This study aimed to investigate the recovery effect of Zuogui Jiangtang Qinggan Prescription on intestinal flora homeostasis control and intestinal mucosal barrier in type 2 diabetes mellitus(T2DM) with nonalcoholic fatty liver disease(NAFLD) induced by a high-fat diet. NAFLD was established in MKR transgenic mice(T2DM mice) by a high-fat diet(HFD), and subsequently treated for 8 weeks with Zuogui Jiangtang Qinggan Prescription(7.5, 15 g·kg~(-1)) and metformin(0.067 g·kg~(-1)). Triglyceride and liver function were assessed using serum. The hematoxylin-eosin(HE) staining and Masson staining were used to stain the liver tissue, while HE staining and AB-PAS staining were used to stain the intestine tissue. 16S rRNA sequencing was utilized to track the changes in the intestinal flora of the mice in each group. Polymerase chain reaction(PCR) and immunofluorescence were used to determine the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-1. The results demonstrated that Zuogui Jiangtang Qinggan Prescription increased the body mass of T2DM mice with NAFLD and decreased the hepatic index. It down-regulated the serum biomarkers of liver function and dyslipidemia such as alanine aminotransferase(ALT), aspartate transaminase(AST), and triglycerides(TG), increased insulin sensitivity, and improved glucose tolerance. According to the results of 16S rRNA sequencing, the Zuogui Jiangtang Qinggan Prescription altered the composition and abundance of the intestinal flora, increasing the relative abundances of Muribaculaceae, Lactobacillaceae, Lactobacillus, Akkermansia, and Bacteroidota and decreasing the relative abundances of Lachnospiraceae, Firmicutes, Deslfobacteria, Proteobacteria, and Desulfovibrionaceae. According to the pathological examination of the intestinal mucosa, Zuogui Jiangtang Qinggan Prescritpion increased the expression levels of the tight junction proteins ZO-1, Occludin, and Claudin-1, promoted intestinal mucosa repair, protected intestinal villi, and increased the height of intestinal mucosa villi and the number of goblet cells. By enhancing intestinal mucosal barrier repair and controlling intestinal microbiota homeostasis, Zuogui Jiangtang Qinggan Prescription reduces intestinal mucosal damage induced by T2DM and NAFLD.
Mice ; Animals ; Non-alcoholic Fatty Liver Disease/metabolism* ; Gastrointestinal Microbiome ; RNA, Ribosomal, 16S ; Diabetes Mellitus, Type 2/metabolism* ; Occludin/pharmacology* ; Claudin-1/metabolism* ; Intestinal Mucosa ; Liver ; Triglycerides/metabolism* ; Diet, High-Fat ; Homeostasis ; Mice, Inbred C57BL

