Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage / 中华危重病急救医学

Objective:To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism.Methods:The LPS induced type Ⅱ alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting.Results:Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2?438.95±64.89, 3?036.41±96.69 vs. 1?736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67?205.60±3?430.40 vs. 56?272.67±7?650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). Conclusion:LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

The molecular mechanism of GSDMB regulating the fate of intestinal epithelial cells / 西安交通大学学报(医学版)

【Objective】 To explore the molecular mechanism of Gasdermin B (GSDMB) regulating the fate of intestinal epithelial cells. 【Methods】 The human GSDMB plasmid was overexpressed into two human intestinal epithelial cell lines (NCM460 and HT-29 cells) and human colon-derived organoids. Western blotting was used to confirm the efficiency of electroporation. Cell counting kit (CCK8), cell apoptosis, and cell cycle by flow cytometry were performed to analyze the effect of GSDMB overexpression on cell function. Transcriptome sequencing was used to analyze the downstream effector molecules of GSDMB. T test was used to compare the data between the two groups. 【Results】 The overexpression of GSDMB protein in the two intestinal epithelial cell lines was successfully reconstructed. The absorbance value (A) of human intestinal epithelial cells overexpressing GSDMB protein [NCM460 cells: (1.17±0.01), HT-29 cells: (0.96±0.06)] was significantly lower than that of blank control cells [NCM460 cells: (1.67±0.12), HT-29 cells: (1.24±0.07)] (t=7.24 and 5.46, P<0.05). The number of apoptotic cells in the GSDMB overexpression group [NCM460 cells: (12.03±1.55), HT-29 cells: (29.30±4.48)] was significantly higher than that in the blank group [NCM460 cells: (4.96±1.74), HT-29 cells: (6.95±3.42)] (t=5.26 and 6.97, P<0.05). Cell cycle analysis showed that the ratio of cells at G0/G1 phase in the GSDMB overexpression group [NCM460 cells: (47.98±5.28)%, HT-29 cells: (38.04±3.45)%] was significantly lower than that in the control group [NCM460 cells: (59.54±3.90) %, HT-29 cells: (63.81±1.76) %] (t=3.05 and 11.53, P<0.05). Transcriptome sequencing results showed that the dual specificity phosphatase 4 and 6 (DUSP4 and DUSP6) genes were significantly upregulated after GSDMB protein expression. Fluorescence quantitative PCR results confirmed that the relative expression levels of DUSP4 (2.45±0.15) and DUSP6 (4.34±0.22) in intestinal epithelial cells transfected with GSDMB were significantly higher than those in the control group (1.06±0.05 and 1.01±0.02) (t=15.08 and 26.52, P<0.05). After GSDMB-expressing NCM460 cells were treated with the DUSP inhibitor BCI, the BCI treatment group had a significantly increased expression level of p-ERK compared to the control group [(1.14±0.17) vs. (0.58±0.12)] (t=5.42, P=0.002); the A value (1.84±0.07) and G0/G1 phase ratio (59.83±2.17)% in the BCI treatment group were significantly higher than those in the non-treatment group [(1.52±0.10) and (52.10±2.23)%] , and the number of apoptosis in the BCI treated group (7.60±0.56) was significantly lower than that in the untreated group (12.57±1.00) (t=4.71, 4.31, 7.52, P<0.05). TUNEL staining in human colon organoids showed a significant increase in apoptotic cells, and the relative expression level of DUSP6 protein (0.85±0.09) was significantly higher than that of the control group (0.21±0.04), accompanied by a decrease in p-ERK levels [(0.83±0.18) vs. (0.19±0.06)] , with statistical significance (t=11.95, P<0.001; t=6.56, P<0.001). 【Conclusion】 GSDMB may inhibit cell proliferation, induce cell cycle arrest, and promote apoptosis by upregulating dual specificity phosphatase DUSP6-mediated ERK phosphorylation, thus affecting the fate of intestinal epithelial cells.

