4.Retrospection,stratagem,and practice on innovative drug research and development of Chinese materia medica
Yifang YANG ; Bicheng YANG ; Lili JIN
Chinese Traditional and Herbal Drugs 1994;0(10):-
Taking the national key scientific and technological project in 11th Five-year and 10th Five-year Plan as its main clue,the innovative Chinese materia medica(CMM)research course has been reviewed in this paper.Based on the enlightenment from the first herbal medicine VeregenTM,which has been approved by FDA,together with the study and comprehension of many great achievements of new drug research,the strategy of innovative CMM research and development(R&D)and its practical route is going to be put forward in this article.With the innovative Compound Prescription of CMM research as the main innovative approach,innovation can be realized by the following three aspects:New substances,including new compound,new effective fraction/group or new components and their combinations;new indication of known compounds or extracts;the secondary development of high-quality Chinese patent medicine.Besides,in order to support the R & D of original drugs with Chinese characteristics,the international experiences in target-dependent screening and evaluation and optimization of leading compounds based on clinical application should be referenced,and the mechanism and targets of traditional Chinese medicine by genetic and molecular level be clarified.
5.Study on methods for detection of multidrug-resistance genes MDR1 C3435T and G2677T/A single nucleotide polymorphisms
Jing FU ; Yirong YANG ; Xiaodong PAN ; Jianjian ZHENG ; Bicheng CHEN
Chinese Journal of Clinical Laboratory Science 2006;0(06):-
0.05).The optimal combination was PCR-CTPP for MDR1 C3435T and PCR-SSP for G2677T/A.Conclusions PCR-CTPP and PCR-SSP are simple,accurate,rapid and economical methods for detection of SNP of MDR1 C3435T and G2677T/A,and can be applied in clinical research.
6.Influence of albumin on expression of NLRP3 inflammasome in renal tubular epithelial cells
Lihong DING ; Bicheng LIU ; Pingsheng CHEN ; Yuxiang GONG ; Minyu YANG
Chinese Journal of Nephrology 2015;31(10):760-765
Objective To investigate the effect of albumin on expression of NLRP3 inflammasome and its downstream cytokines IL-1β and IL-18 in tubular epithelial cells.Methods Thirty mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria were selected, and their renal biopsy samples were stained by PAS and Masson to observe tubular epithelial cells injury and inflammatory cells infiltration.NLRP3, caspase-1, IL-1β and IL-18, as well as different inflammatory cells, were detected by immunohistostaining.In vitro, Western blotting and real-time PCR were employed to detect NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA in HK-2 cells stimulated by bovine serum albumin (BSA) (20 g/L).Results In MsPGN patients with high levels of proteinuria, there were obvious renal tubular epithelial cell injury and inflammatory cells infiltration (all P < 0.05), and the expressions of NLRP3, caspase-1, IL-1β and IL-18 were up-regulated compared to patients with low levels of proteinuria (all P < 0.05).Furthermore, IL-1β and IL-18expressions were positively correlated with the degree of proteinuria (r=0.836, P < 0.05;r=0.901, P <0.05).NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA were significantly increased in HK-2cells stimulated by BSA compared to the control group (all P < 0.05).Conclusions Albumin is able to induce NLRP3 inflammasome activation in tubular epithelial cells, which may be the mechanism of tubulointerstitial injury and inflammation caused by proteinuria.
7.Effect of acute kidney injury on short-and long-term mortality of patients with acute myocardial infarction
Yanbei SUN ; Yuan TAO ; Bicheng LIU ; Min YANG
Chinese Journal of Nephrology 2016;32(11):813-820
Objective To evaluate the role of acute kidney injury (AKI) in predicting the early (30-day) and late (30-day to 5-year) mortality of acute myocardial infarction (AMI) patients during hospitalization.Methods A total of 1371 adult patients diagnosed with AMI in the First People's Hospital of Changzhou from January 2008 to December 2012 were analyzed retrospectively with collecting their relevant clinical data from the hospital's database.AKI was categorized according to the 2012 KDIGO AKI criteria.To compare between death group and non-death group in AMI patients during 30-day and 30-day to 5-year.Different AKI stages of patients were compared,and their all-cause mortality were analyzed by Kaplan-Meier.Using multivariate COX regression analysis with two models to assess the factors for AMI patients in 30-day to 5-year.Results The prevalence of AKI after AMI in death group was higher than that in non-death group (the 30-day prevalence was 72.7% vs 27.4%,P < 0.001;the 5-year prevalence was 36.3% vs 26.2%,P=0.013).In both early (30-day) and late (30-day to 5-year) follow up,the KDIGO grading distribution of AKI was different between death group and non-death group (P < 0.001 in 30-day follow up and P=0.002 in 30-day to 5-year follow up).Among the 1371 AMI patients,410 (29.9%) developed AKI during the hospital stay.The 30-day and 30-day to 5-year mortality rates were 5.6% (77/1371) and 11.3% (146/1294) respectively.All-cause mortality and cardiovascular mortality were significantly higher in patients with AKI-Ⅰ stage,AKI-Ⅱ stage and AKI-Ⅲ stage than those with non-AKI (all P < 0.001),especially in patients with AKI-Ⅲ stage.Further stroke history (HR=3.122,P=0.012),AKI severity (AKI-Ⅰ stage HR=3.034,P=0.028;AKI-Ⅱ stage HR=7.832,P<0.001;AKI-Ⅲ stage HR=9.919,P<0.001),and β-blocker therapy (HR=0.591,P=0.040) were independent predictors of 30-day mortality,while aging (HR=1.061,P < 0.001),albumin (HR=0.943,P=0.023),AKI-Ⅲ stage (HR=3.944,P=0.007),β-blocker therapy (HR=0.660,P=0.041) and percutaneous coronary intervention (HR=0.256,P < 0.001) were independent predictors of 30-day to 5-year mortality.Both at early (30-day) and late (30-day to 5-year) follow-up,AKI with or without baseline renal dysfunction were independent predictors of death in patients with AMI (all P < 0.05).Conclusions AKI strongly correlated with short-and long-term allcause mortality of AMI patients,regardless of the baseline renal impairment.Specifically,the more severe AKI,the higher short-term mortality AMI patients have.