OBJECTIVE: To investigate whether ferroptosis exists in sepsis induced intestinal injury, and to verify the association between ferroptosis in sepsis induced intestinal injury and intestinal inflammation and barrier function by stimulating and inhibiting the nuclear factor E2-related factor 2/glutathione peroxidase 4 (Nrf2/GPX4) pathway. METHODS: Forty-eight SPF grade male Sprague-Darvley (SD) rats with a body weight of 220-250 g were divided into sham operation group (Sham group), sepsis group (CLP group), sepsis+iron chelating agent deferoxamine (DFO) group (CLP+DFO group) and sepsis+ferroptosis inducer Erastin group (CLP+Erastin group) using a random number table method, with 12 rats in each group. The sepsis model was established by cecal ligation and puncture (CLP). The Sham group was only performed with abdominal opening and closing operations. After modeling, the CLP+DFO group received subcutaneous injection of 20 mg/kg of DFO, the CLP+Erastin group was intraperitoneally injected with 20 mg/kg of Erastin. Each group received subcutaneous injection of 50 mg/kg physiological saline for fluid resuscitation after surgery, and the survival status of the rats was observed 24 hours after surgery. At 24 hours after model establishment, 6 rats in each group were selected. First, live small intestine tissue was taken for observation of mitochondrial morphology in smooth muscle cells under transmission electron microscopy and determination of reactive oxygen species (ROS). Then, blood was collected from the abdominal aorta and euthanized. The remaining 6 rats were sacrificed after completing blood collection from the abdominal aorta, and then small intestine tissue was taken. Western blotting was used to detect the expression of intestinal injury markers such as Claudin-1 and ferroptosis related proteins GPX4 and Nrf2. Observe the pathological changes of small intestine tissue using hematoxylin-eosin (HE) staining and complete Chiu score; Detection of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6) levels in serum using enzyme-linked immunosorbent assay (ELISA). The levels of serum iron ions (Fe³⁺), malondialdehyde (MDA), and D-lactate dehydrogenase (D-LDH) were measured. RESULTS: (1) Compared with the Sham group, the 24-hour survival rate of rats in the CLP group and CLP+Erastin group significantly decreased (66.7%, 50.0% vs. 100%, both P < 0.05), while there was no significant difference in the CLP+DFO group (83.3% vs. 100%, P = 0.25). (2) Western blotting results showed that compared with the Sham group, the expressions of GPX4 and Claudin-1 in the small intestine tissue of the CLP group, CLP+DFO group, and CLP+Erastin group decreased significantly, while the expression of Nrf2 increased significantly (GPX4/β-actin: 0.56±0.02, 1.03±0.01, 0.32±0.01 vs. 1.57±0.01, Claudin-1/β-actin: 0.60±0.04, 0.96±0.07, 0.41±0.01 vs. 1.40±0.01, Nrf2/β-actin: 0.88±0.02, 0.72±0.01, 1.14±0.01 vs. 0.43±0.02, all P < 0.05). Compared with the CLP group, the expressions of GPX4 and Claudin-1 were significantly increased in the CLP+DFO group, while the expression of Nrf2 was significantly reduced. In the CLP+Erastin group, the expressions of GPX4 and Claudin-1 further decreased, while the expression of Nrf2 further increased (all P < 0.05). (3) Under the light microscope, compared with the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group showed structural disorder in the small intestinal mucosa and submucosal tissue, significant infiltration of inflammatory cells, and destruction of glandular and villous structures. The Chui score was significantly higher (3.25±0.46, 2.00±0.82, 4.50±0.55 vs. 1.25±0.45, all P < 0.05). (4) Under transmission electron microscopy, compared with the Sham group, the mitochondria in the other three groups of small intestinal smooth muscle cells showed varying degrees of volume reduction, increased membrane density, and reduced or disappeared cristae. The CLP+Erastin group showed the most significant changes, while the CLP+DFO group showed only slight changes in mitochondrial morphology. (5) Compared to the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group had serum levels of TNF-α, IL-1β, IL-6, MDA, D-LDH, and ROS in small intestine tissue were significantly increased, while the serum Fe³⁺ content was significantly reduced [TNF-α (ng/L): 21.49±1.41, 17.24±1.00, 28.66±2.72 vs. 14.17±1.24; IL-1β (ng/L): 108.40±3.09, 43.19±8.75, 145.70±11.00 vs. 24.50±5.55; IL-6 (ng/L): 112.50±9.76, 45.90±6.52, 151.80±9.38 vs. 12.89±6.11; MDA (μmol/L): 5.61±0.49, 3.89±0.28, 8.56±1.17 vs. 1.86±0.41; D-LDH (kU/L): 39.39±3.22, 25.38±2.34, 53.29±10.53 vs. 10.79±0.52; ROS (fluorescence intensity): 90 712±6 436, 73 278±4 775, 110 913±9 287 vs. 54 318±2 226; Fe³⁺ (μmol/L): 22.19±1.34, 34.05±1.94, 12.99±1.08 vs. 51.74±11.07; all P < 0.05]. Compared with CLP group, the levels of TNF-α, IL-1β, IL-6, MDA, D-LDH and ROS in CLP+Erastin group were further increased, and the content of Fe³⁺ was further decreased, the CLP+DFO group was the opposite (all P < 0.05). CONCLUSIONS Ferroptosis exists in the intestinal injury of septic rats, and stimulating or inhibiting ferroptosis through the Nrf2/GPX4 pathway can effectively intervene in the inflammatory state and intestinal mechanical barrier of the body.
Rats ; Male ; Animals ; NF-E2-Related Factor 2 ; Tumor Necrosis Factor-alpha ; Ferroptosis ; Reactive Oxygen Species ; Actins ; Claudin-1 ; Interleukin-6 ; Sepsis/metabolism* ; Iron

