Treatment with Glycyrrhiza glabra Extract Induces Anxiolytic Effects Associated with Reduced Salt Preference and Changes in Barrier Protein Gene Expression.

We have previously identified that low responsiveness to antidepressive therapy is associated with higher aldosterone/cortisol ratio, lower systolic blood pressure, and higher salt preference. Glycyrrhiza glabra (GG) contains glycyrrhizin, an inhibitor of 11ß-hydroxysteroid-dehydrogenase type-2 and antagonist of toll-like receptor 4. The primary hypothesis of this study is that food enrichment with GG extract results in decreased anxiety behavior and reduced salt preference under stress and non-stress conditions. The secondary hypothesis is that the mentioned changes are associated with altered gene expression of barrier proteins in the prefrontal cortex. Male Sprague-Dawley rats were exposed to chronic mild stress for five weeks. Both stressed and unstressed rats were fed a diet with or without an extract of GG roots for the last two weeks. GG induced anxiolytic effects in animals independent of stress exposure, as measured in elevated plus maze test. Salt preference and intake were significantly reduced by GG under control, but not stress conditions. The gene expression of the barrier protein claudin-11 in the prefrontal cortex was increased in control rats exposed to GG, whereas stress-induced rise was prevented. Exposure to GG-enriched diet resulted in reduced ZO-1 expression irrespective of stress conditions. In conclusion, the observed effects of GG are in line with a reduction in the activity of central mineralocorticoid receptors. The treatment with GG extract or its active components may, therefore, be a useful adjunct therapy for patients with subtypes of depression and anxiety disorders with heightened renin-angiotensin-aldosterone system and/or inflammatory activity.

Trefoil factor 3 peptide regulates migration via a Twist-dependent pathway in gastric cell.

Trefoil factor 3 (TFF3) is a member of the TFF-domain peptide family and essential in regulating cell migration and maintaining mucosal integrity in gastrointestinal tract. However, the role of TFF3 and its downstream regulating mechanisms in cancer cell migration remain unclear. We previously reported that TFF3 prolonged the up-regulation of Twist protein to modulate IL-8 secretion in intestinal epithelial cells. In this study, we investigated the role of Twist protein in TFF3-induced migration of SGC7901 cells. While Twist was activated by TFF3, siRNA-mediated knockdown of Twist abolished TFF3-induced cell migration. Furthermore, the migration related marker CK-8 as well as ZO-1 and MMP-9 was also regulated by TFF3 via a Twist-dependent mechanism. Our study suggests that Twist, as an important potential downstream effector, plays a key role in TFF3-modulated metastasis in gastric cancer and can be a promising therapeutic target against intestinal-type gastric cancer.

Effects of electroacupuncture on the microvascular structure and related protein expression in the hippocampus of vascular dementia rats. / ç”µé’ˆå¯¹è¡€ç®¡æ€§ç—´å‘†å¤§é¼ æµ·é©¬å¾®è¡€ç®¡ç»“æž„å’Œç›¸å ³è›‹ç™½è¡¨è¾¾çš„å½±å“.

