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1.
Biol. Res ; 57: 2-2, 2024. ilus, graf
Artículo en Inglés | LILACS | ID: biblio-1550057

RESUMEN

BACKGROUND: Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. RESULTS: Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-ß, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. CONCLUSIONS: Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.


Asunto(s)
Humanos , Interferón Tipo I , alfa-Sinucleína , SARS-CoV-2 , COVID-19 , Replicación Viral , Línea Celular , Células Endoteliales
2.
International Eye Science ; (12): 508-514, 2024.
Artículo en Chino | WPRIM | ID: wpr-1012812

RESUMEN

AIM: To investigate the effect of inhibiting Ca2+/calmodulin-dependent protein kinase Ⅱ(CAMKⅡ)expression in adult retinal pigment epithelial cell line-19(ARPE-19)cells on the migration, invasion, and tube formation of human umbilical vein endothelial cells(HUVECs)in a non-contact co-culture system.METHODS: RNA sequencing was performed on ARPE-19 cells overexpressing CAMKⅡ-δ, and bioinformatics was used to analyze the biological functions of the differentially expressed genes. Transwell inserts was used to construct a non-contact co-culture system of ARPE-19 and HUVECs. The experimental groups included: blank group: only HUVECs were inoculated without ARPE-19 cells; control group: ARPE-19 and HUVECs cells were co-cultured with complete medium; AIP group(CAMKⅡ inhibition group): ARPE-19 cells in AIP(160 nmol/L)were co-cultured with HUVECs in complete medium. The migration, invasion and tube formation abilities of HUVECs were detected. The protein expression levels of CAMKⅡ/AMPK/mTOR/VEGFA were detected by Western blotting.RESULTS:Bioinformatics analysis found that the differentially expressed genes could affect biological processes such as cell growth and death and cell movement. The scratch test and transwell migration test showed that the relative mobility of HUVECs in the AIP group was significantly lower than that in the control group(all P<0.05). However, the invasion and tube formation assay showed that the relative invasion rate and tube formation rate of the AIP group were not significantly different from those of the control group(both P>0.05). Western blotting results showed that the expression levels of CAMKⅡ, P-mTOR, and VEGFA proteins in the AIP group were significantly lower than those in the control group, while the expression level of the P-AMPK protein was significantly higher than that in the control group(all P<0.05).CONCLUSION:In the non-contact co-culture system, inhibition of CAMKⅡ expression in ARPE-19 cells significantly reduced the migration ability of HUVECs, but it cannot change the invasion and tube formation ability, which may be achieved by AMPK/mTOR/VEGFA.

3.
Chinese Pharmacological Bulletin ; (12): 1143-1148, 2023.
Artículo en Chino | WPRIM | ID: wpr-1013902

RESUMEN

Aim To explore the effect of S1P/S1PR1 signaling pathway on high glucose(HG)-induced epithelial-mesenchymal transition of rat renal tubular epithelial cells and its possible mechanism. Methods Cells were treated with different concentrations of glucose, and intracellular S1P expression was detected by ELISA and S1PR1 protein expression was detected by Western blot. The cells were divided into normal control group, HG group and HG + siS1PR1 group. The expression of E-cadherin, Vimentin, Fibronectin and Twist mRNA were detected by RT-qPCR and E-cadherin, α-SMA, Vimentin, NLRP3, ASC and NF-κB protein expression were detected by Western blot, and the levels of reactive oxygen species(ROS) were detected by flow cytometry. The cells were divided into normal control group, S1P group and S1P + siS1PR1 group. Vimentin, Snail, α-SMA, NLRP3, ASC and NF-κB protein expressions were detected by Western blot, and ROS levels were measured by fluorescence microscopy. Results ELISA results showed that the content of S1P in cells increased significantly under high glucose stimulation. Western blot results showed that S1PR1 protein expression was significantly higher at 30 mmol · L

4.
Journal of Prevention and Treatment for Stomatological Diseases ; (12): 370-374, 2023.
Artículo en Chino | WPRIM | ID: wpr-961363

RESUMEN

@#Ankylosis of primary molars is a kind of eruption abnormality of the teeth, where the periodontal membrane disappears, owing to a bony union between bone and root. Studies have shown that the common proportion of ankylosed primary molars is 1.3%~8.9% with an equal occurrence. In the primary dentition, the mandibular first primary molar is the most commonly affected tooth, while in the middle mixed dentition stage of development, the second primary molar is more affected. Its etiology may be related to genetics, signaling pathways of mineralization metabolism of local alveolar bone or cementum, cytokines secreted by epithelial rest cells of Malassez, and enhanced inflammatory reactions during physiological absorption of roots. Ankylosis of primary molars can be diagnosed by clinical symptoms and imaging and is classified as mild, moderate and severe according to the degree of infraocclusion. As it may cause a series of complications, such as occlusal disturbances, delayed exfoliation and incomplete alveolar process development, multidisciplinary treatment, including in the departments of pediatric dentistry, orthodontics, periodontics and prosthodontics, should be adopted, and long-term treatment is determined based on the patient's age, severity of infraocclusion, and presence of permanent teeth. This review summarizes the etiology, diagnosis, complications and treatment of ankylosed primary molars to provide a reference for the clinical diagnosis and treatment of decidual molar fixation.