Differential mRNA expression in fractional low-dose radiation-induced senescence of HBE cells / 中国辐射卫生

Objective To explore the differentially expressed mRNAs and related biological processes and pathways in fractional low-dose ionizing radiation (LDIR)-induced senescence of normal human bronchial epithelial (HBE) cells by high-throughput mRNA sequencing and bioinformatics techniques. Methods Senescence-associated β-galactosidase staining and senescence-associated secretion phenotype gene mRNA and protein expression levels were measured at 24 and 48 h after irradiating HBE cells 7 times at doses of 0, 50, 100, and 200 mGy, respectively. The differentially expressed genes were screened by high-throughput sequencing for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Results The senescence-positive area of fractional low-dose irradiated HBE cells increased in a dose-dependent manner (P < 0.05). The mRNA levels and protein expression of transforming growth factor-β1(TGF-β1) and matrix metalloproteinase-9(MMP-9) genes were increased in the 100 mGy × 7 and 200 mGy × 7 groups at 24 and 48 h after the end of irradiation compared with the control group. High-throughput sequencing showed that there were 882, 475, and 1205 differentially expressed mRNAs in each dose group compared with the control group. GO analysis showed that the differentially expressed mRNAs in each dose group were mainly enriched in biological processes such as cell cycle regulation, regulation of nitrogen compound metabolic process, regulation of cell division and response to stimulus. KEGG analysis showed that the differentially expressed mRNAs were mainly enriched in the pathways of cell cycle, cell senescence, and ferroptosis. Conclusion Fractional LDIR induced senescence in HBE cells, and differentially expressed mRNA-associated biological processes and pathways in senescent cells are related to cell cycle and cell senescence.

Research progress in role of composition and structure of basement membrane in epithelial cell polarization and its mechanism / 吉林大学学报(医学版)

The basement membrane is a specialized extracellular matrix between the epithelium and the mesenchyme.In stratified epithelium,only the basal cells in contact with the basement membrane exhibit the apical-basal polarity,whereas the epithelial cells do being not in contact with the basement membrane do not exhibit the apical-basal polarity.The basement membrane plays an important role in epithelial cell polarization.It is an important extracellular matrix(ECM)structure in the multicellular organisms,is situated between the epithelium and the mesenchyme,and is produced jointly by the epithelial cells and mesenchymal cells.Its components mainly include Laminin,type Ⅳ collagen(Col-Ⅳ),nidogen(NDG),and heparan sulfate proteoglycans(HSPG),and each component plays the different role in influencing the epithelial cell polarity.The network scaffold formed by Col-Ⅳ and Laminin is the main structure of the basement membrane,and the integrity of the structure affects the epithelial cell polarization.This review summarizes the composition and structure of the basement membrane,focuses on its role in epithelial cell polarization and its mechanism,and compiles the current status of biomimetic basement membrane materials that promotes the epithelial cell polarization,and provides the theoretical foundation for further exploration of the establishment and maintenance of epithelial cell polarity.

Epithelial transformation sequence 2 affecting the in vitro metastatic activity of esophageal squamous carcinoma cells by regulating the expression of p33 inhibitor growth-1 / 解剖学报

Objective To investigate the effects of epithelial transformation sequence 2(ECT2)and p33ING1 on the metastatic activity of esophageal squamous cell carcinoma(ESCC)cells.Methods The expressions of ECT2 and p33ING1 in esophageal squamous cell carcinoma tissues and adjacent tissues were detected by immunohistochemistry and Western blotting.Human esophageal squamous carcinoma cell line KYSE140 cells were divided into 4 groups:blank group,negative control(pcDNA 3.1 NC)group,overexpression group(pcDNA 3.1 ECT2)and inhibited expression group(si ECT2).MTT assay and cell colony formation assay were used to study the proliferation and growth ability of cells,Transwell assay and scratch assay used to study the invasion and migration ability of cells,and flow cytometry used to detect apoptosis and cell cycle,Western blotting used to detect the effect of ECT2 on p33ING1 protein.Results ECT2 expression increased and p33ING1 expression decreased in esophageal squamous cell carcinoma tissues.Overexpression of ECT2 significantly increased the growth,colony formation,migration and invasion abilities of KYSE140 cells,and decreased the apoptosis rate and p33ING1 expression of KYSE140 cells.In addition,inhibition of ECT2 expression could reverse the above changes.Conclusion The high expression of ECT2 can promote the growth and metastasis of esophageal squamous cell carcinoma KYSE140 cells and inhibit their apoptosis.The mechanism may be related to the inhibition of p33ING1 expression by ECT2.