OBJECTIVES: To explore the effect of electroacupuncture (EA) at "Baihui" (GV20) and "Shenting" (GV24) on the microvascular structure and related protein expression in the hippocampus of vascular dementia (VD) rat model, and to investigate the mechanism of EA in the treatment of VD. METHODS: A total of 24 SD rats were randomly divided into sham operation, model, EA, and oxiracetam groups, with 6 rats in each group. Multiple cerebral infarction method was used to establish VD model. In the EA group, EA was applied to GV20 and GV24 for 30 min, once daily for 14 days. Rats in the oxiracetam group were treated with oxiracetam (50 mg/kg) by intraperitoneal injection, and the course of treatment was the same as that in the EA group. Learning and memory ability were evaluated by using Morris water maze test and new object recognition experiment. The cerebral blood flow was detected by laser doppler. The microvascular structure in the hippocampus was observed by transmission electron microscopy. The expression of vascular structure related proteins of platelet-derived growth factor receptor (PDGFR)-ß, platelet endothelial cell adhesion molecule-1(CD31), neural cadherin N-Cadherin, Zonula occludens protein-1(ZO-1) in the hippocampus were measured by Western blot. RESULTS: Compared with the sham operation group, the rats in the model group had a significant increase in time of first crossing the platform, a significant decrease in the number of crossing platform and the new object preference index (P<0.05), a significant decrease in cerebral blood flow (P<0.05), and a significant increase in the brain weight (P<0.05). The structure boundary of pericyte and endothelial cells in the microvessels of the hippocampal CA1 area of model group was blurred, accompanied by obvious edema around the vessels and the reduction of tight junctions. The protein expression levels of PDGFR-ß, CD31, N-Cadherin, ZO-1 were significantly decreased in the model group compared with those in the sham operation group (P<0.05). Compared with the model group, the time of first crossing the platform of rats in the EA and oxiracetam group was shortened, the number of crossing platform were increased (P<0.05), the cerebral blood flow was increased (P<0.05), the brain weight was decreased (P<0.05), the morphology and structure of pericyte and endothelial cells in the microvessels of hippocampal CA1 area were intact, accompanied by the increase of tight junctions. Additionally, Compared with the model group, the EA group had a significant increase in the new object preference index (P<0.05), the protein expression levels of PDGFR-ß, CD31, ZO-1 in the EA group were increased (P<0.05), and the expression of PDGFR-ß, N-Cadherin, ZO-1 in the oxiracetam group were increased (P<0.05). CONCLUSIONS: EA at GV20 and GV24 can improve the learning and memory ability of VD rats, and the mechanism may be related to the repair of microvascular structures and improvement of cerebral blood flow.

Fecal microbiota transplantation ameliorates bone loss in mice with ovariectomy-induced osteoporosis via modulating gut microbiota and metabolic function.

Background: Osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass and destruction of bone microstructure, which tends to result in enhanced bone fragility and related fractures. The postmenopausal osteoporosis (PMOP) has a relatively high proportion, and numerous studies reveal that estrogen-deficiency is related to the imbalance of gut microbiota (GM), impaired intestinal mucosal barrier function and enhanced inflammatory reactivity. However, the underlying mechanisms remain unclear and the existing interventions are also scarce. Methods: In this study, we established a mouse model induced by ovariectomy (OVX) and conducted fecal microbiota transplantation (FMT) by gavage every day for 8 weeks. Subsequently, the bone mass and microarchitecture of mice were evaluated by the micro computed tomography (Micro-CT). The intestinal permeability, pro-osteoclastogenic cytokines expression, osteogenic and osteoclastic activities were detected by the immunohistological analysis, histological examination, enzyme-linked immunosorbent assay (ELISA) and western blot analysis accordingly. Additionally, the composition and abundance of GM were assessed by 16S rRNA sequencing and the fecal short chain fatty acids (SCFAs) level was measured by metabolomics. Results: Our results demonstrated that FMT inhibited the excessive osteoclastogenesis and prevented the OVX-induced bone loss. Specifically, compared with the OVX group, FMT enhanced the expressions of tight junction proteins (zonula occludens protein 1 (ZO-1) and Occludin) and suppressed the release of pro-osteoclastogenic cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)). Furthermore, FMT also optimized the composition and abundance of GM, and increased the fecal SCFAs level (mainly acetic acid and propionic acid). Conclusions: Collectively, based on GM-bone axis, FMT prevented the OVX-induced bone loss by correcting the imbalance of GM, improving the SCFAs level, optimizing the intestinal permeability and suppressing the release of pro-osteoclastogenic cytokines, which may be an alternative option to serve as a promising candidate for the prevention and treatment of PMOP in the future. The translational potential of this article: This study indicates the ingenious involvement of GM-bone axis in PMOP and the role of FMT in reshaping the status of GM and ameliorating the bone loss in OVX-induced mice. FMT might serve as a promising candidate for the prevention and treatment of PMOP in the future.

Splenectomy attenuates severe thermal trauma-induced intestinal barrier breakdown in rats.

