Overexpression of Ubiquitin-Specific Protease15 (USP15) Promotes Tumor Growth and Inhibits Apoptosis and Correlated With Poor Disease-Free Survival in Hepatocellular Carcinoma.

USP15 is a member of ubiquitin-specific proteases (USPs, the largest subfamily of deubiquitinases) and functions as a stabilize factor of target proteins in reversible ubiquitiantion progression. Dysregulated expression of USP15 has been observed in various cancers. However the expression profile and regulatory mechanism of USP15 in hepatocellular carcinoma (HCC) remains largely elusive. To exam the USP15 expression changes in the progression of HCC, we performed IHC analysis to test USP15 expression in a series of cancer-prone diseases including 2 normal liver tissues, 6 liver cirrhosis, 16 primary liver lesions and 15 metastases of hepatocellular carcinoma. The expression of USP15 was upregulated in various liver diseases in compared with normal tissue significantly (p < 0.05). Although no significant different of USP15 expression were discovered between cirrhotic tissue and primary tissue, its expression in HCC metastatic tissue was upregulated. Subsequently, we test the USP15 expression profile in a cohort of 66 HCC patients. USP15 expression was positively correlated with the recurrence of HCC significantly (p = 0.004). HCC patients with high USP15 expression had shorter disease free survival time in compare with those with low USP15 expression (56.9% VS 26.7%, P = 0.012). Subsequently, Cox multivariate analyses of clinical factors associated with disease free survival were performed and USP15 expression (p = 0.008) together with tumor size (p = 0.034) were proved to be independent predict factors in HCC. Then, we silenced USP15 expression in HCC cells and the results showed that downregulated USP15 expression resulting proliferation inhibition and apoptosis induction. In conclusion, our results suppose USP15 to be a potential target in HCC.

[The effect on the pro-inflammatory role of N9 microglia exposured to hyperoxia after preconditioning with lipopolysaccharide in vitro].

AIM: To observe the time-dapendcrt expression of TLR4 and TNF-α of N9 microglia exposured to normobaric hyperoxia after preconditioning with lipopolysaccharide in vitro and to explore the role of hyperoxia on the pro-flammation response of microglia and mechanism. METHODS: N9 microglia cell line cultured in vitro was randomly divided into six groups(n=3): normoxia group, sLPS group(100 ng/mL), hLPS group(1 mg/L), hyperoxia group, hyperoxia+sLPS group(100 ng/mL), hyperoxia+hLPS group(1 mg/L). Each of the last two groups, 30 min after pretreatment with different level of LPS, was subjected to 900 mL/L hyperoxia for various times (2 h, 6 h, 12 h, 24 h and 48 h). The remanent groups was cultured in ambient O(2); in which sLPS group and hLPS group respectively was treated with 100 ng/mL and 1 mg/mL LPS in the cell supernatant. After treatment, at each time point, the cells of each group was harvested and TLR4 gene expression were observed by RT-PCR. TLR4 protein expression at 12 h was observed by Western blotting. TNF-α concentrations in the supernatant of cultured microglia N9 cells at different time points were tested with ELISA. RESULTS: After 6 h in hLPS group and 16 h in sLPS group, the expression of TLR4 mRNA was gradually increased(P<0.05), following with increasing time and concentration of LPS, which reached to the maximum at 24 h in hLPS group. Compared with hLPS group, hyperoxia+hLPS group showed downregulation of TLR4 mRNA at each time point after 6 h(P<0.05), especially at 16 h and 24 h. At 12 h, the level of TLR4 protein of hyperoxia+sLPS group and hyperoxia+hLPS group respectively was lower than the corresponding concentration of LPS group. The result of ELISA show that at each time point, compared with the corresponding concentration of LPS group respectively, the expression of TNF-α of hyperoxia+sLPS group and hyperoxia+hLPS significantly increased(P<0.05). CONCLUSION: Hyperoxia enhance the pro-flammation response of N9 microglia triggered by LPS and TLR4 may be the important negative-control target molecule.

Histological study on the effect of electrolyzed reduced water-bathing on UVB radiation-induced skin injury in hairless mice.

Electrolyzed reduced water (ERW), functional water, has various beneficial effects via antioxidant mechanism in vivo and in vitro. However there is no study about beneficial effects of ERW bathing. This study aimed to determine the effect of ERW bathing on the UVB-induced skin injury in hairless mice. For this purpose, mice were irradiated with UVB to cause skin injury, followed by individually taken a bath in ERW (ERW-bathing) and tap water (TW-bathing) for 21 d. We examined cytokines profile in acute period, and histological and ultrastructural observation of skin in chronic period. We found that UVB-mediated skin injury of ERW-bathing group was significantly low compared to TW control group in the early stage of experiment. Consistently, epidermal thickening as well as the number of dermal mast cell was significantly lowered in ERW-bathing group. Defection of corneocytes under the scanning electron microscope was less observed in ERW-bathing group than in TW-bathing group. Further, the level of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-12p70 in ERW group decreased whereas those of IL-10 increased. Collectively, our data indicate that ERW-bathing significantly reduces UVB-induced skin damage through influencing pro-/anti-inflammatory cytokine balance in hairless mice. This suggests that ERW-bathing has a positive effect on acute UVB-mediated skin disorders. This is the first report on bathing effects of ERW in UVB-induced skin injury.

