Anti-fibrosis potential of pirarubicin via inducing apoptotic and autophagic cell death in rabbit conjunctiva.

This study investigated the potential efficacy of pirarubicin (THP) in modulating rabbit conjunctival fibrosis both in vitro and in vivo and characterized the underlying mechanisms. Primary rabbit conjunctival fibroblasts (RCF) were cultured and treated with THP or mitomycin C (MMC) for 5 min, followed by assaying for cell viability, cell cycle distribution, apoptotic and autophagic pathways. The production of reactive oxygen species (ROS) and chemotaxis of macrophages by RCF were evaluated using 2',7'-dichlorofluorescein diacetate (DCFH-DA) labeling and transwell migration assay, respectively. Limbal stem cell excision in combination with alkali burn was performed on the rabbits to establish a model of limbal deficiency and conjunctival fibro-vascular invasion. After three months, the modeled fibro-vascular tissue was excised combined with topical subconjunctival 5-min exposure to THP compared with MMC intraoperatively. The recurrence of postoperative fibrosis and the expression of apoptosis, autophagy, and inflammation markers were evaluated by immunohistochemistry. All modeled rabbits developed conjunctival fibro-vascular lesions, which were similar to human recurrent pterygium (HRP). Both THP and MMC inhibited RCF proliferation and arrested cell cycle at the G0/G1 phase. In particular, 7.5 µmol/L THP remarkably promoted RCF autophagy by upregulating the levels of Beclin 1, Atg 5/12 conjugate, and LC3B, whereas, 15 µmol/L THP significantly triggered a cascade of mitochondrial-associated RCF apoptosis. THP induced the production of ROS and enhanced the chemoattraction of macrophages by RCF. Similar to 600 µmol/L MMC, both 7.5 µmol/L and 15 µmol/L THP attenuated postoperative conjunctival fibrosis in the models; 7.5 µmol/L THP preferentially enhanced autophagy while causing fewer side effects. THP exerted its antifibrotic action by modulating autophagy in RCF, inducing cell cycle arrest, and mitochondrial-mediated apoptosis. THP at the dose of 7.5 µmol/L prevented postoperative conjunctival fibrosis in an animal model.

Eyelid margin cleaning using Deep Cleaning Device for the treatment of meibomian gland dysfunction-associated dry eye: a preliminary investigation.

OBJECTIVE: To evaluate the safety and effectiveness of eyelid margin cleaning using Deep Cleaning Device for the treatment of meibomian gland dysfunction-associated dry eye. METHODS: This was a prospective, randomized, open-label, investigator-masked, and self-controlled study. We randomly assigned one eye of patients with meibomian gland dysfunction-associated dry eye to the treatment group, and the other eye to the control group. Both groups received artificial tears and lid warming; the treatment group received an additional one-time in-office eyelid margin cleaning using Deep Cleaning Device. Non-invasive tear break-up time (NITBUT) and tear meniscus height (TMH) of each eye, and Standard Patient Evaluation for Eye Dryness II (SPEED II) score of each patient were evaluated before and at one week after treatment. RESULTS: Thirty eyes of 15 patients were enrolled. No adverse effects occurred during the treatment. Compared with the baseline values, the SPEED score decreased significantly at one week after treatment (mean±95% confidence interval, 11.00±0.99 vs. 5.67±1.67, P<0.0001), the NITBUT-first in the treatment group increased significantly at one week after treatment ((4.74±1.27) s vs. (7.49±2.22) s, P=0.01). The NITBUT-first was significantly longer in the treatment group ((7.49±2.22) s) than in the control group ((5.17±0.91) s) at one week after treatment (P=0.042). No significant differences were found in other tear film parameters between the two groups. CONCLUSIONS: Eyelid margin cleaning using the novel Deep Cleaning Device is a convenient, effective, and safe treatment for patients with meibomian gland dysfunction-associated dry eye.

