AIM:The study aims to investigate the effects of Huayu-Qutan formula(HYQT)-medicated serum on oleic acid-induced lipid damage and endoplasmic reticulum stress(ERS)in HepG2 cells,and to explore the mecha-nism underlying the prevention and treatment of lipid metabolism disorders by HYQT.METHODS:The HepG2 cells were treated with various concentrations of oleic acid,and CCK8 assay,oil red O staining and ELISA were used to identify the optimal treatment concentration and time of oleic acid and HYQT-medicated serum.Moreover,the cells were divided into control,model,thapsigargin(Tg),and Tg+HYQT groups.Cellular lipid deposition was measured using oil red O staining,while triglyceride(TG)and free fatty acid(FFA)levels were assessed by ELISA.Transmission electron micros-copy was used to examine endoplasmic reticulum structures,and RT-qPCR and Wes fully automated protein quantification analysis system were used to measure the mRNA and protein expression of glucose-regulated protein 78(GRP78),sterol regulatory element binding protein 1c(SREBP1c),acetyl coenzyme A carboxylase(ACC1),fatty acid synthase(FAS)and stearoyl coenzyme A desaturase 1(SCD1)in different groups.RESULTS:Significant lipid deposition was induced in HepG2 cells after treatment with 1 000 μmol/L oleic acid,while treatment with serum containing 10%HYQT for 48 h was found to be optimal.Compared with control group,the cells in model group showed significant deposition of oil red O-stained lipid droplets in the cytoplasm,associated with endoplasmic reticulum expansion,ERS,and nuclear condensa-tion.The TG and FFA levels,and the mRNA and protein expression levels of GRP78,SREBP1c,ACC1,FAS and SCD1 were increased significantly(P<0.01).Compared with model group,the cells treated with HYQT-mediated serum showed marked decrease in the number of lipid droplets in the cytoplasm,with restored endoplasmic reticulum morphology.The TG and FFA levels were significantly reduced(P<0.01),and the mRNA and protein levels of factors related to ERS and de novo fatty acid synthesis were markedly decreased(P<0.01).Treatment with Tg,an ERS agonist,led to greater accu-mulation of cytoplasmic lipid droplets and increased endoplasmic reticulum expansion.Marked variations in the morpholo-gy and size of the endoplasmic reticulum were observed,with fusion and clustering of vacuoles.The TG and FFA levels,and the expression of ERS-and fatty acid synthesis-related factors were increased(P<0.01).Compared with Tg group,the cells treated with Tg+HYQT showed reduced number of cytoplasmic lipid droplets,attenuated endoplasmic reticulum dilation,and decreased number and volume of vacuoles,while the TG and FFA levels and the expression of ERS-and fatty acid synthesis-associated factors were significantly decreased(P<0.01).CONCLUSION:Serum containing HYQT alle-viates oleic acid-induced lipid damage in HepG2 cells by inhibiting ERS-induced de novo synthesis of fatty acids.

AIM:The study aims to investigate the effects of Huayu-Qutan formula(HYQT)-medicated serum on oleic acid-induced lipid damage and endoplasmic reticulum stress(ERS)in HepG2 cells,and to explore the mecha-nism underlying the prevention and treatment of lipid metabolism disorders by HYQT.METHODS:The HepG2 cells were treated with various concentrations of oleic acid,and CCK8 assay,oil red O staining and ELISA were used to identify the optimal treatment concentration and time of oleic acid and HYQT-medicated serum.Moreover,the cells were divided into control,model,thapsigargin(Tg),and Tg+HYQT groups.Cellular lipid deposition was measured using oil red O staining,while triglyceride(TG)and free fatty acid(FFA)levels were assessed by ELISA.Transmission electron micros-copy was used to examine endoplasmic reticulum structures,and RT-qPCR and Wes fully automated protein quantification analysis system were used to measure the mRNA and protein expression of glucose-regulated protein 78(GRP78),sterol regulatory element binding protein 1c(SREBP1c),acetyl coenzyme A carboxylase(ACC1),fatty acid synthase(FAS)and stearoyl coenzyme A desaturase 1(SCD1)in different groups.RESULTS:Significant lipid deposition was induced in HepG2 cells after treatment with 1 000 μmol/L oleic acid,while treatment with serum containing 10%HYQT for 48 h was found to be optimal.Compared with control group,the cells in model group showed significant deposition of oil red O-stained lipid droplets in the cytoplasm,associated with endoplasmic reticulum expansion,ERS,and nuclear condensa-tion.The TG and FFA levels,and the mRNA and protein expression levels of GRP78,SREBP1c,ACC1,FAS and SCD1 were increased significantly(P<0.01).Compared with model group,the cells treated with HYQT-mediated serum showed marked decrease in the number of lipid droplets in the cytoplasm,with restored endoplasmic reticulum morphology.The TG and FFA levels were significantly reduced(P<0.01),and the mRNA and protein levels of factors related to ERS and de novo fatty acid synthesis were markedly decreased(P<0.01).Treatment with Tg,an ERS agonist,led to greater accu-mulation of cytoplasmic lipid droplets and increased endoplasmic reticulum expansion.Marked variations in the morpholo-gy and size of the endoplasmic reticulum were observed,with fusion and clustering of vacuoles.The TG and FFA levels,and the expression of ERS-and fatty acid synthesis-related factors were increased(P<0.01).Compared with Tg group,the cells treated with Tg+HYQT showed reduced number of cytoplasmic lipid droplets,attenuated endoplasmic reticulum dilation,and decreased number and volume of vacuoles,while the TG and FFA levels and the expression of ERS-and fatty acid synthesis-associated factors were significantly decreased(P<0.01).CONCLUSION:Serum containing HYQT alle-viates oleic acid-induced lipid damage in HepG2 cells by inhibiting ERS-induced de novo synthesis of fatty acids.

ObjectiveThe detection of RNA single nucleotide polymorphism (SNP) is of great importance due to their association with protein expression related to various diseases and drug responses. At present, splintR ligase-assisted methods are important approaches for RNA direct detection, but its specificity will be limited when the fidelity of ligases is not ideal. The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection. MethodsIn this study, a dual-competitive-padlock-probe (DCPLP) assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation. To verify the method, we employed dual competitive padlock probe-mediated rolling circle amplification (DCPLP-RCA) to genotype the CYP2C9 gene. ResultsThe specificity was well improved through the competition and strand displacement of dual padlock probe, with an 83.26% reduction in nonspecific signal. By detecting synthetic RNA samples, the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L. Furthermore, clinical samples were applied to the method to evaluate its performance, and the genotyping results were consistent with those obtained using the qPCR method. ConclusionThis study has successfully established a highly specific direct RNA SNP detection method, and provided a novel avenue for accurate identification of various types of RNAs.

To investigate the potential toxic effects and mechanisms of Tris(1; 3-dichloro-2-propyl) phosphate (TDCIPP) on thyroid in female SD rats. Thirty-two 3-weeks-old female SD rats were randomly divided into normal group(treated with corn oil ), and low/moderate/high-dose group treated with TDCIPP (dissolved in corn oil )(n=8). All rats were treated with corn oil or TDCIPP (50, 100, 250 mg/(kg·d)) once a day during a 21-day period. All rats were sacrificed after the last administration. Serum thyroid stimulating hormone (TSH), 3,3',5-triiodothyronine (T3), 3,3',5,5'-tetraiodothyronine (T4), free 3,3',5,5'-tetraiodothyronine (FT4) were detected with ELISA kit. Morphology of thyroid was observed with hematoxylin and eosin (HE) staining. Expressions of genes and proteins correlate with thyroid were measured respectively by real-time fluorescence quantitative PCR and Western blot. Compared with control group, morphology of thyroid showed follicles irregular arrangement, hypocolloid, and follicular hyperplasia in TDCIPP treatment groups. The levels of serum TSH in low-dose TDCIPP group and T3 in high-dose TDCIPP group were significantly higher than those in control group(P＜0.05). Thyroid stimulating hormone receptor (TSHR) mRNA expression was decreased distinctly in low-dose TDCIPP group, while the expression of thyroperoxidase (TPO) mRNA was increased notably in moderate and high-dose TDCIPP groups(P＜0.05,P＜0.01). Compared with control group, the level of TRβ protein was decreased significantly in moderate and high-dose TDCIPP groups, while the expressions of udp-glucuronosyl-transferases (UGTs) and cytochrome-p450-3A1 (CYP3A1) proteins were upregulated notably in TDCIPP treatment groups(P＜0.05). Treated with 50 mg/(kg·d) TDCIPP can cause thyroid hyperplasia, change the levels of thyroid hormones, and disturb thyroid function, therefore, it has toxic effects on the thyroid.