Oxamate alleviates silicotic fibrosis in mice by inhibiting senescence of alveolar type II epithelial cells / 环境与职业医学

Background The senescence of alveolar type II epithelial cells is an important driving factor for the progression of silicotic fibrosis, and the regulatory effects of oxamate on the senescence of alveolar type II epithelial cells is still unclear. Objective To explore whether lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting senescence of alveolar type II epithelial cellsMethods This study was divided into two parts: in vivo experiments and in vitro experiments. In the first part, forty SPF C57BL/6J male mice were randomly divided into four groups with 10 in each group: control group, silicosis model group, low-dose oxamate treatment group, and high-dose oxamate treatment group. The silicotic mouse model was established by intratracheal instillation of 50 μL SiO2 suspension (100 mg·mL−1). The treatment models were prepared by intraperitoneal injection of 100 μL oxamate (225 mmol·L−1 and 1125 mmol·L−1). In the second part, induction of MLE-12 mouse alveolar type II epithelial cells was conducted with SiO2. The in vitro experimental groups were ① SiO2 induction groups: control group, 50 μg·mL−1 SiO2 group, 100 μg·mL−1 SiO2 group, and 200 μg·mL−1 SiO2 group, and ② oxamate treatment groups: control group, SiO2 group (100 μg·mL−1), low-dose oxamate (25 mmol·L−1) treatment group, and high-dose oxamate (50 mmol·L−1) treatment group. Pathological morphology of lung tissues was evaluated after hematoxylin-eosin (HE) staining; deposition of collagen in lung tissues was evaluated after sirius red staining; positive co-expression of prosurfactant protein C (Pro-SPC) and β-galactosidase was detected by immunofluorescence staining; positive expression of β-galactosidase in MLE-12 cells was detected by immunofluorescence staining. The protein expression levels of collagen type I (CoL I), fibronectin1 (FN1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), lactate dehydrogenase A (LDHA), p-ataxia telangiectasia and Rad3-related kinase (ATR), and cyclin-dependent kinase inhibitors p21, and p16 were detected by Western blotting. Results Compared with the control group, the protein expression levels of HK2, PKM2, LDHA, p-ATR, p21, and p16 were significantly upregulated in the silicosis model group and the SiO2-induced MLE-12 cells (P＜0.05). The in vivo studies showed that, compared with the control group, the silicon nodule area, the collagen deposition area, the proportion of β-galactosidase positive cells, and the protein expression levels of CoL I, FN1, LDHA, p-ATR, p21, and p16 were significantly upregulated in the silicosis model group (P＜0.05). Compared with the silicosis model group, the oxamate treatment groups showed significant downregulation of the silicon nodule area, the collagen deposition area, the proportion of β-galactosidase positive cells, and the the CoL I, FN1, LDHA, p-ATR, p21, and p16 protein expression levels, and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group (P＜0.05). The in vitro studies showed that, compared with the control group, the proportion of β-galactosidase positive cells and the protein expression levels of p-ATR, p21, and p16 were significantly upregulated in the SiO2-induced group (P＜0.05). Compared with the SiO2 group, the proportion of β-galactosidase positive cells and the LDHA, p-ATR, p21 and p16 protein expression levels were significantly downregulated in the oxamate treatment groups, and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group (P＜0.05). Conclusion Lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting the senescence of alveolar type II epithelial cells.

Toxic effects of sodium hypochlorite as disinfectants on human bronchial epithelial cells / 环境与职业医学

Background There are a variety of microorganisms in ambient air, and susceptible people can be infected once contact with pathogenic microorganisms in the environment. In order to avoid the spread of pathogenic bacteria, disinfection is the simplest and most effective way of killing pathogenic bacteria in the environment to block the contact between pathogenic bacteria and humans. Sodium hypochlorite (NaClO) is the most widely used disinfectant, but its safety in ambient air disinfection is not clear yet. Objective To establish a model of bronchial epithelial cell (BEAS-2B) injury induced by NaClO, and to explore the mechanism of the toxic effect of NaClO disinfectants on BEAS-2B. Methods Cells were treated with concentration gradients of 0, 25, 50,100, 200, and 400 μmol·L−1 of the diluted NaClO (100 mmol·L−1) standard solution, respectively, and cell activity was measured by cell counting kit-8 (CCK-8) assay after 15 and 30 min. Cells treated with 0, 25, and 50 μmol·L−1 NaClO were selected to observe the cell morphology under an inverted microscope, apoptosis was determined by flow cytometry Annexin V FITC / PI double staining to determine the final experimental concentration. The morphology of organelles such as mitochondria was observed under a transmission electron microscope. Mitochondrial membrane potential of the cells was detected by JC-1 staining. Intracellular Ca2+ concentration was measured with a Fluo-4 AM fluorescent probe. Total cellular reactive oxygen species (ROS) was detected with a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, cell mitochondrial ROS with a dihydroethidium (DHE) fluorescent probe, and lipid peroxidation intermediate malondialdehyde (MDA) with a commercial kit. Results Compared with 0 μmol·L−1, NaClO treatment group, cell morphology did not change a lot after 25 μmol·L−1 NaClO treatment for 30 min, and the cells began to wrinkle and become round after 30 min treatment with 50 μmol·L−1 NaClO, showing about 70% of normal cell viability (P<0.01). So 30 min 50 μmol·L−1 NaClO treatment was selected for the subsequent experiment. The experimental results found that compared with the 0 μmol·L−1 NaClO treatment group, the number of apoptotic cells increased (P<0.05), the mitochondrial membrane potential decreased (P<0.01), the intracellular Ca2+ concentration increased (P<0.05), the cellular ROS level increased (P<0.05), the mitochondrial ROS level increased (P<0.01), and the MDA content increased (P<0.01) in the NaClO treatment group.. Conclusion The study has successfully established a model of BEAS-2B injury induced by NaClO, and found that NaClO can lead to cell damage by inducing apoptosis and oxidative stress in BEAS-2B cells. According to the results, there are two possible reasons. First, NaClO solves in water to form hypochlorous acid (HClO) which is oxidative and increases the intracellular ROS level after entering cells, leading to cellular oxidative stress. Second, HClO enters cells to directly attack the mitochondrial membrane, resulting in the imbalance of potential inside and outside the mitochondrial membrane, and apoptosis caused by Ca2+ efflux.

Effects of inhibiting CAMKⅡ expression in ARPE-19 cells on the migration, invasion, and tube formation of HUVECs in a non-contact co-culture system / 国际眼科杂志(Guoji Yanke Zazhi)

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&#x003C;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&#x003E;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&#x003C;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.

Effect of overexpression of protein phosphatase 2Cm on transcriptome of human renal tubular epithelial cells / 中国组织工程研究

BACKGROUND:A previous study by our group found that protein phosphatase 2Cm(PP2Cm)null mice developed significantly fewer symptoms of renal failure relative to wild-type mice,and thus it was speculated that PP2Cm may play an important protective role in the development of renal fibrosis,however,the molecular mechanisms remain undefined. OBJECTIVE:To investigate the effect of the PP2Cm gene on the transcriptome of human renal tubular epithelial cells. METHODS:Cultured human renal tubular epithelial cells were transfected with the PP2Cm gene into human renal tubular epithelial cells using plasmids.The expression of PP2Cm in the cells was detected by fluorescence quantitative PCR assay and western blot assay,and subsequently,cell RNA was separately extracted for transcriptome sequencing to look for differentially expressed genes between transfected and control groups.The resulting differential genes were further subjected to GO analysis and KEGG analysis using bioinformatics methods. RESULTS AND CONCLUSION:There were 796 differentially expressed genes,553 of which were downregulated genes and 243 upregulated genes,in human renal tubular epithelial cells transfected with the PP2Cm gene compared with untransfected blank cells by sequencing analysis.GO analysis results showed that the upregulated genes were significantly enriched in cellular biosynthetic processes,protein translation,intrinsic apoptotic signaling pathways,and so on.The downregulated expressed genes were significantly enriched in endothelial cell proliferation,cell adhesion and other signaling pathways.KEGG analysis results showed that the significantly up-regulated genes were enriched in metabolism-related signaling pathways such as amino acid metabolism and biosynthesis.The downregulated expressed genes were significantly enriched in signaling pathways such as pantothenate and coenzyme A biosynthesis.Our results show that PP2Cm overexpression can affect a number of signaling pathways related to a range of biological processes in renal tubular epithelial cells,which may be important in metabolism-related signaling pathways such as amino acid metabolism and biosynthesis.

Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

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.

Research progress on ankylosis of primary molars / 口腔疾病防治

@#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.

Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation / 中国血吸虫病防治杂志

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.

Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation / 中国血吸虫病防治杂志

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.

Autophagy-regulated strategies in pre-clinical studies of inflammatory bowel disease / 药学学报

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.

High glucose inhibition of miR-126-5p promotes renal tubular epithelial cell injury / 中国医师杂志

Objective:To explore the differential expression profile of miRN in the development of diabetes nephropathy (DN), and further explore the mechanism of miR-126-5p involved in high glucose induced injury of renal tubular epithelial cells.Methods:Firstly, we downloaded existing chip data from the Gene Expression Integrated Database (GEO) and used GEO2R, miRanda, gene ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to mine differential miRNAs. Subsequently, a high glucose induced HK-2 cell injury model was used and divided into three groups: high glucose model group, si-HOTAIR group, and si HOTAIR+ miR-126-5p inhibitor group. The three groups of cells were sequentially transfected with siRNA-NC, siRNA-HOTAIR, and siRNA-HOTAIR+ miR-126-5p mimic, and cultured in a medium containing 60 mmol/L glucose. Flow cytometry was used to detect changes in apoptosis levels in each group, while cell counting kit-8 (CCK-8) was used to detect changes in cell proliferation.Results:Through data mining analysis using GEO, it was found that compared to ordinary mice, DN mice had 74 upregulated miRNAs and 80 downregulated miRNAs in their kidney tissue. Enrichment analysis results showed that miRNAs could target signaling pathways such as Wnt, PKG, MAPK, and Rap1, and miR-126-5p was significantly downregulated. In the high glucose induced HK-2 cell injury model, the experimental results showed that the inhibitory effect on cell proliferation activity was more significant at a high glucose concentration of 60 mmol/L ( P<0.05); High glucose stimulation significantly reduced the expression of miR-126-5p ( P<0.05). The results of flow cytometry showed that compared with the high glucose model group, the apoptosis rate of the si-HOTAIR group significantly decreased ( P<0.05), while the apoptosis rate of the si-HOTAIR+ miR-126-5p inhibitor group significantly increased ( P<0.05). The CCK-8 experiment showed that compared with the high glucose model group, the cell viability of the si-HOTAIR group significantly increased ( P<0.05); The cell viability of the si-HOTAIR+ miR-126-5p inhibitor group was inhibited ( P<0.05). Conclusions:miR-126-5p can inhibit high glucose induced apoptosis in HK-2 cells and protect them.

Effect of miR200a and SIRT1 expression on epithelial cell apoptosis in age-related cataract lens epithelial cells / 国际生物医学工程杂志

Objective:To investigate the effects of miR200a and SIRT1 expression on epithelial cell apoptosis in age-related cataract lens epithelial cells.Methods:30 anterior capsule samples of age-related cataract patients were enrolled from the ophthalmology outpatient department of Beichen Hospital affiliated with Nankai University from January to August 2022 as the cataract group and another 30 normal anterior capsule samples of corneal transplantation were enrolled during the same period as the control group. Real -time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression levels of miR200a and SIRT1 genes in the cataract group and control group. The oxidative stress level divided human lens epithelial cells SRA01/04 into a negative control group, miR200a overexpression group, and miR200a inhibition group. The negative control group, miR200a overexpression plasmid, and miR200a inhibitor group were transfected, respectively. Cell apoptosis rate and oxidative stress level were detected by flow cytometry, and Western Blot was used to detect the expression of SIRT1 protein and Caspase-3, Bcl-2, and Bcl-2 related X (Bax) proteins containing cysteine in each group of cells. The targeting relationship between miR200a and SIRT1 was validated using dual luciferase reporter gene detection. Results:Compared with the control group, the expression level of miR200a in the cataract group was significantly increased ( P < 0.01), while the expression level of SIRT1 was significantly reduced ( P < 0.01). Compared with the control group, the levels of SOD, GSH, and GSH-Px in the cataract group were significantly reduced (all P < 0.01), and the MDA level was significantly higher than those in the control group ( P < 0.01). The apoptosis rate of the miR200a overexpression group was significantly higher than that of the negative control group ( P < 0.05), and the apoptosis rate of the miR200a inhibition group was significantly lower than that of the negative control group, the differences were statistically significant ( P < 0.05). The level of SIRT1 protein in the miR200a overexpression group was significantly lower than that in the negative control group ( P < 0.05), and the level of SIRT1 protein in cells of the miR200a inhibition group was significantly higher than that of the negative control group ( P < 0.05). Compared with the negative control group, the miR200a overexpression group showed a significant increase in Caspase-3 activity and Bax protein (all P < 0.05), while the Bcl-2 protein level was significantly reduced ( P < 0.05). The miR200a inhibition group showed a significant decrease in Caspase-3 activity and Bax protein (all P < 0.05), while the Bcl-2 protein level was significantly increased ( P < 0.05). The dual luciferase experiment showed that the overexpression group of miR-200a can significantly inhibit the luciferase activity of the wild-type SIRT1 plasmid, which is the target gene of miR-200a. Conclusions:miR220a is highly expressed in the capsule of lens of patients with age-related cataracts, and its mechanism may be that miR220a promotes apoptosis of lens epithelial cells by targeting the expression of SIRT1.

Regulation of the TGF-β type I Receptor/Smad Pathway Inhibits Renal Fibrosis by Shenkang Injection / 世界科学技术-中医药现代化

Objective Chinese herbal injections called Shenkang injections(SKI)have become widely used for treating chronic kidney disease in the clinic.An investigation into the underlying mechanisms of SKI inhibition of renal tubular epithelial cell trans differentiation treated with TGF-β1 was carried out in this study.Methods To create an in vitro model of RF,HK-2 cells were treated with TGF-1(10 ng·mL-1)at 37℃for 48 h.After the cells were treated with SKI for 48 h,the morphology of the cells was observed by electron microscope.And Western blot,RT-PCR and immunofluorescence techniques were used to detect α-Smooth muscle actin(α-SMA),type III collagen(COI-Ⅲ),TGF-β1,Smad3,Smad7 and TβR-I expression changes of proteins and genes.Results SKI can significantly reduce expressed proteins and genes related to renal fibrosis,such as α-Smooth muscle actin(α-SMA)and type Ⅲ collagen(COI-Ⅲ).SKI control the production of proteins associated with the TGF-β/Smad signaling pathway.By downregulating TGF-β1,Smad3,and TβR-I protein expression,as well as upregulating Smad7 protein expression,it is able to prevent cell trans differentiation.Conclusions SKI can inhibit renal tubular epithelial cell mesenchymal trans differentiation.In addition,this drug may prevent chronic kidney disease by downregulating the expression of TβR-I and regulating the TGF-β/Smad pathway-related molecules.

The role of surfactant associated protein-A in silicosis / 中国职业医学

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.

Antagonistic effect of SC79 on high glucose-induced apoptosis of RPE cells and its regulatory mechanism on AKT-XIAP signaling pathway / 中华实验眼科杂志

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.

MicroRNA21-5p alleviates hyperoxia-induced acute lung injury in rats through activating phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway by regulating type Ⅱ alveolar epithelial cell apoptosis / 中华危重病急救医学

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.