Objective To determine the effects of histone deacetylase inhibitor suberoylanilide hydroxamic acid(SAHA) on the cell proliferation and apoptosis of the human hepatic stellate cell line LX-2.The possible underlying mechanisms were also investigated.Methods The LX-2 cells were treated with SAHA in vitro.The morphology of LX-2 cells in different concentrations groups was observed by inverted microscope;the proliferation of LX-2 cells was measured by MTT assay;the Annexin V-FITC and PI staining was used to detect the apoptosis of LX-2 cells by flow cytometry and fluorescence microscope;the expression of α-SMA,collagen Ⅰ,acH3K9,acH3K14 and acH3K18 were detected by Western blot.Results The morphology change of LX-2 cells showed that SAHA inhibited the proliferation rate of LX-2 cells and in a dose dependent manner(P<0.05).The LX-2 cells were sensitive to SAHA along with time increasing,and in a time-dependent manner(P<0.05).Western blot showed that the expression levels of α-SMA and collagen-Ⅰ were significantly lower(P<0.05),on the contrary,the acetylation levels of acH3K9,acH3K14 and acH3K18 were significantly higher (P<0.05).Conclusions The increased acetylation of the histone acH3K9,acH3K14,acH3K18 and the lower expressed α-SMA and collagen-Ⅰ in LX-2 cells may be one of the mechanisms of SAHA.

OBJECTIVE:To establish a method for determining recombinant human calmodulin B subunit(rhCNB)in rat plas-ma,and study its pharmacokinetics characteristics. METHODS:ELISA double-antibody sandwich method was adopted. 1 μg/ml rhCNB monoclonal antibody mAb was wrapped,added to the to-be-test sample,rhCNB polyclonal antibody pAb(dilution ratio of 1∶5 000)and HRP-labeled conjugate of anti-IgG(dilution ratio of 1∶10 000)were added. Using tetramethylbenzidine for develop-ing,microplate reader was conducted in wavelength of 450 nm to determine the absorbance value(OD value)and plasma concen-tration of 6 rats after 2,15,30,60,120,240,480,720 min of iv 2.5 mg/kg rhCNB,and the pharmacokinetic parameters were calculated by BAPP 3.0 software. RESULTS:The linear range of rhCNB were 0.195-12.5 ng/ml(r2=0.995 0),lower limit of quan-titation was 0.195 ng/ml,accuracy were 97.300%-103.622%(RSD<7.5%,n=6);RSDs of within-batch,inter-batch,freezing and thawing 3 times were no higher than 8.5%(n=6,18,15). rhCNB pharmacokinetics characteristics in rat fitted to two-com-partment model,AUC0-720 min was 173.038 mg·min/L and t1/2 was 94.62 min. CONCLUSIONS:The established method has high specificity and sensitivity,good accuracy and precision,which can be used for rhCNB quantitative detection and pharmacokinetics study in biological samples.

AIM:To study the effects of suberoylanilide hydroxamic acid (SAHA) on the apoptosis of hepatic stellate cells (HSCs) and expression of associated proteins, and to investigate the mechanisms of SAHA to induce apoptosis.METHODS:The rat HSCs were isolated by OptiPrep gradient centrifugation method.The effect of SAHA on HSC proliferation was detected by real-time cell analyzer.The morphological changes of HSCs treated with SAHA at different concentrations were observed under inverted microscope.The apoptotic rates of HSCs were analyzed by flow cytometry with Annexin V-FITC/PI staining and fluorescence microscopy.The protein expression of α-smooth muscle actin (α-SMA), collagen I, tissue inhibitor of metalloproteinase 1 (TIMP1), glucose-regulated protein 78 (GRP78) and histone deacetylase 6 (HDAC6) was detected by Western blotting.The interaction of GRP78 with HDAC6 in the HSCs was determined by co-immunoprecipitation.RESULTS:HSCs were successfully isolated and cultured for 14 d, during which the HSCs changed gradually from rest state to active state.SAHA significantly inhibited the proliferation of HSCs in a time-and dose-dependent manner (P<0.05).The results of Western blotting showed that the protein expression levels of α-SMA, TIMP1, collagen-I and HDAC6 were significantly decreased (P<0.05), while GRP78 was significantly increased (P<0.05).Compared with activated HSCs, GRP78 and total acetyl-lysine protein were significantly increased in the co-immunoprecipitated HSCs treated with SAHA, while HDAC6 protein was significantly decreased, indicting that GRP78 formed a complex with HDAC6.CONCLUSION:The anti-hepatic fibrosis effect of SAHA may be related to down-regulation of HDAC6 and up-regulation of acetylated GRP78, thus inducing endoplasmic reticulum stress of HSCs and promoting the apoptosis of HSCs.