5.
Chinese Journal of Schistosomiasis Control ; (6): 271-278, 2023.
Artículo en Chino | WPRIM | ID: wpr-986137

RESUMEN

OBJECTIVE@#To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM).@*METHODS@#Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA.@*RESULTS@#Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells.@*CONCLUSIONS@#CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.


Asunto(s)
Animales , Humanos , Trampas Extracelulares , Neutrófilos , Interleucina-8/metabolismo , Dermatophagoides farinae , Interleucina-6/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células Epiteliales , Interferón gamma/metabolismo , Acetato de Tetradecanoilforbol/farmacología
6.
China Occupational Medicine ; (6): 38-45, 2023.
Artículo en Chino | WPRIM | ID: wpr-988917

RESUMEN

Objective: To investigate the role of surfactant associated protein-A (SP-A) in the development and progression of silicosis, and its mechanism. Methods: Homozygous and heterozygous mice of SP-A knockout of specific pathogen free (SPF) grade were selected for mating, and mice with SP-A-/- genotype were selected for subsequent experiments. SP-A wild-type (SP-A+/+) and SP-A-/- mice were divided into SP-A+/+ control group, SP-A-/- control group, SP-A+/+ silicosis group and SP-A-/- silicosis group with six mice in each group by random number table method. Mice in both silicosis groups were given 20.0 μL 250 g/L silica suspension by tracheal exposure, and mice in both control groups were injected with 0.9% sodium chloride solution at the same volume. On the 28th day after modeling, mice were sacrificed. Lung tissues were used for lung histopathology examination. The apoptosis of alveolar type Ⅱ epithelial cells of mice was detected by TUNEL method. The mRNA expression of B-lymphoblastoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), cysteinyl aspartate specific proteinase (Caspase)-3 and Caspase-9 in lung tissues of mice was detected by quantitative real-time polymerase chain reaction. Results: The histopathological result of mice showed that thickened alveolar septum, scattered silicon nodule and collagen fiber formation were observed in the mice lungs of SP-A+/+ silicosis group, and a large number of inflammatory cells were observed in silicosis nodule, after exposure to silica dust. SP-A-/- silicosis group resulted in a more severe pulmonary inflammation and interstitial fibrosis compared to SP-A+/+ silicosis group. The apoptosis of alveolar type Ⅱ epithelial cells and the mRNA relative expression levels of Bax, Caspase-3 and Caspase-9 in lung tissues of mice in each silicosis groups were increased compared with their control groups (all P<0.05). The above four indexes of mice in SP-A-/- silicosis group were higher than those in SP-A+/+ silicosis group (all P<0.05). There was no significant difference in the expression of Bcl-2 mRNA in lung tissues of these four groups (P>0.05). Conclusion: Knockout of SP-A can aggravate inflammation and pulmonary fibrosis in silicosis model mice, and promote apoptosis of alveolar type Ⅱ epithelial cells. The mechanism may be related to the Bcl-2/Bax/Caspase-3 signaling pathway which affects the apoptosis of mitochondrial pathway.

7.
Chinese Journal of Microbiology and Immunology ; (12): 182-190, 2023.
Artículo en Chino | WPRIM | ID: wpr-995272

RESUMEN

Objective:To isolate and culture WU polyomavirus (WUPyV), and to analyze the genome-wide evolutionary patterns, homology and population dynamics.Methods:Real-time quantitative PCR was used to detect the nasopharyngeal aspirate samples of hospitalized children with respiratory tract infection in Beijing Friendship Hospital during 2020 to 2022. Primary human airway epithelial cells cultured at the air-liquid interface were used to isolate and culture WUPyV. Whole genome sequence of the isolated strain was obtained by Sanger sequencing. For phylogenetic and evolutionary dynamics analysis, the whole genome was compared with the published whole genome sequences in GenBank database.Results:The detection rate of WUPyV was 4.7% (31/659) during 2020 to 2022, and a clinical strain BJ0593 of WUPyV type Ⅲc was successfully isolated. The homology of the whole genome and gene fragments of WUPyV was high. The average evolutionary rate of VP2 gene was about 1.256×10 -4 substitution/site every year, and the population dynamics of WUPyV tended to be flat in the last decade. Conclusions:This study successfully isolated a clinical WUPyV type Ⅲ strain for the first time, which provided the basis for further investigation on the molecular evolution and pathogenicity of WUPyV.

8.
Chinese Journal of Microbiology and Immunology ; (12): 93-101, 2023.
Artículo en Chino | WPRIM | ID: wpr-995261

RESUMEN

Objective:To compare gene expression profiles in normal human cervical epithelial cells (HcerEpic) before and after Chlamydia trachomatis ( Ct) infection. Methods:HcerEpic cells that were pretreated with DEAE-D were infected with Ct serotype E standard strain and then cultured for 44 h. Uninfected HcerEpic cells were used as the control group. Total RNA was extracted from the cells in each group and reverse transcribed to construct a cDNA library. Differences in gene expression profiles between the two groups were analyzed by high-throughput sequencing and the representative genes were selected for verification by qPCR. Results:A total of 23 997 genes were detected, including 125 differentially expressed genes. Among the 125 genes, 119 were up-regulated and six were down-regulated. GO analysis showed that the differentially expressed genes were enriched in several biological processes including defense response to virus, typeⅠinterferon signaling pathway and cellular responses to typeⅠinterferons. KEGG enrichment analysis showed the differentially expressed genes were mainly enriched in the pathways related to virus infections, such as influenza A virus, herpes simplex virus, EB virus and HPV, and NOD-like receptor pathway.Conclusions:There were significant differences in transcriptome profiles of HcerEpic cells before and after Ct infection. The differentially expressed genes were mainly enriched in the interferon pathway, which was closely related to the antiviral processes in cells. qPCR verified the differentially expressed genes and the genes closely related to the interferon pathway, such as ISG15, IFIT2, OASL and UBE2L6.

9.
Chinese Journal of Radiological Medicine and Protection ; (12): 321-327, 2023.
Artículo en Chino | WPRIM | ID: wpr-993092

RESUMEN

Objective:To study the influence of circular RNA hsa_circZDHHC21_004 on the proliferation of human small intestinal epithelial cells HIEC-6 after 60Co γ-rays exposure. Methods:HIEC-6 cells were exposed to 60Co γ-rays at 0, 5, 10, and 15 Gy with a dose rate of 1 Gy/min. The expression level of hsa_circZDHHC21_004 in the irradiated HIEC-6 cell was detected. Hsa_circZDHHC21_004 was knocked-down to investigate the influences of hsa_circZDHHC21_004 on the proliferation of irradiated HIEC-6 cells by CCK-8 assay and colony formation assay. Results:The expression level of hsa_circZDHHC21_004 in HIEC-6 cells was upregulated by (1.00±0.24), (1.34±0.28), (1.85±0.31), and (2.80±0.64) times of control after 0, 5, 10, and 15 Gy irradiation, respectively and there were significant difference between 10 or 15 Gy group and 0 Gy group ( F=10.86, P=0.008). Knockdown of hsa_circZDHHC21_004 significantly increased the proliferation rate of HIEC-6 cells at 24, 48, and 72 h after 10 Gy irradiation compared with non-irradiated control ( t=-6.25, -5.83, -7.75, P < 0.001). Under 2 and 5 Gy irradiation, the clone formation rates of the hsa_circZDHHC21_004 knockdown cells were significantly higher than those of the control ( t=-7.45, -8.83, P<0.01). Conclusions:Hsa_circZDHHC21_004 is increased after irradiation and influenced the proliferation of irradiated HIEC-6 cells.

