OBJECTIVETo investigate the effect of midazolam on human ether-a-go-go (hERG) K+ channels exogenously expressed in human embryonic kidney cells (HEK-293) and the underlying molecular mechanisms.

METHODSWhole-cell patch clamp technique was used to record WT, Y652A and F656C hERG K+ current expressed in HEK-293 cells.

RESULTSMidazolam inhibited hERG K+ current in a concentration-dependent manner, the half-maximum block concentrations (IC50) values were (1.31 ± 0.32) µmol/L. The half-activation voltage (V1/2) were (2.32 ± 0.38) mV for the control and (-1.96 ± 0.83) mV for 1.0 µmol/L midazolam. The half-inactivation voltage (V1/2) was slightly shifted towards negative voltages from (-49.25 ± 0.69) mV in control to (-57.53 ± 0.53) mV after 1.0 µmol/L midazolam (P < 0.05). Mutations in drug-binding sites (Y652A or F656C) of the hERG channel significantly attenuated the hERG current blockade by midazolam.

CONCLUSIONMidazolam can block hERG K+ channel and cause the speed of inactivation faster. Mutations in the drug-binding sites (Y652 or F656) of the hERG channel were found to attenuate hERG current blockage by midazolam.

Dose-Response Relationship, Drug ; Ether-A-Go-Go Potassium Channels ; drug effects ; HEK293 Cells ; Humans ; Midazolam ; pharmacology ; Mutation ; Patch-Clamp Techniques ; Potassium Channel Blockers ; pharmacology

OBJECTIVE To observe the effects of glycogen synthase 3β (GSK-3β) in the regula-tion of NLRP3 inflammasome activation after acute myocardial infarction (MI) in Sprague Dawley(SD) rats. METHODS Ligation of the left anterior descending (LAD) in SD rats was used to establish an acute myocardial infarction model. SD rats were randomly divided into 3 groups (n=10, each group):sham group,MI group,and MI+SB group:the GSK-3β inhibitor(SB216763)was given 1 h by intrave-nous injection(0.6 mg·kg-1·d-1)before surgery.The serum and heart tissue were collected to measure lactate dehydrogenase (LDH) and IL-1β content and mRNA and protein levels of NLRP3, ASC, Cas-pase-1,IL-1β and GSK-3β after 2 days and 7 days operation,respectively.RESULTS The serum levels of LDH and IL-1β in the MI group were significantly higher than those in the sham group(P<0.01),and the MI+SB group was obviously lower than the MI group(P<0.01).In addition,mRNA and protein levels of NNLRP3, ASC, Caspase-1, IL-1β and GSK-3β expressions in MI group were clearly increased (P<0.01) compared with those in sham group.These indicators were significantly decreased in SB+MI group (P<0.01). Interestingly, the indicators were all higher at 7 days than 2 days. CONCLUSION GSK-3β inhibition induces cardioprotection via attenuating the activation of NLRP3 inflammasome after the acute myocardial infarction in rats.

Objective:The paper aims to provide recommendations for improving the essential medicines’ cen-tralized bidding procurement and quantity-based pricing policy. Methods: Based on the documents and literature on essential medicines’ centralized bidding procurement, we analyzed the factors which have a great impact on implemen-tation of the quantity-based pricing in essential medicines’ centralized procurement using the text research, semi-structured interview questionnaire and on-phone interviews. Results:The quantity-based pricing needs to define a ge-neric name and specific dosage form of drugs in the essential medicines’ centralized procurement. Its implementation was mainly influenced by the following factors:the procurement area accessibility, the pharmaceuticals category, dis-ease and drug alternative procurement methods and cycle, the payment and settlement time, and irregularities in the procurement process. Suggestions:During this implementation, we also need to clearly predict the quantity and pro-curement method, set up a proper policy environment for a quantity-based pricing, cancel the price linkage mecha-nism, strictly put into practice the payment deadline, employ a unique billing method and strengthen the information construction for the provincial centralized procurement platform. Some medicines’ quantity-based pricing should be carried out in the chosen pilots for laying a good foundation for its promotion.

Objective: To improve the economic and technical indicator’s evaluation system and the essential medicines’ centralized bidding procurement practice in China. Methods:By using the literature analysis, comparative analysis and field survey, we collected and analyzed the implementation plans and regulations for the essential medicines’ centralized bidding procurement in 30 provinces. Results: The quality level classification lacks in preci-sion. The economic and technical indicator’s concentration grade is low, the score and content in each indicator un-reasonably fluctuates in different provinces and these indicators are of low efficiency in bond with their structures for the drug quality evaluation. The quality level indicator lacks in the distinction degree and the government’s unreason-able interference exists in competition. Conclusions and suggestions: The quality levels’ indicator type and number should be simplified. The economic and technical indicators’ function, content, score, weight value and the structure should be normatively and scientifically set to improve the efficiency during the drug quality evaluation and the gov-ernment should strive to play their role in the market.