Objective To investigate the prevalence of mite sensitivity in patients with urticaria or other skin rashes, and to observe the clinical efficacy of a specific immunotherapy(SIT) by the Injectio dermatophagoidei farinae for the patients.Methods In 7-year period(1998-2005), skin prick test(SPT) with a dust mite(Df) allergen was carried out to detect the prevalence of mite sensitivity in OPD patients suffering from skin rashes.Among the patients sensitive to mite with SPT ≥++ response, 3 groups were established.In group A, routine SIT with Injectio dermatophagoidei farinae was conducted.In 9-week increasing dose phase, three stepwise increasing volumes(0.3ml, 0.6 ml and 1.0 ml) each case was injected subcutaneously with mite concentration of 1 ∶ 100 000(w/v) , 1 ∶ 10 000(w/v) or 1 ∶ 5 000(w/v) respectively once a week, followed by a maintenance dose phase for an injection with 1 ∶ 5 000(w/v) 1.0 ml/wk for 6 weeks.Group B received rush SIT with mite injections.A total of 15 injections in a course of therapy with same concentration and volume was given as those for the routine ones except shortened intervals, namely, 9 initial injections completed in 3 days by three injections of each concentration per day with two 30 min intervals, maintenancedoses were then provided in 6 days with 1 ∶ 5 000(w/v) 1.0 ml/d.Thereafter, both groups A and B were maintained for one year with a dose of 1 ∶ 5 000(w/v) 1.0 ml every 2 wk.Group C received antihistamine treatment as control, the patients received daily oral Ebastine 10 mg in the morning and Cetirizine dihydrochloride 10 mg in the evening for one week course and pro re nata later.Levels of serum tIgE and serum mite sIgE were detected by ELISA in 20 urticaria cases before and after one year mite SIT.Results Altogether, 2 685 cases with skin rashes were detected by Df allergen SPT.The prevalence of urticaria cases sensitive to mite was 70.3%(1 754/2 496), which was higher than that of eczema 63.5%(54/85) and anaphylactoid purpura 60.6%(63/104)(P

Objective:To develop and evaluate the improved Chinese hearing questionnaire for school children(CHQS)for mass epidemiology study on hearing impairment in China.Method:Using the probability proportion to size(PPS) method, 8 412 residents were investigated in 40 clusters in Jiangsu province with the WHO ear diseases and hearing disorders survey protocol.87.9% of the residents aged 7 years and over answered the questionnaire and accepted the pure tone audiometry.Result:The prevalence of hearing impairment was 12.9% by the questionnaire. Compared with golden standard(pure tone audiometry), Sen=58.5%, Spe=96.7%, PV+=78.9%, PV-=91.7%, overall accuracy=90.0% . The sensitivity for women was higher than men.Conclusion:The questionnaire produced high efficiency and specificity values.It could be used in mass hearing screening, particularly in remote and rural area, although the sensitivity was as low as most questionnaires.

