Somatostatin (SST) is an inhibitory polypeptide hormone that plays an important role in a variety of biological processes. Somatostatin receptor 2 (SSTR2) is the most widely expressed somatostatin receptor. However, the specific cell types expressing Sstr2 in the tissues have not been investigated. In this study, we detected the expression pattern of SSTR2 protein in mouse at different development stages, including the embryonic 15.5 days and the postnatal 1, 7, 15 days as well as 3 and 6 months, by multicolour immunofluorescence analyses. We found that Sstr2 was expressed in some specific cells types of several tissues, including the neuronal cells and astrocytes in the brain, the mesenchymal cells, the hematopoietic cells, the early hematopoietic stem cells, and the B cells in the bone marrow, the macrophages, the type Ⅱ alveolar epithelial cells, and the airway ciliated cells in the lung, the epithelial cells and the neuronal cells in the intestine, the hair follicle cells, the gastric epithelial cells, the hematopoietic stem cells and the nerve fibre in the spleen, and the tubular epithelial cells in the kidney. This study identified the specific cell types expressing Sstr2 in mouse at different developmental stages, providing new insights into the physiological function of SST and SSTR2 in several cell types.
Mice ; Animals ; Receptors, Somatostatin/metabolism* ; Hematopoietic Stem Cells/metabolism* ; Epithelial Cells

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.
Humans ; Ferroptosis ; Reactive Oxygen Species/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Sincalide/pharmacology* ; Signal Transduction ; Epithelial Cells/metabolism* ; Glutathione

Objective: To investigate the mechanism of S100A7 inducing the migration and invasion in cervical cancers. Methods: Tissue samples of 5 cases of cervical squamous cell carcinoma and 3 cases of adenocarcinoma were collected from May 2007 to December 2007 in the Department of Gynecology of the Affiliated Hospital of Qingdao University. Immunohistochemistry was performed to evaluate the expression of S100A7 in cervical carcinoma tissues. S100A7-overexpressing HeLa and C33A cells were established with lentiviral systems as the experimental group. Immunofluorescence assay was performed to observe the cell morphology. Transwell assay was taken to detect the effect of S100A7-overexpression on the migration and invasion of cervical cancer cells. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to examine the mRNA expressions of E-cadherin, N-cadherin, vimentin and fibronectin. The expression of extracellular S100A7 in conditioned medium of cervical cancer cell was detected by western blot. Conditioned medium was added into Transwell lower compartment to detect cell motility. Exosomes were isolated and extracted from the culture supernatant of cervical cancer cell, the expressions of S100A7, CD81 and TSG101 were detected by western blot. Transwell assay was taken to detect the effect of exosomes on the migration and invasion of cervical cancer cells. Results: S100A7 expression was positively expressed in cervical squamous carcinoma and negative expression in adenocarcinoma. Stable S100A7-overexpressing HeLa and C33A cells were successfully constructed. C33A cells in the experimental group were spindle shaped while those in the control group tended to be polygonal epithelioid cells. The number of S100A7-overexpressed HeLa cells passing through the Transwell membrane assay was increased significantly in migration and invasion assay (152.00±39.22 vs 105.13±15.75, P<0.05; 115.38±34.57 vs 79.50±13.68, P<0.05). RT-qPCR indicated that the mRNA expressions of E-cadherin in S100A7-overexpressed HeLa and C33A cells decreased (P<0.05) while the mRNA expressions of N-cadherin and fibronectin in HeLa cells and fibronectin in C33A cells increased (P<0.05). Western blot showed that extracellular S100A7 was detected in culture supernatant of cervical cancer cells. HeLa cells of the experimental group passing through transwell membrane in migration and invasion assays were increased significantly (192.60±24.41 vs 98.80±47.24, P<0.05; 105.40±27.38 vs 84.50±13.51, P<0.05) when the conditional medium was added into the lower compartment of Transwell. Exosomes from C33A cell culture supernatant were extracted successfully, and S100A7 expression was positive. The number of transmembrane C33A cells incubated with exosomes extracted from cells of the experimental group was increased significantly (251.00±49.82 vs 143.00±30.85, P<0.05; 524.60±52.74 vs 389.00±63.23, P<0.05). Conclusion: S100A7 may promote the migration and invasion of cervical cancer cells by epithelial-mesenchymal transition and exosome secretion.
Female ; Humans ; Uterine Cervical Neoplasms/pathology* ; HeLa Cells ; Fibronectins/metabolism* ; Culture Media, Conditioned ; Carcinoma, Squamous Cell/metabolism* ; Adenocarcinoma ; Cadherins/metabolism* ; RNA, Messenger/metabolism* ; Cell Movement ; Epithelial-Mesenchymal Transition/genetics* ; Cell Line, Tumor ; Cell Proliferation ; S100 Calcium Binding Protein A7/metabolism*

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.
Animals ; Humans ; Extracellular Traps ; Neutrophils ; Interleukin-8/metabolism* ; Dermatophagoides farinae ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Epithelial Cells ; Interferon-gamma/metabolism* ; Tetradecanoylphorbol Acetate/pharmacology*