OBJECTIVE To explore the effects of perinatal inflammation on the expression of proin-flammatory cytokines and DMEs (drug metabolism enzymes) in offspring mice. METHODS C57BL/6 maternal mice were administrated with single dose 100 μg·kg-1LPS(lipopolysaccharide)or saline(vehicle) during gestation (day 10 after fertilization). Offspring mice were sacrificed at 30 d after birth and liver samples were collected.Real-time PCR was adopted to test the mRNA expression of proinflammatory cytokines (Nrlp3 and IL-1β), nuclear receptors (Pxr and Car), and DMEs (Cyp3a11, 2b10, 1a2, and Ugt1a1).RESULTS Gender different expression of candidate genes was observed.The expression of Car,in the maternal injection of LPS groups,was significantly decreased in both female and male offspring (n=3-8/group, P<0.01). Concomitantly, a significantly lower expression of Cyp3a11 was found in both female and male offspring (P<0.01, P<0.05, respectively). Furthermore, the expression of Ugt1a1 was reduced in male offspring following maternal administration of LPS (P<0.01). In male offspring, Nrlp3 expression was specially decreased(P<0.05).Interestingly,there was an approximately 66% reduction in mRNA level of Cyp1a2 in female offspring (P<0.01), while in male offspring Cyp1a2 expression showed an increased trend (P>0.05) compared with vehicle group. The expression of Pxr, Cyp2b10, and IL-1β was no difference between LPS treatment group and vehicle group(P>0.05).CONCLUSION Maternal LPS administration affects the expression of proinflammatory cytokines, nuclear receptors and DMEs in mouse offspring.

OBJECTIVE To demonstrate the long-or short-term impacts of neonatal Pxr(pregnane X receptor) agonists exposureon DMEs (drug metabolism enzymes) expression in adulthood. METHODS C57BL/6 mice(day 5,postnatal)were injected with different doses(0,50,100,150,200 mg·kg-1·d-1, constitutive 4 d)of PCN(pregnenolone-16a-carbonitrile).Mice at different ages(day 5,10,15,25,postna-tal)were administrated with 200 mg·kg-1·d-1PCN in constitutive 4 d.All mice were sacrificed at day 60 after birth. Liver samples were collected for detecting the expression of Pxr target genes. RESULTS Compared with vehicle group, the significant inductions of Cyp2b10, Cyp3a11 and Pxrwere observed in high dose groups (150, 200 mg·kg-1·d-1, 5-8 d after birth) both in male and female mice (n=4-9/group,P<0.05).Furthermore,high dose groups(200 mg·kg-1·d-1,5-8 d after birth)were found to have higher mRNA expression levels of Cyp2a4,Ugt1a1,Abcc4,and Oatpla4 in female mice,while Papss2 in male mice compared with vehicle groups (n= 4-9/group, P<0.05). Interestingly, a decreased mRNA expression of Sult2a1 was identified in 200 (5-8 d) groups (n=4-9/group, P<0.05). Consistent with these results, the protein expression of Cyp3a11 was only increased in 200 (5-8 d) groups compared with the vehicle groups(n=3/group,P<0.05).Importantly,the persistent impacts on DMEs only occurred in day 5 and day 25 treatment groups,not day 10 and day 15 groups(n=4/group).CONCLUSION Neonatal Pxr activation has a long-term effect on the expression of DMEs in C57BL/6 mice.Dose and treatment exposure time are two key factors involved in this permanent alteration procedure.

Objective To obtain the full length (FL ) and C‐terminal fragment of polymorphic membrane protein I (PmpI) of Chlamydia trachomatis serovar D ,and to study the immunogenicity of these proteins .Methods The target genes of PmpI‐FL and PmpI‐C were amplified by polymerase chain reaction (PCR) and inserted into the prokaryotic plasmid vector pGEX‐6P‐1 .The recombinant plasmids pGEX‐6P‐1/PmpI‐FL and pGEX‐6P‐1/PmpI‐C were separately transformed into Escherichia .coli ( E . coli) DH5αand were identified by enzyme digestion ,sequencing and PCR .After the identification ,the recombinant plasmids were separately transformed into E .coli BL21 and induced to express the proteins . The expected proteins were identified by Coomassie brilliant blue staining and Western blot ,then purified by glutathione S‐transferase (GST) MagBeads .The purified proteins were then injected into BALB/c mice to prepare the polyclonal antibodies against PmpI‐FL or PmpI‐C .Enzyme‐linked immune sorbent assay (ELISA) was used for the quantitative detection of the specific antibody .Results The lengths of cloned target genes PmpI‐FL and PmpI‐C were 2 659 bp and 1 195 bp ,respectively ,and the sequences were consistent with those of Chlamydia trachomatis serovar D in GenBank .The molecular masses of target proteins were 122 000 and 69 000 ,respectively ,which were confirmed by Coomassie brilliant blue staining and Western blot and then purified .The titers of the antibodies (anti‐PmpI‐FL and anti‐PmpI‐C) in sera of immunized mice detected by ELISA were 1∶12 800 and 1∶6 400 ,respectively .Conclusion The PmpI‐FL‐GST and PmpI‐C‐GST fusion proteins with high immunogenicity are successfully expressed and purified , which lays the foundation for further study .

AIMTo learn the electrophysiological interference of levofloxacin (LVFX) to heart in guinea pig.

METHODSHigh, moderate and low doses of LVFX were given to the anesthetic guinea pig via i.p., and QT interval span and corrected QT-interval span in the II leading lines of ECG were recorded and analyzed from 5 min to 360 min after the drug administration. Single ventricular myocytes were obtained and impacted by LVFX solution of different concentrations. Then delayed rectifier potassium currents (I(K)) on single cells were recorded with whole-cell patch clamp technique, and compared with control group(without impact of LVFX).

RESULTS(1) After the administration of LVFX, at the dose of 200 mg/kg. QT-interval span was significantly elongated, and the increasing rate is 19.38% +/- 3.15% (P < 0.05). While at the relatively lower doses of 50 mg/kg and 100 mg/kg, the elongation is of low/no significance (P > 0.05). (2) LVFX inhibited I(K) dose-dependently and time-dependently.

CONCLUSIONLVFX might prolong the QT-interval span by the mechanism of inhibiting I(K), which implies a potential risk in clinical application.

Animals ; Guinea Pigs ; Levofloxacin ; Membrane Potentials ; Myocytes, Cardiac ; drug effects ; physiology ; Ofloxacin ; pharmacology ; Patch-Clamp Techniques

Objective To discuss the personality characteristics and blood lipid levels in patients with infertility and depression,and further analyze the relationship between the degree of depression and the two aspects.Methods We selected 190 infertility patients and they were tested with Self-Rating Depression Scale(SDS) in Tangshan Maternal and Child Health Care Hospital Affiliated to North China University of Science and Technology from December,2015 to December,2016.All the patients were allocated into depression group (with depression symptom) and non-depression group (without depression symptom).Blood lipid levels,including triglycerides (TG),total cholesterol(TC),low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were determined and the Eysenck Personality Questionnaire(EPQ) was applied to evaluate the personality traits in both groups.Results The patients in the depression group were mainly mild-to-moderate depression accouting for 64.4％,and patients with moderate to severe depression accouted for approximately 30.0％.The levels of TG and TC of patients in the depression group were significantly higher than those in the non-depression group(P＜0.01).The scores of neuroticism(N),validity scale(L) and psychoticism(P) of patients in the depression group were significantly higher than those in the non-depression group,while there was no significant difference in exextraversion (E) between the two groups.TG,E dimension,P dimension and L dimension were effective predictors of depression in infertile patients,and the Beta coefficient were 0.199,-0.240,0.424 and 0.220.Pearson partial correlation analysis showed that TG was positively correlated with P,N and L dimensions.Conclusion Infertility patients with depression may have dyslipidemia and personality changes.The degree of depression is positively correlated to the level of TG and the score of P and N.

AIM: To determine the IPP origin of the naphthoquinones (NQs) in Rubia cordifolia, and to evaluate the effects of methyl jasmonate (MeJA) treatment, MEP, and MVA pathway inhibitor treatment on the accumulation of anthraquinones (AQs) and NQs in cell suspension cultures of R. cordifolia. METHODS: Cell suspension cultures of R. cordifolia were established. Specific inhibitors (lovastatin and clomazone) and MeJA were supplied to the media, respectively. Treated cells were sampled every three days. Content determination of purpurin (AQs) and mollugin (NQs) were carried out using RP-HPLC. The yield of the two compounds was compared with the DMSO-supplied group and the possible mechanism was discussed. RESULTS: Lovastatin treatment increased the yield of purpurin and mollugin significantly. Clomazone treatment resulted in a remarkable decrease of both compounds. In the MeJA-treated cells, the purpurin yield increased, meanwhile, the mollugin yield decreased compared with control. CONCLUSION The IPP origin of mollugin in R. cordifolia cell suspension cultures was likely from the MEP pathway. To explain the different effects of MeJA on AQs and NQs accumulation, studies on the regulation and expression of the genes, especially after prenylation of 1,4-dihydroxy-2-naphthoic acid should be conducted.
Acetates ; pharmacology ; Anthraquinones ; metabolism ; Cell Culture Techniques ; Cells, Cultured ; Cyclopentanes ; pharmacology ; Isoxazoles ; pharmacology ; Lovastatin ; pharmacology ; Oxazolidinones ; pharmacology ; Oxylipins ; pharmacology ; Pyrans ; metabolism ; Rubia ; drug effects ; metabolism