[ ABSTRACT] AIM:To investigate the effects of sinapic acid ( SA) on the proliferation and apoptosis of rat vas-cular smooth muscle cells (VSMCs) induced by high glucose (HG).METHODS:Cultured A7r5 cells were randomly di-vided and treated as indicated.The cell viability was determined by MTT assay.DNA synthesis was measured by BrdU as-say.Cell cycle progression and cell apoptotic rate were determined by flow cytometry analysis.The levels of reactive oxygen species (ROS) were detected by ELISA.The protein levels of cyclin D1, P21, P27, phosphorylated protein kinase C (p-PKC), p-P38 andβ-actin were evaluated by Western blot.RESULTS:Compared with control group, the viability of A7r5 cells was significantly enhanced, the DNA synthesis was increased, the cell cycle progression was promoted, the levels of ROS were elevated, the cell apoptotic rate was reduced, the protein expression of P21 and P27 was decreased, and the pro-tein levels of cyclin D1, p-PKC and p-P38 were increased in HG group (all P<0.05).These effects were reversed by SA (0.1, 1 and 10 μmol/L) treatment in a dose-dependent manner (all P<0.05).Both P38 inhibitor SB203580 and PKC inhibitor chelerythrine significantly inhibit HG-induced PKC/P38 activation and cell viability ( P <0.05).CONCLU-SION:SA inhibits HG-induced VSMCs proliferation and promotes cell apoptosis via reducing PKC/P38 activation.

OBJECTIVETo study the autophagy activity between rat bone marrow stem cells (BMSCs) neural differentiation in order to explore the mechanism involve in this process.

METHODSBMSCs were passed by 3 generation, then was induced with the revulsant 2% (DMSO) + 200 µmol/L (BHA), NSE expression was detected by immunocytochemical stain, the mRNA expression of autophagy associated genes L3B, Beclinl, Atg5, Atg7, Atg10 were detected by RT-PCR, the autophagy protein LC3B was examined by Western blot and flow cytometry analysis.

RESULTSBMSCs were passed by 3 generation, the purity of BMSCs could reach more than 90%, the morphology of cells were like fibroblasts, after the revulsant 2% DMSO + 200 µmol/L BRA induced, cells were extended long neurites, like nerve cells, positive rate of NSE staining was (83±5) %, RT-PCR results showed that the expression of autophagy associated genes LC3B, Beclinl, Atg5, Atg7 Atg0 were rised after BMSCs neural differentiation, Western blot analysis showed that the LC3B-II protein expression was increased after neural differentiation and the MFI of L3B was highten by flow cytometry.

CONCLUSIONAutophagy is increased after rat BMSC neural differentiation.

Animals ; Autophagy ; Cell Differentiation ; Cells, Cultured ; Flow Cytometry ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; Rats

BACKGROUND:The production and release of a large amount of inflammatory factors caused by immune system inflammatory response mainly contributes to secondary spinal cord injury. OBJECTIVE:To investigate the effects of umbilical cord Wharton’s jely mesenchymal stem cel transplantation on repair of injured neurological function and expression of inflammatory factors monocyte chemoattractant protein 1 and interleukin 10 in rats with acute spinal cord injury. METHODS: Eighty-one healthy adult male Sprague-Dawley rats were randomly and equaly divided into sham operation, model and cel transplantation groups, with 27 rats per group. Rats in the latter two groups were subjected to hemisection of the spinal cord to establish acute spinal cord injury models. Rat models in the cel transplantation group received umbilical cord Wharton’s jely mesenchymal stem cel injection (1×106)via the tail vein. Rat neurological function was evaluated using the BBB score at different time points after spinal cord injury. The expression of monocyte chemoattractant protein 1 and interleukin 10 in injured spinal cord tissue was detected using ELISA assay at different time points after spinal cord injury. Migration and neuronal differentiation of umbilical cord Wharton’s jely mesenchymal stem cels in the injured spinal cord tissue were determined using immunohistochemical staining method. RESULTS AND CONCLUSION:Compared with the sham operation and model groups, rat neurological function was significantly recovered in the cel transplantation group (P < 0.05). Compared to the model group, monocyte chemoattractant protein 1 level in the serum and monocyte chemoattractant protein 1 mRNA and protein expression in the injured spinal cord tissue were significantly lower (P < 0.05), but interleukin 10 mRNA and protein expression in the injured spinal cord tissue was significantly higher (P < 0.05), in the cel transplantation group. In the cel transplantation group, umbilical cord Wharton’s jely mesenchymal stem cels could migrate to the injured region and express glial fibrilary acidic protein. These findings suggest that umbilical cord Wharton’s jely mesenchymal stem cels promote rat neurological function recovery by regulating the inflammatory response in the injured spinal cord tissue, which is likely to be one of mechanisms by which transplantation of umbilical cord Wharton’s jely mesenchymal stem cels treats spinal cord injury.