10.
Chinese Critical Care Medicine ; (12): 152-157, 2023.
Artículo en Chino | WPRIM | ID: wpr-991994

RESUMEN

Objective:To observe the expression of deleted in malignant brain tumor protein 1 (DMBT1) in rat acute respiratory distress syndrome (ARDS) model induced by sepsis and its relationship with ARDS related biomarkers.Methods:Forty-eight healthy male rats were randomly divided into sham operation group (Sham group) and ARDS model group, and the rats in each group were further divided into three subgroups at 6, 12 and 24 hours after operation, with 8 rats in each subgroup. The rats in the Sham group were exposed to the cecum only, and sepsis induced ARDS model was reproduced by cecal ligation and puncture (CLP) in the ARDS model group. The general performance was observed at 6, 12, 24 hours after operation. Abdominal aortic blood of rats was collected, and the levels of DMBT1, surfactant-associated protein D (SP-D), vascular endothelial growth factor (VEGF), interleukins (IL-6, IL-10) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The lung tissues were collected, and the lung wet/dry weight (W/D) ratio was determined. The lung tissue pathological changes were observed under light microscope after hematoxylin-eosin (HE) staining, and the lung tissue injury score was evaluated. The expression of DMBT1 protein in lung tissue was determined by Western blotting. The relationship between the serum DMBT1 and SP-D, VEGF, IL-6, IL-10, lung tissue injury score were analyzed by Pearson correlation analysis.Results:Rats in the ARDS model group showed obvious pathological manifestations after operation. The alveolar structure destruction, inflammatory cell infiltration, and alveolar hemorrhage were observed under microscope. Compared with the Sham group, the lung tissue injury score and the lung W/D ratio at 12 hours after operation in the ARDS model group were significantly increased (lung tissue injury score: 3.35±0.13 vs. 1.16±0.07, lung W/D ratio: 5.36±0.44 vs. 4.38±0.35, both P < 0.05), and pulmonary edema was present, which suggested that the ARDS model caused by CLP was successfully reproduced. The results of ELISA and Western blotting showed that the levels of serum DMBT1, SP-D, VEGF and IL-6 in the ARDS model group increased gradually with time, while the level of IL-10 increased first and then decreased. Compared with the Sham group, the levels of DMBT1 in serum and the expressions of DMBT1 protein in lung tissue in the ARDS model group were significantly increased from 6 hours after operation [serum (ng/L) : 231.96±19.17 vs. 187.44±10.19, lung tissue (DMBT1/β-actin): 2.05±0.19 vs. 0.93±0.25, both P < 0.05], and the levels of SP-D, VEGF, IL-6 and IL-10 in serum were significantly increased from 12 hours after operation [SP-D (ng/L): 73.35±8.05 vs. 43.28±5.77, VEGF (ng/L): 89.85±8.47 vs. 43.19±5.11, IL-6 (ng/L): 36.01±2.48 vs. 17.49±1.77, IL-10 (ng/L): 84.55±8.41 vs. 39.83±5.02, all P < 0.05]. Pearson correlation analysis showed that serum DMBT1 was positively correlated with serum SP-D, VEGF, IL-6, IL-10 and lung injury score at 12 hours and 24 hours in the ARDS model group (12 hours: r values were 0.946, 0.942, 0.931, 0.936, 0.748, respectively; 24 hours: r values were 0.892, 0.945, 0.951, 0.918, 0.973, respectively; all P < 0.05). Conclusion:DMBT1 is a novel early biomarker of ARDS by affecting alveolar epithelial cell, alveolar capillary permeability and inflammatory response.

11.
Chinese Critical Care Medicine ; (12): 140-145, 2023.
Artículo en Chino | WPRIM | ID: wpr-991992

RESUMEN

Objective:To investigate whether microRNA-21-5p (miR-21-5p) alleviates hyperoxia-induced acute lung injury (HALI) through activating the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway by regulating apoptosis of type Ⅱ alveolar epithelial cell (AECⅡ).Methods:Seventy-two male Sprague-Dawley (SD) rats were divided into normozone-controlled group, HALI group, PI3K/Akt signaling pathway inhibitor LY294002+HALI group (LY+HALI group), miR-21-5p overexpression+LY294002+HALI group (miR-21-5p+LY+HALI group), miR-21-5p overexpression+HALI group (miR-21-5p+HALI group), and dimethyl sulfoxide (DMSO)+HALI group by random number table method with 12 rats in each group. Animal models of HALI were prepared using 95% concentrations of oxygen. The animals in the normozone-controlled group were fed normally under normoxia. Transfection of lung tissue by miR-21-5p adeno-associated viral vector AAV6-miR-21-5p was performed by instillation of 200 μL titer (1×10 12 TU/mL) through a tracheal catheter 3 weeks prior to modeling. DMSO and LY294002 were administered via the tail vein at 0.3 mg/kg 1 hour before modeling. After 48 hours of modeling, carotid artery blood was collected to detect oxygenation index (OI) and respiratory index (RI), and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect miR-21-5p expression. Lung tissue was collected, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA), and the ratio of pulmonary wet/dry weight (W/D) was determined, and the pathological changes of lung histopathology were observed under the light microscopy after hematoxylin-eosin (HE) staining. Each group was purified AECⅡ cells from 6 rats, the apoptosis rate was detected by flow cytometry, and the expression levels of phosphatase and tensin homologous gene (PTEN), and proteins from the PI3K/Akt signaling pathway were detected by Western blotting. Results:Compared with the normozone-controlled group, alveolar septal thickening and massive inflammatory cell infiltration were found after hyperoxia exposure, RI, inflammatory factors, lung W/D ratio, pathological score, AECⅡ cells early apoptosis rate, PTEN protein expression and phosphorylation level of Akt were increased, while OI and miR-21-5p expression were decreased, indicating the successful preparation of the model. After pretreatment, LY294002 could aggravate the pathological injury of lung tissue in HALI rats, RI, inflammatory factors and lung W/D ratio were further increased, and OI was further reduced compared with HALI group. At the same time, it could promote the AECⅡ cell apoptosis, further up-regulate the expression of PTEN, and reduce the phosphorylation of Akt. However, miR-21-5p pretreatment could negatively regulate PTEN, activate PI3K/Akt signal pathway, inhibit AECⅡ cell apoptosis, and reduce HALI, which was shown by the decreased level of inflammatory factors in miR-21-5p+LY+HALI group compared with LY+HALI group [TNF-α (μg/L): 100.33±3.48 vs. 116.55±2.53, IL-6 (ng/L): 141.06±3.70 vs. 161.31±3.59, IL-1β (μg/L): 90.82±3.69 vs. 112.23±2.87, all P < 0.05], RI, lung injury pathology score, lung W/D ratio, and AECⅡ cell early apoptosis rate were significantly decreased [RI: 0.81±0.02 vs. 1.05±0.07, pathology score: 0.304±0.008 vs. 0.359±0.007, lung W/D ratio: 5.29±0.03 vs. 5.52±0.08, apoptosis rate: (27.20±2.34)% vs. (34.17±1.49)%, all P < 0.05], OI and expressions of miR-21-5p were significantly increased [OI (mmHg, 1 mmHg≈0.133 kPa): 266.71±2.75 vs. 230.12±4.04, miR-21-5p (2 -ΔΔCt): 2.21±0.13 vs. 0.33±0.03, both P < 0.05], and PTEN protein expression in AECⅡ cell was significantly reduced (PTEN/GAPDH: 0.50±0.06 vs. 0.93±0.06, P < 0.05), and phosphorylation level of Akt was significantly increased [phosphorylated Akt (p-Akt) protein (p-Akt/GAPDH): 0.86±0.05 vs. 0.56±0.06, P < 0.05]. Conclusion:miR-21-5p attenuates HALI by inhibiting AECⅡ cell apoptosis, possibly through negative regulation of PTEN to activate PI3K/Akt signaling pathway.

