Mechanism study of the protective effects of selective cyclooxygenase-2 enzyme inhibitors on the liver of rats with type 2 diabetes mellitus combined with nonalcoholic steatohepatitis via Rho/ROCK pathway / 中华肝脏病杂志

Objective: To investigate whether the selective cyclooxygenase-2 enzyme inhibitors celecoxib has protective effect on the liver of rats with type 2 diabetes mellitus (T2DM) combined with nonalcoholic steatohepatitis (NASH) via inhibiting the expression of Rho/ROCK pathway. Methods: Forty male SD rats were randomly divided into four groups: type 2 diabetes mellitus combined with nonalcoholic steatohepatitis (T2DM-NASH) group, T2DM-NASH + celecoxib group, control group, and control+celecoxib group. The T2DM-NASH and T2DM-NASH + celecoxib groups were fed with high-sugar and fat diet, and the control group and control + celecoxib group were fed with basal diet (25 kJ/kg). Four weeks later, streptozotocin (STZ, 30 mg/kg) was intraperitoneally injected into the NASH group and T2DM-NASH + celecoxib group to induce T2DM model, and the control group and control + celecoxib group were intraperitoneally injected with isovolumic citric acid-sodium citrate buffer. Four weeks after STZ injection, the T2DM-NASH + celecoxib group and the control + celecoxib group were gavaged with celecoxib (10 mg·kg·d) dissolved in normal saline for 4 weeks, and the remaining two groups of rats were gavaged with isovolumic normal saline for 4 weeks. Animals were sacrificed at the end of the 12- weeks, and the liver tissue was collected. Liver pathological changes were observed by HE staining. The expressions of RhoA, RhoA, ROCK1 and ROCK2 proteins in liver were detected by immunohistochemistry and western blot. The expressional condition of RhoA, ROCK1 and ROCK2 mRNA in liver were detected by real-time quantitative PCR. The differences were compared between protein and mRNA expression among the groups by analysis of variance and t-test. Results: Compared with the control group and the control + celecoxib group, the liver tissue of the T2DM-NASH group and the T2DM-NASH + celecoxib group had severe steatosis, and there was partial inflammatory cell infiltration under the light microscope. The expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were significantly increased (P < 0.05) in each liver tissue, while liver steatosis was reduced to certain extent in T2DM-NASH + celecoxib group than T2DM-NASH group, and the expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were decreased in each liver tissue of T2DM-NASH group (P < 0.05). Conclusion: The selective cyclooxygenase-2 enzyme inhibitors celecoxib has a protective effect on the liver of rats with T2DM-NASH, and its effect may be achieved by inhibiting the expression of Rho/ROCK pathway.

Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin, filamin A and cortactin in activated hepatic stellate cells / 中华肝脏病杂志

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.

Focal adhesion proteins, vinculin and integrin ß5, during early pregnancy in rat uterine epithelial cells: anastrozole favors their normal distribution / Proteínas de adhesión focales, vinculina e integrina ß5, durante el embarazo temprano en células epiteliales uterinas de rata: anastrozol favorece su distribución normal

SUMMARY: An alternative superovulator to replace clomiphene citrate is needed as clomiphene citrate is associated with low pregnancy rates. Anastrozole is an effective superovulator, but it has not been well researched. In order to determine the effectiveness of anastrozole as a superovulator and to compare it with clomiphene citrate in similar situations, this study ascertained the effects of these drugs on the expression of the focal adhesion proteins, vinculin and integrin β5, which are uterine receptivity markers, in the uterine epithelial cells of day 1 and day 6 pregnant Wistar rats. The results show that vinculin and integrin β5 are co-localized at the base of the uterine epithelium at day 1 of pregnancy whereas at day 6, they disassemble from the basal focal adhesions and co-localize and significantly increase their expression apically (p≤0.0001). Moreover, there is a significant difference in the protein expression levels of vinculin and integrin b5 in uterine luminal epithelial cells between untreated (control) and chlomiphene citrate treated rats (p≤0.0001), anastrozole and chlomiphene citrate treated rats at day 6 (p≤0.0001) suggesting the interpretation that anastrozole seems to enhance their expression in order to perhaps assist in the implantation process of the blastocyst. The immunofluorescence experiments agree with the vinculin and integrin β5 gene expression findings in which at day 6 of pregnancy, vinculin and integrin β5 gene expression are significantly upregulated in uterine luminal epithelial cells in the anastrozole treated group relative to the calibrator sample (p≤0.0001). These findings suggest that anastrozole is implantation friendly.

RESUMEN: Es necesario un superovulador alternativo para reemplazar el citrato de clomifeno, debido a que está asociado con bajas tasas de preñez. El anastrozol es un superovulador eficaz, sin embargo es poca su investigación. Con el fin de determinar la efectividad del anastrozol como superovulador y compararlo con citrato de clomifeno en situaciones similares, se determinaron los efectos de estos fármacos sobre la expresión de las proteínas de adhesión focal, vinculina e integrina β5, en marcadores de receptividad uterina en días 1 y 6, en las células epiteliales uterinas de ratas Wistar preñadas. Los resultados muestran que la vinculina y la integrina β5 se co-localizan en la base del epitelio uterino al día 1 de la gravidez mientras que al día 6 se desmontan de las adherencias focales basales, co-localizan y aumentan significativamente su expresión apicalmente (p≤0.0001). Además, existe una diferencia significativa en los niveles de expresión de proteína de vinculina e integrina β5 en células epiteliales luminales uterinas entre ratas no tratadas (control) y tratadas con citrato declomifeno (p≤0.0001), ratas tratadas con anastrozol y citrato declomifeno al día 6 (p≤0,0001) sugiriendo la interpretación de que el anastrozol parece mejorar su expresión con el fin de ayudar en el proceso de implantación del blastocisto. Los experimentos de inmunofluorescencia coinciden con los resultados de la expresión de los genes vinculina e integrina β5 en los cuales al día 6 de la preñez, la vinculina y la integrina β5 están significativamente reguladas en células epiteliales luminales uterinas en el grupo tratado con anastrozol con respecto a la muestra del calibrador (p<0,0001). Estos hallazgos sugieren que el anastrozol es favorable para la implantación.

