trace the metabolism of other FAs,small molecules,or drugs.
2.Expression of STEAP1 in bladder transitional cell carcinoma and its potential diagnostic value
Macao WAN ; Jie BAI ; Xianlong ZHAO ; Bin CHANG ; Jianping WANG ; Lichang YANG
International Journal of Laboratory Medicine 2024;45(3):272-276
Objective To investigate the expression and potential diagnostic value of six-transmembrane epithelial antigen of prostate 1(STEAP1)in bladder transitional cell carcinoma.Methods 52 patients with transitional cell carcinoma of the bladder who underwent surgical treatment at the 940th Hospital of Joint Lo-gistics Support Force of the Chinese People's Liberation Army from June 2021 to December 2022 were select-ed as the observation group.In addition,52 patients with benign tumors of the bladder who matched basic clin-ical data such as age,gender,and disease incidence were selected as the control group.The relative expression levels of STEAP1 and STEAP1 mRNA in bladder tumor tissues of patients in the two groups were deter-mined by enzyme-linked immunosorbent assay and real-time fluorescence quantitative PCR,and the relative expression levels of STEAP1 and STEAP1 mRNA in bladder tumor tissues of patients with different patho-logical parameters were compared.Spearman correlation analysis and binary Logistic regression analysis were used to screen the risk factors for the occurrence and clinical stage of bladder transitional cell carcinoma.Re-ceiver operating characteristic(ROC)curve and area under the curve(AUC)were used to evaluate the diagnos-tic and predictive value of each indicator for bladder transitional cell carcinoma.Results The relative expres-sion levels of STEAP1 and STEAP1 mRNA in bladder tumor tissues in observation group were significantly higher than those in control group,with statistical significance(P<0.05).The relative expression levels of STEAP1 and STEAP1 mRNA in bladder tumor tissues of patients with middle and advanced bladder transi-tional cell carcinoma were significantly higher than those of patients with early bladder transitional cell carci-noma,with statistical significance(P<0.05).Binary Logistic regression analysis showed that the relative ex-pression level of STEAP1 and STEAP1 mRNA in bladder tumor tissues of patients were independent risk fac-tors for the development of bladder transitional cell carcinoma and middle and advanced bladder transitional cell carcinoma(P<0.05).ROC curve analysis showed that the AUC of STEAP1 and STEAP1 mRNA inde-pendently predicting the occurrence of bladder transitional cell carcinoma was 0.841(95%CI:0.760-0.922,P<0.001)and 0.936(95%CI:0.893-0.980,P<0.001),respectively,both of which had high predictive ef-ficacy.Conclusion The relative expression levels of STEAP1 and STEAP1 mRNA in bladder tumor tissues of patients are positively correlated with the occurrence of bladder transitional cell carcinoma and the middle and advanced bladder transitional cell carcinoma,suggesting that STEAP1 can be used as a potential marker for di-agnosis and prediction of the occurrence and development of bladder transitional cell carcinoma.
3.Pro-angiogenic activity of notoginsenoside R1 in human umbilical vein endothelial cells in vitro and in a chemical-induced blood vessel loss model of zebrafish in vivo.
Bin-Rui YANG ; Si-Jia HONG ; Simon Ming-Yuen LEE ; Wei-Hong CONG ; Jian-Bo WAN ; Zhe-Rui ZHANG ; Qing-Wen ZHANG ; Yi ZHANG ; Yi-Tao WANG ; Zhi-Xiu LIN
Chinese journal of integrative medicine 2016;22(6):420-429
OBJECTIVEThis study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish.
METHODSThe in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels.
RESULTSR1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs.
CONCLUSIONR1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.
