To study the effect of different processes of Crotonis Fructus on fatty oil, total protein and intestinal toxicity, three kinds of processed products (heat Crotonis Semen Pulveratum, non-heat Crotonis Semen Pulveratum and diluted Crotonis Semen Pulveratum) were prepared. Mice were orally given Crotonis Fructus. The content of DAO and D-lactic acid in the serum were measured by ELISA to investigate the change of intestinal permeability in mice. Western blot was used to determine the expressions of tight junction proteins (occludin, claudin-1) in different intestinal tract, so as to observe the effect of Crotonis Fructus and its processed products on intestinal epithelial barrier. These results showed that Crotonis Fructus could significantly increase the intestinal permeability and reduce the expression of tight junction proteins in duodenum and jejunum, but with little impact on the ileum and colon. The intestinal permeability and the expression of tight junction proteins became normal after processing. However, the order of the toxicity of Crotonis Semen Pulveratum from high to low was non-heat Crotonis Semen Pulveratum > diluted Crotonis Semen Pulveratum≈4heat Crotonis Semen Pulveratum. According to the results of composition, the composition of fatty oil did not change during the processing, but the content and composition of total protein in Crotonis Semen Pulveratum changed significantly. The order of total protein content from high to low was that non-heat Crotonis Semen Pulveratum > heat Crotonis Semen Pulveratum > diluted Crotonis Semen Pulveratum. The molecular weight distribution of the total protein bands of non-heat Crotonis Semen Pulveratum and diluted Crotonis Semen Pulveratum was consistent, but the composition of total protein of heat Crotonis Semen Pulveratum significantly changed as evidenced by decreased and thin some stripes. This indicated that heating and dilution could reduce the content of total protein, and heating could cause partial protein denaturation and inactivation. In conclusion, both dilution and heating can reduce the toxicity of Crotonis Fructus, but the heating shows a more significant attenuation effect, indicating that heating is the key step in Crotonis Semen Pulveratum preparation.
Animals ; Fruit ; Ileum ; Intestinal Mucosa ; Intestines ; Jejunum ; Mice ; Occludin ; Permeability

Endothelial tight junctions (TJs) serve as an important barrier in vascular endothelial structure and maintain vascular function homeostasis. Occludin, the most representative tight junction protein, is involved in sealing cell connections and maintaining the integrity and permeability of vascular endothelium. Recent studies have shown that alterations in the expression, distribution, and structure of endothelial TJs may lead to many related vascular diseases and pathologies (such as stroke, atherosclerosis, and pulmonary hypertension etc.). Here, we reviewed the research advances on the relationship between occludin and vascular endothelial injury, including the biological information of occludin, the signal pathways that occludin exerts the protective effect of vascular endothelium, and the relationship between occludin and vascular endothelial injury-related diseases.
Endothelium, Vascular ; Occludin/genetics* ; Signal Transduction ; Tight Junctions

BACKGROUND: 'Tight junctions (TJ)' have recently been identified in the granular cell layer of the human epidermis, where they contribute to the normal adhesion between keratinocytes and to the physiologic barrier function of the epidermis. Among the TJ proteins in the epidermis, occludin is an important transmembrane protein, which is considered as a major component among the TJ. OBJECTIVE: The purpose of this study is to investigate whether regional variation exists in the expression of tight junction protein occludin in normal human epidermis. METHODS: The immunofluorescence staining for occludin was performed with specimens taken from different areas of normal skin (4 from each of 7 different anatomical sites, including the scalp, face, posterior neck, upper arm, abdomen, lower back, and inner thigh). The degrees of the expression-intensity in each specimen were estimated with the reciprocals of positive end-point titer of occludin in an immunofluorescence study. RESULTS: The highest degree expression-intensity of the TJ protein occludin among the different areas of normal epidermis was observed on the face and abdomen with a titer of 600. The lowest intensity of expression of occludin was seen in the epidermis from the upper arm. Skin specimens from the scalp, neck, back, and leg demonstrated intermediate degrees of the expression in intensity. CONCLUSION: The expression of occludin in the skin samples obtained from different locations of the body showed a statistically significant variation. This suggests that there is a certain degree of regional variation in the expression-intensity of TJ protein 'occludin' in the human epidermis.
Abdomen ; Arm ; Epidermis ; Fluorescent Antibody Technique ; Humans ; Keratinocytes ; Leg ; Neck ; Occludin ; Proteins ; Scalp ; Skin ; Tight Junctions

OBJECTIVETo investigate the role of cyclic adenosine monophosphate(cAMP) in the regulation of intestinal epithelial barrier function under hypoxia.

METHODSIntestinal epithelial barrier was established by Caco-2 monolayers. Cells were divided into four groups: normoxia (Nx), normoxia plus Forskolin(Nx+FSK), hypoxia(Hx), hypoxia plus SQ22536(Hx+SQ22536). cAMP concentrations of different groups were assessed by cAMP enzyme immunoassay kit. RT-PCR and Western blotting were used to detect the mRNA and protein expressions of claudin-1 and occludin under normoxic and hypoxic condition. Caco-2 monolayers were grown on Millicell filters, and transepithelial electrical resistance(TER) was measured using a Millipore electric resistance system.

RESULTSThe concentration of cAMP under hypoxic conditions(Hx group) was higher compared with Nx group [(6.30±0.50) pmol/L vs. (2.38±0.18) pmol/L, P<0.01]. At the same time, both mRNA and protein expressions of claudin-1 and occluding were lower in Hx group than those in Nx group(all P<0.05). TER decreased by 76.30±0.64(P<0.01). When the monolayers were exposed to hypoxia plus SQ22536 (Hx+SQ22536 group), the concentration of cAMP was(2.12±0.23) pmol/L, which was lower than that under hypoxic conditions(Hx group, P<0.01). Both mRNA and protein expressions of claudin-1 and occludin were higher compared to Hx group (all P<0.01). TER increased by 32.96±2.16 (P<0.05).

CONCLUSIONWhen Caco-2 cells are exposed to hypoxia, barrier function, claudin-1 and occludin expression are diminished in parallel with a high level of intracellular cAMP compared with the normoxic condition. Inhibition of the intracellular cAMP level under hypoxia can maintain the intestinal epithelial function through regulating the claudin-1 and occludin expression and attenuate the permeability of intestinal mucosa.

Adenosine Monophosphate ; Caco-2 Cells ; Claudin-1 ; metabolism ; Humans ; Intestinal Mucosa ; metabolism ; Intestines ; Occludin ; metabolism

OBJECTIVETo investigate the effect of hypoxia on the proliferation and occludin expression of primary rat Sertoli

METHODSWe constructed a primary Sertoli cell system by two-step enzymatic digestion in 18 -22 days old Wistar rats and identified it by oil red O and immunofluorescence methods. We randomly divided the Sertoli cells into five groups to be cultured in oxygen at the concentrations of 20%, 15%, 10%, 5% and 1%, respectively, for 6, 12, 24, 48 and 72 hours. We detected the proliferation of the Sertoli cells by CCK-8 assay, determined the expression of occludin by Western blot, and analyzed the differences among the five groups.

RESULTSOil red O staining revealed red lipid droplets in the cytoplasm of the Sertoli cells, and immunofluorescence showed the positive expression of the FasL protein, with the purity of Sertoli cells over 95% in vitro. Compared with the 20% normoxic group, the proliferation of the Sertoli cells was gradually reduced in the 15% and 10% hypoxia groups, and significantly declined in the 5% and 1% groups (P < 0.01). At 12 hours, the expression of occludin began to decrease with the prolonging of time and reduction of oxygen concentration (P < 0.01).

CONCLUSIONHypoxia suppresses the proliferation of Sertoli cells and reduces the expression of occludin. It could be inferred that hypoxia could damage the integrity of blood-testis barrier and spermatogenesis of the testis.

Animals ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Male ; Occludin ; metabolism ; Rats ; Rats, Wistar ; Sertoli Cells ; metabolism

PURPOSE: Peritumoral brain edema (PTBE) is a serious causative factor that contributes the morbidity or mortality of brain tumors. The development of PTBE is influenced by many factors, including such tight junction proteins as occludin. We evaluated the PTBE volume and survival time with respect to the occludin expression in various pathological types of brain tumors. MATERIALS AND METHODS: Fresh-frozen specimens from sixty patients who had brain tumors were obtained during surgery and the tumors were confirmed pathologically. The occludin expression was investigated by Western blot analysis. The PTBE volume was measured by using preoperative magnetic resonance (MR) imaging, and the survival time in each patient was estimated retrospectively. RESULTS: Occludin was detected in 41 (68.3%) of the cases with brain tumors and it was not expressed in the other 19 (31.7%) cases. Although the lowest expression was revealed in high-grade gliomas, its expression was variable according to the pathology of the brain tumors (p>0.05). The difference of PTBE volume between occludin-positive and negative brain tumors was statistically significant (2072.46+/-328.73 mm3 vs. 7452.42+/-1504.19 mm3, respectively, p=0.002). The mean survival time was longer in the occludin-positive tumor group than in the occludin-negative group (38.63+/-1.57 months vs. 26.16+/-3.83 months, respectively; p=0.016). CONCLUSIONS: This study suggests that the occludin expression is highly correlated to the development of PTBE in brain tumors and it might be a prognostic indicator for patient survival.
Blotting, Western ; Brain Edema ; Brain Neoplasms* ; Brain* ; Edema* ; Glioma ; Humans ; Mortality ; Occludin* ; Pathology ; Retrospective Studies ; Survival Rate ; Tight Junction Proteins

OBJECTIVETo explore the action mechanisms of temperature in male infertility or subfertility by observing the effects of different temperatures on the proliferation of and occludin (OCLN) expression in rat Sertoli cells in vitro.

METHODSWe isolated Sertoli cells from the testis of male Wistar rats, and performed oil red O staining and immunohistochemistry to identify their FasL. We cultured the Sertoli cells at 34 degrees C (control group) and at 35, 36, 37, 38 and 39 degrees C (experimental groups) for 4 days. Then we measured their proliferation by CCK-8 assay, observed their morphology and structure by hematoxylin-eosin staining, and determined their OCLN expression level by Western blotting and immunofluorescence.

RESULTSThe purity of the isolated Sertoli cells was (96.20 +/- 1.95)%. CCK-8 assay indicated that the proliferation of the Sertoli cells was increased between 34 and 36 degrees C, and decreased at 36-39 degrees C. The pyknotic nuclei and fragmentation of the Sertoli cells were more obvious at > 36 degrees C. Western blot and immunofluorescence showed the highest level of OCLN expression at 36 degrees C, which, however, decreased while the temperature rose above 36 degrees C (P < 0. 01).

CONCLUSIONHigh temperature (> 36 degrees C) inhibited the proliferation of rat Sertoli cells in vitro, and decreased the expression of OCLN, which suggests that a higher temperature above 36 degrees C may reduce male fertility by affecting the proliferation of Sertoli cells and integrity of the tight junction among Sertoli cells or Sertoli cells and other cells.

Animals ; Cell Proliferation ; Male ; Occludin ; metabolism ; Rats ; Rats, Wistar ; Sertoli Cells ; cytology ; metabolism ; Temperature ; Testis ; cytology ; metabolism

OBJECTIVE: To explore the expression and distribution of barrier molecules in epithelium of various types of chronic rhinosinusitis and its significance. METHOD: There were four groups including control (13 samples), Eos-CRSwNP (10 samples), nonEos-CRSwNP (14 samples), CRSsNP (11 samples). The method of immunohistochemistry was used to detect the protein expression of E-cadherin and occludin in nasal mucosa. RESULT: There was positive staining extensively distributed among cells in nasal mucosa. There was no significant difference in these groups. However, the occludin mainly located on the top of epithelial cells. In normal nasal mucosa, the positive expression was continuous, however, it was discontinuous both in CRSwNP and CRSsNP groups. CONCLUSION There was no E-cadherin loss in the progression of pathophysiology of chronic rhinosinusitis. But the loss of occludin may correlate to the dysfunction of epithelial barrier, which was beneficial for the pathogen invasion.
Cadherins ; metabolism ; Chronic Disease ; Epithelial Cells ; Epithelium ; metabolism ; Humans ; Immunohistochemistry ; Nasal Mucosa ; Occludin ; metabolism ; Rhinitis ; metabolism ; Sinusitis ; metabolism

Occludin is a cell adhesion molecule that is abundantly expressed in the kidney. However, the expression pattern in various renal epithelial cells is not well established. The purpose of this study was to determine the cellular localization along the tubular epithelial cells in the kidney. Kidneys from adult pigs crossbred of Yorkshire, Landrace and Duroc (three breeds) were processed for immunohistochemistry. Thiazide sensitive sodium chloride cotransporter (TSC), Na+-KATPase bat1, calbindinD28k, and H+-ATPase were used to identify the thick ascending limb, distal convoluted tubule, connecting tubule, and collecting duct, respectively. In the pig kidney, occludin was expressed in the apical domain of the tubular epithelial cells. The immunoreactivity of occludin was strongest in the collecting duct, and then gradually decreased in the connecting tubule, distal convoluted tubule, and thick ascending limb. Occludin expression was weak in the thin limbs of the loop of henle and in the proximal tubule in the pig kidney. These results suggest that occludin may be a major adhesion molecule in distal tubular epithelial cells and play a critical role in maintaining epithelial polarity of these nephron segments.
Adult ; Cell Adhesion ; Epithelial Cells ; Extremities ; Humans ; Immunohistochemistry ; Kidney ; Loop of Henle ; Nephrons ; Occludin ; Sodium Chloride Symporters ; Swine

OBJECTIVE: To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE-cadherin in the cells were examined by qRT-PCR and Western blotting. RESULTS: In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm²), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivalsinfected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P < 0.05). CONCLUSION P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.
Humans ; Cadherins/metabolism* ; Escherichia coli/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Occludin ; Porphyromonas gingivalis/metabolism* ; Umbilical Veins/metabolism*