Determination of Risperidone and 9-hydroxyrisperidone in Human Plasma by LC-MS/MS / 中国药房

OBJECTIVE:To establish the method for simultaneous determination of risperidone (RIS) and 9-hydroxyrisperi-done (9-OH-RIS) in human plasma. METHODS:After liquid-liquid extraction of plasma sample,using AF2672 as internal stan-dard(IS),LC-MS/MS was adopted. The determination was performed on XtimateTM C18 column with mobile phase consisted of ace-tonitrile-10 mmol/L ammonium acetate solution(containing 0.1％ formic acid)(37:63,V/V,pH=3.2)at the flow rate of 0.25 mL/min. The column temperature was 40 ℃,and the sample size was 6 μL. The ESI was equipped and quantitative analysis was operated in positive ion and MRM mode. The mass transition ion-pairs were followed as m/z 411.26→191.02 for RIS,m/z 427.21→206.91 for 9-OH-RIS and m/z 418.00→175.95 for IS. RESULTS:The linear range of RIS was 0.2-50.0 ng/mL(r=0.9997)and 9-OH-RIS was 1.0-200.0 ng/mL (r=0.9987). RSDs of inter-day and intra-day were all lower than 15％,and the method recoveries were 92.42％-104.81％ and 94.51％-100.57％;matrix effects were 98.33％-107.09％ and 91.05％-105.80％;extraction recoveries were 78.11％-92.62％ and 76.32％-85.09％,respectively. The plasma concentrations of RIS and 9-OH-RIS in 78 schizophrenic patients were(13.58±8.31)and(25.62±15.52)ng/mL. CONCLUSIONS:The developed method is simple,sensitive and specific,which is suitable for routine drug monitoring and acute toxic analysis in patients receiving risperidone.

Comparison of Therapeutic Efficacy for Neonatal ABO Hemolytic Disease Treated with Intravenous Immunoglobin G by Different Modes of Administration / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To compare the therapeutic efficacy of patients with neonatal ABO hemolytic disease treated with introvenous immunoglobin G (IVIG) by different modes of administration.</p><p><b>METHODS</b>Ninety-three in patients with neonatal ABO hemolytic disease treated in our hospital were divided into group A (31 cases), B(31 cases) and C (31 cases). Based on basic treatment, the patients in group A were treated by a single high dose of IVIG (1 g/kg), patients in group B were treated by multiple low-dose of IVIG (0.5 g/kg), and the patients in group C treated by placebo without IVIG used as controls. The phototherapy time, jaundice time in 3 groups were observed; the total bilirubin levels in 3 groups were compared before and after treatment; the incidence of anemia, the rate of blood transfusion and the occurrence of bilirubin encephalopathy were compared after treatment between 3 groups.</p><p><b>RESULTS</b>The phototherapy time, jaundice time in group A were statistically significantly shorter than those in the group B and C (P<0.05), but there was not statistical significantly difference between group B and C(P>0.05). Before treatment, serum TBIL level in 3 groups was not significantly different (P>0.05); and after treatment for 24 h and 48 h, the serum TBIL levels in group A were significantly lower than that in group B and C (P<0.05); after treatment for 72 h, the serum TBIL level in group A was all lower than 34.2 µmol/L; before treatment, Hb levels in 3 groups were not significantly different (P>0.05); Hb level in group A was significantly higher than that in group B and C after treatment for 24 h, 48 h and 72 h (P<0.05). The incidence of anemia in group A after treatment was significantly lower than that in group B and C, and that in group B significantly lower than that in group C(P<0.05). The rate of blood transfusion in group A was significantly lower than that in the group B and C (P<0.05); the rate of blood transfusion was not statistically significantly different between group B and C(P>0.05).</p><p><b>CONCLUSION</b>The single high dose of IVIG infusion can effectively reduce the serum TBIL level, shorten treatment time and reduce the incidence of anemia and blood transfusion, so the therapeutic efficacy is significantly improved.</p>

The Correlation between Multidrug Resistance Gene Polymorphism and Plasma Concentration of Risperi-done and Its Metabolite / 中国药房