OBJECTIVESTo investigate the development and the health of reproductive organs of male children and juvenile between the Meng and the Han nationality in the Meng nationality area.

METHODSMale juvenile(4-18 years old) of the Meng nationality (n = 2,315) and the Han nationality (n = 2,832) were divided into four age groups. Height, weight, length and perimeter of penis, volume of left and right testis and reproductive organs illness were examined.

RESULTSIn 13-18 years group, the developmental speed of reproductive organs was faster in Mongolia male juvenile than that in the Han nationality (P < 0.02). After 13 years old, the developmental speed of reproductive organs of male living in town is faster than that in the country (P < 0.05). Illness of male reproductive organs was common such as hernia, varicocle etc.

CONCLUSIONSThere was difference of developmental status and the prevalence rate of reproductive organs of male children and juvenile between the Meng and the Han nationality.

Adolescent ; Body Height ; Body Weight ; Child ; Child, Preschool ; China ; ethnology ; Humans ; Male ; Penis ; physiology ; Testis ; physiology

OBJECTIVETo study the effects of drug plasma of musk and olibanum (DP-M&O) on the release of inflammatory cytokines from monocytes and the expressions of the proteins associated with inflammation of prostatic or endothelial cells induced by prostate antigen (PAg) stimulation.

METHODSWe prepared DP-M&O using SD rats and monocytes and PAgs using BALB/c mice. We pre-treated the monocytes with DP-M&O at the gradient concentrations of 0, 2.5, 5, 10, and 20% for 1 hour, activated them with PAgs, and then cultured them for 96 hours, followed by detection of the release of inflammatory cytokines. We co-cultured the prostate RWPE-1 cells with the endothelial EA. hy926 cells, pre-treated them with the same gradient concentrations of DP-M&O as above for 1 hour, activated with PAgs, and cultured for 96 hours. Then we determined the expression levels of the proteins associated with inflammation of RWPE-1 and EA. hy926 cells by Western blot.

RESULTSDP-M&O decreased the levels of TNF-alpha, IL-1beta, IL-6, and IL-8 and increased that of IL-10 in a concentration-dependent manner. Significant differences were found between the 20% P-M&O and PAg groups in the release of the inflammatory cytokines TNF-alpha (70.8 +/- 22.3 vs. 277.1 +/- 65.5, P < 0.01) , IL-113 (277.5 +/- 22.6 vs. 630.4 +/- 89.7, P <0.01), IL-6 (232.7 +/- 62.7 vs. 994.2 vs. 182.3, P < 0.01), IL-8 (227.3 +/- 79.2 vs. 769.3 +/- 284.1, P < 0.01), and IL-10 (640.2 +/- 201.2 vs. 271.1 +/- 55.8, P < 0.01). Compared with the PAg group, the 10 and 20% P-M&O groups showed remarkable decreases in the protein expression of MCP-1/CCL2 in the RWPE-1 cells (1.12 +/- 0.34 vs. 0.56 +/- 0.11 and 0.34 +/- 0.08) and that of VCAM-1 in the EA. hy926 cells (0.94 +/- 0.22 vs. 0.52 +/- 0.17 and 0.38 +/- 0.12) (P < 0.05 or 0.01).

CONCLUSIONThe compatibility of musk and olibanum can decrease the expression of MCP-1/CCL2 in prostate cells and VCAM-1 in vascular endothelial cells, blocking the adhesion of leucocytes and suppressing inflammatory response.

Animals ; Blotting, Western ; Cytokines ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Fatty Acids, Monounsaturated ; pharmacology ; Frankincense ; pharmacology ; Inflammation ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; Male ; Mice ; Mice, Inbred BALB C ; Monocytes ; drug effects ; metabolism ; Prostate ; cytology ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism