Yixue Xue

To investigate whether the miR-320a could regulate bladder cancer cells invasion by down-regulation of ITGB3. Real-time quantitative PCR was applied to evaluate the expression level of miRNA-320a in bladder transitional cell carcinomas (TCC) and normal bladder transitional cell (NBTC) samples. The invasion ability of miR-320a in TCC T24 cells was analyzed by Transwell assay after pre-miR-320a or anti-miR-125b transfection. For the invasion mechanism analysis of miR-320a on T24 cells, TargetScan, PicTar and miRBase were used to predict the possible target gene of miR-320a. Luciferase activities assay and western blot were used to reveal the predicted target gene of miR-320a were direct and specific. RNA interference technology was used to confirm the invasion inhibition of miR-320a was directly induced by ITGB3. Our study showed that miR-320a was down-regulated in human TCC specimens compared to that in NBTC specimens. Over-expression of miR-320a in T24 cells inhibited TCC invasion and this inhibitory effect on T24 cells could be restore by miR-320a knocked down. Mechanism analysis revealed that ITGB3 was a direct and specific target of miR-320a. The advanced effect of anti-miR-320a on TCC cell invasion was mediated by expression silence of ITGB3. In summary, aberrantly expressed miR-320a contribute to T24 cells invasion partly through directly down-regulating ITGB3 protein expression in TCC and this miRNA signature offers a novel potential therapeutic strategy for TCC.

Background: It has been supposed that green tea polyphenols (GTPs) have neuroprotective effects on brain damage after brain ischemia in animal experiments. Little is known regarding GTPs’ protective effects against the blood-brain barrier (BBB) disruption after ischemic stroke. We investigated the effects of GTPs on the expression of claudin-5, occludin, and ZO-1, and the corresponding cellular mechanisms involved in the early stage of cerebral ischemia.Methods: Male Wistar rats were subjected to a middle cerebral artery occlusion (MCAO) for 0, 30, 60, and 120 min. GTPs (400 mg/kg/day) or vehicle was administered by intragastric gavage twice a day for 30 days prior to MCAO. At different time points, the expression of claudin-5, occludin, ZO-1, and PKCα signaling pathway in microvessel fragments of cerebral ischemic tissue were evaluated.Results: GTPs reduced BBB permeability at 60 min and 120 min after ischemia as compared with the vehicle group. Transmission electron microscopy also revealed that GTPs could reverse the opening of tight junction (TJ) barrier at 60 min and 120 min after MACO. The decreased mRNA and protein expression levels of claudin-5, occludin, and ZO-1 in microvessel fragments of cerebral ischemic tissue were significantly prevented by treatment with GTPs at the same time points after ischemia in rats. Furthermore, GTPs could attenuate the increase in the expression levels of PKCα mRNA and protein caused by cerebral ischemia.Conclusions: These results demonstrate that GTPs may act as a potential neuroprotective agent against BBB damage at the early stage of focal cerebral ischemia through the regulation of TJ and PKCα signaling.

This study was performed to determine whether recombinant human angiopoietin-1 (rhAng-1) decreases the permeability of the blood-brain barrier (BBB) in focal cerebral ischemia and reperfusion rats, whether RhAng-1 opens the BBB by affecting tight junction associated proteins zonnula occludin-1 (ZO-1), occludin and adherens junction protein vascular endothelial (VE)-cadherin. The rats were divided into eight groups randomly( n = 10): (1) sham-operated group; (2) ischemia group; (3)-(5) ischemia/reperfusion (middle cerebral artery occlusion and reperfusion (MCAO/R) 12 h, 48 h, and 7 days) and 0.9% saline groups; (6)-(8) ischemia/reperfusion (MCAO/R 12 h, 48 h, and 7 days) and rhAng-1 groups. The Bee permeability was assessed by Evans blue extravasation. The messenger RNA and protein expressions of ZO-1, occludin, and VE-cadherin were determined by reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry assays. The BBB permeability and the brain infarct volume were significantly decreased after rhAng-1 injection. The expressions of ZO-1, occludin, and VE-cadherin were increased after rhAng-1 injection. rhAng-1 may decrease the permeability of BBB in MCAO/R rats by upregulation of ZO-1, occludin, and VE-cadherin.

Administration of recombinant human erythropoietin (rhEPO) protects neurons from injury after brain ischemia–reperfusion (I/R), which is in part mediated by ameliorating the blood–brain barrier (BBB) leakage. But the mechanism of rhEPO’s protective effects on BBB remains unclear. This study aims to investigate the effects of rhEPO on BBB integrity and the expressions of tight junctions (TJs) associated proteins of zonula occluden-1 (ZO-1), occludin, and claudin-5 in cerebral I/R rats. These rats underwent 2 h of ischemia and then were reperfused for up to 3 and 72 h. Animals were randomly divided into five groups: sham-operated group, I/R 3 h and I/R 72 h group (2 ml saline was injected intraperitoneally just before the onset of ischemia), rhEPO + I/R 3 h, and rhEPO + I/R 72 h group (5,000 U/kg rhEPO diluted in 2 ml saline solution was injected intraperitoneally just before the onset of ischemia). We verified that rhEPO could decrease the BBB leakage induced by I/R injury detected by Evans blue extravasation. 2, 3, 5-Triphenyltetrazolium chloride staining results showed that rhEPO decreased infarct volume after cerebral I/R. TJ integrity was partly restored by rhEPO observed by transmission electron microscopy. The mRNA and protein expression levels of ZO-1, occludin, and claudin-5 were significantly increased compared with I/R groups at the same reperfusion time point by reverse transcriptase-polymerase chain reaction and Western blot assays. The treatment of rhEPO induced the redistribution of ZO-1, occludin, and claudin-5 in cerebral microvessels observed by immunohistochemical staining. Compared with I/R groups, the mRNA level of tumor necrosis factor-alpha (TNF-α) in cerebral microvessels decreased markedly after rhEPO treatment, accompanied with reduced TNF-α protein level and nuclear factor-кB (NF–кB) p65 activation detected by enzyme-linked immunosorbent assay. These results suggested that the protective mechanism of rhEPO on BBB after cerebral I/R injury was associated with the upregulation of TJ-associated proteins. The downregulated TNF-α levels and NF–кB activation induced by rhEPO might be involved in this process.

The strategy for the development of effective and safe neuroprotective agents has great potential to reduce cerebral ischemia–reperfusion injury and improve the functional outcome in stroke patients. Recently, doxycycline, a tetracycline antibiotic, has been shown to have neuroprotective efficiency in reduction of a variety of ischemia–reperfusion injuries as well as ischemic brain damage. We used the rat models of middle cerebral artery occlusion (MCAO) and reperfusion to investigate the effects of treatments with doxycycline against the blood-brain barrier (BBB) leakage at 3, 12, 72, and 120 h of reperfusion. Male Sprague–Dawley rats were subjected to MCAO for 2 h followed by reperfusion for 3, 12, 72, and 120 h and received either doxycycline (45 mg/kg) or saline. The results showed that the treatment of doxycycline significantly reduced the BBB leakage and cerebral infarct volume, which were proved by Evans blue assay and TTC staining. Real-time PCR, immunohistochemistry, and western blot assay verified that the administration of doxycycline significantly up-regulated the expression of tight junction claudin-5, occludin, and ZO-1 from 3 to 120 h after reperfusion. The results of real-time PCR, western blot, and gelatin zymography analyses revealed that the gene and protein expression and activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 were significantly elevated in a different time-dependent manner after ischemia–reperfusion but significantly inhibited by doxycycline treatment. Moreover, doxycycline could also significantly down-regulate the expression of PKCδ mRNA and protein after ischemia–reperfusion. These results suggested that the protective effects of doxycycline against BBB damage induced by reperfusion might be related to the up-regulation of tight junction proteins and inhibition of MMP-2, MMP-9, and PKCδ.

We have previously shown that activation of RhoA by bradykinin (BK) is associated with cytoskeleton rearrangement, tight junction (TJ) protein disassembly, and an increase in blood–tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). Subsequently, we investigated whether Rho-kinases (ROCKs), a family of downstream effectors of activated RhoA known to stimulate F-actin rearrangement, play a key role in the above-mentioned processes in RBMECs. Our study uses primary RBMECs as an in vitro BTB model and a specific ROCK inhibitor (Y-27632) and ROCK II small interfering RNA (siRNA) to establish whether ROCK plays a role in the process of TJ opening by BK. Y-27632 and ROCK II siRNA could partially inhibit endothelial leakage and restored normal transendothelial electric resistance (TEER) values in RBMECs. A shift in occludin and claudin-5 distribution from insoluble to soluble fractions was prevented by Y-27632. Additionally, Y-27632 inhibited BK-induced relocation of occludin and claudin-5 from cellular borders into the cytoplasm as well as stress fiber formation in RBMECs. A time-dependent increase in phosphorylated myosin light chain (p-MLC) and phosphorylated cofilin (p-cofilin) by BK was observed, which was also inhibited by Y-27632. An increase in ROCK activity by BK was inhibited by Y-27632. ROCK’s contribution to BK-induced stress fiber formation is associated with TJ disassembly and an increase in BTB permeability.

This study was performed to determine whether recombinant human angiopoietin-1 (Ang-1) decreases the permeability of the blood–brain barrier (BBB) in focal cerebral ischemia and reperfusion rats, whether Ang-1 opens the BBB by affecting tight junction associated proteins zonula occluden-1 (ZO-1), occludin and adherens junction protein vascular endothelial (VE)-cadherin, and whether the protein kinase C (PKC)α/myosin light chain (MLC) signaling pathway involves in it. The rats were divided into eight groups randomly: (1) sham-operated group, (2) ischemia group, (3–5) ischemia–reperfusion (middle cerebral artery occlusion and reperfusion (MCAO/R) 12 h, 48 h, and 7 days) and 0.9% saline groups, (6–8) ischemia–reperfusion (MCAO/R 12 h, 48 h, and 7 days) and Ang-1 groups. The BBB permeability was assessed by Evans blue extravasation. The messenger RNA and protein expressions of ZO-1, occludin, and VE-cadherin were determined by reverse transcription-polymerase chain reaction, western blot, and immunohistochemistry assays. The BBB permeability was significantly decreased after Ang-1 injection. The expressions of ZO-1, occludin, and VE-cadherin were increased after Ang-1 injection. These were in accordance with the results of immunohistochemistry assays. PKCα and phosphorylated MLC (p-MLC) expressions were decreased after Ang-1 injection. This study demonstrated that Ang-1 may decrease the permeability of BBB in MCAO/R rat by upregulation of ZO-1, occludin, and VE-cadherin. The decreased expressions of PKCα and p-MLC induced by Ang-1 also involved in this process.