OBJECTIVE:To discuss the correlation between multidrug resistance gene (MDR1) polymorphisms and plasma concentration of risperidone,9-hydroxyrisperidone and total active substance in Han patients with schizophrenia. METHODS:78 Han inpatients with schizophrenia in Mental Health Institute of Second Xiangya Hospital of Central South University from Dec. 2011 to Jan. 2013 were selected,LC-MS/MS was conducted to determine the plasma concentration of risperidone,9-hydroxyrisperi-done and total active substance,PCR-LDR was adopted to determine the genotyping of C1236T,G2677T and C3435T of patients, and one-way ANOVA was used to detect the correlation between C1236T,G2677T and C3435T polymorphisms and plasma concen-tration/dose calibration ratio (C/D) value of risperidone,9-hydroxyrisperidone and total active substance. RESULTS:The average plasma concentrations of risperidone,9-hydroxyrisperidone and total active substance for 78 patients were(13.58±8.31),(25.62± 15.52)and(39.24±25.76)ng/ml,respectively;the distribution frequencies of C1236T,G2677T and C3435T met the Hardy-Wein-berg equilibrium(P>0.05);the risperidone C/D value of patients with C12367T CT and TT genotype were higher than those with CC genotype,risperidone and total active substance C/D values of patients with G2677T TT genotype were higher than those with GG genotype,the differences were statistically significant (P0.05). CONCLUSIONS:The MDR1 C1236T and G2677T polymorphisms are associated with plasma concentration of risperidone and total active substance in Han patients with schizophrenia.

Effect of CYP4F2 Gene Polymorphism on Warfarin Maintenance Dose in Chinese Population:a Systematic Review / 中国药房

OBJECTIVE:To systematic review the relationship between CYP4F2 gene polymorphism and warfarin maintenance dose in Chinese population,and to provide evidence-based reference for clinical treatment. METHODS:Retrieved from Cochrane library,PubMed,EMBase,CJFD,VIP and Wanfang database,studies about the relationship between CYP4F2 gene polymorphism and warfarin maintenance dose in Chinese population were collected,and Meta-analysis was performed by using Rev Man 5.0 statis-tics software. RESULTS:A total of 13 studies were included,involving 2 958 patients. The type of gene was type TT,type CT and type CC. Results of Meta-analysis showed the relationship between 3 gene polymorphism and warfarin maintenance dose was type TT>type CT>type CC,and there were significant differences among groups. CONCLUSIONS:CYP4F2 gene polymorphism in Chinese population has significant correlation with warfarin maintenance dose. However,due to the limit of methodological quali-ty,large-scale and high quality studies are required for further validation of the conclusions.

Quantitative analysis of trazodone in human plasma by using HPLC-fluorescence detector coupled with strong cation exchange chromatographic column: application to a pharmacokinetic study in Chinese healthy volunteers.

A simple, selective, and sensitive high performance liquid chromatography (HPLC) procedure has been developed for determination of trazodone in human plasma. Prazosin was employed as the internal standard (IS). Sample preparation involved liquid-liquid extraction by methyl tert-butyl ether after alkalinization with ammonia. The HPLC separation was performed on a CAPCELL PAK SCX column (250mm×4.6mm, 5.0µm, Shiseido, Japan) with a mobile phase of acetonitrile/80mmol/L ammonium phosphate (pH adjusted to 6.0) (60:40, v/v) at a flow rate of 1.2mL/min. The peaks were detected by using fluorescence detector (excitation wavelength 320nm and emission wavelength 440nm). The extraction recovery was 72.6-88.3% and the method was over the concentration range of 5.0-2486ng/mL with a lower limit of quantitation (LLOQ) of 5.0ng/mL using 300µL of plasma. The intra- and inter-day accuracy of the method at three concentrations ranged from 96.7% to 104.2% for trazodone with precision of 2.9-3.7%. This validated method was successfully applied to a pharmacokinetic study enrolling 12 Chinese volunteers administered a single oral trazodone hydrochloride extended-release tablet of 75mg.

A novel molecular mechanism of interferon alpha-regulated expression of retinoic acid-induced gene G / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms by which IFN-alpha regulated retinoic acid-induced gene G (RIG-G) expression.</p><p><b>METHODS</b>The expression of STAT1, p-STAT1 and RIG-G in IFN-alpha-treated NB4 cells was detected by Western blot. The roles of STAT1, STAT2 and IRF-9 in IFN-alpha-induced RIG-G expression were analyzed in STAT1-null U3A cells by cell transfection, reporter gene assay, co-immunoprecipitation and chromatin immunoprecipitaion.</p><p><b>RESULTS</b>In U3A cells, only when STAT2 and IRF-9 were co-transfected, the luciferase activities of RIG-G promoter-containing reporter gene could be highly increased about 8-fold compared with that in the control group. Moreover, in the absence of IFN-alpha, similar effects were observed in either IRF-9 co-transfected with wild type or mutant form of STAT2, whereas IFN-alpha could increase the transactivation activity of wild type STAT2 and IRF-9 by 6-fold compared with that without IFN-alpha, but had no effect on mutant STAT2. In addition, STAT2 could interact with IRF-9 and bind to the RIG-G promoter.</p><p><b>CONCLUSION</b>STAT2 may interact with IRF-9 in a STAT1-independent manner. The complex STAT2/IRF-9 is the key factor mediating the expression of RIG-G gene regulated by IFN-alpha. This is a novel signal transduction cascade for IFN which is different from the classical JAK-STAT pathway.</p>

Regulation mechanism for rig-g gene expression induced by all-trans retinoic acid / 中国实验血液学杂志

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.

Role of the interferon-stimulated response elements I/II in expression regulation of the retinoic acid induced gene G / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the regulatory role of interferon-stimulated response elements (ISREs) located on the retinoic acid-induced gene G (RIG-G) promoter in RIG-G expression.</p><p><b>METHODS</b>By using point mutation technique, the authors constructed the wide type and site mutant reporter gene plasmids according to the ISRE sequence on RIG-G promoter, and detected the functional activities by luciferase reporter assay.</p><p><b>RESULTS</b>Mutation in ISRE II alone had no obvious effect on the expression of the reporter gene, whereas mutation in ISRE I dramatically inhibited the transactivity of RIG-G promoter. Mutation in both ISRE I and ISRE II resulted in complete loss of its response to the transcription factors for the reporter gene.</p><p><b>CONCLUSION</b>Both ISRE I and ISRE II on the RIG-G promoter are the binding sites for the complex of transcription factors. They are required for RIG-G expression, and ISRE I has a preferential role over ISRE II.</p>

Effects of PML-RARalpha on cAMP-induced AML cell differentiation / 中国实验血液学杂志

To explore the molecular mechanisms of acute promyelocytic leukemia cell differentiation induced by cAMP combined with low-dose As2O3, the PR9 cell line, which was stably transfected by PML-RARa fusion gene, was used as in vitro model. The effects of PML-RARa on cAMP-induced AML cell differentiation were evaluated according to cell growth, cell morphology, cell surface antigen as well as luciferase reporter gene assay, in the cells before and after the treatment with cAMP and/or As2O3. The results showed that cAMP alone could slightly increase the expression of CD11b in the PR9 cells expressing the PML-RARa fusion protein, but could not induce these cells to differentiate. The cells presented the terminal differentiation morphology and significantly increased CD11b expression only under the treatment of cAMP combined with As2O3. In addition, PML-RARa had strong inhibitory activity on the transcription of the reporter gene containing cAMP response elements. In conclusions, the PML-RARa fusion protein could dramatically block the signaling pathway of cAMP during the AML cell differentiation.

Study on mechanisms of the expression regulation of interferon-induced gene RIG-G / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms of the expression regulation of retinoic acidinduced gene G (RIG-G) by interferon alpha (IFNalpha).</p><p><b>METHODS</b>RIG-G promoter region was analyzed by bioinformatics. The functional activities of RIG-G promoter with or without IFNalpha were detected by luciferase reporter assay and electrophoretic mobility shift assay (EMSA).</p><p><b>RESULTS</b>RIG-G promoter region contained two well-conserved IFN-stimulated response elements (ISREs). Both ISRE I and ISRE II showed their effective binding abilities with signal transducer and activator of transcription 1 (STAT1). In HT1080 cells, in contrast with the empty plasmid pXP2, pXP2-A reporter construct containing intact ISRE I and ISRE II showed a significant higher baseline expression (1741.2 +/- 517.5) which could be further enhanced up to three-four folds by IFNalpha (5338.7 +/- 1226.9, P < 0.05). However, the luciferase activity of pXP2-A as well as its IFNalpha inducibility could be abrogated in STAT1-deficient U3A cells (from 1741.2 +/- 517.5 to 406.1 +/- 103.2, P < 0.05), indicating that the STAT1 protein was a prerequisite for the activities of ISRE I and ISRE II.</p><p><b>CONCLUSION</b>ISREs present in RIG-G promoter region are molecular basis of IFNalpha induced RIG-G expression. RIG-G is a target gene directly regulated by STAT1 protein and should play a key role in IFNalpha signaling pathways.</p>