Background: Thrombolysis with tissue plasminogen activator (tPA) remains the only approved drug therapy for acute ischemic stroke. However, delayed tPA treatment is associated with an increased risk of brain hemorrhage. In this study, we assessed whether QiShenYiQi (QSYQ), a compound Chinese medicine, can attenuate tPA-induced brain edema and hemorrhage in an experimental stroke model.Methods: Male mice were subjected to ferric chloride-induced carotid artery thrombosis followed by mechanical detachment of thrombi. Then mice were treated with QSYQ at 2.5 h followed by administration of tPA (10 mg/kg) at 4.5 h. Hemorrhage, infarct size, neurological score, cerebral blood flow, Evans blue extravasation, FITC-labeled albumin leakage, tight and adherens junction proteins expression, basement membrane proteins expression, matrix metalloproteinases (MMPs) expression, leukocyte adhesion, and leukocyte infiltration were assessed 24 h after tPA administration.Results: Compared with tPA alone treatments, the combination therapy of QSYQ and tPA significantly reduced hemorrhage, infarction, brain edema, Evans blue extravasation, albumin leakage, leukocyte adhesion, MMP-9 expression, and leukocyte infiltration at 28.5 h after stroke. The combination also significantly improved the survival rate, cerebral blood flow, tight and adherens junction proteins (occludin, claudin-5, junctional adhesion molecule-1, zonula occludens-1, VE-cadherin, α-catenin, β-catenin) expression, and basement membrane proteins (collagen IV, laminin) expression. Addition of QSYQ protected the downregulated ATP 5D and upregulated p-Src and Caveolin-1 after tPA treatment.Conclusion: Our results show that QSYQ inhibits tPA-induced brain edema and hemorrhage by protecting the blood-brain barrier integrity, which was partly attributable to restoration of energy metabolism, protection of inflammation and Src/Caveolin signaling activation. The present study supports QSYQ as an effective adjunctive therapy to increase the safety of delayed tPA thrombolysis for ischemic stroke.
Coccidiosis is an important parasitic disease of poultry with great economic importance. Due to drug resistance issues, the study was conducted to investigate how probiotics (Lactobacillus plantarum or L. plantarum) affected oocysts per gram of feces (OPG), fecal scores, feed conversion ratio (FCR), immunomodulatory effect in terms of the cell-mediated and humoral immune response. Serum chemistry (ALT, AST, LDH, and creatinine) was measured in different treated chicken groups. mRNA expression levels of antioxidant enzymes (SOD 1 and CAT), peptide transporter 1 (PepT 1), and tight junction proteins (ZO and CLDN 1) were also examined in chicken groups infected with Eimeria tenella (E. tenella). Chickens supplemented with L. plantarum 1 × 108 CFU (colony-forming unit) showed an improved cell-mediated and humoral immune response, compared with the control group (p < 0.05). Probiotics also enhanced the performance of antioxidant enzymes, PepT 1, and tight junction proteins, and improved serum chemistry (AST, ALT, and LDH), compared with control-infected, non-medicated chickens. However, no significant difference (p > 0.05) was observed in CLDN 1 expression level and creatinine in all treated chicken groups. These findings demonstrated that probiotics supplementation in the feed can protect the birds against E. tenella infection.
Tight junction proteins play roles beyond permeability barriers functions and control cell proliferation and differentiation. The relation between tight junctions and the signal transduction pathways affects cell growth, invasion and migration. Abnormality of tight junction proteins closely contributes to epithelial mesenchymal transition (EMT) and malignancy of various cancers. Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) forms tricellular contacts that has a barrier function. Downregulation of angulin-1/LSR correlates with the malignancy in various cancers, including endometrioid-endometrial carcinoma (EEC). These alterations have been shown to link to not only multiple signaling pathways such as Hippo/YAP, HDAC, AMPK, but also cell metabolism in ECC cell line Sawano. Moreover, loss of angulin-1/LSR upregulates claudin-1, and loss of apoptosis stimulating p53 protein 2 (ASPP2) downregulates angulin-1/LSR. Angulin-1/LSR and ASPP2 concentrate at both midbody and centrosome in cytokinesis. In EEC tissues, angulin-1/LSR and ASPP2 are reduced and claudin-2 is overexpressed during malignancy, while in the tissues of endometriosis changes in localization of angulin-1/LSR and claudin-2 are seen. This review highlights how downregulation of angulin-1/LSR promotes development of endometriosis and EEC and discusses about the roles of angulin-1/LSR and its related proteins, including claudins and ASPP2.
