Objective To determine the effect of microRNA-146a (miR-146a) on the life cycle of hepatitis B virus (HBV) and investigate the underlying mechanisms.Methods The miRNA expression profiles were compared by miRNA array between HepG2 and HepG2.2.15 cells.Then miR-146a was chosen as objective,and its expression level was further confirmed by RT-PCR.After miR-146a mimic and inhibitor were transfected into HepG2.2.15 cells respectively,the quantification of HBV replication was determined by RT-PCR,and the levels of HBsAg and HBeAg in the supernatant were measured by ELISA,and the expression of HS3ST3B1 at mRNA and protein levels were tested by RT-PCR and Western blotting.Dualluciferase reporter assay was used to detect the interaction between miR-146a and potential target HS3ST3B1.Results The expression levels of totally 72 miRNAs were changed in HepG2.2.15 cells,with 27 upregulated and 45 down-regulated.RT-PCR showed the expression level of miR-146a was significantly higher in HepG2.2.15 cells than HepG2 cells (1.55-± 0.13 vs 1.00 ± 0.01,P ＜ 0.05).Transfection of miR-146a mimic into HepG2.2.15 cells resulted in significantly increased HBV replication and levels of HBsAg and HBeAg (P ＜ 0.05),while the transfection of its inhibited caused opposite results (P ＜ 0.05).Bioinformatic analysis showed that HS3ST3B1 was a potential target of miR-146a.The reporter luciferase reporter system indicated that the reported fluorescence intensity of HS3ST3B1 wild type vector was significantly lower than that of the control group (P ＜ 0.05),but showed no significant difference between HS3ST3B1 mutant vector and control group (P ＞0.05).The mRNA level of HS3ST3B1 was not significantly changed in HepG2.2.15 cells transfected with miR-146a mimic (P ＞ 0.05),but its protein level was significantly decreased (P ＜ 0.05).Conclusions miR-146a affects the life cycle of HBV,which may be through suppressing the translation of HBV inhibitory factor HS3ST3B1 3'UTR.

BACKGROUND:It has been reported that mesenchymal stromal cells(MSCs)are capable of differentiating into cells of multilineage.Different methods and reagents have been used to induce the differentiation of MSCs,but most inducing systems contain serum and cytokines.OBJECTIVE:To investigate the gene expression of mash-1 and ngn-1 in differentiation of SD rat bone marrow-derived mesenchymal stem cells induced by salvia mitiorrhiza. DESIGN:Controlled experiment in vitro with repeated observation and measurement based on cells.SETTING:Department of Anatomy,School of Preclinical and Forensic Medicine,Sichuan University. MATERIALS:This study was performed in the Department of Anatomy,School of Preclinical and Forensic Medicine,Sichuan University from October 2004 to December 2005.SD male rats weighing 160-200 g were purchased from the Animal Center of Sichuan University.The experimental animals were disposed according to ethical criteria.Parenteral solution of salvia mitiorrhiza purchased from Tianyang Medicine Company Limited of Anhui(batch number:20050411).METHODS:The nucleated cells were separated from rat bone marrow through gradient centrifugation and cell adherent method,and then MSCs differentiated into neurcyte-like cells induced by salvia mitiorrhiza.Cells in the control group were cultured with salvia mitiorrhiza-free serum-free culture media.The expression of neuron specific enolase(NSE)and glial fibrillary acidic protein(GFAP)were detected by immunohistochemistry.RT-PCR was used to detect the mRNA of mash-1 and ngn-1. MAIN OUTCOME MEASURES:①mash-1 and ngn-1 expressions were detect by the RT-PCR method.②NSE and GFAP expressions were detected by immunohistochemistry.RESULTS:①The MSCs were well adherent to walls.Most of the cells transformed in dipolar-like or multipolar-like.Axon-like or dentrite-like process was developed and these processes synapse with each other.② RT-PCR showed that ngn-1 and mash-1 mRNA were negative before induction,but positive after induction.③Immunohistochemistry indicated that NSE and GFAP expressions were positive after induction but negative in the control group. CONCLUSION:MSCs can be induced to differentiate into neurocyte-like cells by salvia mitiorrhiza.

Objective To explore the role of interleukin (IL)-1β in the pathogenesis of gout.Methods Real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay(ELISA) were used to measure the expression of IL-1β mRNA in peripheral blood mononuclear cells (PBMCs) and IL-1β in plasma samples from 120 acute gouty (AG) arthritis,70 chronic gouty (CG) arthritis,80 intercritical gouty (IG) arthritis patients and 96 healthy control subjects respectively.Results The expression of PBMCs IL-1β mRNA and plasma concentration of IL-1β were both much higher in gout patients than those in controls (P ＜ 0.01,respectively).And the plasma levels of IL-1β mRNA and IL-1β significantly increased in the AG group compared with CG and IG groups (P ＜ 0.01,respectively) and much higher in the CG group than those in the IG group.Positive correlations existed between plasma concentration of IL-1β and the levels of white blood cell,neutrophil,monocyte,erythrocyte sedimentation rate,blood uric acid,globulin and PBMCs IL-1β mRNA (P ＜ 0.01,respectively) while negative correlation between plasma IL-1β and plasma level of apolipoprotein in gout patients (P ＜ 0.05).Conclusion Elevated plasma level of IL-1β may be involved in the pathogenesis of acute and chronic gouty arthritis.

Objective To investigate the development of rat heart and the expressions of TGF-?1,SMAD4 and Bax protein to detect the location and mechanism of action in different developmental periods of rat heart. Methods Histology and immunohistochemistry of rat embryonic hearts from day11 to day19(E11～E19) in paraffin-embedded were used to analyze the heart development and TGF-?1,SMAD4 and Bax protein expressions. Results The muscular part of interventricular septum appeared on E12.5,and the partition of the ventricle finished on E16.The positive expression of TGF-?1 can be seen in the rat embryonic heart during E11～E19.The positive staining was increased to E15 and then declined significantly.The expression of SMAD4 was enhanced gradually and the positive signals were strong on E17,and a spatial difference was found in the expression on E13.The expression peaks of the Bax protein appeared on E15 then subsided to a stable.Conclusion The critical period of cardiac muscle cell differentiation and heart moulding was E15.TGF-?1,Smad4 and Bax protein play important roles during the development of rat embryonic heart.

Objective:To explore the expression and clinical significance of late autophagy in per-ipheral blood mononuclear cells (PBMCs) of patients with primary gouty arthritis (GA).Methods:Peripheral blood, clinical data, and laboratory tests were collected from 30 patients with acute gout (AG), 30 patients with intermittent gout (IG), and 50 healthy controls (HC). Quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression levels of autophagy-related genes (ATG5, ATG12, ATG16, ATG3, ATG7, ATG10, ATG4B, LC3-2/LC3B). Measurement data conformed to normal distribution were tested using t test or analysis of variance (ANOVA), and non-normal distribution data were tested using Mann-Whitney test or Kruskal-Wallis H test. SNK was used for pairwise comparison among the three groups. Correlation between variables was tested by Spearman correlation analysis. Results:① The expression level of ATG5 mRNA,ATG12 mRNA, ATG16 mRNA, ATG10 mRNA and LC3-2 mRNA in the AG group was lower than that of the IG group and the HC group, and the expression level of the IG group was lower than that of the HC group[9.16×10 -3(6.04×10 -3, 15.00×10 -3) vs 14.48×10 -3(9.95×10 -3, 21.38×10 -3) vs 0.08×10 -3(12.21×10 -3, 42.79×10 -3), H=19.377, P<0.001; 18.89×10 -3(13.85×10 -3, 24.92×10 -3) vs 21.13×10 -3(12.11×10 -3, 28.06×10 -3) vs 33.57×10 -3(13.11×10 -3, 49.89×10 -3), H=7.545, P=0.023; 8.72×10 -3(4.96×10 -3, 13.74×10 -3) vs 10.62×10 -3(7.48×10 -3, 24.71×10 -3) vs 20.07×10 -3(11.99×10 -3, 39.56×10 -3), H=20.962, P<0.001; 1.05×10 -3(0.73×10 -3, 1.84×10 -3) vs 1.60×10 -3(0.93×10 -3, 2.58×10 -3) vs 1.69×10 -3(1.05×10 -3, 3.54×10 -3), H=8.193, P=0.017; 2.31×10 -3(1.22×10 -3, 3.53×10 -3) vs 2.78×10 -3(1.68×10 -3, 5.96×10 -3) vs 3.68×10 -3(2.00×10 -3, 5.67×10 -3) , H=7.135, P=0.028]. The expression level of ATG4B mRNA in the AG and IG group was higher than that in HC group, and there was significant difference between IG group and AG group, IG group and HC group[9.95×10 -3(6.32×10 -3, 12.23×10 -3) vs 10.86×10 -3 (8.80×10 -3, 17.03×10 -3) vs 8.07×10 -3(5.52×10 -3, 11.63×10 -3), H=8.531, P=0.014]. There was no significant difference between the ATG3 mRNA and ATG7 mRNA groups ( H=0.539, 3.739, bothall P values >0.05). ② The results of Spearman correlation analysis suggested that in patients with acute gout, ATG3 was negatively correlated with PDW and MPV ( r=-0.499, P=0.006; r=-0.463, P=0.011); ATG4B was positively correlated with HDL-C ( r=0.408, P=0.048); ATG7 was negatively correlated with GLOB ( r=-0.554, P=0.001); ATG10 was positively correlated with ALB ( r=0.412, P=0.024) and negatively correlated with Crea and hsCRP ( r=-0.459, P=0.011; r=-0.375, P=0.045); ATG12 was negatively correlated with MO ( r=-0.434, P=0.017); ATG16 was negatively correlated with ALT and AST ( r=-0.389, P=0.034; r=-0.366, P=0.047); LC3-2 was positively correlated with UA ( r=0.381, P=0.041) and negatively correlated with MPV and PDW ( r=-0.413, P=0.026; r=-0.449, P=0.015). In patients with intermittent gout, ATG3 and ATG4B were negatively correlated with apoB100 ( r=-0.555, P=0.011; r=-0.462, P=0.040); ATG5 was negatively correlated with Crea ( r=-0.456, P=0.011); ATG10 was negatively correlated with TC, LDL-C, and apoB100 ( r=-0.526, P=0.017; r=-0.556, P=0.011; r=-0.515, P=0.020). Conclusion:Autophagy is involved in the development of gout, and is correlated with ibflammatory and metabolic indicators, suggesting that autophagy is an important feature in the pathogenesis of GA.

Objective To evaluate the histologic changes of dorsal root ganglion (DRG) and pain threshold following implantation of iodine-125(125I) seeds in the lumbar intervertebral foramina in rats.Methods Thirtysix adult male SD rats aged 6 months weighing 220-250 g were randomly assigned into 3 groups (n =12 each):group sham operation (group A) and groups low and high radioactive 125I seeds (groups B and C).125I seeds of 0.4 and 0.8 mCi were implanted in L4,5 and L5,6 intervertebral foramina in groups B and C respectively.Paw withdrawal threshold to von Frey filament stimulation (MWT) and paw withdrawal latency to thermal nociceptive stimuli (TWL) were measured 2 days before (T1,baseline) and at 2,7,14,30 and 60 days after implantation (T2-6) in 6 animals in each group.The remaining 6 animals in each group were sacrificed at 14 and 60 days after 125I seeds implantation for microscopic examination of L5 DRG.Results MWT and TWL were significantly decreased after 125I seeds implantation at T4 compared with the baseline values at T1 in both groups,and returned to the baseline in group B and were significantly increased in group C at T6.The neurons were severely injured in DRG in groups B and C in a time-and dose-dependent manner.Conclusion Implantation of 125I seeds in intervertebral foramen can induce radiation resection of DRG.

Objective To evaluate the efficacy of destruction of dorsal root ganglia with local doxorubicin injection guided by CT for postherpetic neuralgia involving thoracic back region. Methods One hundred and fifty patients suffering from postherpetic neuralgia in thoracic back region were randomly divided into 3 groups ( n = 50 each): group A oral medicine; B and C groups undergoing local injection under the guidance of C-arm and CT respectively + oral medicine. Three spinal segments severely affected by herpes virus were chosen for paravertebral puncture. 1% lidocaine 1 ml was injected at each segment as test dose. Fifteen minutes later doxorubicin 3.3 mg and betamethasone compound 4.7 mg were injected at each segment if no side-effect occurred. All 3 groups were given oral medicine according to the intensity of pain after local injection. The number of patients who exited from the study because of the side effects of oral medicine was recorded. VAS, sleep interference score (SIS) and a short form of McGill pain questionnaire (SF-MPQ) were used to evaluate the efficacy of the treatment the day before (baseline), 24 h, 1 week, 1, 3 and 6 months after local injection. The dosage of oxycodone extended-release tablets and gabapentin was recorded, and also the incidence of pneumothorax within 12 h after local injection. Results The exit rate, VAS, SIS and SF-MPQ scores, dosage of oxycodone extended-release tablet and gabapentin were significantly lower in B and C groups than in group A, but there was no significant difference between the 2 groups. The incidence of pneumothorax was 10% in group B but no pneumothorax developed in group C.Conclusion Destruction of dorsal root ganglia with local doxorubicin injection guided by CT is more effective for the treatment of postherpetic neuralgia.