OBJECTIVES: Restoration of blood circulation within "time window" is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO). METHODS: Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting. RESULTS: The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO. CONCLUSIONS Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.
Animals ; Rats ; Blood-Brain Barrier/metabolism* ; Brain Ischemia ; Claudin-5/metabolism* ; Infarction, Middle Cerebral Artery/metabolism* ; Ischemic Stroke/metabolism* ; Occludin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Receptor, Fibroblast Growth Factor, Type 1/metabolism* ; Reperfusion Injury/metabolism*

Cold-heat combination is a common method in the treatment of ulcerative colitis, which is represented by classic drug pair, Coptidis Rhizoma and Zingiberis Rhizoma.The present study explored the synergetic effects of berberine and 6-shogaol, the primary components of Coptidis Rhizoma and Zingiberis Rhizoma, respectively, on intestinal inflammation and intestinal flora in mice with ulcerative colitis to reveal the effect and mechanism of cold-heat combination in the treatment of ulcerative colitis.The ulcerative colitis model was induced by dextran sulfate sodium(DSS) in mice.The model mice were administered with berberine(100 mg·kg~(-1)), 6-shogaol(100 mg·kg~(-1)), and berberine(50 mg·kg~(-1)) combined 6-shogaol(50 mg·kg~(-1)) by gavage, once per day.After 20 days of drug administration, mouse serum, colon tissues, and feces were sampled.Hematoxylin-eosin(HE) staining was used to observe histopathological changes in colon tissues.Alcian blue/periodic acid-Schiff(AB/PAS) staining was used to observe the changes in the mucus layer of colon tissues.Enzyme-linked immunosorbent assay(ELISA) was employed to detect the serum content of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6).Immunohistochemical method was adopted to detect the protein expression of macrophage surface markers F4/80, mucin-2, claudin-1, and zonula occludens-1(ZO-1) in colon tissues.High-throughput Meta-amplicon library sequencing was used to detect changes in the intestinal flora of mice.The results indicated that the 6-shogaol group, the berberine group, and the combination group showed significantly relieved intestinal injury, reduced number of F4/80-labeled positive macrophages in colon tissues, increased protein expression of mucin-2, claudin-1, and ZO-1, and decreased serum le-vels of TNF-α, IL-1β, and IL-6.Shannon, Simpson, Chao, and Ace indexes of the intestinal flora of mice in the 6-shogaol group and the combination group significantly increased, and Chao and Ace indexes in the berberine group significantly increased.As revealed by the bioinformatics analysis of intestinal flora sequencing, the relative abundance of Verrucomicrobia at the phylum, class, and order levels decreased significantly in all treatment groups after drug administration, while that of Bacillibacteria gradually increased.In the 6-shogaol group and the combination group, Akkermansia muciniphila completely disappeared, but acid-producing bacillus still existed in large quantities.As concluded, both 6-shogaol and berberine can inhibit intestinal inflammation, reduce the infiltration and activation of macrophages, relieve intestinal damage, reduce intestinal permeability, improve the structure of flora, and promote intestinal microecological balance.The combined application of berberine and 6-shogaol has a significant synergistic effect.
Animals ; Berberine/therapeutic use* ; Catechols ; Claudin-1/therapeutic use* ; Colitis/metabolism* ; Colitis, Ulcerative/metabolism* ; Colon ; Dextran Sulfate/metabolism* ; Disease Models, Animal ; Drugs, Chinese Herbal/pharmacology* ; Inflammation/metabolism* ; Interleukin-6/metabolism* ; Mice ; Mice, Inbred C57BL ; Mucin-2/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism*

This study aims to observe the therapeutic effect of salidroside on cerebral ischemia-reperfusion(I/R) model rats, and to specifically explore the protection of salidroside on endothelial cell barrier after I/R and the mechanism. In the experiment, SD rats were randomized into sham group, model group, and high-, medium-, and low-dose(10, 5, and 2.5 mg·kg~(-1)) salidroside groups. The suture method was used to induce I/R in rats. The infarct area, neurobehavioral evaluation, and brain water content were used to evaluate the efficacy of salidroside. As for the experiment on the mechanism, high-dose and low-dose salidroside groups were designed. The pathological morphology was observed based on hematoxylin and eosin(HE) staining, and ultrastructure of vascular endothelial cells based on transmission electron microscopy. The content of nitric oxide(NO) in serum, four indexes of blood coagulation, and the content of von Willebrand factor(vWF) in plasma were measured. Western blot(WB) and immunofluorescence(IF) were employed to determine the expression of tight junction proteins(ZO-1, occluding, and claudin-1) and matrix metalloproteinase 9(MMP-9) in the cortex. The results showed that the model group had obvious neurological deficit, obvious infarct in the right brain tissue, and significant increase in water content in brain tissue compared with the sham group. Compared with the model group, high-dose and low-dose salidroside groups showed decrease in neurobehavioral score, and the high-, medium-, and low-dose salidroside groups demonstrated obviously small infarct area and significant decrease in water content in brain tissue. The results of HE staining and transmission electron microscopy showed that rats had necrosis of neurons, damage of original physiological structure of endothelial cells, and disintegration of the tight junction between endothelial cells after I/R compared with the sham group. Compared with the model group, the high-dose and low-dose salidroside groups showed alleviation of neuron injury and intact physiological structure of endothelial cells. The model group had significantly lower serum level of NO, significantly higher plasma levels of vWF and fibrinogen(FIB), and significantly shorter thrombin time(TT) and prothrombin time(PT) than the sham group. Compared with model group, the high-dose and low-dose salidroside groups increased the serum content of NO in serum, decreased the plasma levels of FIB and vWF, and significantly prolonged TT and PT. WB and IF results showed that the model group had significantly lower levels of ZO-1, occluding, and claudin-1 among endothelial cells and significantly higher level of MMP-9 than the sham group. Compared with the model group, high-dose and low-dose salidroside significantly increased the levels of ZO-1, occluding, and claudin-1 in the cortex. The above experimental results show that salidroside has clear therapeutic effect on I/R rats and protects the brain. To be specific, it alleviates the damage of endothelial cells by increasing NO synthesis in endothelial cells, inhibiting coagulation reaction and MMP-9 expression, up-regulating the expression of ZO-1, occludin, and claudin-1, thereby protecting the brain.
Animals ; Rats ; Matrix Metalloproteinase 9/metabolism* ; Endothelial Cells/metabolism* ; Reperfusion Injury/metabolism* ; Blood-Brain Barrier ; Claudin-1/therapeutic use* ; von Willebrand Factor/therapeutic use* ; Rats, Sprague-Dawley ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Reperfusion ; Water/metabolism*

To investigate the expressions of mucosal barrier proteins in colon cell line DLD-1 under hypoxic environment and its mechanism. Methods After DLD-1 cells were treated separately with hypoxia(l% O),vitamin D(100 nmol/L),or vitamin D plus hypoxia for 48 hours,the expressions of vitamin D receptor(VDR),tight junction proteins zonula occludens-1(ZO-1),occludin,Claudin-1,and adherent junction protein(E-cadherin)were determined by Western blot.Stable VDR knock-down(Sh-VDR)DLD-1 cell line and control DLD-1 cell line were established by lentivirus package technology and the protein expressions after hypoxia treatment were detected. Results Compared with control group,the expressions of occludin,Claudin-1,and VDR increased significantly after hypoxia treatment(all <0.001).In addition to the protein expressions of occludin,Claudin-1 and VDR,the expressions of ZO-1 and E-cadherin were also obviously higher in vitamin D plus hypoxia group than in single vitamin D treatment group(all <0.001).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of ZO-1(<0.001),occludin(<0.05),Claudin-1(<0.01)and E-cadherin(<0.001)when compared with untreated Sh-VDR cell line. Conclusion VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD-1 colon cell line,indicating that VDR pathway may be another important protective mechanism for gut barrier in low-oxygen environment.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Hypoxia ; Cell Line ; Claudin-1 ; metabolism ; Colon ; cytology ; Humans ; Occludin ; metabolism ; Receptors, Calcitriol ; metabolism ; Tight Junctions ; Vitamin D ; pharmacology ; Zonula Occludens-1 Protein ; metabolism

This paper aimed to observe the protective effect of catalpol on the high glucose induced destruction of tight junctions of rat primary brain microvascular endothelial cells (BMECs). Catalpol co-administrated with high glucose increased BMECs survival, decreased its ET-1 secretion, and improved transmembrane electrical resistance in a time-dependent manner. Furthermore, transmission electron microscopy was used to observe catalpol's protective effect on tight junction. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and up-regulated the tight junction proteins claudin-5 and ZO-1, which were further demonstrated by the mRNA expression levels of claudin-5, occludin, ZO-1, ZO-2, ZO-3, -actintin, vinculin and cateinins. This study indicated that catalpol reverses the disaggregation of cytoskeleton actin in BMECs and up-regulates the expression of tight junction proteins, such as claudin-5, occludin, and ZO-1, and finally alleviates the increase in high glucose-induced BMECs injury.
Actin Cytoskeleton ; drug effects ; Actins ; metabolism ; Animals ; Brain ; cytology ; Cells, Cultured ; Claudin-5 ; metabolism ; Endothelial Cells ; drug effects ; Glucose ; Iridoid Glucosides ; pharmacology ; Phosphoproteins ; Rats ; Tight Junctions ; drug effects ; Zonula Occludens-1 Protein ; metabolism

BACKGROUNDLeakage of the intestinal mucosal barrier may cause translocation of bacteria, then leading to multiorgan failure. This study hypothesized that rhubarb monomers might protect the gut mucosal barrier in sepsis through junction proteins.

METHODSHealthy male Sprague-Dawley rats (weighing 230-250 g) under anesthesia and sedation were subjected to cecal ligation and perforation (CLP). After surgical preparation, rats were randomly assigned to eight groups (n = 6 or 8 each group): sham group (Group A: normal saline gavage); sepsis group (Group B: normal saline gavage); Group C (intraperitoneally, dexamethasone 0.5 mg/kg) immediately after CLP surgery; and rhubarb monomer (100 mg/kg in normal saline)-treated groups (Group D: rhein; Group E: emodin; Group F: 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid; Group G: 1-O-caffeoyl-2-(4-hydroxy-O-cinnamoyl)-D-glucose; and Group H: daucosterol linoleate). Animals were sacrificed after 24 h. Intestinal histology, lactulose, mannitol concentrations were measured, and zonula occludens (ZO)-1, occludin and claudin-5 transcription (polymerase chain reaction), translation (by Western blot analysis), and expression (by immunohistochemistry) were also measured.

RESULTSIntestinal histology revealed injury to intestinal mucosal villi induced by sepsis in Group B, compared with Group A. Compared with Group A (0.17 ± 0.41), the pathological scores in Groups B (2.83 ± 0.41, P < 0.001), C (1.83 ± 0.41, P < 0.001), D (2.00 ± 0.63, P < 0.001), E (1.83 ± 0.41, P < 0.001), F (1.83 ± 0.75, P < 0.001), G (2.17 ± 0.41, P < 0.001),and H (1.83 ± 0.41, P < 0.001) were significantly increased. Lactulose/mannitol (L/M) ratio in Group B (0.046 ± 0.003) was significantly higher than in Group A (0.013 ± 0.001, P< 0.001) while L/M ratios in Groups C (0.028 ± 0.002, P< 0.001), D (0.029 ± 0.003, P< 0.001), E (0.026 ± 0.003, P< 0.001), F (0.027 ± 0.003, P< 0.001), G (0.030 ± 0.005, P< 0.001), and H (0.026 ± 0.002, P< 0.001) were significantly lower than that in Group B. ZO-1, occludin and claudin-5 transcription, translation, and expression in Group B were significantly lower than that in Group A (P < 0.001), but they were significantly higher in Groups C, D, E, F, G, and H than those in Group B (P < 0.05).

CONCLUSIONRhubarb monomer treatment ameliorated mucosal damage in sepsis via enhanced transcription, translation, and expression of junction proteins.

Animals ; Claudin-5 ; metabolism ; Intestinal Mucosa ; drug effects ; metabolism ; Lactulose ; metabolism ; Male ; Mannitol ; metabolism ; Occludin ; metabolism ; Plant Extracts ; chemistry ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Rheum ; chemistry ; Sepsis ; drug therapy ; metabolism ; Zonula Occludens-1 Protein ; metabolism

Objective: To explore the role of TGF-β1 in the proliferation and apoptosis of Sertoli cells and its effect on the expressions of tight junction-related proteins and genes in rats. METHODS: Rat Sertoli cells were isolated in vitro, primarily cultured, and divided into groups A (blank control), B (TGF-β1 receptor blocker), C (TGF-β1), and D (TGF-β1 + receptor blocker). The proliferation and apoptosis of the cells were detected by CCK-8 and flow cytometry, respectively. After establishment of the dual-chamber model for the primary culture of Sertoli cells, the trans-epithelia electrical resistance (TER) value was measured and the relative expressions of Occludin, ZO-1 and Claudin Ⅱ determined by RT-PCR and Western blot. RESULTS: The OD value of the proliferation of the Sertoli cells was markedly higher in group C than in groups A and D (0.79 ± 0.04 vs 0.66 ± 0.05 and 0.68 ± 0.02, P<0.05), with statistically significant differences among the four groups (F = 5.05, P <0.05). However, no remarkable difference with found among the four groups in the apoptosis rate of the cells (F = 1.13, P >0.05). The TER value was dramatically decreased in group C as compared with groups A and D (［176.37 ± 16.61］ vs ［281.42 ± 9.83］ and ［254.37 ± 13.55］ /cm2, P<0.01), with statistically significant differences among the four groups (F = 38.99, P<0.01). There were no remarkable differences among the four groups in the mRNA expressions of ZO-1 and Claudin Ⅱ (F = 0.49 and 0.93, P>0.05) or their protein expressions (F = 0.28 and 1.31, P>0.05). Both the mRNA and protein expressions of Occludin were markedly lower in group C than in A and D (P<0.01 and P<0.05), with statistically significant differences among the four groups (F = 6.86 and 6.87, P<0.01). CONCLUSIONS TGF-β1 can promote the proliferation of Sertoli cells in rats and act on the tight junction of the cells by regulating the expression of Occludin.
Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Claudin-2 ; metabolism ; Male ; Occludin ; metabolism ; RNA, Messenger ; Rats ; Sertoli Cells ; cytology ; physiology ; Tight Junction Proteins ; metabolism ; Tight Junctions ; genetics ; metabolism ; Transforming Growth Factor beta1 ; physiology ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVETo culture rat prostate glandular epithelial cells and study their barrier functions in vitro.

METHODSRat prostate glandular epithelial cells were cultured in vitro. The expression of the tight junction protein claudin-1 was determined by immunohistochemistry, the structure and composition of the epithelial cells observed under the inverted microscope and transmission electron microscope. The transepithelial electrical resistances (TEERs) were monitored with the Millicell system. The permeability of the prostate glandular epithelial cells was assessed by the phenol red leakage test.

RESULTSCompact monolayer cell structures were formed in the prostate glandular epithelial cells cultured in vitro. Immunohistochemistry showed the expression of the tight junction protein claudin-1 and transmission electron microscopy confirmed the formation of tight junctions between the adjacent glandular epithelial cells. The TEERs in the cultured prostate glandular epithelial cells reached the peak of about (201.3 ± 3.5) Ω/cm2 on the 8th day. The phenol red leakage test manifested a decreased permeability of the cell layers with the increase of TEERs.

CONCLUSIONThe structure and function of rat prostate glandular epithelial cells are similar to those of brain capillary endothelial cells, retinal capillary endothelial cells, and intestinal epithelial cells. In vitro cultured prostate glandular epithelial cells have the barrier function and can be used as a model for the study of blood prostate barrier in vitro.

Animals ; Cell Membrane Permeability ; Cells, Cultured ; Claudin-1 ; metabolism ; Electric Impedance ; Epithelial Cells ; pathology ; physiology ; ultrastructure ; In Vitro Techniques ; Male ; Microscopy, Electron, Transmission ; Phenolsulfonphthalein ; pharmacokinetics ; Prostate ; metabolism ; pathology ; Rats ; Tight Junctions