This study aims to investigate the effects of exposure to different dosages of zearalenone (ZEA) on cecal physical barrier functions and its mechanisms based on the TGF-β1/Smads signaling pathway in weaned piglets. Thirty-two weaned piglets were allotted to four groups and fed a basal diet supplemented with ZEA at 0, 0.15, 1.5, and 3.0 mg/kg, respectively. The results showed that 1.5 and 3.0 mg/kg ZEA damaged cecum morphology and microvilli, and changed distribution and shape of M cells. Moreover, 1.5 and 3.0 mg/kg ZEA decreased numbers of goblet cells, the expressions of TFF3 and tight junction proteins, and inhibited the TGF-β1/Smads signaling pathway. Interestingly, the 0.15 mg/kg ZEA had no significant effect on cecal physical barrier functions but decreased the expressions of Smad3, p-Smad3 and Smad7. Our study suggests that high-dose ZEA exposure impairs cecal physical barrier functions through inhibiting the TGF-β1/Smads signaling pathway, but low-dose ZEA had no significant effect on cecum morphology and integrity through inhibiting the expression of smad7. These findings provide a scientific basis for helping people explore how to reduce the toxicity of ZEA in feeds.
Claudins (CLDNs) are the most important tight junction proteins, which are mainly expressed in endothelial cells or epithelial cells in a tissue-specific manner. As a member of the CLDNs family, CLDN6 is highly expressed in fetal tissues such as the stomach, pancreas, lung, and kidney, but is not expressed in corresponding adult tissues. The expression of CLDN6 is regulated by a variety of factors, including but not limited to stimuli and transcription factors, DNA methylation, and post-translational modifications. CLDN6 has been found to have a key role in the formation of barriers, especially the lung epithelial barrier and the epidermal permeability barrier (EPB). Importantly, the roles of CLDN6 in cancers have gained focus and are being investigated in recent years. Strong evidence indicates that the altered expression of CLDN6 is linked to the development of various cancers. Malignant phenotypes of tumors affected by CLDN6 include proliferation and apoptosis, migration and invasion, and drug resistance, which are regulated by CLDN6-mediated key signaling pathways. Given the important role in tumors and its low or no expression in normal tissues, CLDN6 is an ideal target for tumor therapy. This review aims to provide an overview of the structure and regulation of CLDN6, and its traditional barrier function, with a special emphasis on its emerging roles in cancers, including its impact on the malignant phenotypes, signal-modulating effects, the prognosis of tumor patients, and clinical applications in cancers.
Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA is not fully understood. In this study, in vivo and in vitro models were used to explore the effects of ZEA on the blood–testis barrier (BTB) and related molecular mechanisms. First, male BALB/C mice were administered ZEA orally (40 mg/kg·bw) for 5–7 d. Sperm motility, testicular morphology, and expressions of BTB junction proteins and autophagy-related proteins were evaluated. In addition, TM4 cells (mouse Sertoli cells line) were used to delineate the molecular mechanisms that mediate the effects of ZEA on BTB. Our results demonstrated that ZEA exposure induced severe testicular damage in histomorphology and an ultrastructural, time-dependent decrease in the expression of blood–testis barrier junction-related proteins, accompanied by an increase in the expression of autophagy-related proteins. Additionally, similar to the in vitro results, the dose-dependent treatment of ZEA increased the level of cytoplasmic Ca2+ and the levels of the autophagy markers LC3-II and p62, in conjunction with a decrease in the BTB junction proteins occludin, claudin-11, and Cx43, with the dislocation of the gap junction protein Cx43. Meanwhile, inhibition of autophagy by CQ and 3-MA or inhibition of cytoplasmic Ca2+ by BAPTA-AM was sufficient to reduce the effects of ZEA on the TM4 cell BTB. To summarize, this study emphasizes the role of Ca2+-mediated autophagy in ZEA-induced BTB destruction, which deepens our understanding of the molecular mechanism of ZEA-induced male reproductive disorders.
Disruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear.
Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test.
Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.
This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.