Phenotype-specific down-regulation of nicotinic acetylcholine receptors in the pelvic ganglia of castrated rats: implications for neurogenic erectile dysfunction.

Pelvic ganglia (PG) play critical roles in relaying sympathetic and parasympathetic information from the spinal cord to the penile vasculature and, controlling the penile reflex. Animal studies have shown that androgen deprivation by castration causes erectile dysfunction (ED). Until now, however, neural mechanisms underlying castration-induced ED remain unclear. Therefore, we examined whether androgen deprivation down-regulates nicotinic acetylcholine receptors (nAchRs), which mediate fast excitatory synaptic transmission in the PG. Toward this end, neurogenic ED was demonstrated by measuring the intracavernous pressure in castrated rats. Real-time PCR analysis revealed that the transcripts encoding nAchR α3/α5/ß4 subunits were significantly down-regulated in the PG neurons. In addition, down-regulation of the nAchR subunits was reversed by replacement of testosterone. Patch-clamp experiments showed that the nAchR currents were selectively attenuated in the parasympathetic PG neurons innervating the penile vasculature, activation of which elicits penile erection. Taken together, our data suggest that phenotype-specific down-regulation of nAchRs in the PG neurons may contribute to the neurogenic ED in castrated rats.

Essential involvement of cross-talk between IFN-gamma and TNF-alpha in CXCL10 production in human THP-1 monocytes.

Interferon (IFN)-gamma-induced protein 10 (IP-10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN-gamma depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN-gamma and TNF-alpha have not been adequately elucidated in human monocytes. In this study, we showed that TNF-alpha had more potential than IFN-gamma to induce CXCL10 production in THP-1 monocytes. Furthermore, IFN-gamma synergistically enhanced the production of CXCL10 in parallel with the activation of NF-kappaB in TNF-alpha-stimulated THP-1 cells. Blockage of STAT1 or NF-kappaB suppressed CXCL10 production. JAKs inhibitors suppressed IFN-gamma plus TNF-alpha-induced production of CXCL10 in parallel with activation of STAT1 and NF-kappaB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF-kappaB, but not that of STAT1. IFN-gamma-induced phosphorylation of JAK1 and JAK2, whereas TNF-alpha induced phosphorylation of ERK1/2. Interestingly, IFN-gamma alone had no effect on phosphorylation and degradation of IkappaB-alpha, whereas it significantly promoted TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha. These results suggest that TNF-alpha induces CXCL10 production by activating NF-kappaB through ERK and that IFN-gamma induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF-alpha-induced NF-kappaB may be the primary pathway contributing to CXCL10 production in THP-1 cells. IFN-gamma potentiates TNF-alpha-induced CXCL10 production in THP-1 cells by increasing the activation of STAT1 and NF-kappaB through JAK1 and JAK2.

The adenylyl cyclase-cAMP system suppresses TARC/CCL17 and MDC/CCL22 production through p38 MAPK and NF-kappaB in HaCaT keratinocytes.

Patients with atopic dermatitis (AD) have significantly reduced plasma cAMP levels, and the cAMP level is correlated with the immunopathogenesis of AD. The production of thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) in keratinocytes is significantly enhanced in patients with AD. In the present study, we investigated the in vitro effects of the adenylyl cyclase-cAMP system on IFN-gamma and TNF-alpha-stimulated production of TARC and MDC in human HaCaT keratinocytes. Both forskolin (a direct activator of adenylyl cyclase) and dibutyryl-cAMP (DBcAMP, a permeable analog of cAMP) suppressed production of TARC and MDC in parallel with the activation of NF-kappaB in IFN-gamma and TNF-alpha-stimulated HaCaT cells. Moreover, inhibition of NF-kappaB suppressed TARC and MDC production induced by IFN-gamma plus TNF-alpha. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the secretion of these chemokines. An inhibitor of p38 MAPK suppressed the production of TARC and MDC in parallel to the activation of NF-kappaB in HaCaT cells. Of note, the IFN-gamma plus TNF-alpha-stimulated activation of p38 MAPK was suppressed following incubation with forskolin or DBcAMP alone. These results indicate that the adenylyl cyclase-cAMP system has an inhibitory role in IFN-gamma plus TNF-alpha-stimulated production of TARC and MDC in HaCaT keratinocytes by inhibiting NF-kappaB activation through p38 MAPK pathway, implying that the adenylyl cyclase-cAMP system could be a candidate therapeutic target of Th2-skewed skin inflammation such as AD.

[Analysis on neuropsychological characteristics of subtypes of attention deficit hyperactivity disorder].

OBJECTIVE: To explore the neuropsychological characteristics of children with attention deficit hyperactivity disorder (ADHD). METHODS: Neuropsychological tests, including visual working memory, Stroop test, digits inverse reciting, vocabulary fluency, Wisconsin card sort test (WCST), and Temporal discounting were used to evaluate the capacity of response inhibition, phonological working memory, visual working memory executive function and delayed satisfying capacity of subjects. RESULTS: 1. The ADHD children spent longer time [ADHD-I (84(20), ADHD-C: 98 (31), normal: 70 (28)] to accomplish color naming and made more errors [ADHD-I: 3 (3), ADHD-C: 6 (19), normal: 2 (5)] than the normal control when the color was inconsistent with the word meaning in Stroop test (P < 0.01). 2. The scores of digits reciting [ADHD-I: 3 (3), ADHD-C: 3 (4), normal 4 (4)] inverse was lower in ADHD than in normal control (P < 0.01). 3. The representation of ADHD was poorer than normal control in visual working memory [ADHD-I: 21 (3), ADHD-C: 20 (5), Normal: 20 (3)], and in delayed visual memory [ADHD-I: 19 (5), ADHD-C: 19 (5), Normal: 20 (5)] (P < 0.01). 4. The scores of vocabulary fluency [ADHD-I: 1 (1), ADHD-C: 2 (1), normal: 0 (0)] was lower in ADHD than in normal control (P < 0.01). 5. In WCST, the ADHD children made more errors [ADHD-I :15 (17), ADHD-C: 15 (15), normal: 13 (13)] and less classification [ADHD-I: 5 (4), ADHD-C: 5 (4), normal: 5 (3)] than normal control (P < 0.01). 6. In Temporal discounting, the ADHD children showed significantly more impairments than normal control did (P < 0.01). 7. There was significant difference between the two subtype groups on some tests (P < 0.01). CONCLUSIONS: Obvious cognitive impairments were found in children with ADHD, involving poor response inhibition, impaired working memory, dysfunction of planning and set-shifting, and there was no significant difference between the two subtype groups.

[Study on the correlation between adolescents' emotional and behavioral problems and life events].

OBJECTIVE: To study the life events which are correlated with adolescent's emotion and behavior problems, and to provide evidence for clinicians and school staff to develop intervention for those problems. METHODS: Youth's Self Report (YSR) and Adolescent Self-Rating Life Events Check List (ASLEC) were used to assess adolescent's emotional and behavioral problems and life events by 'spot' study. The referred group consisted of 585 patients from 11 to 18 years old in a Mental Health Centre of West China Hospital of Sichuan University from July in 2002 to March in 2004. Level of IQ was above 5th grade of primary school among the study subjects who were willing to fill in the YSR and ASLEC. The non-referred group was selected in a 1280 students cluster-sample from the schools of Chengdu city, whose sex, age and father's career were matched with the referred group. Variance inflation factor (VIF) was used to verify that there was no collinearity to each other in the 6 factors of ASLEC: interpersonal relationship, learning pressure, being punished, losing good adaptation and other. Linear stepwise regression was adopted. RESULTS: The YSR scores in referred group were higher than those in non-referred group, and the referred group had more emotional and behavioral problems than the non-referred one. Partial correlations ranged from 0.124 to 0.418 in referred group, and from 0.104 to 0.388 in non-referred group. Unsatisfied interpersonal relationship, heavy learning pressure, having been punished and poor adaptation were likely to increase the risk of youth's emotional and behavioral problems. CONCLUSION: More attention should be paid to help adolescents in the following areas: solving intrapersonal affairs, relieving pressure from learning, avoiding punishment, and improving ability to fit themselves to their surroundings.

[An epidemiological study on mental problems in adolescents in Chengdu, China].

OBJECTIVE: To study the mental health problems in the adolescents aged 11-18 years in Chengdu, China. METHODS: Youth Self-Report (YSR) and the General Information Questionnaire were administered to 1802 students who were randomly selected from schools in Chengdu City. RESULTS: The overall rate of mental problems was 15.1% in the adolescents, 15.2% in boys and 14.9% in girls if 90th percentile of total scores and/or 98th percentile of syndromes scores were assigned as the cutpoint of norms. The overall rate of mental problems was 27.2% in the adolescents, 27.6% in boys and 26.8% in girls if 75th percentile of total scores and/or 98th percentile of syndromes scores were assigned as the cutpoint of norms. The rates of mental problems were not significantly different between boys and girls (chi-squared = 0.042, P = 0.838; chi-squared = 0.126, P = 0.723). Mental problems tended to be increasing with age (P = 0.000). There were 29 items that the percentiles were higher than 50% but 5 items were under 10% in all the 101 items of YSR. 3.1% of the adolescents often had suicidal intention, and 6.1% of the adolescents often thought of becoming opposite sex identity. CONCLUSION: The rates of mental problems in adolescents aged 11-18 years in Chengdu, Chana were 27.2% or 15.1% respectively,if 75th or 90th percentile of the total scores were assigned as the cutpoint of total scores. The mental problems in the adolescents tended to be increasing along with age.

Nerve injury alters profile of receptor-mediated Ca2+ channel modulation in vagal afferent neurons of rat nodose ganglia.

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague-Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of alpha2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of micro-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury.