The role of Nrf2 in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated MMP9 / 中华劳动卫生职业病杂志

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.

The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma

Abstract Objectives Metastasis is one of the biggest challenges in the management of Esophageal Squamous Cell Carcinoma (ESCC), of which molecular mechanisms remain elusive. The present study aimed to explore the roles and underlying mechanisms of Transmembrane protein 26 (TMEM26) in ESCC. Method TMEM26 expressions in tumorous and adjacent tissues from patients with ESCC and in normal esophageal epithelial and ESCC cell lines were detected by immunostaining and western blotting, respectively. The Epithelial-Mesenchymal Transition (EMT), a critical process during metastasis, was investigated by wound healing and Transwell assays, and EMT-related proteins were examined after the TMEM26 alteration in ESCC cell lines. NF-κB signaling activation and Tight Junction (TJ) protein expression were analyzed by western blotting and immunofluorescence, respectively. In vivo verification was performed on the liver metastatic murine model. Results Compared with non-cancerous esophageal tissues and cells, the TMEM26 expression level was higher in ESCC samples and cell lines, where the plasma membrane localization of TMEM26 was observed. The EMT-related processes of ESCC cells were suppressed by RNAi depletion of TMEM26 but aggravated by TMEM26 overexpression. Mechanistically, TMEM26 promoted NF-κB signaling to accelerate EMT in ESCC cells. The plasma membrane presentation and assembly of TJ proteins were impaired by TMEM26. Conclusion Overall, TMEM26 acts as a critical determinant for EMT in ESCC cells by disrupting TJ formation and promoting NF-κB signaling, which may be a potential therapeutic target for treating metastatic ESCC.

Damage of retinal pigment epithelial cells function by paclitaxel and its potential mechanism / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the potential toxic effects of paclitaxel(PTX)on the proliferation, apoptosis, cell cycle, morphology, and blood-retinal barrier(BRB)of human retinal pigment epithelial cells(ARPE-19). <p>METHODS: ARPE-19 cells were cultured <i>in vitro</i> and divided into two groups: Control group(Control)and drug plus group(PTX). ARPE-19 cells were treated with different concentrations of PTX(0.005, 0.05, 0.5, 5mg/L)for a certain period of time(12, 24, 36, 48, 72h), and CCK8 assay and flow cytometry were used to detect the effects of drug on proliferation and apoptosis of ARPE-19 cells at different concentrations and time points. The same time, the cell cycle was detected by flow cytometry. Morphological changes of cells were observed by immunofluorescence. Expressions of apoptosis-related proteins and barrier function-related proteins were detected by Western blot. The effect of the drug on the cell barrier was measured by measuring the transepithelial resistance of the cells. <p>RESULTS: PTX reduced the proliferation ability of ARPE-19 cells. After 36h of treatment with low concentration of 0.005mg/L paclitaxel, cell proliferation began to be affected. At the same time, PTX accelerated cell apoptosis was dependent on drug concentration and time. Flow cytometry showed that the cells were arrested in the G2-M phase. In addition, PTX causes significant morphological changes in cells, with normal cells fusiform or irregular. In the PTX group, the number of cells decreased and the cell shape tended to be round. PTX affected retinal barrier function, and the transepithelial resistance of cells was significantly decreased after treatment, and the expression of tight junction proteins ZO-1 and Occludin were significantly decreased compared with the control group(<i>P</i><0.05). The expression levels of Cleaved-caspase-3 and Bax were significantly increased compared with the control group, while the expression levels of Bcl-2 were significantly decreased(<i>P</i><0.05)and was dependent on drug concentration and time. <p>CONCLUSION: PTX can affect the proliferation and apoptosis of ARPE-19 cells, and it depends on time and concentration. In addition, PTX affected the cell cycle and morphology of ARPE-19 cell. At the same time PTX can destroy the barrier function of the retina,suggesting that anti-tumor drugs have a potential toxic effect on the retina.