8.The application of universal stem loop primer for microRNA scanning and quantification
Bicheng CHEN ; Silu WANG ; Yongheng BAI ; Yunxiu YANG ; Yong CAI ; Peng XIA ; Shaoling ZHENG ; Yirong YANG
Chinese Journal of Laboratory Medicine 2011;34(10):926-930
Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ~ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV < 2.5% ).Compared with the traditional stem loop primers,our method saved 75% cost of primers ( 1 917 bp vs 7 851 bp) and 60% test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.
9.Angiotensin Ⅱ induces lipid accumulation in human renal proximal tubular epithelial cells via the disruption of low density lipoprotein receptor pathway
Kunling MA ; Jie NI ; Changxian WANG ; Jing LIU ; Yang ZHANG ; Bicheng LIU
Chinese Journal of Nephrology 2013;(4):293-297
Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) stimulating on cholesterol influx in human renal proximal tubular epithelial cells (HK-2) and the relation to low-density lipoprotein receptor (LDLr) pathway.Methods HK-2 cells were cultured and divided into the control group (incubated with serum-free medium) and Ang Ⅱ group (treated by 10-7 mol/L of Ang Ⅱ for 24 hours).The effects of Ang Ⅱ on lipid accumulation were examined by Oil red O staining and a quantitative assay of intracellular cholesterol.The expression of LDLr,sterol regulatory elementbinding protein (SREBP) cleavage activating protein (SCAP) and SREBP-2 mRNA and protein were examined by real-time PCR and Western blotting.The cotranslocation of SCAP-SREBP-2 from endoplasmic retieulum to Golgi in HK-2 cells was examined by immunofluorescent staining under confocal microscopy.Results Ang Ⅱ treatment increased intracellular lipid accumulation in HK-2 cells,which was associated with increased mRNA and protein expression of LDLr,SCAP,and SREBP-2 in HK-2 cells induced by Ang Ⅱ.Furthermore,results from confocal microscopy observation demonstrated that Ang Ⅱ increased the translocation of SCAP/SREBP-2 complex from endoplasmic reticulum to Golgi,thereby up-regulating LDLr gene transcription.Conclusion Ang Ⅱ disrupts LDLr feed-back regulation to increase cholesterol uptake and induce intracellular lipid accumulation.
10.Effect of PM10 in Moxa Smoke on Apoptosis of Human Lung Adenocarcinoma Cell Line-A549 Cells
Yuhai HUANG ; Ping LIU ; Bicheng YANG ; Yingxue CUI ; Chang HUANG ; Juntian LIU ; Baixiao ZHAO
Chinese Journal of Information on Traditional Chinese Medicine 2014;(7):53-57
Objective To observe the effect of PM10 (inhalable particles) in moxa smoke on apoptosis of human lung adenocarcinoma cell line-A549 cells, and explore the possible mechanisms of inducing apoptosisMethods The A549 cells were studied in vitro experiment method, which were stained by Hoechest33258 staining method. Their morphological changes of apoptosis were observed by the fluorescence microscopy. The levels of intracellular Ca2+ and reactive oxygen species (ROS), and expression of NF-κB p65 were also measured.Results Some apoptotic cells were observed after treated with moxa smoke PM10 in the concerntration of 400μg/mL. After 4 hours intervention by moxa smoke PM10 in A549 cells, the intracellular Ca2+ level increased significantly (P<0.01). Compared with the blank control group, the expression of NF-κB p65 decreased significantly (P<0.05) after intervention of moxa smoke PM10 with different concerntrations for 4 hours. When the A549 cells were cultured with moxa smoke PM10 for 20 hours, the expression of p65 decreased significantly (P<0.05) in the concerntrations of 70, 280μg/mL, while there was no significant change in the concerntration of 140μg/mL (P>0.05). Compared with the blank control group, ROS level was significantly lower (P<0.05) in A549 cells after intervention of moxa smoke PM10.Conclusion PM10 in moxa smoke could induce apoptosis of A549 cells, could increase cytosolic Ca2+ level.