Animals ; Blood Vessels ; pathology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Collagen ; pharmacology ; Disease Models, Animal ; Drug Combinations ; Ginsenosides ; chemistry ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; physiology ; Humans ; Laminin ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Proteoglycans ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Zebrafish
4.Panax Notoginseng Flower Extracted by Methanol on Platelet Function in Healthy Human in Vitro
Nan QIN ; Qing LI ; Xiao ZUO ; Pei-fu ZOU ; Li-juan MA ; Jian-bo WAN ; Yan YANG ; Hong-yun JIA
Journal of Sun Yat-sen University(Medical Sciences) 2019;40(2):204-210
【Objective】To evaluate the effects of methanol extract of Panax Notoginseng flower(PNFM)on platelet function in healthy human.【Methods】Platelet rich plasma were separated from venous blood of healthy volunteers and incubated with different concentrations(0,100,300 and 500 μg/mL)of PNFM for 20 min. After using ADP as agonist, granule-secretion were tested by CD62P expression and ATP release;integrin-αIIbβ3 activation was examined by PAC-1; Test platelet aggregation by turbidimetry ;Immunofluorescence examine platelet spreading on fibrinogen ;Changes in cytoplasmic calcium was studied using Fluo 3-AM,calcium ionophore. 【Results】After using ADP as agonist ,PNFM significantly inhibited platelet aggregation,compared to the control group(72.00±6.08),the 500μg/mL group decreased to 35.67±3.78(P<0.01);Compared to the control group(30.05±6.48),PNFM reduced the CD62P expression on platelet surface,the 500 μg/mL group decreased to 2.66±0.90(P<0.001);PNFM inhibited the expression of PAC-1 as a marker of the integrin- αIIbβ3 comformation,compared to the control group(33.37 ± 8.12),the 500 μg/mL group decreased to 11.89±6.12(P<0.01);Compared to the control group(1.93±0.47),all dose groups attenuated platelet ATP release,the 500 μg/mL group decreased to 35.67±3.78(P<0.01);Results demonstrated that 500 μg/mL PNFM markedly decreases the surface area of the spreading platelets(89.57±17.34 to 25.12±3.52,P<0.001),and all doses were affected;The Ca2 + mobilization was also reduced by all PNFM doses,compared to the control group(183.87 ± 11.59),the 500 μg/mL group was decreased to 71.25±5.33(P<0.001).【Conclusions】PNFM attenuated platelet activation,spreading,and aggregation; Our results provided new ideas for prevention and treatment of cardiovascular and cerebrovascular diseases.
5.Hypogonadism and the quality of life in male patients with type-2 diabetes mellitus.
Lu-Yao ZHANG ; Wei HE ; Jian-Xin WAN ; Qi-Qi YIN ; Zhen CHENG ; Guan-Ming CHEN ; Wen JI ; Hai LI ; Yan-Bing LI ; Zhi-Hong LIAO
National Journal of Andrology 2016;22(12):1088-1094
ObjectiveTo compare the level of testosterone between type-2 diabetes mellitus (T2DM) patients and healthy controls and to investigate the status of hypogonadism and the influence of hypopgonadism on the quality of life.
METHODSWe collected serum total testosterone (TT), free testosterone (FT), sex hormone-binding globulin (SHBG), and other clinical data from 166 T2DM patients aged over 30 years and 186 age-matched healthy controls. We investigated the quality of life (QoL) of the two groups of subjects using the questionnaires of Androgen Deficiency in Aging Males (ADAM), Aging Male Symptoms (AMS), 36-Item Short-Form Health Survey (SF-36), and Special Quality of Life for Diabetes Mellitus (DSQL).
RESULTSThe level of calculated FT (cFT) was remarkably lower in the T2DM patients than in the healthy controls (P<0.05), but no statistically significant differences were observed between the two groups in the levels of TT, bio-available testosterone (Bio-T), and SHBG. The T2DM males with hypogonadism showed significant differences from those without in age, height, systolic blood pressure, and creatinine (P<0.05). Based on the criteria of cFT <0.3 nmol/L and AMS score ≥27, the incidence rate of hypogonadism was 51.81% in the T2DM patients, 31.58% in the 30-39 yr group, 32.50% in the 40-49 yr group, 50% in the 50-59 yr group, 69.23% in the 60-69 yr group, and 77.27% in the ≥70 yr group, elevated by 77.4% with the increase of 10 years of age (OR = 1.774, P<0.001). The AMS score was significantly correlated with the scores of DSQL (r = 0.557, P<0.001) and SF-36 (r = -0.739, P<0.001) in the T2DM patients.
