No abstract available.
Animals ; Apoptosis/*physiology ; Claudins/*metabolism ; Colitis/*physiopathology ; Intestinal Mucosa/*physiopathology ; Mannose-Binding Lectin/*immunology

BACKGROUNDClaudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported.

METHODSIn this study, HCMECs were stimulated with CoCl2to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs.

RESULTSWe found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription.

CONCLUSIONSThe results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.

Cell Hypoxia ; Cells, Cultured ; Claudins ; analysis ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Histones ; metabolism ; Humans ; Myocardium ; cytology ; metabolism ; Promoter Regions, Genetic

ObjectiveTo study the expression of CLAUDIN-11 in the testis tissue of non-obstructive azoospermia (NOA) patients with different severities and investigate its clinical significance.

METHODSSixty-two NOA patients were divided into a hypospermatogenesis (HS) group (n = 30) and a Sertoli cell only syndrome (SCO) group (n =32). The expression of CLAUDIN-11 in the testicular tissue of the patients was detected by immunohistochemistry, that of CLAUDIN-11 mRNA determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the levels of serum reproductive hormones measured by chemiluminescent immunoassay.

RESULTSImmunohistochemistry showed that the expression of CLAUDIN-11 was mainly in the cytoplasm of the Sertoli cells around the seminiferous tubule wall in the HS group, but diffusely distributed in the membrane of the Sertoli cells in the SCO group. RT-qPCR revealed a significantly lower expression of CLAUDIN-11 mRNA in the HS than in the SCO group (0.008 ± 0.001 vs 0.013 ± 0.002, t = 10.616, P<0.01). The level of serum luteotropic hormone (LH) was also markedly lower in the HS than in the SCO group (［3.62 ± 1.34］ vs ［4.96 ± 3.10］ IU/L, P<0.05) and so was that of follicle-stimulating hormone (FSH) (［5.36 ± 2.80］ vs ［10.65 ± 9.18］ IU/L, P<0.05).

CONCLUSIONSThe up-regulated expression of CLAUDIN-11 in Sertoli cells may play an important role in the development and progression of spermatogenic dysfunction in NOA patients.

Azoospermia ; genetics ; metabolism ; Claudins ; metabolism ; Follicle Stimulating Hormone ; metabolism ; Humans ; Male ; Oligospermia ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Seminiferous Tubules ; metabolism ; Sertoli Cell-Only Syndrome ; genetics ; metabolism ; Sertoli Cells ; metabolism ; Spermatogenesis ; Testis ; metabolism

OBJECTIVETo investigate the role of claudin-11, a tight junction component of Sertoli cells, in spermatogenic dysfunction induced by oxidative stress in mice exposed to local radiation.

METHODSWe randomly allocated 48 male Kunming mice to a blank control group (A) and three radiation groups (B, C and D) of equal number, the latter three exposed to local radiation of the lower abdomen with 2 Gy, 6 Gy and 10 Gy of 60Co-gamma-ray, respectively, to induce oxidative stress. Four weeks later, we killed the animals, obtained their body and testis weights, observed the histological changes of the testis by HE staining, measured the levels of serum FSH, testosterone and LH by ELISA, and determined the mRNA levels of claudin-11 and inhibin beta B in Sertoli cells by real time quantitative PCR.

RESULTSAfter exposure to 60Co-gamma-ray radiation, the testis weights were (129.4 +/- 10.81), (87.5 +/- 16.83) and (56.1 +/- 12.36) mg in groups B, C and D, significantly decreased as compared with (182.9 +/- 8.43) mg in group A (P < 0.05); the testis indexes were (3.39 +/- 0.57), (2.46 +/- 0.46) and (1.63 +/- 0.44) mg/g in groups B, C and D, remarkably lower than (4.28 +/- 0.31) mg/g in group A (P < 0.01). Histological analysis revealed obviously decreased diameters of seminiferous tubules, reduced seminiferous epithelia and disarranged spermatogenic cells in the three radiation groups. The tubule differentiation indexes (TDI) were markedly lower in groups B, C and D than in A (P < 0.01). The levels of serum FSH were (6.74 +/- 1.95), (8.41 +/- 2.44) and (10.93 +/- 3.16) IU/L in groups B, C and D, 1.9 times higher in D than in A. With increased dose of radiation, the mRNA levels of inhibin beta in the testis tissue were descended, while the transcription levels of claudin-11 elevated, significantly higher in groups C and D than in A (P < 0.01).

CONCLUSIONLocal radiation-induced testicular oxidative stress can decrease the mRNA level of inhibin beta , increase serum FSH, damage Sertoli cells and elevate the expression of claudin-11 in the testis tissue. Increased claudin-11 and serum FSH may delay the cyclical restitution of hemo-testicular barrier and reduce the number of meiotic spermatocytes in the seminiferous epithelium, which consequently leads to male infertility.

Animals ; Claudins ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Oxidative Stress ; radiation effects ; RNA, Messenger ; genetics ; Seminiferous Tubules ; metabolism ; radiation effects ; Sertoli Cells ; metabolism ; Spermatocytes ; metabolism ; radiation effects ; Spermatogenesis ; Testis ; metabolism ; radiation effects

BACKGROUND/AIMS: This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. METHODS: Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. RESULTS: The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%+/-1.2% in controls and was significantly increased to 7.2%+/-1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. CONCLUSIONS: There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability.
Animals ; Apoptosis/*physiology ; Claudins/*metabolism ; Colitis/chemically induced/immunology/*physiopathology ; Colon/immunology/physiopathology ; Dextran Sulfate ; Intestinal Mucosa/*physiopathology ; Lactulose/metabolism ; Mannitol/metabolism ; Mannose-Binding Lectin/*immunology ; Permeability ; Rats ; Rats, Sprague-Dawley ; Sucrose/analogs & derivatives/metabolism ; Up-Regulation

Clinical studies reported hypomagnesaemia in long-term omeprazole usage that was probably due to intestinal Mg2+ wasting. Our previous report demonstrated the inhibitory effect of omeprazole on passive Mg2+ transport across Caco-2 monolayers. The present study aimed to identify the underlying mechanism of omeprazole suppression of passive Mg2+ absorption. By using Caco-2 monolayers, we demonstrated a potent inhibitory effect of omeprazole on passive Mg2+, but not Ca2+, transport across Caco-2 monolayers. Omeprazole shifted the %maximum passive Mg2+ transport-Mg2+ concentration curves to the right, and increased the half maximal effective concentration of those dose-response curves, indicating a lower Mg2+ affinity of the paracellular channel. By continually monitoring the apical pH, we showed that omeprazole suppressed apical acid accumulation. Neomycin and spermine had no effect on passive Mg2+ transport of either control or omeprazole treated monolayers, indicating that omeprazole suppressed passive Mg2+ transport in a calcium sensing receptor (CaSR)-independent manner. The results of western blot analysis showed that omeprazole significantly suppressed claudin (Cldn)-7 and -12, but not Cldn-2, expression in Caco-2 cells. By using apical solution of pH 5.5, 6.0, 6.5, and 7.0, we found that apical acidity markedly increased passive Mg2+ transport, Mg2+ affinity of the paracellular channel, and Cldn-7 and -12 expression in Caco-2 monolayers. Apical acidity abolished the inhibitory effect of omeprazole on passive Mg2+ transport and Cldn-7 and -12 expression. Our results provided the evidence for the regulation of intestinal passive Mg2+ absorption by luminal acidity-induced increase in Cldn-7 and -12 expression.
Absorption/drug effects ; Caco-2 Cells ; Calcium/metabolism ; Claudins/genetics/*metabolism ; Dose-Response Relationship, Drug ; Gene Expression/drug effects ; Humans ; Hydrogen-Ion Concentration ; Magnesium/*metabolism ; Omeprazole/*pharmacology ; Proton Pump Inhibitors/*pharmacology ; Receptors, Calcium-Sensing/metabolism

OBJECTIVETo investigate the effects of the combination of musk and olibanum on the tight junction protein expressions in prostatic epithelial cells of normal and chronic prostatitis (CP) rats.

