A new method was developed for simultaneous detection of total 19 kinds of organophosphate esters(OPEs)and their diester metabolites(di-OPEs)in human serum(1.0 mL)and urine(1.5 mL)with low volume of samples.The target compounds were determined using ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)after acetonitrile liquid-liquid extraction combined with purification using an ENVI-18 solid-phase extraction(SPE)column.OPEs and di-OPEs were separated using a Shim-pack GIST C18 column(100 mm×2.1 mm,2 μm)with a Shim-pack GIST-HP(G)C18 guard column.An electrospray ionization source(ESI)was employed in mass spectrometry analysis,with positive/negative ion mode using the multiple reaction monitoring(MRM).All target compounds were separated within 15 min,and exhibited good linear relationships in the concentration range of 2-100 ng/mL,with correlation coefficients(R2)above 0.994.The method detection limits(MDL)in serum ranged from 0.001 to 0.178 ng/mL and the MDL in urine ranged from 0.001 to 0.119 ng/mL.The recoveries of the analytes spiked in serum and urine matrices at two concentration levels were 30.5%-126.8%,with the relative standard deviations(RSDs)ranged from 1%to 23%.In addition,paired serum and urine samples from 11 patients were analyzed.For all samples tested,the internal standards of OPEs exhibited recoveries between 61%and 114%,whereas the internal standards for di-OPEs had recoveries ranging from 43%to 103%.OPEs and di-OPEs exhibited high detection frequencies in 22 serum and urine samples.Triethyl phosphate(TEP),tributyl phosphate(TBP),tris(2-ethylhexyl)phosphate(TEHP),tris(2-butoxyethyl)phosphate(TBEP),tris(1-chloro-2-propyl)phosphate(TCIPP),triphenyl phosphate(TPHP),tri-m-tolyl-phosphate(TMTP)and 2-ethylhexyl diphenyl phosphate(EHDPP)were universally detected in all serum samples.TCIPP was identified at the highest concentrations(median 0.548 ng/mL)in serum samples.In urine samples,the detection frequency for 12 kinds of target compounds reached 100%.Notably,TBP emerged as the predominant OPE in urine,demonstrating a median concentration of 0.506 ng/mL.Regarding di-OPEs,bis(2-chloroethyl)phosphate(BCEP)and bis(2-butoxyethyl)hydrogen phosphate(BBOEP)were the most abundant in urine,with median concentrations of 6.404 and 2.136 ng/mL,respectively.The total concentrations of OPEs and di-OPEs in serum and urine were 1.580-3.843 ng/mL and 5.149-17.537 ng/mL,respectively.These results not only confirmed the effectiveness of the method in detection of OPEs and di-OPEs in biological matrices,but also revealed the widespread presence of OPE compounds in human body and pointed to potential exposure risks.

OBJECTIVES: To study the factors influencing the short-term (28 days) efficacy of initial adrenocorticotropic hormone (ACTH) therapy for infantile epileptic spasms syndrome (IESS), as well as the factors influencing recurrence and prognosis. METHODS: The clinical data were collected from the children with IESS who received ACTH therapy for the first time in the Department of Pediatric Neurology, Xiangya Hospital of Central South University, from April 2008 to January 2018 and were followed up for ≥2 years. The multivariate logistic regression analysis was used to evaluate the factors influencing the short-term efficacy of ACTH therapy, recurrence, and long-term prognosis. RESULTS: ACTH therapy achieved a control rate of seizures of 55.5% (111/200) on day 28 of treatment. Of the 111 children, 75 (67.6%) had no recurrence of seizures within 12 months of follow-up. The possibility of seizure control on day 28 of ACTH therapy in the children without focal seizures was 2.463 times that in those with focal seizures (P<0.05). The possibility of seizure control on day 28 of ACTH therapy in the children without hypsarrhythmia on electroencephalography on day 14 of ACTH therapy was 2.415 times that in those with hypsarrhythmia (P<0.05). The possibility of recurrence within 12 months after treatment was increased by 11.8% for every 1-month increase in the course of the disease (P<0.05). The possibility of moderate or severe developmental retardation or death in the children without seizure control after 28 days of ACTH therapy was 8.314 times that in those with seizure control (P<0.05). The possibility of moderate or severe developmental retardation or death in the children with structural etiology was 14.448 times that in those with unknown etiology (P<0.05). CONCLUSIONS Presence or absence of focal seizures and whether hypsarrhythmia disappears after 14 days of treatment can be used as predictors for the short-term efficacy of ACTH therapy, while the course of disease before treatment can be used as the predictor for recurrence after seizure control by ACTH therapy. The prognosis of IESS children is associated with etiology, and early control of seizures after ACTH therapy can improve long-term prognosis.
Child ; Humans ; Infant ; Adrenocorticotropic Hormone/therapeutic use* ; Spasms, Infantile/drug therapy* ; Treatment Outcome ; Seizures ; Electroencephalography/adverse effects* ; Spasm/drug therapy*

