ABSTRACT
Objective To investigate whether GLT25D2 gene regulates autophagy in acetaminophen (APAP)-induced hepatotoxicity injury.Methods GLT25D2+/+ wild-type C57BL/6J mice and GLT25D2-/- C57BL/6J mice were selected as subjects. ①In vivo experiment: 20 for wild-type mice and 20 for GLT25D2-/- mice were respectively divided into phosphate buffer (PBS) control group and APAP intervention group according to random number table, with 10 mice in each group. The hepatotoxicity injury model of mice was reproduced by intraperitoneal injection of 25 g/L APAP solution 500 mg/kg. The PBS control group was intraperitoneally injected with the same amount of PBS. The mice were sacrificed immediately after model reproduction, and the liver tissues were harvested. Western Blot was used to detect the expressions of autophagy-related proteins ATG5, ATG7, microtubule-associated protein 1 light chain 3 (LC3) and P62. The ultrastructural changes in liver tissue were observed under electron microscope to observe the level of autophagy. ②In vitro experiment: primary hepatocytes extracted by two-step collagen perfusion from one GLT25D2+/+wild-type mouse and one GLT25D2-/- mouse were divided into two parts respectively. One part was treated with 5 mmol/L APAP solution. The cells were harvested at 0, 8, and 12 hours, and the expressions of autophagy-related proteins ATG5, ATG7, LC3, and P62 were determined by Western Blot. The other part was transfected with the green fluorescent protein-LC3 plasmid (GFP-LC3) for 24 hours. The cells were cultured with PBS (PBS control group) or 5 mmol/L APAP (APAP intervention group) for 12 hours, and the positive expression of GFP-LC3 was observed under the fluorescence microscope, thereby reflecting the expression of autophagosomes.Results ①In vivo experiment: compared with the corresponding PBS control group, the expressions of the positive-associated proteins ATG5, ATG7 and LC3-Ⅱ in liver tissue of the APAPintervention group were down-regulated in the wild-type and GLT25D2-/- mice, while the expression of the negative correlation protein P62 was up-regulated, indicating that the overall level of autophagy decreased after treatment with APAP. Compared with wild-type mice, the expressions of autophagy positive correlation proteins ATG5 and ATG7 were up-regulated in GLT25D2-/- mice (ATG5/β-actin: 1.21±0.29 vs. 0.84±0.19, ATG7/β-actin:1.29±0.14 vs. 1.54±0.40, bothP > 0.05), LC3-Ⅱ expression was slightly down-regulated (LC3-Ⅱ/β-actin: 0.52±0.06 vs. 0.58±0.06,P > 0.05), while negative correlation protein P62 was down-regulated (P62/β-actin: 1.13±0.94 vs. 1.54±0.40,P > 0.05), indicating that the expression of autophagy in GLT25D2-/- mice was higher than that in wild-type mice. Ultrastructural observation under electron microscope showed that the number of autophagosomes in the liver tissue of wild-type mice did not change significantly after APAP intervention as compared with that in PBS control group, but the number of autophagosomes in GLT25D2-/- mice was increased. ②In vitro experiment: with the prolongation of APAP intervention, the expressions of ATG5 and ATG7 in the primary hepatocytes of wild-type and GLT25D2-/-mice were up-regulated, LC3 was slightly fluctuated, and the expression of negative-related protein P62 was gradually down-regulated. The peak value or the trough value reached at 12 hours. It was indicated that the expression of autophagy in APAP-stimulated cells was enhanced with a time-dependent manner. Compared with wild-type mice, the expressions of autophagy correlation proteins ATG5, ATG7, LC3-Ⅱ and P62 were up-regulated in GLT25D2-/- mice at 12 hours (ATG5/β-actin: 0.93±0.09 vs. 0.74±0.06, ATG7/β-actin: 0.80±0.09 vs. 0.65±0.10, LC3-Ⅱ/β-actin:1.35±0.30 vs. 1.15±0.20, P62/β-actin: 0.36±0.02 vs. 0.31±0.03, allP > 0.05), indicating that the expression of autophagy was enhanced after gene knockout. Fluorescence microscopy showed that GFP-LC3 positive cells in both wild-type and GLT25D2-/- mice hepatocytes were significantly increased after APAP intervention as compared with those of PBS control group, and the proportion of GFP-LC3 positive cells in GLT25D2-/- mice was significantly higher than that in wild-type mice (0.64±0.08 vs. 0.36±0.05,P < 0.05).Conclusions GLT25D2 is a negative regulator of autophagy. Knockout of GLT25D2 gene can enhance the autophagy level of APAP-induced hepatotoxicity injury in mice.
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Objective To investigate the local or systemic effects of initial periodontal therapy on peritoneal dialysis in patients with chronic kidney disease and periodontitis. Methods Sixty-one patients with both periodontitis and regular peritoneal dialysis were selected in this study and were randomly divided into observation group (n=31) and control group (n=30). Patients in observation group were given periodontal initial therapy (ultrasonic supragingival scaling and ultrasonic subgingival irrigation, drug, root planing, oral health education) and peritoneal dialysis treatment. Patients in control group were given only peritoneal dialysis treatment. Clinical parameters including plaque index (PLI), gingival index (GI) and probing depth (PD) were detected before and one month after treatment in two groups. The concentrations of high-sensitivity C-reactive protein (hs-CRP) in gingival sulcus fluid and serum samples were measured by immune transmission turbidity method in two groups. Results There were no significant differences in PLI, GI, PD, hs-CRP of gingival sulcus fluid, and serum hs-CRP before treatment between the two groups. After one-month initial therapy, all the indexes were decreased in observation group, which were significantly different compared with those before treatment (P<0.05). And all the indexes were significantly lower in observation group than those of control group. There were no significant differences in PLI, PD, GI, hs-CRP of gingival sulcus fluid, and serum hs-CRP between control group and observation group before treatment. Conclusion Periodontal initial treatment can improve the periodontal inflammation in patients with chronic kidney disease, and reduce the concentration of systemic inflammatory factor hs-CRP, decreasing the risk of infection.
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Objective To evaluate effects of glucocorticoids on maxillary bone mineral density in rats with acute adriamycin-induced nephrotoxicity (ADR). Methods Forty rats were randomly divided into four groups, control group, glucocorticoids- treated group, ADR group and ADR + glucocorticoids- treated group. ADR group and ADR +glucocorticoids-treated group were given 4 mg/kg adriamycin injection via tail vein to establish ADR model. Control group and glucocorticoids-treated group were given 4 mg/kg saline injection via tail vein. After establishment of ADR model, glucocorticoids-treated group and ADR + glucocorticoids-treated group were intragastric administration of 30 mg/(kg · d) methylprednisolone for 10 weeks, and control group and ADR group were given the same volumes of normal saline. Values of bone calcium pigment (BGP), type Ⅰ collagen, N-terminal pro-peptide (PINP), β-Ⅰ type collagen C-terminal cross-linked telopeptide (CTX) were detected by ELISA. The micro-CT scan was used to measure Tb.Th, Tb.Sp, Tb.N, BVF and bone mineral density (BMD). Results Compared with other three groups, the levels of BGP and PINP were significantly decreased, and CTX were significantly increased in ADR + glucocorticoids-treated group (P<0.05). Micro-CT analysis showed that there was significant maxillae osteoporosis, including changes of porous micro architecture, lower BMD, decreased BVF, lower Tb.Th and widening Tb.Sp in ADR + glucocorticoids-treated group (P<0.05). There was no significant difference in Tb.N between four groups. Conclusion There is imbalanced bone metabolism in rat model of ADR. High-dose hormone therapy can accelerate the occurrence of osteoporosis, decrease bone metabolism, and affect bone structure.