New insights into the integrity of intercellular junctions between retinal pigment epithelial cells / 中华实验眼科杂志

Retinal pigment epithelium (RPE) is composed of a layer of highly specialized hexagonal pigment epithelial cells.The apical surface of RPE interacts with the photoreceptor, and RPE basal surface interacts with Bruch membrane and choroidal capillaries to maintain the function of retinal photoreceptor.A variety of junction proteins distributed between RPE cells are the basis for RPE to perform normal functions, ensuring the integrity and physiological function of RPE.Under pathological conditions, the abnormal function of RPE is first manifested by the abnormal junctional protein, which leads to the loss of adhesion between cells, cells and basement membrane, and then a series of abnormal biological behaviors, such as dissociation, migration, transdifferentiation and protein expression changes in RPE cells, which have become an important cause of many fundus diseases.The role of RPE junctional complexes during normal and pathological conditions, as well as their role in proliferative vitreoretinopathy, age-related macular degeneration and diabetic vitreoretinopathy was reviewed in this article from the composition and correlation of junctional proteins between RPE cells.

The protective effects of disialyllacto-N-tetraose of neonatal intestinal barriers in the prevention of necrotizing enterocolitis / 中国新生儿科杂志

Objective:To study the expression of zonula occludens-1(ZO-1) in neonates with necrotizing enterocolitis (NEC) and to explore the effects of disialyllacto-N-tetraose (DSLNT), a compound extracted from human milk, on the intestinal barriers in rat model of NEC.Methods:(1) Human study: From Feb 2013 to Dec 2020, the pathological samples of ileum tissue from 21 neonates (12 patients with NEC and 9 with intestinal atresia) from Pathology Department of our hospital were collected. The expressions of ZO-1 in these samples were examined using immunohistochemistry (IHC) method. (2) Animal study: A total of 28 newborn rats were randomly assigned into control group ( n=8), NEC group ( n=10) and DSLNT+NEC group ( n=10). Experimental NEC model was established based on hypoxia (95%N 2 10 min) /cold exposure (4 ℃ 10 min) three times a day for consecutive 3 days. DSLNT+NEC group were fed with formula+DLSNT (300 μmol/L) during hypoxia/cold exposure. All the surviving rats were sacrificed at the end of the experiment (72 h) and the terminal ileum tissues were collected. Hematoxylin-Eosin (HE) staining was used to evaluate tissue damage and Western blotting was used to determine the expressions of ZO-1. (3) Cellular study: Bacterial lipopolysaccharide (LPS) was used to establish a cellular inflammation model in human intestinal epithelial cell lines (Caco-2) and DSLNT (300 μmol/L) was applied to this model. Thiazolyl blue assay was used to examine cell viabilities and immunofluorescence assay was used to detect ZO-1 expression. The effects of DSLNT on cell growth and tight junctions of Caco-2 cells were analyzed. Results:(1)Human study: The villi of mucous layer of the lesion were damaged in NEC patients. ZO-1 expressions at the epithelial junction of NEC patients were decreased compared with intestinal atresia patients and non-lesion intestines of NEC patients. (2)Animal study: Apical extrusion, necrosis and shedding of epithelial cell were seen at the lesions in NEC group. The expression of ZO-1 in NEC group was significantly lower than the control group and DSNLT+NEC group ( P<0.05).DSNLT+NEC group had higher survival rates (8/10 vs. 6/10) and lower ileum inflammatory pathological scores [2.0(1.0, 2.8) vs. 3.5(3.0, 4.0)] than NEC group. (3) Cellular study: Caco-2 cells exposed to LPS showed inhibited cell growth and decreased ZO-1 immunofluorescence staining. Caco-2 cells in the DSLNT+LPS group showed better viability than LPS group and were comparable with the control group. The expression of ZO-1 was significantly increased in the DSLNT+LPS group. Conclusions:Tight junction injury of the intestinal epithelial cell is an important characteristic of NEC. ZO-1 is a potential target for the prevention and treatment of NEC. DSLNT may protect the neonatal intestines by modulating the expression of ZO-1 and keeping tight junction integrity.

Caludin-5: target of regulating blood brain barrier in the treatment of ischemic stroke / 国际脑血管病杂志

The blood brain barrier (BBB) is a physical and metabolic barrier that maintains central nervous system homeostasis and protects brain tissues from potentially hazardous circulating substances. This article reviews the biological characteristics of caludin-5 during cerebral ischemia, its role in BBB integrity and permeability, as well as the research progress of related drug therapy based on calludin-5.