The severe local thermal trauma activates a number of systemic inflammatory mediators, such as TNF-α, NF-κB, resulting in a disruption of gut barrier. The gastrointestinal tight junction (TJ) is highly regulated by membrane-associated proteins including zonula occludens protein-1 (ZO-1) and occludin, which can be modulated by inflammatory cytokines. As splenectomy has been shown to reduce secretion of cytokines, we hypothesized that (1) severe scald injury up-regulates TNF-α and NF-κB, meanwhile down-regulates expression of ZO-1 and occludin, leading to the increased intestinal permeability, and (2) splenectomy can prevent the burn-induced decrease in ZO-1 and occludin expression, resulting in improved intestinal barrier. Wistar rats undergoing a 30% total body surface area (TBSA) thermal trauma were randomized to receive an accessorial splenectomy meanwhile or not. Intestinal injury was assessed by histological morphological analysis, and serum endotoxin levels, TNF-α, NF-κB, ZO-1 and occludin levels were detected by Western blotting in the terminal ileum mucosal tissue. 30% TBSA burn caused a significant increase in serum endotoxin levels, but NF-κB, and TNF-α, and the average intestinal villus height and mucosal thickness were decreased significantly. Burn injury could also markedly decrease the levels of ZO-1 and occludin in terminal ileum mucosal tissue (all P<0.01). Splenectomy at 7th day after burn significantly reversed the burn-induced breakdown of ZO-1 and occludin (all P<0.01). The results of this study suggest that severe thermal injury damages the intestinal mucosal barrier. Splenectomy may provide a therapeutic benefit in restoring burn-induced intestinal barrier by decreasing the release of inflammatory cytokines and recovering TJ proteins.

Effect of salvianolate on intestinal epithelium tight junction protein zonula occludens protein 1 in cirrhotic rats.

AIM: To study the effect of salvianolate on tight junctions (TJs) and zonula occludens protein 1 (ZO-1) in small intestinal mucosa of cirrhotic rats. METHODS: Cirrhosis was induced using carbon tetrachloride. Rats were randomly divided into the untreated group, low-dose salvianolate (12 mg/kg) treatment group, medium-dose salvianolate (24 mg/kg) treatment group, and high-dose salvianolate (48 mg/kg) treatment group, and were treated for 2 wk. Another 10 healthy rats served as the normal control group. Histological changes in liver tissue samples were observed under a light microscope. We evaluated morphologic indices of ileal mucosa including intestinal villi width and thickness of mucosa and intestinal wall using a pathological image analysis system. Ultrastructural changes in small intestinal mucosa were investigated in the five groups using transmission electron microscopy. The changes in ZO-1 expression, a tight junction protein, were analyzed by immunocytochemistry. The staining index was calculated as the product of the staining intensity score and the proportion of positive cells. RESULTS: In the untreated group, hepatocytes showed a disordered arrangement, fatty degeneration was extensive, swelling was obvious, and disorganized lobules were divided by collagen fibers in hepatic tissue, which were partly improved in the salvianolate treated groups. In the untreated group, abundant lymphocytes infiltrated the fibrous tissue with proliferation of bile ducts, and collagen fibers gradually decreased and damaged hepatic lobules were partly repaired following salvianolate treatment. Compared with the untreated group, no differences in intestinal villi width between the five groups were observed. The villi height as well as mucosa and intestinal wall thickness gradually thickened with salvianolate treatment and were significantly shorter in the untreated group compared with those in the salvianolate treatment groups and normal group (P < 0.01). The number of microvilli decreased and showed irregular lengths and arrangements in the untreated group. The intercellular space between epithelial cells was wider. The TJs were discontinuous, which indicated disruption in TJ morphology in the untreated group. In the treated groups, the microvilli in the intestinal epithelium were regular and the TJs were gradually integrated and distinct. The expression of ZO-1 decreased in the small intestine of the untreated cirrhotic rats. The high expression rate of ZO-1 in ileal mucosa in the untreated group was significantly lower than that in the medium-dose salvianolate group (21.43% vs 64.29%, χ(2) = 5.25, P < 0.05), high-dose salvianolate group (21.43% vs 76.92%, χ(2) = 8.315, P < 0.01) and normal group (21.43% vs 90%, χ(2) = 10.98, P < 0.01). CONCLUSION: Salvianolate improves liver histopathological changes, repairs intestinal mucosa and TJ structure, and enhances ZO-1 expression in the small intestinal mucosa in cirrhotic rats.

Splenectomy attenuates severe thermal trauma-induced intestinal barrier breakdown in rats / 华中科技大学学报（医学）（英德文版）

The severe local thermal trauma activates a number of systemic inflammatory mediators, such as TNF-α, NF-κB, resulting in a disruption of gut barrier. The gastrointestinal tight junction (TJ) is highly regulated by membrane-associated proteins including zonula occludens protein-1 (ZO-1) and occludin, which can be modulated by inflammatory cytokines. As splenectomy has been shown to reduce secretion of cytokines, we hypothesized that (1) severe scald injury up-regulates TNF-α and NF-κB, meanwhile down-regulates expression of ZO-1 and occludin, leading to the increased intestinal permeability, and (2) splenectomy can prevent the burn-induced decrease in ZO-1 and occludin expression, resulting in improved intestinal barrier. Wistar rats undergoing a 30% total body surface area (TBSA) thermal trauma were randomized to receive an accessorial splenectomy meanwhile or not. Intestinal injury was assessed by histological morphological analysis, and serum endotoxin levels, TNF-α, NF-κB, ZO-1 and occludin levels were detected by Western blotting in the terminal ileum mucosal tissue. 30% TBSA burn caused a significant increase in serum endotoxin levels, but NF-κB, and TNF-α, and the average intestinal villus height and mucosal thickness were decreased significantly. Burn injury could also markedly decrease the levels of ZO-1 and occludin in terminal ileum mucosal tissue (all P<0.01). Splenectomy at 7th day after burn significantly reversed the burn-induced breakdown of ZO-1 and occludin (all P<0.01). The results of this study suggest that severe thermal injury damages the intestinal mucosal barrier. Splenectomy may provide a therapeutic benefit in restoring burn-induced intestinal barrier by decreasing the release of inflammatory cytokines and recovering TJ proteins.

Tight junctions in Hailey-Hailey and Darier's diseases.

Hailey-Hailey disease (HHD) and Darier's disease (DD) are caused by mutations in Ca(2+)-ATPases with the end result of desmosomal disruption and suprabasal acantholysis. Tight junctions (TJ) are located in the granular cell layer in normal skin and contribute to the epidermal barrier. Aberrations in the epidermal differentiation, such as in psoriasis, have been shown to lead to changes in the expression of TJ components. Our aim was to elucidate the expression and dynamics of the TJ proteins during the disruption of desmosomes in HHD and DD lesions. Indirect immunofluorescence and avidin-biotin labeling for TJ, desmosomal and adherens junction proteins, and subsequent analyses with the confocal laser scanning microscope were carried out on 14 HHD and 14 DD skin samples. Transepidermal water loss (TEWL) was measured in normal and lesional epidermis of nine HHD and eight DD patients to evaluate the function of the epidermal barrier in HHD and DD skin. The localization of TJ proteins claudin-1, claudin-4, ZO-1, and occludin in perilesional HHD and DD epidermis was similar to that previously described in normal skin. In HHD lesions the tissue distribution of ZO-1 expanded to the acantholytic spinous cells. In agreement with previous findings, desmoplakin was localized intracellularly. In contrast claudin-1 and ZO-1 persisted in the cell-cell contact sites of acantholytic cells. TEWL was increased in the lesional skin. The current results suggest that TJ components follow different dynamics in acantholysis of HHD and DD compared to desmosomal and adherens junction proteins.

THE EXPRESSION AND ASSOCIATION OF CONNEXIN 36 WITH ZONULA OCCLUDENS PROTEIN 1 IN HeLa CELLS / 解剖学报

Objective To investigate the expression and association of Cx36 with ZO-1 in Cx36 transfected HeLa cells. Methods RT-PCR,PCR products cloning into PCR2.1TOPO vector,Cx36-pcDNA3 expression vector construction,cell culture,transient transfection using lipofectamine 2000,stable clone screening with G418,Western blotting,double immunofluorescence,and immunoprecipitation(IP) were used. Results Cx36-pcDNA3 expression vector was constructed and transfected into HeLa cells.Using homogenates from Cx36 transfected HeLa cells,Western blotting showed Cx36 band.By immunofluorescence microscopy,Cx36 transfected HeLa cells displayed punctate immunolabeling for Cx36 between cells.Irmmunolabeling for ZO-1 in these cells exhibited similar distribution to Cx36.By laser scanning confocal microscopy after double labeling with Cx36 and ZO-1 antibody revealed a high degree of Cx36 and ZO-1 colocalization at sites of cell-cell contact.Cx36 transfected HeLa cells were taken for IP with ZO-1 antibody,blots of IP protein probed with Cx36 antibody showed detectin of Cx36.Blots showed detection of ZO-1 after IP with Cx36 antibody from Cx36 transfected HeLa cells.Conclusion Cx36 was expressed in Cx36 transfected HeLa cells,Cx36 colocalizated and associated with ZO-1 in Cx36 transfected HeLa cells.