METHODSEighty male SD rats were randomly divided into 8 groups of equal number: normal control, normal musk, normal olibanum, normal musk + olibanum, CP model control, CP model musk, CP model olibanum, and CP model musk + olibanum. At 60 days after modeling, the rats in the control, musk, olibanum, and musk + olibanum groups were treated intragastrically with normal saline, musk (0.021 g per kg body weight per day), olibanum (1.05 g per kg body weight per day), or musk + olibanum respectively, all for 3 days. Then, all the rats were sacrificed and their prostate tissues harvested for detection of the expressions of the tight junction proteins Claudin-1, Claudin-3, Occludin, and ZO-1 in the prostatic epithelial cells by immunohistochemical staining.

RESULTSIn the CP models, only the expression of Claudin-1 was significantly increased. In the normal rats, the expression of Claudin-1 was remarkably upregulated after treated with musk (824.6 ± 393.3, P < 0.05), olibanum (982.0 ± 334.0, P < 0.05), and musk + olibanum (1088.1 ± 640.2, P < 0.01); that of Claudin-3 was elevated markedly by olibanum (1 009.5 ± 243.6, P < 0.05) and insignificantly by musk (597.5 ± 80.7), but the increasing effect of olibanum was reduced by musk + olibanum (678.4 ± 255.1). No statistically significant differences were found in the expression of Occludin among the rats treated with musk (693.0 ± 424.8), olibanum (732.1 ± 302.0), and musk + olibanum (560.2 ± 202.3), or in that of ZO-1 in the animals treated with musk (290.0 ± 166.8) and olibanum (419.7 ± 108.1), but the latter was markedly decreased in the musk + olibanum group (197.7 ± 98.2, P < 0.05). In the CP rat models, both the expressions of Claudin-1 (823.0 ± 100.1, P < 0.01) and Occludin (1160.0 ± 32.2, P < 0.05) were significantly increased. The expression of Claudin-1 was remarkably down-regulated by musk (764.9 ± 179.0), olibanum (468.4 ± 220.4), and musk + olibanum (335.1 ± 204.0) (all P < 0.05), but that of Claudin-3 up-regulated by musk (744.6 ± 94.5) and olibanum (700.1 ± 223.7) (both P < 0.05). The expression of Occludin was reduced by musk (615.0 ± 221.0), olibanum (749.6 ± 321.7), and musk + olibanum (505.8 ± 523.7), while that of ZO-1 increased by olibaum (443.2 ± 44.9) and decreased by musk + olibanum (213.5 ± 24.9, P < 0.05).

CONCLUSIONIn physiological and pathological conditions, the combination of musk and olibanum acts on the expressions of tight junction proteins in prostate epithelial cells in a selective and dual-targeting manner, promoting their permeability by down-regulating the expression of ZO-1 and maintaining their structural stability by regulating the expressions of Claudin-1, Claudin-3, and Occludin.

Animals ; Claudins ; metabolism ; Down-Regulation ; Epithelial Cells ; drug effects ; Fatty Acids, Monounsaturated ; chemistry ; Frankincense ; chemistry ; Male ; Occludin ; metabolism ; Prostate ; cytology ; Prostatitis ; Rats ; Rats, Sprague-Dawley ; Tight Junction Proteins ; metabolism ; Up-Regulation

OBJECTIVETo explore the regulatory effect of CpG methyltransferase (M.SssI) on expression of claudin-7 and claudin-8, promoting apoptosis and inhibiting proliferation of human colorectal cancer HT-29 cells.

METHODSHT-29 cells were treated with M.SssI (50 U/ml) for 24 hours. The methylation status of claudin-7 and claudin-8 gene promoters was assayed by bisulfite sequencing PCR (BSP). Real-time PCR with SYBR green I technique was used to detect the relative expression of claudin-7 and -8 mRNA, and claudin-7 and claudin-8 proteins were tested by cell immunofluorescence and Western blotting, while the effect on cell apoptosis was assessed by Hoechst 33342 fluorescence and flow cytometry. Inhibition of cell proliferation was measured by MTT assay.

RESULTSThe amounts of methylated claudin-7 and claudin-8 gene CpGs were 25, 10 in the M.SssI group, 9 and 5 in the PBS group, 0 and 3 in the 5-azacytidine group, respectively. Compared with the PBS group, Claudin-7 and -8 were significantly reduced by M.SssI (P < 0.05), but increased by 5-azacytidine (P < 0.05) at both mRNA and protein levels. Hoechst 33342 staining revealed that HT-29 cells treated with PBS and 5-azacytidine were not significantly different, showing even blue fluorescence, round shape and same cell volume. But the M.SssI group presented more apoptotic cells with intensive white fluorescence intensity. Cytometry indicated that early apoptotic index of the M.SssI group was increased by 84.7%, compared with that of the PBS group (P = 0.002). Measurement of MTT optical density demonstrated that cell growth of the M.SssI group was significantly lower than that of the PBS group (P = 0.002), with an inhibition rate of 32.1%, whereas the proliferation of 5-azacytidine group was similar to that of the PBS group (P = 0.084).

CONCLUSIONSOur findings suggest that M.SssI can down-regulate claudin-7, -8 mRNA and proteins in the human colon cancer HT-29 cells by up-regulating methylation status of claudin-7 and -8 gene promoters, and finally induce apoptosis and inhibit proliferation of the tumor cells.

Apoptosis ; physiology ; Cell Proliferation ; Claudins ; metabolism ; Colonic Neoplasms ; DNA-Cytosine Methylases ; metabolism ; Down-Regulation ; physiology ; Flow Cytometry ; Gene Expression Regulation ; HT29 Cells ; Humans ; RNA, Messenger ; Real-Time Polymerase Chain Reaction

OBJECTIVETo explore the role of estrogen in the regulation of the expression of claudin-6 and biological behavior in MCF-7 cells.

METHODSRT-PCR and immunocytochemistry were conducted to analyze the expression and localization of claudin-6 in MCF-7 cells treated with 17β-estradiol. CCK-8 kit assay and Scratch Test were conducted to analyze the capability of proliferation and migration of 17β-estradiol treated MCF-7 cells.

RESULTSRT-PCR analysis and immunocytochemistry showed that 17β-estradiol induced a concentration-and time-dependent effect on claudin-6. At 5 nmol/L and at 24 h, 17β-estradiol treatment led to an increased level of claudin-6, which was located in the membrane of MCF-7 cells. CCK-8 analysis showed a significant decrease in the capability of proliferation of MCF-7 cells compared with the control group (P < 0.05). Cells Scratch Test showed decreased migration capability of MCF-7 cells compared with the control group (P > 0.05).

CONCLUSIONS17β-E2 might regulate the expression of claudin-6 and inhibit the proliferation and migration of MCF-7 cells. The inhibitory effects of 17β-E2 on growth and migration of MCF-7 cells may be mediated by claudin-6 expression regulation.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Claudins ; Dose-Response Relationship, Drug ; Estradiol ; administration & dosage ; pharmacology ; Female ; Humans ; Membrane Proteins ; metabolism

BACKGROUNDClaudin-6 is a protein component of tight junctions and its expression could downregulate the malignant phenotype of breast carcinoma. Here we investigated the mechanisms of claudin-6 induced human MCF-7 breast cancer cells apoptosis, invasion, and migration.

METHODSTerminal deoxyribonucleotide transferase-mediated nick-end labeling assay and Annexin-V/PI double stain assay were carried out to evaluate apoptosis. Inhibitors of each pathway were used to inactivate the signaling pathways. The expression of claudin-6 and phosphate p38, Erk 1/2 and Akt protein levels was confirmed by Western blotting analysis. Invasive and migratory traits of claudin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay.

RESULTSCells with high-level expression of claudin-6 had a higher rate of apoptosis than control cells. Western blotting assay showed that by contrast to control groups, p38 pathways were more activated in claudin-6 expressing cells. However, after inhibitor SB203580 treatment, the activation status could be significantly counteracted. Furthermore, by applying inhibitors to the apoptotic rate, invasive and migratory traits were also recovered in cells with claudin-6 expression.

CONCLUSIONClaudin-6 may function through p38 mitogen-activated protein kinase pathway, of which inhibition may reverse claudin-6-induced cell apoptosis, invasion, and migration.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Movement ; genetics ; physiology ; Claudins ; genetics ; metabolism ; Humans ; In Situ Nick-End Labeling ; MAP Kinase Signaling System ; genetics ; physiology ; MCF-7 Cells ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism