Effect of Wenyang Huazhuo Tongluo formula on Sema3A/Nrpl signaling pathway in systemic sclerosis mouse model / 中国药理学通报

Aim To study the effeet of Wenyang Hua- Nrpl signaling pathway in systemic sclerosis ( SSc ) zhuo Tongluo formula ( WYHZTLF) on the Sema3A/ mouse model, and to explore its mechanism in the treatment of SSe.Methods The systemic sclerosis mouse model was constructed and divided into control group, model group, WYHZTLF low (21 g • kg 1 ) , medium (42 g • kg 1 ) , high (84 g • kg 1 ) dose group and the Sema3A inhibitor epigallocatechol gallate (EGCG) group.All groups were given intragastric administration for four weeks, and the control and model groups were treated with saline.Histopathology and dermal thickness were detected by HE, VEGFA pro-tein expression levels were detected by immunohisto- chemistry; the protein expression levels of Sema3A, Nrpl and VEGFA were determined by Western blot; Sema3A expression levels in mouse serum were detec-terl by ELISA.Results Comparer] with model group, WYHZTLF significantly alleviated the skin lesions in systemic sclerosis mouse model, significantly inhibited the dermal thickness, inhibited the protein expression levels of VEGFA, Sema3A and Nrpl , and reduced the expression levels of serum Sema3A.Conclusion WYHZTLF can improve the vascular injury of SSc, and its mechanism may be related to the inhibition of VEGFA and Sema3A/Nrpl signaling 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.

Suppression of experimental abdominal aortic aneurysm by saturated hydrogen saline: a preliminary study with rats / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of saturated hydrogen saline on the prevention of abdominal aortic aneurysm (AAA) induced by calcium chloride in a rat model.</p><p><b>METHODS</b>In healthy male Sprague-Dawley rats, AAA was induced by infiltration of abdominal arota with 0.5 mol/L calcium chloride. Saturated hydrogen saline (5 ml·kg(-1)·d(-1)) or saline was administred intraperitoneally once daily. Twenty-eight days later, the diameter of the aorta was measured, and the aortic tissue was exercised for histological examination. Pro-inflammatory cytokines (tumor necrosis factor α (TNF-α), IL-1β) in AAA tissue were detected with ELISA. The protein expression and mRNA expression of matrix metalloproteinase 2 (MMP-2) and MMP-9 in AAA tissue were observed by immunohistochemistry staining and real-time PCR.</p><p><b>RESULT</b>The aorta diameter of the experiment group and control group were (2.2 ± 0.3) mm and (3.4 ± 0.5) mm, the tissue IL-1β levels were (81 ± 29) ng/L and (165 ± 51) ng/L, the tissue TNF-α levels were (109 ± 46) ng/L and (360 ± 51) ng/L, the relative mRNA expressions were 2.4 ± 1.0 and 11.8 ± 2.9, the relative mRNA expressions were 2.9 ± 0.6 and 6.7 ± 1.0 (t = 4.055 to 10.406, P < 0.05). Compared with the control group, the infiltration of inflammation, the injury of elastic fibers in the vessel wall, and the positive expression of MMP-2 and 9 protein of the experiment group were all reduced.</p><p><b>CONCLUSIONS</b>Saturated hydrogen saline prevents the degradation of elastin in vessel wall and ameliorates the formation and development of AAA, which may be associated with its anti-inflammatory effects, thereby reduces the MMP-2 and 9 mRNA and protein expression.</p>

Real-time elastography for quantitative assessment of liver fibrosis in a rat model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of real-time elastography for quantitative evaluation of liver fibrosis in a rat model.</p><p><b>METHODS</b>A total of 70 male Wistar rats were included in the group for dimethylnitrosamine (DMN)-induced liver injury, and 10 saline-injected rats were used as normal control. Hepatic injury was induced by a single intraperitoneal injection of DMN at a dose of 50 mg/kg of body weight. Nine or ten rats in the group with DNM injected and one or two rats in the normal control group were randomly selected and sacrificed at each of the following post-injection time: day 5, 7, 10, 14, 21, 24, and 28. And their livers were taken for pathology analysis. All the rats underwent real-time elastography before sacrificed in order to acquire area ratio of low-strain region (% AREA) and liver fibrosis index (LF index) which were compared with the stage of liver fibrosis and grade of necroinflammatory pathologically. By the different data, Spearman correlation analysis, rank-sum test or receiver operating characteristic curve was used.</p><p><b>RESULTS</b>Among 58 successfully modeled rats, there were nine, 13, 14 and 12 rats of S1, S2, S3 and S4 liver fibrosis on pathology, respectively, which were with or without mild necroinflammatory. The other 10 rats were found to be S0 with severe necroinflammatory. Values of LF index and % AREA both increased with liver fibrosis stage (P less than 0.05). There was certain correlation between LF index and liver fibrosis stage (r=0.643, P=0.000), so was % AREA and liver fibrosis stage (r=0.662, P=0.000). As for LF index, Areas under the receiver operating characteristic curve (Az) was 0.943, 0.890, 0.743 and 0.821 for the diagnosis of hepatic fibrosis S1 or higher, S2 or higher, S3 or higher and S4, respectively; as for % AREA, they were 0.948, 0.883, 0.772 and 0.842, respectively. However, we found a significant difference for LF index or % AREA between S0 with and without severe inflammatory activity rats (P=0.005 and P=0.017).</p><p><b>CONCLUSION</b>Real-time elastography is available for quantitative assessment of liver fibrosis in rats induced by DMN, but severe inflammatory activity can affect its accuracy.</p>

