四君子汤对溃疡性结肠炎小鼠模型occludin和claudin-1表达的影响

目的:探讨四君子汤对溃疡性结肠炎(UC)小鼠模型结肠粘膜中occludin、claudin-1表达的影响。方法:采用右旋葡聚糖硫酸钠(DSS)诱导UC小鼠模型,实验分为五组,即正常组、模型组、四君子汤低剂量治疗组、中剂量治疗组、高剂量治疗组、西药组,共治疗7天。对小鼠肠黏膜的大体形态和组织病理变化进行观察,使用RT-PCR和Western blot检测occludin、claudin-1 m RNA和蛋白的表达。结果:与模型组相比较,四君子汤低、中、高剂量治疗组以及西药组小鼠的饮食、体重、精神、活动度、脓血便等一般情况有所改善,黏膜层缺损、隐窝破坏、炎症细胞浸润等病理表现有所缓解。与模型组相比较,高剂量治疗组小鼠结肠组织中occludin、claudin-1蛋白和m RNA的表达升高(P0.05),低剂量和中剂量治疗组也有不同程度的升高。与西药组相比较,低、中、高剂量治疗组小鼠结肠组织中occludin、claudin-1蛋白和m RNA的表达无统计学差异(P0.05)。结论:四君子汤可以改善脓血便等症状,缓解肠粘膜的损伤,上调occludin和claudin-1的表达,对UC小鼠有治疗作用。     

Evaluation of Caco-2 and human intestinal epithelial cells as in vitro models of colonic and small intestinal integrity

Although the colonic cell line Caco-2 is widely used as a model of the small intestinal barrier function, it has limitations such as overestimated transepithelial electrical resistance (TEER) compared to in vivo conditions. Therefore, we investigated Human Intestinal Epithelial Cells (HIECs) as an alternative in vitro model.We explored whether cell seeding number of HIEC-6, and the number of incubation days for HIEC and Caco-2 cells had an impact on TEER, and tight junction expression was examined for both cell lines via immunofluorescence in the presence and absence of probiotic bacteria.We observed no significant difference in TEER readings for either cell lines when cultured for different days. Further, the HIEC TEER readings did not change with increased seeding number and were not significantly different from a control with no cells. HIECs expressed Claudin-1 and Zonula Occludens-1 but not Occludin. Caco-2 co-culture with probiotic bacteria demonstrated a significant increase in TEER, particularly for the lactobacillus strains, whereas HIEC TEER did not respond to bacterial co-incubation.Our study shows that although HIECs express certain TJ proteins, a significant TEER was not observed, likely due to the embryonic origin of the cells, which limits the application of this cell line as a suitable model for small intestinal barrier function.     

Disruption of the blood-testis barrier integrity by bisphenol A in vitro: Is this a suitable model for studying blood-testis barrier dynamics?

Bisphenol A, an estrogenic environmental toxicant, has been implicated to have hazardous effects on reproductive health in humans and rodents. However, there are conflicting reports in the literature regarding its effects on male reproductive function. In this study, it was shown that in adult rats treated with acute doses of bisphenol A, a small but statistically insignificant percentage of seminiferous tubules in the testes displayed signs of germ cell loss, consistent with some earlier reports. It also failed to disrupt the blood-testis barrier in vivo. This is possibly due to the low bioavailability of free bisphenol A in the systemic circulation. However, bisphenol A disrupted the blood-testis barrier when administered to immature 20-day-old rats, consistent with earlier reports concerning the higher susceptibility of immature rats towards bisphenol A. This observation was confirmed using primary Sertoli cells cultured in vitro with established tight junction-permeability barrier that mimicked the blood-testis barrier in vivo. The reversible disruption of Sertoli cell tight junction barrier by bisphenol A was associated with an activation of ERK, and a decline in the levels of selected proteins at the tight junction, basal ectoplasmic specialization, and gap junction at the blood-testis barrier. Studies by dual-labeled immunofluorescence analysis and biotinylation techniques also illustrated declining levels of occludin, connexin 43, and N-cadherin at the cell–cell interface following bisphenol A treatment. In summary, bisphenol A reversibly perturbs the integrity of the blood-testis barrier in Sertoli cells in vitro, which can also serve as a suitable model for studying the dynamics of the blood-testis barrier.     

Tight junction claudins and the kidney in sickness and in health

The epithelial cell tight junction has several functions including the control of paracellular transport between epithelial cells. Renal paracellular transport has been long recognized to exhibit unique characteristics within different segments of the nephron, functions as an important component of normal renal physiology and has been speculated to contribute to renal related pathology if functioning abnormally. The discovery of a large family of tight junction associated 4-transmembrane spanning domain proteins named claudins has advanced our understanding on how the paracellular permeability properties of tight junctions are determined. In the kidney, claudins are expressed in a nephron-specific pattern and are major determinants of the paracellular permeability of tight junctions in different nephron segments. The combination of nephron segment claudin expression patterns, inherited renal diseases, and renal epithelial cell culture models is providing important clues about how tight junction claudin molecules function in different segments of the nephron under normal and pathological conditions. This review discusses early observations of renal tubule paracellular transport and more recent information on the discovery of the claudin family of tight junction associated membrane proteins and how they relate to normal renal function as well as diseases of the human kidney.     

Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma

Junctional adhesion molecule-A (JAM-A) is one component of tight junctions which are involved in important processes like paracellular permeability, cell polarity, adhesion, migration, and angiogenesis. Here we describe JAM-A expression in distal convoluted tubule, connecting tubule, and in cells of the collecting duct of the healthy human kidney. In addition, JAM-A was weakly expressed in cells of the proximal tubule. Using immunofluorescence, FACS and Western blot analysis we investigated JAM-A expression in tubular cells in vitro. Interestingly, treatment of HK-2 cells with IFN-γ and TNF-α resulted in a metalloproteinase mediated downregulation of JAM-A. Importantly, in a tissue micro-array JAM-A protein expression was significantly downregulated in patients with clear cell renal cell carcinoma. Furthermore, knockdown of JAM-A with JAM-A specific siRNA induced the migration of RCC4 cells. In summary, downregulation of JAM-A is an early event in the development of renal cancer and increases the migration of renal cancer cells.     

Occludin expression in goldfish held in ion-poor water

With an emphasis on the tight junction protein occludin, the response of goldfish following abrupt exposure (0–120 h) as well as long-term acclimation (14 and 28 days) to ion-poor water (IPW) was examined. Both abrupt and long-term exposure to IPW lowered serum osmolality, [Na⁺] and [Cl⁻], and elevated serum glucose. After abrupt exposure to IPW, gill tissue exhibited a prompt and sustained decrease in Na⁺–K⁺–ATPase activity, and a transient increase in occludin expression that returned to control levels by 6 h. Following 14 and 28 days in IPW, gill occludin expression was markedly elevated, while Na⁺–K⁺–ATPase activity was only significantly different (elevated) at day 14. Kidney tissue exhibited an elevation in both Na⁺–K⁺–ATPase activity and occludin expression after 28 days; however, in the intestine, occludin expression declined at day 14 but did not differ from FW fish at day 28. These studies demonstrate that goldfish can tolerate abrupt as well as sustained exposure to ion-poor surroundings. Data also suggests that occludin may play an adaptive role in fishes acclimated to ion-poor conditions by contributing to the modulation of epithelial barrier properties in ionoregulatory tissues. Helen Chasiotis and Jennifer C. Effendi contributed equally to this work.     

Difructose anhydride III and sodium caprate activate paracellular transport via different intracellular events in Caco-2 cells

A nondigestible disaccharide, difructose anhydride (DFA) III, is known to activate calcium transport via tight junctions (TJs); however, the characteristics of and mechanisms for the increase in paracellular transport induced by DFAIII have not been clarified. We compared the effect of DFAIII with that of sodium caprate (C10), a well-known enhancer of TJ permeability, on the changes in TJ proteins, transport of paracellular markers, and effects of nine cellular signaling blockers using Caco-2 monolayers. The addition of DFAIII (0-100mmol/L) and C10 (0-10mmol/L) to the apical medium of the Caco-2 monolayers dose-dependently decreased transepithelial electrical resistance (TER), which is an indicator of TJ permeability. The reduction with C10 was much faster than that with DFAIII. Transport of the paracellular markers of various molecular weights (182-43,200) was elevated by the addition of 100mmol/L DFAIII and 10mmol/L C10. The transport rates were much in the presence of C10 than of DFAIII, while the reduction in TER by two treatments was similar (from 1000 to 300Omega cm(2)). Treatment with DFAIII and C10 changed the distribution of actin filament and claudin-1, but not occludin, junctional adhesion molecule-1, or zonula occludens-1; however, alterations in the patterns of the TJ proteins differed according to treatment. An inhibitor of myosin light chain kinase and a chelator of intracellular calcium ion ([Ca(2+)](i)) attenuated the TER reduction by C10, but not by DFAIII. These data demonstrate that the increase in TJ permeability induced by DFAIII results from the alterations to actin and claudin-1 via [Ca(2+)](i)-independent mechanisms.     

A Collagen Substrate Enhances the Sealing Capacity of Tight Junctions of A6 Cell Monolayers

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 × 10⁻⁷ S/cm for cells grown on collagen and 30.3 × 10⁻⁷ S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands. Received: 18 September 1996/Revised: 17 June 1997