Effect of herb-partitioned moxibustion in improving tight junctions of intestinal epithelium in Crohn disease mediated by TNF-α-NF-κB-MLCK pathway / 针灸推拿医学（英文版）

Objective: To explore the effect of herb-partitioned moxibustion (HPM) on tight junctions (TJs) of intestinal epithelial cells in Crohn disease (CD) mediated by tumor necrosis factor-α (TNF-α)-nuclear factor kappa B (NF-κB)-myosin-light- chain kinase (MLCK) pathway. Methods: Forty-eight male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an HPM group and a mesalazine (MESA) group, with 12 rats in each group. Trinitrobenzene sulfonic acid (TNBS) was administered to establish CD models. When the model was confirmed a success, the HPM group rats were treated with HPM at Tianshu (ST 25) and Qihai (CV 6), while the MESA group rats were given MESA solution by lavage. When the intervention finished, the colonic epithelial tissues were separated, purified and cultured in each group to establish the intestinal epithelial barrier model in vitro, and TNF-α was added (100 ng/mL) in the culture medium and maintained for 24 h to establish an increased epithelial permeability model. Transepithelial electrical resistance (TEER) was used to examine the permeability of the barrier; Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway; immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium. Results: After TNF-α induction, compared with the MC+TNF-α group, the TEER value increased significantly in the HPM+TNF-α and MESA+TNF-α groups (both P<0.001); the expressions of nuclear factor kappa B (NF-κB) p65, MLCK, myosin light chain (MLC), tumor necrosis factor receptor-associated factor 6 (TRAF6) and receptor interaction protein-1 (RIP1) decreased significantly (P<0.01 or P<0.05), and the expression of zinc finger protein A20 (A20) increased significantly (P<0.01); the expressions of occludin, claudin-1, zonula occludens protein 1 (ZO-1) and F-actin also increased significantly (all P<0.01). Compared with the MESA+TNF-α group, the expressions of MLC, occludin, claudin-1, ZO-1 and F-actin increased significantly in the HPM+TNF-α group (P<0.01 or P<0.05). Conclusion: HPM can protect or repair the damage of intestinal epithelial barrier in CD rats, which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.

Integrins as attractive targets for cancer therapeutics

Integrins are transmembrane receptors that have been implicated in the biology of various human physiological and pathological processes. These molecules facilitate cell-extracellular matrix and cell-cell interactions, and they have been implicated in fibrosis, inflammation, thrombosis, and tumor metastasis. The role of integrins in tumor progression makes them promising targets for cancer treatment, and certain integrin antagonists, such as antibodies and synthetic peptides, have been effectively utilized in the clinic for cancer therapy. Here, we discuss the evidence and knowledge on the contribution of integrins to cancer biology. Furthermore, we summarize the clinical attempts targeting this family in anti-cancer therapy development.

Cancer-specific calcium nanoregulator suppressing the generation and circulation of circulating tumor cell clusters for enhanced anti-metastasis combinational chemotherapy

Tumor metastasis is responsible for chemotherapeutic failure and cancer-related death. Moreover, circulating tumor cell (CTC) clusters play a pivotal role in tumor metastasis. Herein, we develop cancer-specific calcium nanoregulators to suppress the generation and circulation of CTC clusters by cancer membrane-coated digoxin (DIG) and doxorubicin (DOX) co-encapsulated PLGA nanoparticles (CPDDs). CPDDs could precisely target the homologous primary tumor cells and CTC clusters in blood and lymphatic circulation. Intriguingly, CPDDs induce the accumulation of intracellular Ca

Claudin 14/15 play important roles in early wallerian degeneration after rat sciatic nerve injury / 中华创伤杂志(英文版)

PURPOSE@#Wallerian degeneration (WD) is an antegrade degenerative process distal to peripheral nerve injury. Numerous genes are differentially regulated in response to the process. However, the underlying mechanism is unclear, especially the early response. We aimed at investigating the effects of sciatic nerve injury on WD via CLDN 14/15 interactions in vivo and in vitro.@*METHODS@#Using the methods of molecular biology and bioinformatics analysis, we investigated the molecular mechanism by which claudin 14/15 participate in WD. Our previous study showed that claudins 14 and 15 trigger the early signal flow and pathway in damaged sciatic nerves. Here, we report the effects of the interaction between claudin 14 and claudin 15 on nerve degeneration and regeneration during early WD.@*RESULTS@#It was found that claudin 14/15 were upregulated in the sciatic nerve in WD. Claudin 14/15 promoted Schwann cell proliferation, migration and anti-apoptosis in vitro. PKCα, NT3, NF2, and bFGF were significantly upregulated in transfected Schwann cells. Moreover, the expression levels of the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK signaling pathways were also significantly altered.@*CONCLUSION@#Claudin 14/15 affect Schwann cell proliferation, migration, and anti-apoptosis via the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK pathways in vitro and in vivo. The results of this study may help elucidate the molecular mechanisms of the tight junction signaling pathway underlying peripheral nerve degeneration.

