RESUMO
<p><b>OBJECTIVE</b>In this study, lipopolysaccharides (LPS) was used to damage human periodontal ligament cells (hPDLCs) and consequently investigate the protective effects of hydrogen on reducing oxidative stress and cell apoptosis rate.</p><p><b>METHODS</b>hPDLCs were isolated, and then cultured with normal medium+1 μg·mL⁻¹ LPS or with hydrogen-rich medium+ 1 μg·mL⁻¹ LPS. Cell proliferation activity was assessed using a cell counting kit-8 (CCK-8), and lactic dehydrogenase (LDH) release was also detected. The activities of superoxide dismutase (SOD) and catalase (CAT), and the level of malonaldehyde (MDA) in supernatants were also measured. Cell apoptosis was detected by flow cytometry at 24 h after LPS stimulation.</p><p><b>RESULTS</b>CCK-8 results showed that hydrogen could significantly improve hPDLCs growth and decrease cell apoptosis under LPS stimulation (P<0.05). However, no significant difference in LDH release was found between the two groups. The CAT levels significantly increased at 6 and 12 h in the hydrogen-rich medium as compared with the normal medium group (P<0.05, P<0.01, respectively). However, SOD levels were not significant different at each time point. At 6 h after LPS stimulation, the MDA levels in the cell supernatant of hydrogen-rich medium group were significantly reduced as compared with those in the normal medium group (P<0.05).</p><p><b>CONCLUSIONS</b>The hydrogen-rich medium can effectively improve hPDLCs proliferation activity and antioxidant capacity and reduce apoptosis and oxidative stress under LPS stimulation.</p>
RESUMO
Objective To investigate the effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction of human intestinal epithelial (Caco2) cells. Methods Caco2 cells (passages 28-35) were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences in Shanghai, China, and they were cultured in Dulbecco minimum essential medium (DMEM) containing 20% fetal bovine serum. These cells were randomly divided into four groups: control group (group A), hydrogen-rich medium group (group B), LPS group (group C) and LPS + hydrogen-rich medium group (group D). Cells were cultured with normal medium in group A and group C or with hydrogen-rich medium in group B and group D. Meanwhile, 1 g/L LPS was simultaneously added into group C and group D, while an equivalent volume of normal saline was added into group A and group B instead. In vitro intestinal epithelial models were reproduced with monolayer filter-grown Caco2 and intestinal epithelium. The trans-epithelial electrical resistance (TEER) in models of each group was measured at different incubation times (0, 3, 6, 12, 24 and 48 hours). Cell viability and cytotoxicity were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay, respectively, after incubation for 24 hours. The expression levels of claudin-1 and occludin were respectively determined at 6, 12 and 24 hours of incubation by Western Blot assay. The morphological structure of claudin-1 and occludin was respectively observed after incubation for 24 hours with immunofluorescence staining. Results There was no statistical significance in variables between group A and group B. Compared with group A, it was shown that TEER was time-dependently decreased in groups C and D after 6 hours. Compared with group C, TEER in group D was increased after 6 hours. Compared with group A, the cell viability was significantly reduced in group C [(67.2±7.9)% vs. (100.0±0.0)%, P < 0.05] and cell injury was obvious [LDH release rate: (38.5±2.1)% vs. (1.2±0.3)%, P < 0.05]; the expression levels of claudin-1 and occludin at 6, 12, 24 hours were significantly down-regulated [claudin-1 (gray value): 0.351±0.079, 0.272±0.075, 0.190±0.049 vs. 0.518±0.030; occludin (gray value): 0.416±0.044, 0.290±0.062, 0.226±0.019 vs. 0.602±0.038, all P < 0.05], and the structure of claudin-1 and occludin were profoundly disrupted. Compared with group C, it was shown that the cell viability was significantly increased in group D [(88.8±7.4)% vs. (67.2±7.9)%, P < 0.05] and cell injury was significantly abated [LDH release rate: (16.4±4.3)% vs. (38.5±2.1)%, P < 0.05]; the expression levels of claudin-1 and occludin were significantly up-regulated at 24 hours [claudin-1 (gray value): 0.428±0.046 vs. 0.190±0.049, occludin (gray value): 0.466±0.071 vs. 0.226±0.019, both P < 0.05]; the disrupted structures of claudin-1 and occludin were partially recovered. Conclusion Hydrogen-rich medium can effectively attenuate LPS-induced dysfunction of intestinal epithelial barrier in human Caco2 cells by ameliorating cell viability as well as regulating claudin-1 and occludin expression and structure.