CONCLUSIONST2DM patients have lower levels of cFT than healthy men, accompanied with a higher incidence of hypogonadism. Age is a main risk factor of hypogonadism. Severer testosterone deficiency symptoms are associated with lower scores of QoL in T2DM males.
6.Toward Evidence-Based Chinese Medicine: Status Quo, Opportunities and Challenges.
Yao-Long CHEN ; Chen ZHAO ; Li ZHANG ; Bo LI ; Chuan-Hong WU ; Wei MU ; Jia-Ying WANG ; Ke-Hu YANG ; You-Ping LI ; Chiehfeng CHEN ; Yong-Yan WANG ; Chen WANG ; Zhao-Xiang BIAN ; Hong-Cai SHANG
Chinese journal of integrative medicine 2018;24(3):163-170
How to test the treatments of Chinese medicine (CM) and make them more widely accepted by practitioners of Western medicine and the international healthcare community is a major concern for practitioners and researchers of CM. For centuries, various approaches have been used to identify and measure the efficacy and safety of CM. However, the high-quality evidence related to CM that produced in China is still rare. Over the recent years, evidence-based medicine (EBM) has been increasingly applied to CM, strengthening its theoretical basis. This paper reviews the past and present state of CM, analyzes the status quo, challenges and opportunities of basic research, clinical trials, systematic reviews, clinical practice guidelines and clinical pathways and evidence-based education developed or conducted in China, pointing out how EBM can help to make CM more widely used and recognized worldwide.
Critical Pathways
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Evidence-Based Medicine
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Humans
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Medicine, Chinese Traditional
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Practice Guidelines as Topic
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Randomized Controlled Trials as Topic
7.A four-component combination derived from Huang-Qin Decoction significantly enhances anticancer activity of irinotecan.
Dou-Dou XU ; Xiao-Ying HOU ; Ou WANG ; Di WANG ; Dan-Ting LI ; Si-Yuan QIN ; Bo LV ; Xiao-Min DAI ; Zun-Jian ZHANG ; Jian-Bo WAN ; Feng-Guo XU
Chinese Journal of Natural Medicines (English Ed.) 2021;19(5):364-375
Huang-Qin Decoction (HQD) is a classic prescription for diarrhea in Chinese medicine treatment. Recent studies have demonstrated that HQD and its modified formulation PHY906 could ameliorate irinotecan (CPT-11) induced gastrointestinal (GI) toxicity and enhance its anticancer therapeutic efficacy. Nevertheless, which constituents in HQD are effective is still unclear so far. The study aims to screen out the key bioactive components combination from HQD that could enhance the anticancer effect of CPT-11. First, the potential bioactive constituents were obtained through system pharmacology strategy. Then the bioactivity of each constituent was investigated synthetically from the aspects of NCM460 cell migration, TNF-α release of THP-1-derived macrophage and MTT assay in HCT116 cell. The contribution of each constituent in HQD was evaluated using the bioactive index E
8.Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway.
Ai-Jun WU ; Nai-Qing CHEN ; Li-Hua HUANG ; Ran CHENG ; Xiao-Wan WANG ; Chuang LI ; Wei MAO ; Qing-Ming HUANG ; Peng XU ; Rui-Min TIAN
China Journal of Chinese Materia Medica 2023;48(8):2176-2183
To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.
Humans
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Ferroptosis
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Reactive Oxygen Species/metabolism*
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NF-E2-Related Factor 2/metabolism*
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Sincalide/pharmacology*
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Signal Transduction
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Epithelial Cells/metabolism*
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Glutathione