With the progress of globalization, the public health emergencies represented by major infectious diseases have become a major challenge for the public health management in China. The article briefly describes the emergency response capability assessment tools in China, and introduces two emergency response assessment tools with complete content structure and wide application in the world. Then the advantages and disadvantages of the tools are compared and discussed in order to provide reference for improvement of the assessment tools for public health emergency response capability in China.
China ; Disaster Planning ; Humans ; Public Health ; Public Health Administration

OBJECTIVE: To characterize the molecular mechanism underlying the antineoplastic activity of Celastrus orbiculatus Thunb. extracts (COE). METHODS: The human hepatocellular carcinoma HepG2 cells with mammalian target of rapamycin (mTOR) knockdown expressed (HepG2/mTOR) were constructed using molecular biological technology. In vitro, the HepG2/mTOR cells were treated with COE at various concentrations (10, 20, 40, 80, 160 and 320 µ g/mL). Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. According to the half-maximal inhibitory concentration (IC) value (140 mg/L), the concentrations of COE in the subsequent experiment was set to alleviate cytotoxicity. The HepG2/mTOR cells were divided into 5 groups: negative control (untreated), COE treatment groups (40, 80, 120 mg/L COE) and positive control group (cisplatin, DDP, 2 mg/L), respectively. Wild-type HepG2 cells were used as a blank control. The effects of COE on the cell apoptosis were analyzed by flow cytometry and transmission electronic microscopy (TEM), respectively. The protein expression levels of mTOR signal pathways were determined by Western blotting. In vivo, HepG2/mTOR cells (2 × 10 cell/mice) were subcutaneously injected into the right flank of nude mice. Thirty-six female nude mice were randomly assigned to 6 groups according to body weight (6 mice per group) as follows: solvent vehicle control, Banmao Capsule treated group (BM, 195 mg/kg), Tegafur, Gimeracil and Oteracil Potassium Capsules (10 mg/kg) treated group, and different dosages of COE (10, 20, 40 mg/kg) groups. Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues. RESULTS: COE inhibited the proliferation significantly in a concentration-dependent manner in HepG2/mTOR cells (P<0.01). COE significantly induced the apoptosis of HepG2/mTOR cells (P<0.01), and the apoptotic bodies can be observed under TEM. COE significantly inhibits the proteins expression of mTOR-related signal pathways. In vivo, COE significantly inhibited tumor growth in nude mice (P<0.01). Moreover, the results showed that COE down-regulated the expression of Bcl-2 and Bcl-xL, and up-regulated the levels of Bax and caspase-3 protein (P<0.01). CONCLUSION COE was a potential chemotherapeutic drug in HCC treatments via targeting mTOR signal pathway.

OBJECTIVETraditional Chinese medicine (TCM) is regarded as an important treatment for gastric cancer patients, especially for those in advanced stage. To evaluate the effects of TCM treatment on gastric cancer patients, the authors performed a retrospective study to report the result of the integrated treatment of TCM with chemotherapy for stage IV non-surgical gastric cancer.

METHODSIn this study, 182 patients with stage IV and non-surgical gastric cancer were retrospectively analyzed to evaluate the effects of TCM integrated with chemotherapy. Among the 182 cases, 88 cases received integrated therapy consisting of TCM and chemotherapy, while 94 cases received chemotherapy alone. The overall survival and Karnofsky performance status (KPS) score were measured as the main outcome.

RESULTSThe median overall survival of the integrated therapy group and chemotherapy group were 16.9 and 10.5 months, respectively. The 1-, 3- and 5-year survival rates of integrated therapy group vs. chemotherapy group were 70% vs. 32%, 18% vs. 4%, and 11% vs. 0%, respectively. There was a significant difference between the two groups (χ= 42.244, P > 0.001). After six-month treatment, KPS scores of the integrated therapy group and the chemotherapy group were 75.00 ± 14.78 and 60.64 ± 21.39, respectively (P > 0.001). The Cox regression analysis showed that TCM treatment is a protective factor for patients' overall survival.

CONCLUSIONThis study demonstrated that TCM integrated with chemotherapy may prolong overall survival and improve survival rate and life quality of patients with stage IV non-surgical gastric cancer.

Bentysrepinine (Y101), a derivative of phenylalanine dipeptide, is a novel drug candidate for the treatment of hepatitis B virus (HBV) infection. Our previous preclinical pharmacokinetic study showed that its in vivo absorption and distribution characteristics were probably related to transmembrane transport after Y101 was administered intragastically in rats. In this study, Caco-2 and MDCK-MDR1 cell models were used to investigate interactions between Y101 and P-gp through the apparent permeation coefficient (P_app) and efflux ratio (R_E); the results showed that Y101 was a substrate of P-gp. In addition, gene-transfected cell models, HEK293-hOATP1B1, HEK293-hOATP2B1 and CHO-PEPT1 were used to evaluate the affinity to OATP1B1,

Bentysrepinine (Y101), a derivative of phenyalanine dipeptide, has a novel mechanism in the treatment of hepatitis B virus (HBV) infection with a good anti-HBV effect. In the present study, a fluorometric-based high throughput method using cytochrome P450 (CYP) screening kit was adopted to evaluate in vitro inhibition potential of Y101 on CYP isoenzymes by calculating remaining enzyme activities and inhibitory potential (IC₅₀ values) using the determined values of fluorescence intensity. The result showed that Y101 exhibited little activity in the inhibition of CYP1A2, CYP3A4, CYP2C9, CYP2C19 and CYP2D6 (IC₅₀ > 100 μmol·L^-1). Y101 was used to treat human primary hepotocytes for 72 h, and the enzyme activities of CYP1A2, CYP2B6 and CYP3A4 were determined with a cocktail of probe substrates for the three CYP isoforms. The metabolites were simultaneously determined using a LC-MS/MS method. Y101 had no activity in the induction of CYP1A2, CYP2B6 and CYP3A4 on the basis of the following results:① The ratio of enzyme activities between test and control groups were all below than 1 (varied from 0.662 to 0.928); ② The induction potential of Y101 were lower than forty percent compared with that of positive groups. The above results suggest that Y101 has little activity in the regulation of metabolic drug-drug interactions based on the CYP isoform changes following co-administration of drugs.

OBJECTIVETo explore the effect of Modified Sijunzi Decoction (MSD) on the bone metabolism of prednisone intervened adriamycin-induced nephropathy rats.

METHODSThe adriamycin-induced nephropathy rat model was prepared. Totally 50 SD rats were randomly divide into five groups, i.e., the model group, the hormone group, the Chinese medicine (CM) group, the CM + hormone group, and the normal control group. The 24-h urine samples were collected on the 7th, 21st, and 35th day after modeling. The 24-h urine protein was measured by biuret colorimetry. Serum levels of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL), osteocalcin (BGP), and tartrate-resistant acid phosphatase (TRACP) were determined by ELISA. Expressions of OPG and RANKL in the tibia tissue were detected using real-time quantitative PCR and Western blot.