Correlation between vascular endothelia injury factors and occupational hand-arm vibration disease / 中国职业医学

OBJECTIVE: To analyze the correlation between vascular endothelia injury factors and occupational hand-arm vibration disease( HAVD). METHODS: A judge sampling method was used to select 23 male patients with HAVD as the HAVD group,61 male workers who exposed to hand-arm vibration without HAVD as the vibration exposure group,64 male workers without hand-arm vibration exposure as the control group. The plasma levels of myosin light chain 2( MLC2),endothelin-1( ET-1) and vinculin( VCL) were detected by enzyme-linked immunosorbent assay. Logistic regression analysis was used to analyze the related indicators of HAVD for building the new multivariable model index Y. The indicators of HAVD were screened and judged by receiver operating characteristic( ROC) curves. RESULTS: There was significant difference in plasma levels of MLC2 among the three groups( P < 0. 05). The levels from high to low was as follows: HAVD group > vibration exposure group > control group. The plasma level of ET-1 in HAVD group was lower than that in the control group( P < 0. 05),but there was no significant difference between vibration exposure group and HAVD group( P > 0. 05). There was no significant difference among the three groups in the plasma level of VCL( P > 0. 05).The logistic regression analysis results showed that after adjusting confounding factors such as age,length of service,smoking,alcohol drinking and subjective symptoms,the higher MLC2 plasma level,the higher risk of HAVD( P < 0. 01),and the lower ET-1 plasma level,the higher risk of HAVD( P < 0. 05). According to ROC curve analysis,the area under the ROC curve( A_Z) value of the plasma levels of MLC2 and ET-1 were 0. 820 and 0. 524,respectively( P < 0. 01). The predictive probability index Y built with MLC2 and ET-1 by logistic regression model was used to judge the A_Z value of HAVD to be 0. 799( P < 0. 01). The A_Z values from high to low was as follows: MLC2 > Y> ET-1( P < 0. 01).CONCLUSION: The plasma levels of MLC2 and ET-1 are correlated with HAVD. The efficacy of MLC2 as a biomarker for screening HAVD is better than that of ET-1. No association was found between VCL and HAVD.

Characterization of the ultrastructure and cytoskeleton of bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To explore ultrastructure and cytoskeleton characteristics of bone marrow-deftved mesenchymal stem cells (MSCs) in patients with systemic lupus erythematosus (SLE).Methods MSCs were isolated from bone marrow of 2 SLE patients and 2 healthy controls.Their ultrastrnctures were examined by transmission electron microscope (TEM).The expression pattern of actin and vinculin was assessed by laser confocal microscopy (LCM).Results MSCs in patients with SLE presented with signs of ageing and lots of autophagosome could be found in most of the cells.F-actin was aggregated and condensed at the:border of cytoplasm.Vinculin was arranged disorderly and condensed in the cytoplasm.Conclusion The change of uhrastructure and cytoskeleton patterns of bone marrow derived mesenchymal cells of SLE patients may play an important role in the abnormal proliferation of these cells in vitro.

Expression of Vinculin in Stomach Intraepithelial Neoplasia and Clinical Significance / 医学研究杂志

Objective To study the expression of vinculin in gastric carcinoma and stomach intraepithelial neoplasia and its clinical significance.Methods The expression of vinculin proteins in carcinoma and stomach intraepithelial neoplasia was examined by immunohistochemical staining. The data was analyzed by SAS9.0 statistical software.Results The higher expression of vinculin appeared in nomal gastric mucosa.With the lesion progressing,the expression was decreased.The expression in gastric carcinoma was remarkably lower than that in stomach intraepithelial neoplasia (P

EFFECTS OF ATP ON U937 CELL APOPTOSIS / 解剖学报

Objective To study the mechanism of U937 cell (human histocytic lymphema) apoptosis induced by ATP. Methods ATP (0^23?g/L) was added into U937 cell cultured medium as experiment group, no ATP adding group as the control. Using immunofluorescent cytochemistry method to demonstrat the expresses of Cx43, F\|actin, vinculin. Results ATP induced U937 cell to form apoptotic bodies. Expresses of Cx43, F\|actin, vinculin were increased in ATP treated U937 cells.Conclusion ATP could induce U937 cell apoptosis. Apoptotic bodies formed from ATP treated U937 cells was correlated closely with express of Cx43, F\|actin, vinculin, It indicats that Cx43, F\|actin, vinculin play important roles in U937 cell apoptosis induced by ATP.

Role of cytokine RhoA in the formation of hepatic fibrosis in rats / 中华消化杂志

Objective To observe the expression changes of cytokine RhoA,stress fiber and Vinculin in the formation of hepatic fibrosis in rats, and investigate the role of RhoA in hepatic fibrosis. Methods Forty male SD rats were divided into two groups:model group and control group. The rats in the model group were given 60% CCl 4 by subcutaneous injection(1 ml/kg). The liver tissue was taken out for research at 1st、4th and 8th week respectively, RhoA and Vinculin mRNAs were detected by RT PCR, Vinculin and F actin proteins were detected by immunohistochemistry and immunofluorescence respectively, RhoA and Vinculin proteins were detected by Western blotting. Results Compared with control group, the expressions of mRNA of RhoA and Vinculin, the proteins of Vinculin, F actin and RhoA were all significantly increased( P