ABSTRACT
Background Osteoclast stimulatory transmembrane protein (OC-STAMP) is involved in silicosis fibrosis induced by silicon oxide (SiO2) exposure. Its role in silicosis fibrosis by inducing ferroptosis of alveolar type II epithelial cells and its related mechanism remain unclear. Objective To explore the effect and possible mechanism of OC-STAMP on ferroptosis of alveolar type II epithelial cells and silicosis fibrosis in rats under SiO2 exposure. Methods Twenty male Wistar rats of SPF grade were randomly divided into two groups: control (Sham) group and SiO2 group, 15 rats in each group. Rats in the SiO2 group were given 1 mL of 50 mg·L−1 SiO2 suspension at one time through the non-exposed intratracheal instillation method to establish an animal model of silicosis, and rats in the Sham group were give 1 mL of 0.9% sodium chloride solution in the same way. Rats were sacrificed after 8 weeks. Samples of lung tissue were fixed in glutaraldehyde or paraformaldehyde for observing ultrastructure of mitochondria by transmission electron microscopy; HE, Masson, VG, and Prussian blue were used to observe changes in lung tissue structure and iron deposition. The expression level of OC-STAMP and the degree of lung fibrosis were evaluated by immunohistochemistry and immunofluorescence. The expression level of OC-STAMP in rat lung tissue was detected and the transfection effect of OC-STAMP was verified by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). Overexpression (OCS group) and inhibition expression (SI-OC group) models were constructed by OC-STAMP plasmid and OC-STAMP small interfering RNA (siRNA) transfection to cultured MLE-12 cells, respectively. The relative expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and other proteins in lung tissue and MLE-12 were detected by Western blotting. Results The results of HE, Masson, and VG staining showed that the silicosis modeling was successful after 8 weeks of SiO2 exposure. The immunofluorescence results showed that OC-STAMP and ATP binding cassette subfamily A member 3 (ABCA3) co-localized in alveolar type II epithelium. The immunohistochemical results showed that the levels of OC-STAMP and collagen I in the SiO2 group were significantly higher than those in the Sham group (P<0.01). The RT-PCR results showed that the OC-STAMP mRNA in the lung tissue of the SiO2 group was significantly higher than that of the Sham group (P<0.01). The Prussian blue staining in the lung tissue of the SiO2 group showed positive brownish-yellow particles. Compared with the Sham group which showed normal mitochondrial structure, the mitochondrial structure was generally swollen and the mitochondrial cristae dissolved and disappeared in the SiO2 group by transmission electron microscope observation. The Western blotting results showed that the expression levels of SLC7A11 and GPX4 both decreased in the lung tissue of the SiO2 group (P<0.05, P<0.01), and the expression level of Vimentin increased (P<0.01). In the transfected MLE-12 cells, compared with the Sham group, the expression levels of SLC7A11 and GPX4 in the OCS group were significantly reduced (P<0.05, P<0.01). Conclusion OC-STAMP may affect the expression of proteins related to ferroptosis, and promote lung fibrosis induced by SiO2 exposure.
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Objective To investigate the effect of silenced RBM8A gene on the biological behavior (proliferation, migration, and apoptosis) of human endometrial cancer HEC-1A cells and its possible mechanism. Methods The hairpin shRNA targeted by the RBM8A gene was designed, and the best shRNA silencing fragment was screened. The recombinant lentiviral interference vector carrying the target gene was constructed and used to infect HEC-1A cells. Cells with stable knockdown of RBM8A gene were screened by puromycin as the experimental group (shRBM8A), while the shRNA of nonsense sequence was designed as the control group (shControl). CCK-8 method was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Transwell assay was used to detect cell migration and invasion. Western blot was used to analyze the expression of apoptosis-related proteins and EMT signal transduction pathway related proteins. Results In comparison with the shControl group, after RBM8A knockdown, HEC-1A cell proliferation was reduced, apoptosis was increased, migration and invasion ability were significantly inhibited (P < 0.05), the expression of apoptosis-related proteins cleaved caspase 9 and caspase 3 increased, EMT-related protein E-cadherin expression increased, and Vimentin expression decreased. Conclusion RBM8A gene silencing can inhibit the proliferation, migration, and invasion and promote the apoptosis of endometrial cancer cells. The inhibition of EMT signal transduction pathway may be its mechanism.
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[ ABSTRACT] AIM: To investigate whether L-carnitine ( LC) inhibits the eryptosis effect of uremic serum on erythrocytes.METHODS:Erythrocyte suspension (2%) was cultured and divided into 3 groups in vitro: control group ( C group) , uremic serum group ( U group, 30%uremic serum) , and uremic serum+LC group ( L group, 30%uremic serum+200 μmol/L LC) .Erythrocytes were collected at 24 h and 48 h.Eryptosis ( phosphatidylserine expression repre-sents eryptosis) was estimated by flow cytometry with Annexin V staining.The content of reactive oxygen species ( ROS) was also detected.Glutathione ( GSH) was measured by ELISA.RESULTS:Eryptosis in C group was increased as the in-cubating time extended.Eryptosis in U group was higher than that in C group, while that in L group was lower than that in U group.Meanwhile, ROS content was higher and GSH was lower in U group than those in C group.ROS content was low-er and GSH was higher in L group than those in C group.CONCLUSION:LC inhibits uremic serum-induced eryptosis by decreasing ROS and increasing GSH, thus attenuating oxidative stress.