Objective:To observe the ameliorative effects of exogenous miR-146a-5p on lipopolysaccharide (LPS)-induced embryonic resorption and fetal mouse dysplasiamice, and to preliminarily investigate its mechanism of action.Methods:1) After 36 healthy adult female mice were mated with male mice, uterine tissues were collected from females on day (D) 0 (D0/not pregnant), D0.5 (the day of embryo observed), D4.5, D7.5, D9.5 and D13.5 of gestation, and the expression levels of miR-146a-5p and its target gene TRAF6 protein in uterine tissues of mice at different gestation periods were detected by real-time fluorescent quantitative PCR (qPCR) and Western blotting. 2) The mice on D7.5 of pregnancy were treated with intraperitoneal injection of saline (control, COL group), intraperitoneal injection of 250 μg/kg LPS (named LPS250 group), LPS combined with tail vein injection of 10 nmol miR-146a-5p unrelated sequence (negative control, NC, named LPS250+NC group), or LPS combined with tail vein injection of 10 nmol miR-146a-5p agonist (miR-146a-5p agomir, named LPS250+miR-146a-5p agomir group). The total number of embryos and the number of absorbed embryos in the uterus of pregnant mice were measured and statistically analyzed on D8.5, and the expression levels of TNFα mRNA and TRAF6 protein in uterine tissues were detected by qPCR and Western blotting. 3) Then we reduced the dosage of LPS to 50 μg/kg and treated the same groups, named LPS50+NC group, LPS50+miR-146a-5p agomir group, respectively. The total number of fetal mice/embryos, the number of absorbed embryos, the number of surviving fetal mice, the weight of surviving fetal mice and the weight of the placenta were measured and statistically analyzed on D16.5. 4) Primary mouse bone marrow-derived macrophages (BMDM) were isolated and cultured. Mouse BMDM was inducted to M1 polarization by LPS stimulation, and then was transient transfected miR-146a-5p mimics or their NC fragments. The expression levels of TNFα mRNA and pSTAT1 protein were detected by qPCR and Western blotting. Results:The expression level of miR-146a-5p was significantly higher in the implantation sites of D7.5, D9.5 and D13.5 pregnant mice than in the non-implantation sites ( P=0.013, P=0.012, P=0.003), and the protein expression level of TRAF6 was significantly lower in the implantation site of D13.5 pregnant mice than in the non-implantation site ( P=0.012). After intraperitoneal injection of 250 μg/kg of LPS into D7.5 pregnant mice, the embryo absorption rate of the LPS group on D8.5 was 43.13%±3.31%, which was significantly higher than that of COL group (0%, P=0.002), while the embryo absorption rate of the LPS250+miR-146a-5p agomir group (13.50%±0.87%) was significantly lower than that of the LPS250+NC group (59.33%±4.04%, P=0.001). After intraperitoneal injection of 50 μg/kg of LPS combined with tail vein injection of NC or miR-146a-5p agomir to D7.5 pregnant mice, the fetal mouse weight [(0.29±0.09) g] and placental weight [(0.06±0.02) g] of surviving fetal mice in the LPS50+NC group on D16.5 and the LPS50+miR-146a-5p agomir group were statistically significant [(0.46±0.06) g, P<0.001; (0.07±0.02) g, P=0.021], and the differences in the number of absorbed embryos and embryo uptake rate between the two groups were not statistically significant (all P>0.05). The expression levels of both pSTAT1 protein and TNFα mRNA were significantly downregulated in BMDM transfected with miR-146a-5p mimics compared with those transfected with NC ( P=0.012, P=0.039). Conclusion:miR-146a-5p expression levels were significantly increased at the maternal-fetal interface during the late stage of mouse embryo implantation and placental development. Exogenous miR-146a-5p could effectively improve LPS-induced mouse embryo resorption and fetal mouse dysplasia. miR-146a-5p could inhibit the M1 polarization activity of mouse macrophages, suggesting that miR-146a-5p may inhibit the M1 polarization activity of mouse macrophages by suppressing M1 polarization of mouse maternal-fetal interface macrophages to safeguard the normal establishment and maintenance of pregnancy.

Objective:To observe the ameliorative effects of exogenous miR-146a-5p on lipopolysaccharide (LPS)-induced embryonic resorption and fetal mouse dysplasiamice, and to preliminarily investigate its mechanism of action.Methods:1) After 36 healthy adult female mice were mated with male mice, uterine tissues were collected from females on day (D) 0 (D0/not pregnant), D0.5 (the day of embryo observed), D4.5, D7.5, D9.5 and D13.5 of gestation, and the expression levels of miR-146a-5p and its target gene TRAF6 protein in uterine tissues of mice at different gestation periods were detected by real-time fluorescent quantitative PCR (qPCR) and Western blotting. 2) The mice on D7.5 of pregnancy were treated with intraperitoneal injection of saline (control, COL group), intraperitoneal injection of 250 μg/kg LPS (named LPS250 group), LPS combined with tail vein injection of 10 nmol miR-146a-5p unrelated sequence (negative control, NC, named LPS250+NC group), or LPS combined with tail vein injection of 10 nmol miR-146a-5p agonist (miR-146a-5p agomir, named LPS250+miR-146a-5p agomir group). The total number of embryos and the number of absorbed embryos in the uterus of pregnant mice were measured and statistically analyzed on D8.5, and the expression levels of TNFα mRNA and TRAF6 protein in uterine tissues were detected by qPCR and Western blotting. 3) Then we reduced the dosage of LPS to 50 μg/kg and treated the same groups, named LPS50+NC group, LPS50+miR-146a-5p agomir group, respectively. The total number of fetal mice/embryos, the number of absorbed embryos, the number of surviving fetal mice, the weight of surviving fetal mice and the weight of the placenta were measured and statistically analyzed on D16.5. 4) Primary mouse bone marrow-derived macrophages (BMDM) were isolated and cultured. Mouse BMDM was inducted to M1 polarization by LPS stimulation, and then was transient transfected miR-146a-5p mimics or their NC fragments. The expression levels of TNFα mRNA and pSTAT1 protein were detected by qPCR and Western blotting. Results:The expression level of miR-146a-5p was significantly higher in the implantation sites of D7.5, D9.5 and D13.5 pregnant mice than in the non-implantation sites ( P=0.013, P=0.012, P=0.003), and the protein expression level of TRAF6 was significantly lower in the implantation site of D13.5 pregnant mice than in the non-implantation site ( P=0.012). After intraperitoneal injection of 250 μg/kg of LPS into D7.5 pregnant mice, the embryo absorption rate of the LPS group on D8.5 was 43.13%±3.31%, which was significantly higher than that of COL group (0%, P=0.002), while the embryo absorption rate of the LPS250+miR-146a-5p agomir group (13.50%±0.87%) was significantly lower than that of the LPS250+NC group (59.33%±4.04%, P=0.001). After intraperitoneal injection of 50 μg/kg of LPS combined with tail vein injection of NC or miR-146a-5p agomir to D7.5 pregnant mice, the fetal mouse weight [(0.29±0.09) g] and placental weight [(0.06±0.02) g] of surviving fetal mice in the LPS50+NC group on D16.5 and the LPS50+miR-146a-5p agomir group were statistically significant [(0.46±0.06) g, P<0.001; (0.07±0.02) g, P=0.021], and the differences in the number of absorbed embryos and embryo uptake rate between the two groups were not statistically significant (all P>0.05). The expression levels of both pSTAT1 protein and TNFα mRNA were significantly downregulated in BMDM transfected with miR-146a-5p mimics compared with those transfected with NC ( P=0.012, P=0.039). Conclusion:miR-146a-5p expression levels were significantly increased at the maternal-fetal interface during the late stage of mouse embryo implantation and placental development. Exogenous miR-146a-5p could effectively improve LPS-induced mouse embryo resorption and fetal mouse dysplasia. miR-146a-5p could inhibit the M1 polarization activity of mouse macrophages, suggesting that miR-146a-5p may inhibit the M1 polarization activity of mouse macrophages by suppressing M1 polarization of mouse maternal-fetal interface macrophages to safeguard the normal establishment and maintenance of pregnancy.

OBJECTIVETo investigate histone acetylation modification of topoisomerase enzyme Ⅱα (TOPOⅡα) promoter regulation factors in patients with chronic benzene poisoning, to explore the possible regulatory mechanism of TOPOⅡα involved in toxicity of chronic benzene poisoning;

METHODSThe bone marrow samples were from 25 chronic benzene poisoning cases and 25 controls. The Chromatin Immunoprecipitation (ChIP) assay was carried out to study the possible mechanism of TOPOⅡα promoter regulation factors expression changes. TOPOⅡα promoter regulation factors mRNA were detected by RT-PCR technique.

RESULTS(1) Compared with the control, the histone H4 acetylation, histone H3 acetylation level of TOPOⅡα promoter regulation factors SP1, ATF-2, SP3, NF-YA, P53, C-MYB, ICBP90, NF-M in chronic benzene poisoning patients decreased, with the significant difference (P<0.05) , except for C-JUN (P>0.05) ; (2) The mRNA expression of TOPOⅡαpromoter regulation factors SP1, NF-YA, C-MYB, C-JUN and NF-M were significantly lower than in the control with the significant difference (P<0.05) , while the expression of SP3、P53 mRNA increased (P<0.05) , ATF-2、ICBP90 mRNA wasn't changed (P>0.05) .

CONCLUSION(1) Chronic benzene poisoning TOPO Ⅱα promoter regulation factors histone modification changes accompanied with mRNA level changed. (2) Histone acetylation modification of topoisomerase enzyme Ⅱα promoter regulation factors takes important role in the benezen's Hematopoietic toxicity.

Acetylation ; Antigens, Neoplasm ; metabolism ; Benzene ; poisoning ; Case-Control Studies ; Chromatin Immunoprecipitation ; Chronic Disease ; DNA Topoisomerases, Type II ; metabolism ; DNA-Binding Proteins ; metabolism ; Histones ; metabolism ; Humans ; Poisoning ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism

Objective To observe the activity of histone deacetylase and the mRNA expression level of HDAC1 and HDAC2 in human bone marrow mononuclear cells,which induced by hydroquinone and exposed to hydroquinone plus Trichostatin as a histone deacetylase inhibitor for 10 hours respectively.Methods Collect the bone marrow mononuclear cells suspension,divided into control group,HQ group (3 h,6 h,12 h,24 h),HQ+ TSA 10 h group and HQ 10 h group.Extract the nuclear proteins and RNA,test the activity of histone deacetylase with the colorimetric HDAC assay kit and detect the mRNA expression level of HDAC 1 and HDAC2 by real-time Polymerase Chain Reaction(PCR).Results The HDAC activity of HQ3 h group,HQ6 h group and HQ12 h group were 1.31 times,1.53 times and 1.148 times than that of control group respectively.And the difference was statistically significant (P＜0.05).Except the HQ24 h group (P＞0.05),the HDAC1 mRNAexpression of HQ3 h group,HQ6 h group and HQ12 h group were 1.173 times,1.901 times and 2.348 times than that of control group respectively.And the difference was statistically significant (P＜0.05).The HDAC2 mRNA expression of HQ6 h group and HQ12 h group were 1.426 times and 1.766 times than that of the control group respectively.And the difference was statistically significant (P＜0.05).No significant difference was observed between HQ3 h group,HQ24 h group and control group(P＞0.05).The cells were treated by hydroquinone plus TSA for 10 hours.The HDAC activity of HQ+TSA 10h group was reduced by 25.6％ than that of HQ 10 h group (P＜0.05) and rised 13.0％ compared to the control group (P＜0.05).And the difference was statistically significant between groups (P＜0.05).The cells were treated by hydroquinone plus TSA for 10 hours.The HDAC 1 mRNA expression of the HQ+TSA 10h group is reduced by 26.9％ than that of HQ10h group.The HDAC2 mRNA expression is reduced by 19.3％ compared to the HQ 10h group.And the difference was statistically significant between groups (P＜0.05).The HDAC1 and HDAC2 mRNA expression is obviously higher than the control group,the difference was statistically significant (P＜0.05).Conclusion Treatment of hydroquinone,the histone deacetylase activity and the mRNA expression of HDAC1 and HDAC2 were increased in a certain time range.The histone deacetylase inhibitor (TSA) can reduce the histone deacetylase activity and the mRNA expression level of HDAC 1 and HDAC2 in the bone marrow mononuclear cell induce by hydroquinone.It can be confirmed that hematopoietic damage induced by the benzene metabolites is related to the histone acetylation modification level.

Objective To observe the activity of histone deacetylase and the mRNA expression level of HDAC1 and HDAC2 in human bone marrow mononuclear cells,which induced by hydroquinone and exposed to hydroquinone plus Trichostatin as a histone deacetylase inhibitor for 10 hours respectively.Methods Collect the bone marrow mononuclear cells suspension,divided into control group,HQ group (3 h,6 h,12 h,24 h),HQ+ TSA 10 h group and HQ 10 h group.Extract the nuclear proteins and RNA,test the activity of histone deacetylase with the colorimetric HDAC assay kit and detect the mRNA expression level of HDAC 1 and HDAC2 by real-time Polymerase Chain Reaction(PCR).Results The HDAC activity of HQ3 h group,HQ6 h group and HQ12 h group were 1.31 times,1.53 times and 1.148 times than that of control group respectively.And the difference was statistically significant (P＜0.05).Except the HQ24 h group (P＞0.05),the HDAC1 mRNAexpression of HQ3 h group,HQ6 h group and HQ12 h group were 1.173 times,1.901 times and 2.348 times than that of control group respectively.And the difference was statistically significant (P＜0.05).The HDAC2 mRNA expression of HQ6 h group and HQ12 h group were 1.426 times and 1.766 times than that of the control group respectively.And the difference was statistically significant (P＜0.05).No significant difference was observed between HQ3 h group,HQ24 h group and control group(P＞0.05).The cells were treated by hydroquinone plus TSA for 10 hours.The HDAC activity of HQ+TSA 10h group was reduced by 25.6％ than that of HQ 10 h group (P＜0.05) and rised 13.0％ compared to the control group (P＜0.05).And the difference was statistically significant between groups (P＜0.05).The cells were treated by hydroquinone plus TSA for 10 hours.The HDAC 1 mRNA expression of the HQ+TSA 10h group is reduced by 26.9％ than that of HQ10h group.The HDAC2 mRNA expression is reduced by 19.3％ compared to the HQ 10h group.And the difference was statistically significant between groups (P＜0.05).The HDAC1 and HDAC2 mRNA expression is obviously higher than the control group,the difference was statistically significant (P＜0.05).Conclusion Treatment of hydroquinone,the histone deacetylase activity and the mRNA expression of HDAC1 and HDAC2 were increased in a certain time range.The histone deacetylase inhibitor (TSA) can reduce the histone deacetylase activity and the mRNA expression level of HDAC 1 and HDAC2 in the bone marrow mononuclear cell induce by hydroquinone.It can be confirmed that hematopoietic damage induced by the benzene metabolites is related to the histone acetylation modification level.

AIM:To investigate the histone modification changes of topoisomerase Ⅱα( TOPOⅡα) promoter regulatory factor Sp1 in the patients with chronic benzene poisoning .METHODS:The bone marrow samples were collect-ed from 25 chronic benzene poisoning cases and 25 controls.The chromatin immunoprecipitation assay was carried out to study the possible mechanism of TOPOⅡαpromoter regulatory factor Sp 1 expression changes .The mRNA expression of Sp1 was detected by RT-PCR.RESULTS:Compared with the controls , the histone H4 acetylation and histone H3 acetyla-tion of Sp1 in the chronic benzene poisoning patients significantly decreased (P<0.01), and histone H3K9 methylation level of Sp1 increased (P<0.01), but the histone H3K4 methylation level of SP1 was not obviously changed (P>0.05). The mRNA expression of Sp1 in the chronic benzene poisoning patients was significantly lower than that in the controls (P<0.05).CONCLUSION:In chronic benzene poisoning patients , the histone acetylation and methylation modification changes of TOPOⅡαpromoter regulatory factor Sp 1 accompanied with the changes of mRNA level are observed .Histone H4 and H3 acetylation and H3K9 methylation modification of Sp1 may play an important role in the benzene ’s hematopoiet-ic toxicities.

Objective To investigate the roles of fiberoptic bronchoscopy in diagnosis and treatment for infants with refractory and persistent wheezing. Methods From Jun. 2012 to Dec. 2013, 52 hospitalized children with age between four 4 months and 1 year old were recruited for ifberoptic bronchoscopy, who had been wheezing for at least four weeks and treated ineffectively with conventional anti-inlfammatory agents:budesonide and compound ipratropium bromide solution. Then, the pathogenesis of refractory and persistent wheezing was summarized based on clinical features, detection of CT imaging of three-dimensional airway reconstruction and cardiac CT, results of bronchoscopy inspection, and bronchoalveolar lavage lfuid culture. Results Among the 52 cases, 40 were with ground glass-like changes (76.92%) in pulmonary spiral CT testing, 4 with mosaic perfusion syndrome (7.69%), 8 with segmental pulmonary consolidation (15.38%), 8 with obstructive pulmonary emphysema (15.38%), and 1 with left primary bronchial foreign body. In addition, through bronchofibroscopy, there were 52 cases with imlfammation (100%),3 with tracheal stenosis (5.77%), 3 with left and/or right main bronchus stenosis of the external pressure, 18 with bronchomalacia(34.62%), 2 cases with foreign body (3.84%), one in trachea (1.92%), the other in left main bronchus (1.92%), 10 with bronchial mucus plug (19.23%), and 8 (15.38%) with congenital airway malformations (including 3 at tracheal bronchus, 1 at left upper lobe bronchial stenosis and 1 at bronchial Bridge). The culture of bronchoalveolar lavage lfuid were conducted for all patients. The positive rate of bronchoalveolar lavage lfuid was 9.62%(5/52 cases), including 2 cases with tip Escherichia coli, 2 with Haemophilus inlfuenzae, and 1 with Acinetobacter baumannii. Conclusions First, infection is the primary cause of refractory and persistent wheezing, which is persistent in airway resulted from multi-drug resistant bacteriua. Second, refractory and persistent wheezing is often caused by multi-factors including infection, congenital airway malformations, the endogenous and exogenous foreign body, cardiovascular malformation, etc. These factors often lead to dififcult wheezing control. The last, the diagnosis rate of the refractory and persistent wheezing can be improved by combination of ifberoptic bronchoscopy and lung spiral CT.