12.
Chinese Journal of Experimental Ophthalmology ; (12): 226-232, 2023.
Artículo en Chino | WPRIM | ID: wpr-990836

RESUMEN

Objective:To investigate the antagonistic effect and potential mechanism of specific AKT activator SC79 on the apoptosis of human retinal pigment epithelial (ARPE)-19 cells induced by high glucose in vitro. Methods:The ARPE-19 cells were cultured in high glucose medium (containing 30 mmol/L glucose) plus 5, 10 or 20 μg/ml SC79, respectively.After 6-, 12- and 24-hour culture, the optimal experimental concentration and timing were determined according to cell proliferation rate.Then ARPE-19 cells were divided into four groups, normal control group cultured in normal medium containing 5.6 mmol/L glucose for 48 hours, mannitol group cultured in medium containing 5.6 mmol/L glucose and 24.4 mmol/L mannitol for 48 hours, high glucose group cultured in high glucose medium for 48 hours, and high glucose+ SC79 group cultured in normal medium containing 10 μg/ml SC79 for 12 hours plus in high glucose medium for 36 hours.The proliferation rate of APRE-19 cells was detected by MTS assay.The apoptosis rate was measured by flow cytometry.The relative expression levels of phosphorylated protein kinase B (p-Akt), X-linked inhibitor of apoptosis protein (XIAP), caspase-9, caspase-3 and its active fragments (active-caspase-3) were assayed by Western blot.The ARPE-19 cells were divided into Neg-shRNA group, AKT shRNA group and blank control group and were treated with the corresponding transfection complex and serum-free medium.The AKT mRNA expression was detected by real-time PCR.The transfected ARPE-19 cells were divided into Neg-shRNA+ SC79 group and AKT shRNA+ SC79 group and were cultured according to the culturing method of high-glucose+ SC79 group.The apoptosis rate of the two groups was tested by flow cytometry.Results:Among different concentrations of SC79 and treatment times, the proliferation rate of cells treated with 10 μg/ml SC79 for 12 hours was the highest.The proliferation rate of ARPE-19 cells in high-glucose group was significantly lower than that in normal control group, mannitol group and high-glucose+ SC79 group, and the differences were statistically significant (all at P<0.01). The apoptosis rate of cells in the high-glucose group was (52.27±3.21)%, which was significantly higher than (3.90±0.71)% in normal control group and (20.70±3.62)% in high-glucose+ SC79 group (both at P<0.01). The relative expression levels of p-Akt, XIAP, caspase-9 and caspase-3 were significantly lower and the relative expression level of active-caspase-3 was significantly higher in high glucose group than those in normal control group and high-glucose+ SC79 group (all at P<0.05). The relative expression level of AKT mRNA in normal control group, Neg-shRNA group and AKT shRNA group was 0.60±0.07, 0.59±0.03 and 0.11±0.10, respectively, showing a statistically significant difference among the groups ( F=30.44, P<0.01). The apoptosis rate of cells in the AKT shRNA+ SC79 group was significantly higher than that in high-glucose+ SC79 group and Neg-shRNA+ SC79 group (both at P<0.001). Conclusions:SC79 can partially antagonize the apoptosis of ARPE-19 cells induced by high glucose, which is related to the activation of AKT/XIAP pathway and the inhibition of the caspase family.