Bioengineered miR-124-3p prodrug selectively alters the proteome of human carcinoma cells to control multiple cellular components and lung metastasis

With the understanding of microRNA (miRNA or miR) functions in tumor initiation, progression, and metastasis, efforts are underway to develop new miRNA-based therapies. Very recently, we demonstrated effectiveness of a novel humanized bioengineered miR-124-3p prodrug in controlling spontaneous lung metastasis in mouse models. This study was to investigate the molecular and cellular mechanisms by which miR-124-3p controls tumor metastasis. Proteomics study identified a set of proteins selectively and significantly downregulated by bioengineered miR-124-3p in A549 cells, which were assembled into multiple cellular components critical for metastatic potential. Among them, plectin (PLEC) was verified as a new direct target for miR-124-3p that links cytoskeleton components and junctions. In miR-124-3p-treated lung cancer and osteosarcoma cells, protein levels of vimentin, talin 1 (TLN1), integrin beta-1 (ITGB1), IQ motif containing GTPase activating protein 1 (IQGAP1), cadherin 2 or N-cadherin (CDH2), and junctional adhesion molecule A (F11R or JAMA or JAM1) decreased, causing remodeling of cytoskeletons and disruption of cell-cell junctions. Furthermore, miR-124-3p sharply suppressed the formation of focal adhesion plaques, leading to reduced cell adhesion capacity. Additionally, efficacy and safety of biologic miR-124-3p therapy was established in an aggressive experimental metastasis mouse model

Acid-functionalized single-walled carbon nanotubes alter epithelial tight junctions and enhance paracellular permeability

The T(EB4)Nta, T(IBj5)Nta, and T(B362i)NtA strains were constructed by introgressing the insertionaltranslocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma. Theprogeny from crosses of T(IBj5)Nta and T(B362i)NtA with opposite mating-type derivatives of the standard N.tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavoredhomokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A ? mat a] dikaryons produced following either segregation. Further, crosses of the T(IBj5)Nta and T(B362i)NtA strains with the Eight spore (E) mutant showed anunusual ascus phenotype called ‘max-4’. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 ofthe homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryoticprogeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.

Mechanism of Fushen Granule on improving intestinal epithelial barrier injury / 中草药

Objective: To investigate the protection mechanism of Fushen Granule on LPS-induced intestinal epithelial cell (IEC-6) barrier injury. Methods: IEC-6 were divided into control group, model group, Fushen granule group, and p38MAPK inhibitor group. In addition to the control group, 1 μg/mL LPS was added to the other groups, 10% drug-containing serum and 10 μmol/L SB203580 were respectively added to the administration group. The apoptosis of IEC-6 were detected by flow cytometry; The permeability and transmembrane resistance of IEC-6 barrier were detected by Transwell assay; The mRNA levels of ICAM-1 and IL-1β were assessed by Real-time PCR; The expressions of occludin, ZO-1, p38MAPK, and DUSP1 were detected by western blotting. Results: Compared with the control group, the apoptosis of IEC-6 were significantly increased in model group (P < 0.01), the levels of ICAM-1 and IL-1β were significantly increased (P < 0.01), the occludin and ZO-1 were significantly decreased (P < 0.01), the intestinal permeability was significantly increased (P < 0.05), the mRNA levels of ICAM-1 and IL-1β in IEC-6 were significantly inhibited, the permeability of intestinal mucosal barrier was reduced (P < 0.05), the expressions of DUSP1, occludin and ZO-1were increased (P < 0.05, 0.01), the expression of p38MAPK was inhibited (P < 0.01), which showed a similar effect with p38MAPK inhibitor. Conclusion: Fushen Granule could regulate DUSP1 which inhibits the activation of p38MAPK signaling pathway, up-regulate Occludin and ZO-1, improve the tight junction of IEC-6, thereby improving the cell barrier function damage caused by LPS.

The relationship between adherens junction and tight junction and clinical symptoms in patients with diarrhea predominant irritable bowel syndrome / 中华内科杂志

Objective@#To investigate the association between adherens junction proteins E-cadherin and β-catenin and tight junction protein claudin-2 and clinical symptoms in patients with diarrhea predominant irritable bowel syndrome (IBS-D).@*Methods@#Cecal biopsy tissues were collected from IBS-D patients (n=26) according to Rome Ⅲ criterion and healthy controls (n=26). The duration of symptoms, abdominal pain score and mean weekly bowel movements were recorded. Colorectal dilatation combined with restraint stress were applied to establish visceral hypersensitivity rat model. Abdominal contraction reflex (AWR) was applied to assess the visceral sensitivity in rats. The stool frequency within 1 hour was recorded after establishing the rat model. The expression of E-cadherin、β-catenin and claudin-2 were assessed by Western blot and immunofluorescence microscopy. Intercellular ultrastructure was observed by transmission electron microscopy.@*Results@#Compared with the healthy controls, the protein expression of E-cadherin and β-catenin in cecal epithelium in IBS-D patients were significantly lower (P=0.015 and P=0.005, respectively), while claudin-2 was significantly higher (P=0.000). Reduced E-cadherin and β-catenin expression was associated high abdominal pain score (r=-0.463, P=0.017 and r=-0.407, P=0.039). The lower expression of β-catenin was associated with longer duration of symptoms (r=-0.458, P=0.019). The protein expression of E-cadherin and ß-catenin in the cecal epithelium of the visceral hypersensitivity rats were significantly lower (P=0.004 and P=0.003, respectively), while claudin-2 was significantly higher (P=0.008). Reduced E-cadherin and ß-catenin expression was associated high visceral sensitivity in IBS-D rats (r=-0.639, P=0.047 and r=-0.888, P=0.001).@*Conclusions@#Intercellular ultrastructure alterations well as cecal β-catenin and E-cadherin protein expression decrease and are associated with high abdominal pain score in IBS-D patients and hypersensitivity rats. β-catenin is further associated with prolonged duration of symptoms in IBS-D patients. The expression of E-cadherin and β-catenin may play a vital role in visceral sensitivity and intestinal barrier dysfunction in IBS-D.

Alanyl-glutamine protects the intestinal barrier function in trained rats against the impact of acute exhaustive exercise

Strenuous exercise triggers deleterious effects on the intestinal epithelium, but their mechanisms are still uncertain. Here, we investigated whether a prolonged training and an additional exhaustive training protocol alter intestinal permeability and the putative effect of alanyl-glutamine (AG) pretreatment in this condition. Rats were allocated into 5 different groups: 1) sedentary; 2 and 3) trained (50 min per day, 5 days per week for 12 weeks) with or without 6 weeks oral (1.5 g/kg) AG supplementation; 4 and 5) trained and subjected to an additional exhaustive test protocol with or without oral AG supplementation. Venous blood samples were collected to determine gasometrical indices at the end of the 12-week protocol or after exhaustive test. Lactate and glucose levels were determined before, during, and after the exhaustive test. Ileum tissue collected after all experimental procedures was used for gene expression analysis of Zonula occludens 1 (ZO-1), occludin, claudin-2, and oligopeptide transporter 1 (PepT-1). Intestinal permeability was assessed by urinary lactulose/mannitol test collected after the 12-week protocol or the exhaustive test. The exhaustive test decreased pH and base excess and increased pCO2. Training sessions delayed exhaustion time and reduced the changes in blood glucose and lactate levels. Trained rats exhibited upregulation of PEPT-1, ZO-1, and occludin mRNA, which were partially protected by AG. Exhaustive exercise induced intestinal paracellular leakage associated with the upregulation of claudin-2, a phenomenon protected by AG treatment. Thus, AG partially prevented intestinal training adaptations but also blocked paracellular leakage during exhaustive exercise involving claudin-2 and occludin gene expression.

The liver injury following ischemia and reperfusion is worse in experimental knockout heterozygote mouse model for expression of connexin 43

Abstract Purpose: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. Methods: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. Results: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. Conclusions: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.

ROS Scavenger, Ebselen, Has No Preventive Effect in New Hearing Loss Model Using a Cholesterol-Chelating Agent

BACKGROUND AND OBJECTIVES: The antioxidant ebselen will be able to limit or prevent the ototoxicity arising from 2-hydroxypropyl-β-cyclodextrin (HPβCD). Niemann-Pick Type C (NPC) disease is a disorder of lysosomal storage manifested in sphingolipidosis. Recently, it was noted that experimental use of HPβCD could partially resolve the symptoms in both animals and human patients. Despite its desirable effect, HPβCD can induce hearing loss, which is the only major side effect noted to date. Understanding of the pathophysiology of hearing impairment after administration of HPβCD and further development of preventive methods are essential to reduce the ototoxic side effect. The mechanisms of HPβCD-induced ototoxicity remain unknown, but the resulting pathology bears some resemblance to other ototoxic agents, which involves oxidative stress pathways. To indirectly determine the involvement of oxidative stress in HPβCD-induced ototoxicity, we tested the efficacy of an antioxidant reagent, ebselen, on the extent of inner ear side effects caused by HPβCD. MATERIALS AND METHODS: Ebselen was applied prior to administration of HPβCD in mice. Auditory brainstem response thresholds and otopathology were assessed one week later. Bilateral effects of the drug treatments also were examined. RESULTS: HPβCD-alone resulted in bilateral, severe, and selective loss of outer hair cells from base to apex with an abrupt transition between lesions and intact areas. Ebselen co-treatment did not ameliorate HPβCD-induced hearing loss or alter the resulting histopathology. CONCLUSIONS: The results indirectly suggest that cochlear damage by HPβCD is unrelated to reactive oxygen species formation. However, further research into the mechanism(s) of HPβCD otopathology is necessary.

DL-3-n-butylphthalide protects the blood-brain barrier against ischemia/hypoxia injury via upregulation of tight junction proteins / 中华医学杂志(英文版)

Background@#The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia.@*Methods@#A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0 μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1% O2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3β (GSK-3β)/GSK-3β, and β-catenin/β-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy.@*Results@#In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ± 0.9 μg/g vs. 12.3 ± 1.9 μg/g, P = 0.005; 6.7 ± 0.6 μg/g vs. 12.3 ± 1.9 μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 vs. 0.41 ± 0.06, P < 0.01; 0.97 ± 0.07 vs. 0.41 ± 0.06, P < 0.01), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with the corresponding values in the hypoxia group. NBP treatment also elevated the relative expression levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and β-catenin/β-actin in comparison with the corresponding values in the hypoxia group (all P < 0.05).@*Conclusion@#NBP improves the barrier function of BBB against ischemic injury by upregulating the expression of TJ proteins, possibly by reducing oxidative stress and activating the Akt/GSK-3β/β-catenin signaling pathway.

Formation of gap junctions between adipose stem cells-derived Schwann cells in a rat model of dyskinesia induced by brain injury / 南方医科大学学报

OBJECTIVE@#To investigate the formation of gap junctions between Schwann cells derived from differentiated adipose stem cells implanted in a rat model of dyskinesia induced by brain injury and its positive effect in promoting functional recovery of the rats.@*METHODS@#In a rat model of hemiplegia induced by motor cortex injury, adipose stem cells or Schwann cells differentiated from adipose stem cells, either with or without RNAi-mediated silencing of Cx43, were transplanted orthotopically in the lesion. The recovery of the motor function of the rats was observed and scored after the transplantation. Rat brain tissues were sampled to detect the expressions of nerve growth factor (NGF) using Western blotting and RT-PCR.@*RESULTS@#All the 3 cell transplantation therapies obviously improved the motor function scores of the rats as compared with the control rats. The expression of NGF in the brain tissue was significantly lower in the control group than in the cell transplantation groups. NGF expression in the brain tissues of rats receiving transplantation of Schwann cells with Cx43 gene silencing was lower than that in rats receiving Schwann cells without Cx43 silencing, and was similar with that in rats transplanted with adipose stem cells. The results of RT-PCR showed that NGF mRNA level in the control group was significantly lower than that in the other 3 groups. NGF mRNA expression was the highest in Schwann cell group without Cx43 silencing, followed by adipose stem cell group, and then by Schwann cell group with Cx43 silencing.@*CONCLUSIONS@#In the rat model of dyskinesia induced by brain injury, transplantations of adipose stem cells and adipose stem cells-derived Schwann cells both promote the functional recovery of brain damage, in which gap junction protein Cx43 plays an important role to promote functional gap junction formation possibly by enhancing NGF expression.

Tight junctions-associated proteins:a novel therapeutic target for cholestatic liver injury / 中国药科大学学报

@#The occurrence of cholestatic liver injury is accompanied by the alterations of hepatocyte polarization and bile acid homeostasis. Located in epithelial cells, tight junctions(TJs)are a special barrier structure which are important in maintaining permeability and bile acid homeostasis. Based on the fully analysis and discussion of TJs, the latest therapeutic drugs for cholestasis were summaried, which may provide new perspectives and potential therapeutic agents for the treatment of cholestatic liver injury.