The mechanisms of inhibitory effect of adenovirus-mediated wild-type PTEN gene on the proliferation in activated hepatic stellate cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Using an adenoviral vector, the wild-type PTEN gene was transduced into activated hepatic stellate cell (HSC) cultured in vitro and cell cycle markers and were detect. Thereby, the potential mechanisms of inhibitory effect of the wild-type PTEN overexpression on the proliferation in activated HSC was investigated.</p><p><b>METHODS</b>The wild type PTEN gene was transduced into activated HSC (HSC-T6 ) cultured in vitro mediated by adenoviral vector. PTEN expression in HSC was measured by Western blot and Real-time fluorescent quantitation PCR. Flow cytometry (FCM) was then used to detect cell cycle phase of activated HSC. And the expressions of cyclinD1 and cyclin dependent kinase 4 (CDK4) in HSC were determined by Western blot.</p><p><b>RESULTS</b>The data showed that exogenous wild type PTEN gene was successfully transduced and expressed in activated HSC cultured in vitro. The over-expression of wild type PTEN resulted in the increased number of HSC at G0/G1 phase ( P less than 0.01), and the number of HSC at S phase and G2/M phase were decreased significantly, P less than 0.01. Furthermore, there were decreased cyclinD1 and CDK4 expression in HSC infected with Ad-PTEN, P less than 0.01.</p><p><b>CONCLUSION</b>The over-expression of wild type PTEN inhibit transition of activated HSC in vitro from G1 to S phase and arrest cell cycle of them at G0/G1 phase via the down-regulated expressions of cyclinD1 and CDK4, and then inhibit HSC proliferation.</p>

The influence of down-regulation of focal adhesion kinase by RNA interference on the adhesion and migration of rat hepatic stellate cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC).</p><p><b>METHODS</b>Two recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN.</p><p><b>RESULTS</b>The recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively.</p><p><b>CONCLUSIONS</b>FAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.</p>

The dynamic expression of PTEN in fibrogenic rat liver tissues and its relation to the activation and proliferation of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic expression of PTEN in fibrogenic liver tissue of rats and its effect on the activation and proliferation of hepatic stellate cells (HSC).</p><p><b>METHODS</b>A rat model of hepatic fibrosis was established by common bile duct ligation (BDL). The expressions of PTEN in the rat liver tissues were detected by immunohistochemical staining, Western blot and real-time PCR assay. The expressions of PTEN in activated HSC in the rat liver tissues were detected by immunofluorescence double labeling confocal laser scanning microscopy. The alpha-SMA in the rat liver tissues was determined by immunohistochemical staining.</p><p><b>RESULTS</b>The immunohistochemical staining indicated that there was extensive expression of PTEN in the liver tissues of normal rats, it was expressed mainly in the cytoplasm of the HSC. With the aggravation of hepatic fibrosis, the expression of PTEN in the hepatic tissues decreased gradually (P less than 0.01), while the alpha-SMA positive cells in the hepatic tissues increased significantly (P less than 0.01). The expressions of PTEN protein and mRNA in the rat liver tissues at week 1, 2, 3 and 4 after BDL were all lower than those in the sham operation group (P less than 0.01), and the expressions gradually decreased with the development of hepatic fibrosis (P less than 0.01). Immunofluorescence double labeling confocal laser scanning microscopy showed that PTEN were expressed extensively in activated HSC, especially in the cytoplasm, and with the development of hepatic fibrosis, the PTEN-expressing activated HSC accounted for an increasingly smaller percentage of total activated HSC.</p><p><b>CONCLUSION</b>The expressions of PTEN mRNA and protein in rat fibrogenic liver tissues were downregulated, and their expressions in HSC in vivo also decreased. The dynamic expressions of PTEN in liver tissues had a significant negative correlation with the activation and proliferation of HSC.</p>

Time Variation Regularity of TVOC in Indoor Air in Newly Decorated Houses / 环境与健康杂志

Objective To investigate the pollution condition and concentration variability of TVOC in different time after the decoration finished. Methods 57 newly decorated houses were collected in two districts in Guiyang and TVOC monitoring was conducted for 6 months. Results At the first monitoring time, the content range of TVOC was 0.532-23.560 mg/m3 and the median was 2.573 mg/m3. The content range of high, middle and low concentration after determination was 5.520-23.560 mg/m3, 2.004-4.993 mg/m3 and 0.532-1.905 mg/m3 respectively and the median was 10.150, 2.320, 1.074 mg/m3 respectively. 147 days after the decoration the concentration decreased to 0.6 mg/m3. Conclusion The pollution of TVOC in the newly decorated houses is severe. 147 days after the decoration, the concentration of TVOC may decrease and reaches to under the standard limit.