ABSTRACT
Objective:Rat model of RA complicated with pulmonary fibrosis were constructed to observe the degree of improvement of pulmonary fibrosis in RA rats by JAK2 inhibitor CEP33779 and the possible mechanisms.Methods:①The RA models were constructed by subcutaneous injection of 0.2 ml (1 mg/ml) of bovine type Ⅱ collagen into the tail of the rats on the day of modeling development (d0); intratracheal injection of 100 μl bleomycin (2.5 mg/kg) was used to induce pulmonary fibrosis model at d13. In vivo study: model rats were randomly divided into the normal group, pulmonary fibrosis group, pulmonary fibrosis CEP treatment group, RA complicated with pulmonary fibrosis group, and RA complicated pulmonary fibrosis CEP treatment group. Rats in the treatment group was given CEP (10 ml/kg) qd by gavage from d14 to week 4. The right hind foot of the rats was measured for joints swelling and the arthritis index score were measured, lung compliance (Cst) and lung specific gravity were measured. In addition, the pathological changes of the left lung were observed by HE and Masson staining, and the extracellular matrix level of the right lung was measured by protein immunoblotting (WB). ② In vitro study: TGF-β 1 (10 ng/ml) was applied to stimulate human embryonic lung fibroblasts (HFL1) for 24 h and 48h, and p-JAK2 expression was detected by immunofluorescence. After HFL1 inoculation of culture plates, the control group, TGF-β 1 stimulation group, TGF-β 1+ LY2109761 (TGFβ-R1/2 inhibitor group, 0.5 mmol/L and 2 mmol/L) group, TGF-β 1+CEP (0.1 mmol/L and 1.0 mmol/L) group were co-incubated for 48 h, and the expression levels of TGFβ-R2, α-SMA, JAK2, and Col 1 were measured by WB. Comparisons between multiple groups were made by Tukey′s test, and comparisons between the two groups were analyzed by independent t-test. Results:① In vivo study, compared with the control group (1.45±0.04), joint swelling was increased at d13 [(2.54±0.16) in RA+PF+Vehicle group, t=16.02, P<0.001], and the mean arthritis index score and toe volume were decreased 3 days after CEP treatment(d16) [(2.89±0.11), t=5.78, P<0.001; (1.92±0.13), t=6.85, P<0.001]. For rats with pulmonary fibrosis, all had different degrees of lung enlargement, increased lung specific gravity, decreased Cst, and increased lung inflammation and fibrosis[(0.96±0.06), t=19.76, P<0.001; (0.26±0.09), t=17.64, P<0.001; (3.63±1.51), t=6.00, P<0.001; (1.75±0.71), t=5.84, P<0.001]. After CEP gavage, rats that had RA complicated with pulmonary fibrosis had reduced lung swelling, decreased lung specific gravity, increased Cst [(0.82±0.05), t=5.76, P<0.001; (0.43±0.18), t=2.31, P=0.038], and the scores of pulmonary fibrosis and inflammation [(3.00±1.00); (1.56±0.52)] all showed a trend of decrease, but did not reach statistical difference CEP inhibited the expression of TGF-β 1, TGFβ-R2, α-SMA, Fn and JAK2 in lung tissue of pulmonary fibrosis rats, and the differences among the five groups were statistically significant ( F=9.02, P=0.017; F=4.86, P=0.048; F=6.57, P=0.032; F=11.26, P=0.010; F=13.32, P=0.007). ② In vitro study, TGF-β 1 stimulated HFL1 showed stronger phosphorylated JAK2 (p-JAK2) fluorescent signal WB showed a significant increase in the expression of TGFβ-R2, α-SMA, JAK2 and Col1, and after LY and CEP intervention, the above proteins were reduced in a concentration-dependent manner, with statistically significant differences among all five groups ( F=337.30, P<0.001; F=20.61, P<0.001; F=100.60, P<0.001; F=180.90, P<0.001). Conclusion:JAK2 inhibitors can ameliorate RA-related pulmonary fibrosis, and the mechanism may be through interfering with the "crosstalk" between JAK2 and TGF-β 1 signaling pathway.
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Objective:To investigate the expression of activator of basal transcription 1 (ABT1) protein in gastric cancer tissue and its relationships with the clinical parameters and prognosis of gastric cancer patients, and to clarify the role of ABT1in the occurrence and development of gastric cancer.Methods:A total of 100cases of cancer tissue of the gastric cancer patients and 80pairs of adjacent tissue were selected.The expressions of ABT1in cancer tissue and adjacent tissue were detected by immunohistochemistry and the proportion of stained cells and the degree of staining in the immunohistochemistry results were analyzed using semi-quantitative analysis.The relationships between the semi-quantitative analysis results and the clinical parameters of gastric cancer patients were statistically analyzed.Kaplan-Meier method was used to analyze the correlation between the ABT1 protein expression level and the survival of gastric cancer patients.Results:ABT1-positive staining was observed in the nucleus and cytoplasm of gastric cancer tissue and adjacent gastric tissue.The expression level of ABT1in gastric cancer tissue was lower than that in adjacent tissue (P=0.021) .The ABT1protein expression level in gastric cancer tissue was significantly negatively correlated with the pathological grade (r=-0.224, P=0.026) .The Kaplan-Meier analysis results of the survival curve showed that the high expression of ABT1was associated with good prognosis in the gastric cancer patients (HR=1.483, P＜0.01) .The survival rate of gastric cancer patients with high ABT1expression was significantly higher than that of the patients with low ABT1expression (HR=2.411, P=0.0272) .Conclusion:The expression of ABT1in gastric cancer tissue is lower, indicating that ABT1can be used one of the markers of good prognosis of gastric cancer.
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Objective: To investigate the effects of overexpression of microspherule protein 1 (MCRS1) in the gastric cancer cells on the invasion and migration of gastric cancer cells, and to elucidate their possible mechanisms. Methods: The gastric cancer BGC-823 cells, SGC-7901 cells and the normal gastric mucosal epithelial GES-1 cells were cultivated. Western blotting method was used to detect the expressions of MCRS1 in three kinds of cells. The result showed that MCRS1 protein had the lowest expression in the BGC-823 cells, so the gastric cancer BGC-823 cells were selected for next experiments. The recombinant plasmid of MCRS1 was constructed, and the gastric cancer BGC-823 cells in the logarithmic growth phase were selected. Blank group, empty vector transfection group and MCRS1 transfection group were established, and the plasmid was transfected into the BGC-823 cells using Lipo3000. The expression levels of epithelial cadherin (E-cadherin) protein, neuronal cadherin (N-cadherin) protein and Snail protein were detected by Western blotting method. The cell scratch assay and Transwell assay were used to detect the migration and invasion of gastric cancer BGC-823 cells. Results: Compared with the normal gastric epithelial GES-1 cells, the expression level of MCRS1 protein in the gastric cancer BGC-823 cells was decreased (P < 0 . 01), but the expression level of MCRS1 protein in the SGC-7901 cells was increased (P < 0 . 01). The PCR identification and sequencing analysis showed that the MCRS1 recombinant plasmid was successfully constructed. Compared with blank group and empty vector transfection group, the expression levels of MCRS1 protein and E-cadherin protein in MCRS1 transfection group were increased (P