We assessed the role of diabetes mellitus (DM) on treatment effects in drug-susceptible initial pulmonary tuberculosis (PTB) patients. A prospective study was conducted in eight provinces of China from October 2008 to December 2010. We enrolled 1,313 confirmed drug-susceptible initial PTB patients, and all subjects received the treatment regimen (2H3R3E3Z3/4H3R3) as recommended by the national guidelines. Of the 1,313 PTB patients, 157 (11.9%) had DM; these patients had more sputum smear-positive rates at the end of the second month [adjusted odds ratios (aOR) 2.829, 95% confidence intervals (CI) 1.783-4.490], and higher treatment failure (aOR 2.120, 95% CI 1.565-3.477) and death rates (aOR 1.536, 95% CI 1.011-2.628). DM was a contributing factor for culture-positive rates at the end of the second month and treatment failure and death of PTB patients, thus playing an unfavorable role in treatment effects of PTB.
Antitubercular Agents ; therapeutic use ; China ; epidemiology ; Diabetes Mellitus ; epidemiology ; therapy ; Female ; Humans ; Male ; Mycobacterium tuberculosis ; drug effects ; Tuberculosis, Pulmonary ; complications ; drug therapy ; epidemiology ; microbiology

The objective of this prospective study of the risks of treatment failure in patients with drug-susceptible pulmonary tuberculosis (PTB) was to provide reference data to help develop a disease control strategy. Participants were recruited in eight provinces of China from October 2008 to December 2010. A total of 1447 patients with drug-susceptible PTB and older than 15 years of age were enrolled. Demographic characteristics, bacteriological test results, and patient outcome, i.e., cure or treatment failure were recorded and compared using the chi-square or Fisher's exact tests. Multivariate logistic regression was used to identify factors associated with risk of treatment failure. Of the 1447 patients who were enrolled, 1349 patients (93.2%) were successfully treated and 98 (6.8%) failed treatment. Failure was significantly associated with age 365 years [odds ratio (OR)=2.522, 95% confidence interval (CI): (1.097-5.801)], retreatment [OR=2.365, 95% CI: (1.276-4.381)], missed medicine [OR=1.836, 95% CI: (1.020-3.306)], treatment not observed [OR=1.879 95% CI: (1.105-3.195)], and positive culture result after the first [OR=1.971, 95% CI: (1.080-3.597)] and second month [OR=4.659, 95% CI: (2.590-8.382)]. The risk factors associated with treatment failure were age 365 years, retreatment, missed medication, treatment not observed, and positive culture at the end of month 1 or month 2. These risk factors should be monitored during treatment and interventions carried out to reduce or prevent treatment failure and optimize treatment success.
Adolescent ; Adult ; Aged ; Antitubercular Agents ; therapeutic use ; China ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; drug effects ; physiology ; Prospective Studies ; Retreatment ; Risk Factors ; Treatment Failure ; Tuberculosis, Multidrug-Resistant ; drug therapy ; epidemiology ; microbiology ; Tuberculosis, Pulmonary ; drug therapy ; epidemiology ; microbiology ; Young Adult

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A novel fibrinolytic enzyme was produced from Rhizopus chinensis 120, which was screened from the starter for brewing rice wine in the South of China, by solid fermentation, and purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the alpha-, beta- and gamma-chains of fibrinogen simultaneously, and it also activated plasminogen to plasmin. The enzyme hydrolyzed N-Succinyl-Ala-Ala- Pro-Phe-pNA, and Km was 0.23 mmol/L and Kcat 16.36 s(-1). The optimal temperature of the enzyme for hydrolying fibrin was 45 degrees C, and the optimal pH range of 6.8 - 8.8. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5 +/- 0.1. The enzyme was a glycoprotein. EDTA, PCMB, PMSF inhibited the activety of the enzyme, and SBTI, Lys, TPCK, Aprotinine had none obvious inhibition, which suggested that the activity centre of the enzyme had hydrosulfuryl, metal and serine. The first 12 amino acids of the N-termimal sequence of the enzyme were NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly, and had none homology with that of other fibrinolytic enzyme from other microbes. The novel fibrinolytic enzyme from Rhizopus chinensis 12# has potential to become a therapeutic agent in thrombosis.
Enzyme Stability ; Fermentation ; Fibrinolysin ; metabolism ; Fibrinolysis ; Fibrinolytic Agents ; chemistry ; Humans ; Plasminogen ; metabolism ; Rhizopus ; enzymology