AIM:To investigate the effect of curcumin derivatives B06(B06) on the synthesis of testosterone from type 2 diabetic rats.METHODS: Male Sprague-Dawley rats were evenly divided into 5 groups randomly: normal control group (C group), high fat group (H group), high fat treatment group (HT group), diabetes mellitus group (D group) and diabetes treatment group ( DT group) .The rats in the later 4 groups were fed with high fat diet, after 4 weeks of high fat diet feeding, the rats from D group and DT group were injected with low dose of streptozotocin intraperitoneally to induce diabetes mellitus, while the rats in HT group and DT group were gavaged with B06 at the dose of 0.2 mg· kg-1 · d-1 for 8 weeks.The blood glucose was detected by glucometer, blood insulin was assayed by ELISA and the insulin resist-ance index was calculated.The morphology of testes were observed by light and transmission electron microscopy.Serum testosterone and estradiol were measured by radioimmunoassay.The protein expression of steroidogenic acute regulatory pro-tein ( StAR) was detected by immunohistochemistry.The mRNA expression of StAR, cholesterol side-chain cleavage en-zyme (P450scc), cytochrome P450 17A1 (P450c17), cytochrome P450 aromatizing enzyme (P450arom), 3β-hydroxyste-roid dehydrogenase ( HSD) and 17β-HSD was detected by RT-PCR.RESULTS:The levels of blood glucose and insulin resistance index were increased in H group and D group, and serum testosterone was decreased, all of which were reversed after the treatment of B06.Testicular seminiferous tubule was distorted, spermatogenic cells were dropped in H group and D group.In addition, leydig cells were found to have swelling mitochondria in H group and D group, endoplasmic reticu-lum was reduced, and there was karyopyknosis accompany with sparse chromatin, all of which were ameliorated by B06. The protein expression of StAR was decreased in D group.The mRNA expression of StAR and P450scc was decreased in H group and D group, all of which were increased in B06 treatment group.There was no significant difference in the mRNA expression of P450c17, P450arom, 3β-HSD and 17β-HSD.CONCLUSION: B06 may increase serum testosterone and relieve the damage of testes from type 2 diabetic rats.B06 improves metabolic disorder by up-regulating mRNA expression of StAR and P450scc.

Objective To investigate the association of TLR4 gene polymorphisms with susceptibility to primary gouty arthritis (GA) in Chinese Han population.Methods A total of 459 patients with GA and 459 healthy control subjects were enrolled into this study.All the genotyping assays of TLR4 gene polymorphisms loci [rs4986790 (Asp299Gly),rs4986791 (Thr399Ile),rs2 1 49356T＞G] were measured using TaqMan probes that specifically target the alternate alleles.Results All the subjects were found to be homozygous for the wild-type TLR4 alleles (Asp/Asp,Thr/Thr) on TLR4 Asp299Gly and Thr399Ile genotyping.There was no significant deviation from HWE both in GA and controlsin the rs2149356 (x2=0.778,1.295; P＞0.05,respectively).Significant differences were observed between the GA and control groups with respect to genotype and allele frequencies of TLR4 gene rs2149356 (x2=16.23,17.08; P＜0.01,respectively),and TT genotype was the risk factor for gout (adjusted OR=2.09).Conclusion The TLR4 gene rs2149356 SNP may be associated with GA susceptibility,and TT genotype may be the risk factor for developing gout.

Objective To detect the variation of vascular endothelial growth factor (VEGF) in the splanchnic vessels under portal hypertension (PHT) and explore its mechanism and influence on the process of hyperdynamic circulation.Methods In humans,the level of VEGF pathway related proteins were detected in the splenic artery of PHT patients in clinical trials.In animal experiments,the following parameters were observed for rats in the control group (group N,n =7) and the CCl4 induced portal hypertension group (group PHT,n=7):portal vein diameter,portal vein blood flow velocity (PBV),portal vein blood flow (PBF),portal vein pressure (PP),norepinephrine (NE) reactivity in the isolated mesenteric artery microcirculation,contractile reactivity and degree of endothelial ni tric oxide synthase (eNOS) activation in the mesenteric artery by selectively inhibiting the VEGF signal pathway with SU5416.In cell experiments,primary culturing of arterial endothelial cells in vitro were used to verify the effects of VEGF on eNOS activation.Results The results showed that VEGF expression levels in the splenic artery of PHT patients significantly increased.In animal experiments,there was not a significant difference in portal vein diameter between group N and group PHT.How ever,the PBV and PBF of group PHT were lower than those of group N,and SU5416 had no clear effect on PBV and PBF in group PHT.PP of group PHT was much higher than that of group N,and SU5416 had little influence on reducing PP in group PHT.The contractile response of mesenteric artery microcirculation to NE in group PHT decreased significantly,EC50 increased a lot,and SU5416 improved this hypoergia partially.The protein levels of VEGF,VEGFR-2,eNOS,and p-eNOS in the mesentery artery of group PHT raised quite a lot compared to group N,and SU5416 decreased the protein level of VEGFR-2 and activation of eNOS significantly.Experiments in vitro confirmed that VEGF could promote the activation of eNOS.Conclusion The excessive VEGF produced in visceral arteries under PHT may participate in the process of hyperdynamic circulation partially through promoting the synthesis and activation of eNOS and then reducing visceral arteries' response to NE.