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OBJECTIVE@#To investigate enterovirus 71 (EV71)-induced of autophagy, apoptosis and the related signaling pathways in THP-1 macrophages.@*METHODS@#THP-1 macrophages were infected with EV71 at the multiplicity of infection (MOI) of 0.1 for 2, 8 or 16 h, and the cell proliferation and toxicity were analyzed using CCK-8 kit. The intracellular viral nucleic acid in THP-1 macrophages were detected by fluorescence quantitative PCR, and the ultrastructural changes of the cells were observed using transmission electron microscopy. Cell apoptosis induced by EV71 infection was detected using Hoechst 33342 staining and AnnexinV/PI double staining. Western blotting was performed for analysis of changes in autophagy and apoptosis of the cells and in the expressions of the related proteins. The effect of EV71 infection on apoptosis of THP-1 macrophages incubated with 3-MA and Ac-DEVD-CHO inhibitor for 2 h was assessed using Western blotting.@*RESULTS@#EV71 infection significantly lowered the cell survival rate of THP-1 macrophages at 2, 8 h and 16 h after the infection ( < 0.05). The total copy number of viral nucleic acid in THP-1 macrophages incubated with EV71 increased significantly and progressively over time ( < 0.01). Intracellular autophagosomes and virions could be seen in EV71-infected THP-1 macrophages. The total apoptotic rate of the infected cell also increased significantly over time ( < 0.01). EV71 infection significantly increased LC3 conversion (LC3-Ⅱ/ LC3-I) and the expression of cleaved caspase 3 protein and decreased the protein expressions of p62, Bcl-2 and caspase-3 ( < 0.01) without causing obvious changes in cleaved caspase-8 (>0.05). 3-MA significantly inhibited the EV71-induced autophagy of THP-1 macrophages and reduced LC3 conversion (LC3-Ⅱ/LC3-I) and p62 protein expression at 8 h after EV71 infection ( < 0.01). Compared with DMSO, Ac-DEVD-CHO significantly inhibited EV71-induced apoptosis of THP-1 macrophages (15.5% 7.7%, < 0.01).@*CONCLUSIONS@#EV71 not only can infect and replicate in THP-1 macrophages, but also induces autophagy and cell apoptosis possibly by activating LC3/p62 autophagy pathway and caspase apoptosis pathway.
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Humans , Apoptosis , Autophagy , Cell Line , Enterovirus A, Human , MacrophagesABSTRACT
Objective To observe the changes of renal tubular injury and the extent of interstitial fibrosis in the C57BL/6 mouse models of chronic kidney disease(CKD),and provide experimental animal evidence for study of the pro-gression of acute kidney injury(AKI)to chronic kidney disease as well as its mechanisms. Methods Twenty-four 8-week-old male C57BL/6 mice were randomly and equally divided into control group, low-dose, medium-dose, and high-dose cisplatin groups,6 mice in each group. Mice in the cisplatin groups were administrated with 5,7 or 10 mg/kg cispla-tin by intraperitoneal injection once a week for 4 weeks. Plasma creatinine and 24-hour urinary protein were detected to as-sess the renal function. The mice were sacrificed, and plasma and kidney samples were collected for subsequent tests. Pathological changes were observed using periodic acid-Schiff(PAS)staining. To evaluate renal tubules injury, the ex-pression of kidney injury molecule 1(KIM-1)was examined by immunohistochemistry and the level of urinary N-Acetyl-β-D-glucosaminidase was detected with a commercial kit. The infiltration of CD3-positive T cells and F4/80-positive macro-phages was observed by immunohistochemistry(IHC)and immunofluorescence. The expression of collagen I and α-smooth muscle actin(α-SMA)were tested by immunohistochemistry to assess the renal fibrosis, while total kidney collagen was detected by Picrosirius red staining. Results In contrast to the normal control group,the kidney injury became more seri-ous in the cisplatin-treated mice as cisplatin concentration increased. Particularly,significant kidney damage was observed in the high-dose cisplatin group. Compared with the control group,the plasma creatinine and 24-hour urinary protein were significantly increased in the high-dose cisplatin group(P<0.05 and P<0.001)indicating impaired renal function. Mor-phologically,numerous clear vacuoles and necrosis were present in renal tubule epithelial cells in the high-dose cisplatin group. The expression of KIM-1 was markedly up-regulated and the level of urinary NAG was elevated. Infiltration of CD3-positive T cells and F4/80-positive macrophages was enhanced in the mice of high-dose cisplatin group. Data from immuno-histochemistry and picrosirius red staining showed that mice of the high-dose cisplatin group developed renal fibrosis evi-denced by markedly up-regulated expression of collagen I and α-SMA. Conclusions Repeated administration of 10 mg /kg cisplatin for 4 weeks can induce chronic renal insufficiency in mice,which may serve as a novel model for the research on underlying mechanisms of progression from acute kidney injury to chronic kidney disease.
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Objective:To investigate the activation of peripheral basophils from patients with rheumatoid arthritis ( RA) and its mechanisms. Methods:The activation markers including CD203c and the proportion of IL-4 positive rate of peripheral basophils from RA patients and healthy controls were detected by flow cytometry. Serum levels of IgE in RA patients and healthy controls were detected by electrochemilu minescence immunoassay. Basophils were negatively isolated and co-cultured with or without purified IgE,anti-IgE as positive control,then,the expression of CD203c and propotion of IL-4 positive basophils were detected by flow cytometry. Results:The expression of CD203c and IL-4 positive rate of basophils from RA patients were higher than that of healthy controls (P<0. 05). Serum levels of IgE in RA were higher than that of healthy controls (P<0. 05). After co-cultured with isolated IgE from RA patients,basophils negatively isolated from healthy controls were activated,and higher expression of CD203c and proportion of IL-4 (P<0.05). Conclusion:Basophil activation is related with development of RA,and its activation is mainly mediated by IgE. Targeting basophil and its activation pathway would be expected to provide new strategies for the treatment of RA.
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Objective To investigate the effect of cyclosporine A (CsA) on autophagylysosomal pathway in tubular epithelial cells.Methods Human renal tubular epithelial cell line (HK-2 cell) was treated with different concentrations (3,5 and 10 μmol/L) of CsA for 24 h.Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV-PI staining followed by flow cytometry analysis,respectively.Autophagy-related protein LC3-Ⅱ and p62 were detected by immunofluorescence assay.Autophagic flux was analyzed in HK-2 cells transfected with a tandem mRFP-GFP fluorescent-tagged LC3 (ffLC3) plasmid by laser confocal microscope.The lysosomal degradation was evaluated by DQ-ovalbumin staining followed by flow cytometry analysis.Results The viability of HK-2 cells was significantly decreased with CsA stimulation when compared with control group (P ＜ 0.01),but the number of apoptotic cells was markedly increased by CsA treatment (P ＜ 0.05).Compared with the control group,different doses of CsA dramatically increased the expressions of LC3-Ⅱ (P ＜ 0.01) and p62 (P ＜ 0.05) in HK-2 cells.Moreover,HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes.In HK-2 cells,exposured to CsA caused a decrease in lysosomal degradation by DQ-ovalbumin staining when compared with control group (P ＜ 0.01).Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.
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[ ABSTRACT] AIM:To investigate the effects of pathological products, urinary proteins and advanced glycosyla-tion end products ( AGE) produced in the progression of chronic kidney disease ( CKD) , on the structure and function of lysosomes in renal tubular epithelial cells ( TECs ) , and try to find a novel approach for preventing or delaying CKD. METHODS:The renal specimens of the untreated patients with minimal change nephrotic syndrome (MCNS), diabetic nephropathy (DN) or normal kidney were collected.The expression of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B ( CB) was studied in TECs by indirect immunofluorescent staining.Human renal tubular epithelial cell line HK-2 was incubated with 8 g/L urinary proteins or 100 mg/L AGE.The expression of LAMP1 and CB was investigated by indirect immunofluorescence and the activity of CB and cathepsin L ( CL) was measured by biochemical and enzymatic as-says.The degradation of DQ-ovalbumin was also determined.RESULTS: The lysosomal membrane permeabilization oc-curred in the TECs of MCNS and DN patients.After treatment with urinary proteins or AGE-BSA, the lysosomal membrane permeabilization of the HK-2 cells was increased.The activity of CB and CL and degradation of DQ-ovalbumin were de-creased as compared with normal control group.CONCLUSION:The digestive function of lysosome was decreased and ly-sosomal membrane permeabilization occurred in the TECs exposed to urinary proteins and AGE, which might be a key factor to induce the tubulointerstitial fibrosis.
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Objective:To investigate levels of autophagy in T cells and B cell of patients with systemic lupus erythematosus ( SLE) and its clinical significance.Methods: 68 SLE patients without treatment within 4 weeks were enrolled in this study.We accessed the levels of autophagy in T cells and B cells of 23 healthy controls and 68 patients before and after treatment by flow cytometry,and analyzed their correlations with serum levels of C3 and anti-dsDNA antibodies,SLEDAI score,et al.Results: Before treatment,a significantly increased levels of LC3-Ⅱ was observed in SLE patients than healthy controls, the active group ( SLEDAI score≥10) was significantly higher than the stable group(SLEDAI score0.05 ) . Conclusion:Levels of autophagy in T and B lymphocytes of SLE patients are abnormal compared to healthy controls,and these changes are associated with disease activity.Also,these changes are expected to be the indicators of disease activity and potential therapeutic targets in SLE.
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Objective To investigate the role of basophils in the imbalance of Th 1/Th2 response in mouse models of collagen-induced arthritis(CIA).Methods 4-6-weeks old C57/BL6 mice were immunized with collagen at multiple points on the back and foot twice (0 and 3 weeks) to establish a mouse model of collagen-induced arthritis.Blood samples were collected before the first immunization and 1, 3, 6 and 9 weeks after immunization , and cells from lymph nodes were collected.Flow cytometry and ELISA were employed to detect the levels of basophils and IL-4, and the joint swelling was scored.Results Mouse model of CIA was successful established .The ratio of IL-4/IFN-γof the CIA group was significantly lower than that in the mice before CIA modeling and the control group , indicating a Th2-dominant response .At the same time, the peripheral basophils counting and percentage of IL-4 positive basophils of the CIA group were significantly higher than those of the control group .While, the IL-4/IFN-γratio of the CIA group was significantly higher than that of the control group , indicating a Th1-dominant response .The peripheral basophils counting of the CIA group was slightly lower than that of the control group .Conclusion Basophils may participate in the development of CIA in mouse models through affecting the imbalance of Th 1/Th2 response.