13.
Chinese Journal of Schistosomiasis Control ; (6): 271-278, 2023.
Artículo en Chino | WPRIM | ID: wpr-978515

RESUMEN

Objective To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). Methods Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA. Results Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells. Conclusions CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

14.
Acta Pharmaceutica Sinica ; (12): 1-8, 2023.
Artículo en Chino | WPRIM | ID: wpr-964304

RESUMEN

Inflammatory bowel disease (IBD) is a group of chronic idiopathic colorectal inflammatory diseases with a progressive and unpredictable course, including ulcerative colitis (UC) and Crohn's disease (CD). Abnormal intestinal inflammation and immune response contribute to the pathogenesis of IBD. Autophagy as an essential catabolic process in cells, has been demonstrated to have associations with a variety of inflammatory diseases including IBD. Here, we review the relationship between autophagy dysfunction and the process of IBD. The progress of several autophagy regulators for intestinal epithelial cells and macrophages is highlighted (inflammasome inhibitors, intestinal flora regulators, and other signal regulators) in the current studies on IBD.

15.
Arq. bras. oftalmol ; 85(4): 389-398, July-Aug. 2022. tab, graf
Artículo en Inglés | LILACS-Express | LILACS | ID: biblio-1383816

RESUMEN

ABSTRACT Purpose: To systematically examine the dynamic changes and time sequence in corneal epithelial cell apoptosis after excessive ultraviolet B irradiation. Methods: Ultraviolet B (144 mJ/cm2) was used to irradiate rat corneal epithelial cells for 2 h. Cell morphology was observed on differential interference contrast microscopy, and the numbers of the different kinds of apoptotic cells were counted using the ImageJ software. Cell viability was measured with the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide method. Cell apoptotic rate and loss of mitochondrial membrane potential were detected using flow cytometric analyses. The expression levels of 3 apoptotic genes were measured with real-time quantitative polymerase chain reaction at different time points within 0-24 h after irradiation. Results: After 144-mJ/cm2 ultraviolet B irradiation for 2 h, the expression levels of caspase-8 and Bax were highest at 0 h; furthermore, the mitochondrial membrane potential decreased at 0 h and remained constant for 6 h in a subsequent culture. At 6 h, caspase-3 was activated. The decrease in cell viability and increase in apoptotic rate peaked at 6 h. The caspase-3 expression level decreased within 12-24 h, which led to a decline in apoptotic rate and change in apoptotic stage. Conclusions: The corneal epithelial cells exhibited rapid apoptosis after ultraviolet B irradiation, which was associated with both extrinsic and intrinsic pathways.


RESUMO Objetivos: Explorar sistematicamente as mudanças dinâmicas e a sequência temporal no processo de apoptose de células epiteliais corneanas após excesso de irradiação com ultravioleta B. Métodos: A radiação ultravioleta B (144 mJ/cm2) foi utilizada para irradiar células epiteliais da córnea de rato durante 2h. A morfologia celular foi observada por meio de microscópio de contraste de interferência diferencial, e os números de diferentes tipos de células apoptóticas foram contados e registrados pelo software ImageJ. A viabilidade celular foi medida pelo método brometo de 3- (4, 5-dimetil-2-tiazolil) -2, 5-difenil-2-H-tetrazólio. A taxa apoptótica celular e a perda do potencial da membrana mitocondrial foram detectadas por meio de análises citométricas de fluxo. Os níveis de expressão de três genes apoptóticos foram medidos por reação em cadeia da polimerase quantitativa em tempo real em diferentes momentos dentro de 0-24 h após a irradiação. Resultados: Após 144 mJ/cm2 de irradiação com ultravioleta B por 2h, os níveis de expressão de caspase-8 e Bax foram maiores em 0h; o potencial da membrana mitocondrial diminuiu a 0h e permaneceu constante por 6h na cultura subsequente. Às 6h, a caspase-3 foi ativada. A diminuição da viabilidade celular e o aumento da taxa apoptótica atingiu o pico em 6h. A expressão de caspase-3 diminuiu dentro de 12 - 24 h, levando a um declínio na taxa apoptótica e alteração no estágio apoptótico. Conclusões: As células epiteliais da córnea apresentaram uma apoptose rápida após excesso de irradiação com ultravioleta B, e esse processo foi associado tanto à via extrínseca como à via intrínseca.

16.
Chinese Journal of Biochemistry and Molecular Biology ; (12): 809-815, 2022.
Artículo en Chino | WPRIM | ID: wpr-1015696

RESUMEN

Breast cancer is a malignant tumor with high mortality, and multidrug resistance (MDR) mediated by ABCG2 (ATP-Binding cassette G2) is an important cause of chemotherapy failure. It is an urgent problem to explore the mechanism of ABCG2-mediated drug resistance and its key molecules. Epithelial cell adhesion molecule (EpCAM) is involved in multiple tumor drug resistance and is closely related to breast cancer MDR. However, its role in ABCG2-mediated breast cancer drug resistance has not been clarified. The purpose of this study was to explore the regulation of EpCAM on ABCG2-mediated MDR in breast cancer cells and its mechanism. CCK8 cytotoxicity assays confirmed that the drug resistance of MCF-7/MX cell line to mitoxantrone (MX) was significantly increased compared with MCF-7 drug-sensitive strain of human breast cancer. Western blotting results showed that ABCG2 was highly expressed and EpCAM was up-regulated in MCF-7/MX cells compared with MCF-7. SiRNA knockdown of EpCAM in MCF-7/MX cells down-regulated ABCG2 expression and restored sensitivity to MX. Cell morphology was observed under an inverted microscope, and it was found that knocking down EpCAM reduced cell-cell connections between MCF-7/MX cells. The co-localization of EpCAM and claudin 1 in MCF-7/MX cells was observed by immunofluorescence. Furthermore, Western blotting results showed that EpCAM knockdown reduced claudin 1 expression in MCF-7/MX cells. In conclusion, EpCAM may promote ABCG2-mediated mMDR in breast cancers by enhancing intercellular tight junctions through interaction with claudin 1.

17.
Chinese Pharmacological Bulletin ; (12): 1147-1155, 2022.
Artículo en Chino | WPRIM | ID: wpr-1014027

RESUMEN

Aim To study the effect of different hepa- ry.Methods First, heparin derivatives with different rin sulfation patterns on bleomycin induced lung inju- sulfation patterns,6-desulfated heparin (6-DeH) and N-acetvlated heparin ( N-AcH ) , were synthesized.Secondly, the effect of these compounds on BLM-in¬duced bronchial epithelial cell ( BEARS-2B) injury was evaluated via lactate dehydrogenase activity, MTT experiment, Annexin V/ PI staining and Hoechst 33258 staining.Then , immunofluorescence staining and West¬ern blotting were used to investigate the shedding of Svndecan-1 and the activation of c-Met by 6-DeH/Akt j j signaling pathway.Finally, a BLM-induced lung injury mouse model was used to further verify the protective effect of 6-DeH by HE staining, Svndecan-1 immunos- taining,bodv weight change,and survival rate.Results In the BLM-induced BEARS-2B injury model, 6- DeH was selected as the best candidate, which exerted their effect by competitively binding to BLM, thereby reducing the damage of heparan sulfate barrier (Svnde- can-1 ) on cell surface, and improving cell survival by activating the downstream c-Met/Akt pathway.In the BLM-induced lung injury mouse model, it was further confirmed that 6-DeH reduced the shedding of Svnde- can-1 in the early stage, and delayed the lung injury and fibrosis process.Conclusions 6-DeH protects the bronchial epithelial cells against BLM-induced lung in¬jur)' through inhibiting the shedding of Svndecan-1 and activating the c-Met/Akt signaling pathway.

18.
Journal of Environmental and Occupational Medicine ; (12): 708-713, 2022.
Artículo en Chino | WPRIM | ID: wpr-960469

RESUMEN

Fine particulate matter (PM2.5) has attracted more and more attention in the field of environmental pollution and public health. Previous studies have found that PM2.5 can be inhaled and deposit in the airway and alveoli, and even spread to the whole-body tissues and organs through blood, resulting in various toxic effects. The malignant transformation of lung epithelial cells associated with long-term exposure to PM2.5 may play an important role in the occurrence and development of lung cancer. This paper reviewed recent studies on the mechanisms of malignant transformation of lung epithelial cells associated with PM2.5 exposure, and discussed the main biological mechanisms, including epigenetics, tumor microenvironment, and other biological pathways. Besides, the potential research directions of malignant transformation of lung epithelial cells associated with PM2.5 exposure were proposed. This work aims to provide a scientific basis and reference for public health management and air quality assessment.

19.
International Eye Science ; (12): 373-377, 2022.
Artículo en Chino | WPRIM | ID: wpr-920402

RESUMEN

@#AIM: To investigate the synthesis method of curcumin nanoparticles grafted with deoxycholic acid and the effect of curcumin nanoparticles on human retinal pigment epithelial(hRPE)cells. <p>METHODS: The synthesis and performance analysis of Cur/Chit-DC. The relationship between FITC/Chit-DC and hRPE cells was observed under an inverted fluorescence microscope after treating hRPE cells with FITC(FITC/Chit-DC)and Cur/Chit-DC(FITC/Cur/Chit-DC)for 24h, keeping them in dark for 1, 3 and 5d respectively.<p>RESULTS: By mixing Cur and Chit-DC, the nanoparticles containing chitosan derivatives were light yellow. The drug release from the nanoparticles reached equilibrium after 96h, and the cumulative drug release amount was 31.6%. After FITC/Chit-DC was treated with hRPE cells for 1d, most of Chit-DC nanoparticles were still located near the cell membrane. After 3d, the nanoparticles gradually converged towards the nucleus and most of them were located around the nucleus. After 5d, it was observed that Chit-DC nanoparticles had entered the nucleus and were partially degraded under the action of intracellular lysosomes. The relationship between Cur/Chit-DC and cellular action is roughly the same as the relationship between Chit-DC and cellular action. <p>CONCLUSION: Cur can be continuously released from Cur/Chit-DC nanoparticle, which has long-lasting sustained-release function.