RESULTS(1) Compared with the normal control group, the 24-h urine protein increased in each group on the 7th, 21st, and 35th day (P < 0.05, P < 0.01). Compared with the model group, the 24-h urinary protein decreased in the hormone group and the CM + hormone group (P < 0.05, P < 0.01). The decrement was more obvious along with the treatment time went by (P < 0.05, P < 0.01). There was statistical difference in the reduction of urine protein on the 35th day between the CM group and the model group (P < 0.05). (2) Compared with the 21st-day of the same group, the serum levels of TRACP and RANKL increased (P < 0.05, P < 0.01). Compared with the model group, the serum levels of the TRACP and RANKL increased (P < 0.05, P < 0.01), OPG and BGP decreased (P < 0.05, P < 0.01) in the hormone group. Compared with the CM group at the same period, serum OPG level decreased and the RANKL level increased in the hormone group and the CM + hormone group (P < 0.05, P < 0.01). Besides, the serum level of TRACP increased and BGP decreased (P < 0.05, P < 0.01). Compared with the hormone group at the same period, OPG and BGP increased (P < 0.05, P < 0.01), RANKL decreased (P < 0.01) in the CM + hormone group. On the 35th day TRACP decreased (P < 0.01). (3) Compared with the normal group, mRNA expressions of OPG and RANKL on the 21st day increased (P < 0.05, P < 0.01), mRNA expressions of OPG and RANKL on the 35th day decreased in the model group (P < 0.01). Compared with the CM group at the same period, OPG mRNA expression decreased (P < 0.01) and RANKL mRNA expression increased in the hormone group (P < 0.05). OPG mRNA expression decreased in the CM +hormone group (P < 0.05). (4) Compared with the hormone group on the 21st day, the OPG level decreased and the RANKL protein increased (both P < 0.05). RANKL decreased in the CM + hormone group (P < 0.05). Compared with the model group at the same period, OPG decreased and RANKL increased in the hormone group (P < 0.01). Compared with the CM group at the same period, OPG decreased (P < 0.01), RANKL increased (P < 0.01) in the hormone group and the CM + hormone group. Compared with the hormone group at the same period, OPG increased and RANKL decreased in the CM + hormone group (both P < 0.01).

CONCLUSIONSPrednisone could induce osteoporosis through the OPG/RANKL/RANK pathway. MSZ could slow down the formation of prednisone-induced osteoporosis through promoting osteoblast differentiation, and inhibiting osteoclastogenesis.

Acid Phosphatase ; metabolism ; Animals ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Isoenzymes ; metabolism ; Male ; Nephrosis ; chemically induced ; metabolism ; Osteocalcin ; metabolism ; Osteoprotegerin ; metabolism ; Prednisone ; pharmacology ; RANK Ligand ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tartrate-Resistant Acid Phosphatase ; Tibia ; metabolism

Animals ; Decompression ; Decompression Sickness ; physiopathology ; Electrocardiography ; Electroencephalography ; Electromyography ; Electrophysiology ; Male ; Rabbits

OBJECTIVETo prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes.

METHODSShort hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2E1-silenced hepatocytes were selected; real-time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1-silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE (0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L). The cell viability and half maximal inhibitory concentration (IC50) of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR.

RESULTSThe IC50s of TCE for L02 hepatocytes and CYP2E1-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2E1-silenced hepatocytes hada significantly lower apoptotic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE (P < 0.05 or P < 0.01). The mRNA expression level of bcl-2 (anti-apoptosis gene) in CYP2E1-silenced hepatocytes was 15% ∼ 60% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of caspase-3 and caspase-9 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30% ∼ 60% lower than those in L02 hepatocytes (P < 0.01). The mRNA expression level of p53 (cancer suppressor gene) in CYP2E1-silenced hepatocytes was 81 - 278% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of c-fos and k-ras (oncogenes) in CYP2E1-silenced hepatocytes were 20-68% lower than those in L02 hepatocytes (P < 0.01).

CONCLUSIONCYP2E1-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology. Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes, suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.

Apoptosis ; drug effects ; genetics ; Cell Line ; Cell Survival ; drug effects ; genetics ; Cytochrome P-450 CYP2E1 ; genetics ; metabolism ; Genetic Vectors ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lentivirus ; genetics ; RNA Interference ; Trichloroethylene ; toxicity