OBJECTIVE: To explore the mechanism of Yifei Jianpi recipe for improving cigarette smoke- induced inflammatory injury and mucus hypersecretion in cultured human bronchial epithelial cells. METHODS: Serum samples were collected from 40 SD rats treated with Yifei Jianpi recipe (n=20) or normal saline (n=20) by gavage. Cultured human bronchial epithelial 16HBE cells were stimulated with an aqueous cigarette smoke extract (CSE), followed by treatment with the collected serum at different dilutions. The optimal concentration and treatment time of CSE and the medicated serum for cell treatment were determined with CCK-8 assay. The expressions of TLR4, NF-κB, MUC5AC, MUC7, and muc8 at both the mRNA and protein levels in the treated cells were examined with RT- qPCR and Western blotting, and the effects of TLR4 gene silencing and overexpression on their expressions were assessed. The expressions of TNF-α, IL-1 β, IL-6 and IL-8 in the cells were detected using ELISA. RESULTS: At the optimal concentration of 20%, treatment with the medicated serum for 24 h significantly lowered the mRNA and protein expressions of TLR4, NF- κB, MUC5AC, MUC7, and MUC8 in CSE- exposed 16HBE cells, and these effects were further enhanced by TLR4 silencing in the cells. In 16HBE cells with TLR4 overexpression, the expressions of TLR4, NF-κB, MUC5AC, MUC7, and MUC8 were significantly increased after CSE exposure and were lowered following treatment with the medicated serum (P < 0.05). The medicated serum also significantly lowered the levels of TNF-α, IL-1β, IL-6 and IL-8 in CSE-exposed 16HBE cells (P < 0.05). CONCLUSIONS In the 16HBE cell model of chronic obstructive pulmonary disease (COPD), treatment with Yifei Jianpi recipe-medicated serum improves inflammation and mucus hypersecretion possibly by reducing MUC secretion and inhibiting the TLR4/NF-κB signaling pathway.
Humans ; Rats ; Animals ; NF-kappa B/metabolism* ; Toll-Like Receptor 4/metabolism* ; Interleukin-8/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Cigarette Smoking/adverse effects* ; Interleukin-6/metabolism* ; Rats, Sprague-Dawley ; Pulmonary Disease, Chronic Obstructive/drug therapy* ; Signal Transduction ; Epithelial Cells/metabolism* ; Mucus/metabolism* ; RNA, Messenger/metabolism*

Humans ; Nerve Tissue Proteins ; Epithelial-Mesenchymal Transition/genetics* ; Constriction, Pathologic ; Receptors, Immunologic ; Epithelial Cells/metabolism* ; Smad3 Protein/metabolism* ; Transforming Growth Factor beta1/metabolism*

Animals ; Humans ; Thymic Stromal Lymphopoietin ; Pyroglyphidae/metabolism* ; Cytokines/metabolism* ; Asthma/metabolism* ; Respiratory System ; Epithelial Cells/metabolism*

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.
Animals ; Mice ; Epithelial Cells ; Lung ; Mucin 5AC/metabolism* ; Mycoplasma pneumoniae/metabolism* ; Signal Transduction

Olfactory epithelium, which detects and transmits odor signals, is critical for the function of olfactory system. Olfactory epithelium is able to recover spontaneously after injury under normal circumstances, but this ability is dampened in certain diseases or senility, which causes olfactory dysfunction. The olfactory epithelium consists of basal cells, sustentacular cells and olfactory sensory neurons. In order to develop an olfactory epithelial organoid containing multiple olfactory cell types in vitro, we used three-dimensional culture model and small molecules screening. This organoid system consists of horizontal basal-like cells, globose basal-like cells, sustentacular-like cells and olfactory sensory neurons-like cells. Through statistical analysis of clone diameter, immunofluorescence staining and qPCR detection of the expression level of related marker genes. We identified a series of growth factors and small molecule compounds that affected the proliferation, composition and gene expression of the organoids. CHIR-99021, an activator of Wnt signaling pathway, increased the colony formation and proliferation rate of olfactory epithelial organoids and the expression level of marker genes of olfactory sensory neurons-like cells. In addition, each factor in the culture system increased the proportion of c-Kit-positive globose basal-like cell colonies in organoids. Moreover, EGF and vitamin C were both beneficial to the expression of horizontal basal-like cell marker genes in organoids. The established olfactory epithelial organoid system mimicked the process of olfactory epithelial stem cells differentiating into various olfactory epithelial cell types, thus providing a research model for studying olfactory epithelial tissue regeneration, the pathological mechanism of olfactory dysfunction and drug screening for olfactory dysfunction treatment.
Humans ; Olfactory Mucosa/metabolism* ; Epithelial Cells ; Organoids/metabolism* ; Olfaction Disorders/metabolism*

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.
Rats ; Animals ; Matrix Metalloproteinase 9/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Tight Junction Proteins/metabolism* ; Occludin/pharmacology* ; Choroid Plexus/metabolism* ; Reactive Oxygen Species/metabolism* ; Lanthanum/pharmacology* ; Epithelial Cells ; Zonula Occludens-1 Protein/metabolism* ; Phosphoproteins/pharmacology*