20.
Chinese Critical Care Medicine ; (12): 1132-1137, 2022.
Artículo en Chino | WPRIM | ID: wpr-991929

RESUMEN

Objective:To investigate the role of cholinergic anti-inflammatory pathway in the regulation of peptide transporter 1 (PepT1) expression in small intestinal epithelium of septic rats by Ghrelin.Methods:One hundred adult male Sprague-Dawley (SD) rats were randomly divided into sham operation group, sepsis group, sepsis+vagotomy group, sepsis+Ghrelin group, and sepsis+vagotomy+Ghrelin group, with 20 rats in each group. In the sham operation group, the cecum was separated after laparotomy, without ligation and perforation. In the sepsis group, the rats received cecal ligation puncture (CLP). In the sepsis+vagotomy group, the rats received CLP and vagotomy after laparotomy. In the sepsis+Ghrelin group, 100 μmol/L Ghrelin was intravenously injected after CLP immediately. The rats in the sepsis+vagotomy+Ghrelin group received CLP and vagotomy at the same time, then the Ghrelin was intravenously injected immediately with the same dose as the sepsis+Ghrelin group. Ten rats in each group were taken to observe their survival within 7 days. The remaining 10 rats were sacrificed 20 hours after the operation to obtain venous blood and small intestinal tissue. The condition of the abdominal intestine was observed. The injury of intestinal epithelial cells was observed with transmission electron microscopy. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in serum and small intestinal tissue were detected by enzyme-linked immunosorbent assay (ELISA). The brush border membrane vesicle (BBMV) was prepared, the levels of mRNA and protein expression of PepT1 in the small intestinal epithelium were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting.Results:All rats in the sham operation group survived at 7 days after operation. The 7-day cumulative survival rate of rats in the sepsis group was significantly lower than that in the sham operation group (20% vs. 100%, P < 0.05). The cumulative survival rate of rats after Ghrelin intervention was improved (compared with sepsis group: 40% vs. 20%, P < 0.05), but the protective effect of Ghrelin was weakened after vagotomy (compared with sepsis+Ghrelin group: 10% vs. 40%, P < 0.05). Compared with the sham operation group, in the sepsis group, the small intestine and cecum were dull red, the intestinal tubules were swollen and filled with gas, the intestinal epithelial cells were seriously injured under transmission electron microscopy, the levels of TNF-α and IL-1β in serum and small intestinal were significantly increased, and the expression levels of PepT1 mRNA and protein in the small intestinal epithelium were significantly decreased. It indicated that the sepsis rat model was successfully prepared. After vagotomy, the intestinal swelling and gas accumulation became worse in septic rats, leading to the death of all rats. Compared with the sepsis group, the abdominal situation in the sepsis+Ghrelin group was improved, the injury of intestinal epithelial cells was alleviated, the serum and small intestinal TNF-α and IL-1β were significantly decreased [serum TNF-α (ng/L): 253.27±23.32 vs. 287.90±19.48, small intestinal TNF-α (ng/L): 95.27±11.47 vs. 153.89±18.15, serum IL-1β (ng/L): 39.16±4.47 vs. 54.26±7.27, small intestinal IL-1β (ng/L): 28.47±4.13 vs. 42.26±2.59, all P < 0.05], and the expressions of PepT1 mRNA and protein in the small intestinal epithelium were significantly increased [PepT1 mRNA (2 -ΔΔCt): 0.66±0.05 vs. 0.53±0.06, PepT1 protein (PepT1/GAPDH): 0.80±0.04 vs. 0.60±0.05, both P < 0.05]. Compared with the sepsis+Ghrelin group, after vagotomy in the sepsis+vagotomy+Ghrelin group, the effect of Ghrelin on reducing the release of inflammatory factors in sepsis rats was significantly reduced [serum TNF-α (ng/L): 276.58±19.88 vs. 253.27±23.32, small intestinal TNF-α (ng/L): 144.28±12.99 vs. 95.27±11.47, serum IL-1β (ng/L): 48.15±3.21 vs. 39.16±4.47, small intestinal IL-1β (ng/L): 38.75±4.49 vs. 28.47±4.13, all P < 0.05], the up-regulated effect on the expression of PepT1 in small intestinal epithelium was lost [PepT1 mRNA (2 -ΔΔCt): 0.58±0.03 vs. 0.66±0.05, PepT1 protein (PepT1/GAPDH): 0.70±0.02 vs. 0.80±0.04, both P < 0.05], and the injury of small intestinal epithelial cells was worse. Conclusion:Ghrelin plays a protective role in sepsis by promoting cholinergic neurons to inhibit the release of inflammatory factors, thereby promoting the transcription and translation of PepT1.

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