Effect of netrin-1 on high glucose induced injury of renal tubular epithelial cells / 中国病理生理杂志

AIM:To study the effect of netrin-1 on the damage of renal tubular epithelial cells induced by high glucose.METHODS:Human renal tubular epithelial HK-2 cells were treated with high glucose.Real-time PCR and Western blot were used to detect the expression level of netrin-1 in the cells.HK-2 cells were infected with netrin-1-overexpressing lentivirus, and the effect of netrin-1 over-expression on the HK-2 cells treated with high glucose was observed.The apoptosis rate was analyzed by flow cytometry.The protein level of cleaved caspase-3 was determined by Western blot.lactate dehydrogenase (LDH) activity in the culture medium was measured by 2, 4-binitrobenzene hydrazine method.The content of malondialdehyde (MDA) in the culture medium was detected by thiobarbituric acid method.The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture medium were measured by ELISA.RE-SULTS:The expression of netrin-1 at mRNA and protein levels in the HK-2 cells after high glucose treatment was significantly lower than that in the control cells (P<0.05).Infection with netrin-1-over-expressing lentivirus up-regulated the expression of netrin-1 in the HK-2 cells treated with high glucose.High glucose promoted the secretion of IL-1βand TNF-α, decreased the levels of LDH and MDA in the cell culture supernatant, and induced apoptosis and activation of caspase-3 in renal tubular epithelial cells (P<0.05).After the HK-2 cells with up-regulation of netrin-1 were induced by high glucose, the IL-1βand TNF-αsecretion, the levels of LDH and MDA in the culture medium, the apoptosis, and the level of activated caspase-3 protein in the cells were all decreased, as compared with the control cells (P<0.05).CONCLU-SION:Up-regulation of netrin-1 expression attenuates oxidative damage and inflammatory injury, and reduces apoptosis induced by high glucose in renal tubular epithelial cells.

Involvement of sodium nitrite in the regulation of cancer stem cell properties in human hepatoma cells / 药学学报

Increasing evidence suggests that hepatocellular carcinomas (HCCs) are sustained by a distinct subpopulation of self-renewing cells known as cancer stem cells (CSC). However, our understanding of their regulation is limited. Rapid reversible changes of CSC-like cells within tumors may result from the effect of biological mediators found in the tumor microenvironment. This paper aims to explore how nitrite, a key cellular modulator whose level is elevated in many tumors, affects CSC-like phenotypes of human hepatoma cells SMMC-7721 cells. The SMMC-7721 cell line was cultured under serum-free conditions to produce floating spheres. The distribution of cell cycle was analyzed by flow cytometry, the capability of cells self-renew was detected by colony-forming capabilities and spheroid-formation assay, the expression of stemness protein such as CD133, CD90 and EpCAM were determined by flow cytometry and Western blot, cell invasion was analyzed by transwell assay, and viability of SMMC-7721 parental cells and spheroids cancer cells was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Xenograft tumor models were established by subcutaneously injecting SMMC-7721 spheroids cancer cells, the transplanted tumor tissue ROS levels was detected by reactive oxygen species (ROS) test kits, the expression of HIF-1α was observed by immunofluorescence. Our results showed that the SMMC-7721 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, SMMC-7721 parental cells and spheroids cancer cells were treated with 150 μmol·L-1 sodium nitrite for 6 days, compared with control cells, an increased accumulation of G0/G1 phase cells was observable in treatment cells. Indeed, our data demonstrated that in parent cells and spheres cells that were treated with sodium nitrite for different time, the cells' ability to chemoresistance and invasion, clone-forming efficiencies and the spheres forming ability were significantly higher than that of control cells. Exposure of sodium nitrite regulated CSC-like phenotype, indicated by increased expression of known CSC markers, CD133, CD90 and EpCAM in the exposed parental cells, as well as in dormant spheroids cancer cells. Compared with the parent cells, the above effects of nitrite on the spheres cells were significantly enhanced. In vivo data also presented a more significant promotion of tumor xenograft growth from the nitrite treatment than from either of the control. Mechanistic analysis indicated that nitrite induced the upregulation of HIF-1α as well as the downregulation of ROS in the tumor microenvironment. These results suggest that nitrite increases the invasiveness of SMMC-7721 cells through up-regulation of tumor stemness.

Exposure of human hepatoma cells to nitrite and ammonia promotes invasive activity through activation of ROS/ODC pathway / 药学学报

Recent studies have demonstrated that nitrite and ammonia levels are higher in the tumor environment, but their effects on cancer cells remains unclear. The present study was designed to determine the effects of nitrite and ammonia on tumor invasion and the role of reactive oxygen (ROS)/ornithine decarboxylase (ODC) pathway. SMMC-7721 cells were treated with sodium nitrite, ammonium chloride, sodium nitrite and ammonium chloride mixture for 24 h, the cell viability was analyzed using the MTT assay, cell invasion was analyzed with the transwell assay, the intracellular ROS levels were detected with a reactive oxygen species (ROS) test kits, the expression of intracellular ODC was examined with immunofluorescence and Western blot, the expression of matrix metallopeptidase-2(MMP-2) and MMP-9 were analyzed by Western blot. Compared with the control group, SMMC-7721 cells exhibited an increase in cell viability, invasion ability, ROS levels and ODC protein after exposure to 150 μmol·L-1 sodium nitrite and ammonium chloride mixture for 24 h. The invasive activity was reduced by ROS scavenger N-acetycysteine (NAC) in SMMC-7721 cells. The specific ODC inhibitor difluoromethylornithine (DFMO) increased ROS levels and weakened the ability of sodium nitrite and ammonium chloride mixture in the regulation of invasion of SMMC-7721 cells. These data demonstrated that sodium nitrite and ammonium chloride mixture promote invasion of SMMC-7721 cells by enhancing ROS/ODC pathway.

Sodium nitrite enhanced the potentials of migration and invasion of human hepatocellular carcinoma SMMC-7721 cells through induction of mitophagy / 药学学报

Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg·L-1 sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COXⅠ and COXⅣ mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg·L-1) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.

Sodium nitrite enhanced the potentials of migration and invasion of human hepatocellular carcinoma SMMC-7721 cells through induction of mitophagy / 药学学报

Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1 α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg x (-1) sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COX I and COXIV mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg x L(-1)) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.

Homer protein expression after focal brain contusion in rats / 法医学杂志

OBJECTIVE@#To investigate Homer protein expression after focal brain contusion and explore the relationship between expression and injury time.@*METHODS@#Focal brain contusion in rats was established and Homer protein expression in brain at different injury intervals after contusion was detected by immunohistochemistry and Western blotting.@*RESULTS@#A small amount of Homer positive expression cells were detected in control group, sham operated group and experimental group (0.5 h after contusion). The amount of Homer positive expression cells increased after 3 h and reached peak 12 h after contusion. The amount of positive cells continued to decrease 1 d after contusion and to the base level 7 d after contusion. Homer protein expression based on immunohistochemistry and Western blotting had statistical difference among adjacent groups.@*CONCLUSION@#Expression of Homer protein near the focal contusion area shows time dependence after brain contusion in rats.

Role of long non-coding RNAs in gene regulation and oncogenesis / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>This article aims to review recent studies on the biological characteristics of long non-coding RNAs (lncRNAs), transcription regulation by lncRNAs, and the results of recent studies on the mechanism of action of lncRNAs in tumor development.</p><p><b>DATA SOURCES</b>The data cited in this review were mainly obtained from the articles listed in PubMed and HighWire that were published from January 2002 to June 2010. The search terms were "long non-coding RNA", "gene regulation", and "tumor".</p><p><b>STUDY SELECTION</b>The mechanism of lncRNAs in gene expression regulation, and tumors concerned with lncRNAs and the role of lncRNAs in oncogenesis.</p><p><b>RESULTS</b>lncRNAs play an important role in transcription regulation by controlling chromatin remodeling, transcriptional control, and post-transcriptional controlling. lncRNAs are involved in many kinds of tumors and play key roles as both suppressing and promoting factors.</p><p><b>CONCLUSION</b>lncRNAs could perfectly regulate the balance of gene expression system and play important roles in oncogenic cellular transformation.</p>

A cross-sectional survey of occult hepatitis B virus infection in HIV-infected patients in acquired immune deficiency syndrome area / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To assess the prevalence of occult HBV infection in HIV-infected patients inacquired immune deficiency syndrome area.</p><p><b>METHODS</b>Serum samples were obtained from 97 HIV-infected patients who transmitted by paid blood donation. ELISA was used to detect HBV erologic markers (HBsAg, Anti-HBs, HBeAg, anti-HBe and anti-HBc) and HCV antibody. Flow Cytometry were used to detect CD4+ T cell count. Nested PCR was used to amplify surface protein region of HBV DNA.</p><p><b>RESULTS</b>Ninety two patients were HBsAg negative in the 97 HIV-infected patients (94.85%). Twenty seven patients were co-infected with occult hepatitis B virus infection in the 92 HBsAg negative patients (29.35%). Seventy three patients were co-infected with HCV in the 92 HBsAg negative patients(79.35%). CD4 cell count of subjects with occult HBV infection were significantly lower (212.11 +/- 133.1 cells/mm3 versus 318.9 +/- 172.2 cells/mm3, respectively, P < 0.01). A significantly higher prevalence of isolated anti-HBc was observed in HIV-infected subjects co-infectioned with occult HBV infection [62.96% (13 of 27) versus 18.46% (15 of 65), P < 0.01]. No statistical significant association could be established between the age, sex and whether co-infected with HCV.</p><p><b>CONCLUSION</b>It is found that occult HBV infection did occurs in HIV-infected patients. Individuals co-infected with HIV and occult HBV infection are more likely to have isolated anti-HBc than subjects with HIV alone. Co-infection with HIV and occult HBV is more likely to occue in subjects with lower CD4.</p>

Expression of matrix metalloproteinase-3 after brain contusion in rats / 法医学杂志

OBJECTIVE@#To investigate the expression of matrix metalloproteinase-3 after brain contusion and its applicability for estimating the age of brain contusion.@*METHODS@#Rats had been divided into three groups: control group, sham operation group and brain contusion group. The expression of matrix metalloproteinase-3 at different time was detected by immunohistochemistry and Western blot.@*RESULTS@#By the immunohistochemistry, no staining was observed in control and sham operation groups. The positive staining of MMP-3 appeared 6 hours after contusion, increased gradually in 24 hours and peaked 5 days after contusion, then started to decrease, 14 days after contusion still could be observed. By the Western blot analysis, no expression of MMP-3 was detected in control and sham groups. The positive staining of MMP-3 appeared 6 hours after contusion, increased gradually and maximized 5 days after contusion, then started to decrease, 14 days after contusion still could be found.@*CONCLUSION@#Time-order expression of MMP-3 could be used for estimating the age of brain contusion in forensic pathology.

Study on the association between chronic hepatitis B cirrhosis and HLA-DRB1 * 1301,1302 allele / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the association between the chronic hepatitis B cirrhosis and HLA-DRB1 * 1301,1302 gene.</p><p><b>METHODS</b>HLA-DRB1 * 1301,1302 allele in 27 patients with chronic hepatitis B cirrhosis and 30 patients with chronic hepatitis B was analyzed by using the polymerase chain reaction/sequence specific primer (PCR-SSP) technique.</p><p><b>RESULTS</b>The frenquency of HLA-DRB1 * 1301,1302 allele in the chronic hepatitis B cirrhosis group was markly higher than that in the chronic hepatitis B group.</p><p><b>CONCLUSION</b>HLA-DRB1 * 1301,1302 is closely associated with the suseptibility to chronic hepatitis cirrhosis.</p>

Study on the association between severe chronic hepatitis B and HLA-DRB1 gene polymorphism / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the association between HLA-DRB1 gene polymorphism and severe chronic hepatitis B.</p><p><b>METHODS</b>26 patients with severe chronic hepatitis B were investigated for HLA-DRB1 gene polymorphism by polymerase chain reaction-sequence specific primers technique. The results were compared with those from 45 normal healthy people by use of chi2-test of Microsoft SPSS 13.0.</p><p><b>RESULTS</b>The frequency of HLA-DRB1 * 1301/1302 allele in severe chronic hepatitis B group was significantly higher than the frequency in the control group, while the frequencies of HLA-DRB1 * 1201/1202, 1501/1502 allele were not significantly different.</p><p><b>CONCLUSION</b>HLA-DRB1 * 1301,1302 is closely associated with the suseptibility to severe chronic hepatitis B, While HLA-DRB1 * 1201/1202, 1501/1502 have no association with severe chronic hepatitis B.</p>

Analysis on the decline of hepatitis C virus RNA in the blood using a mathematical model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a mathematical model of hepatitis C virus (HCV) replication and develop a working theory for antiviral therapy in order to understand the dynamics of HCV replication.</p><p><b>METHODS</b>Peripheral blood cells of 4 hepatitis C patients were cultured. Quantities of the HCV were detected every 15 min by real-time PCR. The data were analyzed using SPSS software. A mathematical functional relationship between HCV RNA and the time lapse was established.</p><p><b>RESULTS</b>The quantity of HCV RNA declined and it fell into a mathematical model: Y=3E+0.8e(-0.5467x) (r=0.9547). The estimated virion half-life was 45 min on the average.</p><p><b>CONCLUSIONS</b>The decline of HCV RNA in the blood is not of a linear trend and the HCV RNA lasts a longer time although the speed of the decline is faster than that in vivo.</p>