Intercellular Tight Junctions Research Articles

AFDN deficiency promotes liver tropism of metastatic colorectal cancer.

Liver metastasis is a major cause of morbidity and mortality in patients with colorectal cancer. A better understanding of the biological mechanisms underlying liver tropism and metastasis in colorectal cancer could help to identify improved prevention and treatment strategies. In this study, we performed genome-side CRISPR loss-of-function screening in a mouse colorectal cancer model and identified deficiency of AFDN, a protein involved in establishing and maintaining cell-cell contacts, as a driver of liver metastasis. Elevated AFDN expression was correlated with prolonged survival in patients with colorectal cancer. AFDN-deficient colorectal cancer cells preferentially metastasized to the liver but not in the lungs. AFDN loss in colorectal cancer cells at the primary site promoted cancer cell migration and invasion by disrupting tight intercellular junctions. Additionally, CXCR4 expression was increased in AFDN-deficient colorectal cancer cells via the JAK-STAT signaling pathway, which reduced the motility of AFDN-deficient colorectal cancer cells and facilitated their colonization of the liver. Collectively, these data shed light on the mechanism by which AFDN deficiency promotes liver tropism in metastatic colorectal cancer.

Octreotide attenuates intestinal barrier damage by maintaining basal autophagy in Caco2 cells.

The intestinal mucosal barrier is of great importance for maintaining the stability of the internal environment, which is closely related to the occurrence and development of intestinal inflammation. Octreotide (OCT) has potential applicable clinical value for treating intestinal injury according to previous studies, but the underlying molecular mechanisms have remained elusive. This article is based on a cell model of inflammation induced by lipopolysaccharide (LPS), aiming to explore the effects of OCT in protecting intestinal mucosal barrier function. A Cell Counting Kit‑8 assay was used to determine cell viability and evaluate the effectiveness of OCT. Gene silencing technology was used to reveal the mediated effect of somatostatin receptor 2 (SSTR2). The changes in intestinal permeability were detected through trans‑epithelial electrical resistance and fluorescein isothiocyanate‑dextran 4 experiments, and the alterations in tight junction proteins were detected using immunoblotting and reverse transcription fluorescence‑quantitative PCR technology. Autophagosomes were observed by electron microscopy and the dynamic changes of the autophagy process were characterized by light chain (LC)3‑II/LC3‑I conversion and autophagic flow. The results indicated that SSTR2‑dependent OCT can prevent the decrease in cell activity. After LPS treatment, the permeability of monolayer cells decreased and intercellular tight junctions were disrupted, resulting in a decrease in tight junction protein zona occludens 1 in cells. The level of autophagy‑related protein LC3 was altered to varying degrees at different times. These abnormal changes gradually returned to normal levels after the combined application of LPS and SSTR2‑dependent OCT, confirming the role of OCT in protecting intestinal barrier function. These experimental results suggest that OCT maintains basal autophagy and cell activity mediated by SSTR2 in intestinal epithelial cells, thereby preventing the intestinal barrier dysfunction in inflammation injury.

The silent threat: Nanopolystyrene and chrysene pollutants disrupt the intestinal mucosal barrier, new insights from juvenile Siniperca chuatsi

The intestinal mucosal barrier—comprising microbial, mechanical, chemical, and immunological barriers—is critical to protection against pathogens and maintenance of host health; however, it remains unclear whether it is affected by environmental contaminants. Therefore, the present study assessed whether exposure to ambient concentrations of nanopolystyrene (NP) and chrysene (CHR)—two ubiquitous environmental pollutants in the aquatic environment—affect the intestinal mucosal barrier in juvenile Siniperca chuatsi. After exposure for 21 days, S. chuatsi exhibited intestinal oxidative stress and imbalance of intestinal microbial homeostasis. NP and/or CHR exposure also disrupted the intestinal mechanical barrier, as evidenced by the altered intestinal epithelial cell morphology, disrupted structure of intercellular tight junctions, and decreased expression of tight junction proteins. Damage to the intestinal chemical barrier manifested as thinning of the mucus layer owing to the loss and damage of goblet cells. Furthermore, the intestinal immunological barrier was impaired as indicated by the loss of intestinal intraepithelial lymphocytes and increase in pro-inflammatory cytokines, chemokines, and immunoglobulins. These findings collectively suggest that the intestinal mucosal barrier was damaged. This study is, to the best of our knowledge, the first to report that exposure to NP and/or CHR at environmentally relevant concentrations disrupts the intestinal mucosal barrier in organisms and highlight the significance of nanoplastic/CHR pollution for intestinal health.

Andrographolide Improves ApoE4-Mediated Blood-Brain Barrier Injury by Alleviating Inflammation.

The pathological and physiological studies of Alzheimer's disease (AD) have been in-depth, and apolipoprotein E4 (ApoE4) has been proven to be highly correlated with AD, and clinical and experimental data show that ApoE4 can cause blood-brain barrier (BBB) injury, and the change of BBB permeability is an important factor affecting the development of AD. Andrographolide (Andro), as the active component of the natural plant Andrographis paniculata, has been proven to have anti-inflammatory and antioxidant effects, which have potential neuroprotective effects. To verify the protective effect of Andro on BBB in a short term, our research group used atorvastatin (Atorva)-mediated zebrafish brain injury model and the ApoE4-mediated cell co-culture model of BBB injury to explore the protective effects and mechanisms of Andro on BBB injury. Studies have shown that Andro can inhibit the activation of CypA/NF-κB/MMP-9 signaling pathway and has achieved the effect of antagonizing the inhibition of ApoE4 on intercellular tight junction proteins (occludin, claudin-5, and ZO-1). At the same time, Andro can inhibit the secretion of cell adhesion molecules (VCAM-1 and ICAM-1) in cells, thereby delaying the occurrence and progression of neuroinflammation and playing a protective role in BBB. In conclusion, Andro is a potent natural product which can protect the blood-brain barrier.

Evaluation of drug interactions of Saposhnikoviae Radix and its major components with astragaloside IV and paeoniflorin using in vitro and in vivo experiments

Saposhnikoviae Radix (SR) may enhance the pharmacodynamics of Huangqi Chifeng Tang (HQCFT) in the treatment of cerebral infarction according to our previous research, but the underlying mechanism is unknown. Herein, an in vivo pharmacokinetic assay in rats and in vitro MDCK-MDR1 cell assays were used to investigate the possible mechanism of SR, its main components, and its interactions with Astragali Radix (AR) and Paeoniae Radix (PR). An ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC‒MS/MS)-based analytical method for quantifying astragaloside IV (ASIV) and paeoniflorin (PAE) in microdialysis and transport samples was developed. The pharmacokinetic parameters of SR were determined using noncompartmental analyses CCK-8 assays were used to detect the cytotoxicity of ASIV, PAE, cimifugin (CIM), prim-o-glucosylcimifugin (POG) and their combinations. Moreover, drug transport was studied using MDCK-MDR1 cells. Western blotting was performed to measure the protein expression levels of P-GP and MRP1. Claudin-5, ZO-1, and F-actin expression was determined via immunohistochemical staining of MDCK-MDR1 cells. harmacokinetic studies revealed that, compared with those of Huangqi Chifeng Tang-Saposhnikoviae Radix (HQCFT-SR), the Tmax of ASIV increased by 11.11 %, and the MRT0-t and Tmax of PAE increased by 11.19 % and 20 %, respectively, in the HQCFT group. Transport studies revealed that when ASIV was coincubated with 28 μM CIM or POG, the apparent permeability coefficient (Papp) increased by 71.52 % and 50.33 %, respectively. Coincubation of PAE with 120 μM CIM or POG increased the Papp by 87.62 % and 60.95 %, respectively. Moreover, CIM and POG significantly downregulated P-gp and MRP1 (P < 0.05), inhibited the expression of Claudin-5, ZO-1, and F-actin (P < 0.05), and affected intercellular tight junctions (TJs). In conclusion, our study successfully established a selective, sensitive and reproducible UPLC‒MS/MS analytical method to detect drug‒drug interactions between SR, AR and PR in vivo and in vitro, which is beneficial for enhancing the therapeutic efficacies of AR and PR. Moreover, this study provides a theoretical basis for further research on the use of SR as a drug carrier.

Umbilical therapy for promoting transdermal delivery of topical formulations: Enhanced effect and underlying mechanism

Umbilical paste therapy is a promising method to promote transdermal drug delivery of topical formulations. This work investigated the effect and mechanism of transdermal drug delivery through the umbilical skin. The transdermal permeation studies showed the phenomenon of higher cumulative penetration and faster penetration rates for drug through the umbilical skin compared with non-umbilical skin, namely umbilical pro-permeability. This special transdermal permeability of drugs is influenced by their molecular weight, logP value, ability to form hydrogen bonds, and molecular volume. The underlying mechanism of umbilical pro-permeability was elucidated from unique structure and regulation the effect of drugs on microcirculation in the umbilical skin. Mechanistic studies revealed that this phenomenon was not only associated with the structural and physiological properties of the skin but also to the interactions between drugs and different skin layers. The umbilical pro-permeation is attributed to the thinner stratum corneum layer, differences in stratum corneum lipid composition and keratin structure, and lower levels of intercellular tight junction proteins in the viable epidermis and dermis layer of the skin. Our research indicated that umbilical paste therapy enhanced the transdermal delivery and absorption of drugs by stimulating local blood flow through mast cell activation. Surprisingly, skin temperature modulation and calcitonin gene-related peptide and substance P levels did not appear to significantly affect this process. In conclusion, umbilical drug administration, as a straightforward and non-invasive approach to enhance transdermal drug delivery, presents novel concepts for continued investigation and practical implementation of transdermal drug delivery systems.

Latest Findings on Phase Separation of Cytomechanical Proteins

The cellular response to mechanical stimuli depends largely on the structure of the cell itself and the abundance of intracellular cytomechanical proteins also plays a key role in the response to the stimulation of external mechanical signals. Liquid-liquid phase separation (LLPS) is the process by which proteins or protein-RNA complexes spontaneously separate and form two distinct "phases", ie, a low-concentration phase coexisting with a high-concentration phase. According to published findings, membrane-free organelles form and maintain their structures and regulate their internal biochemical activities through LLPS. LLPS, a novel mechanism for intracellular regulation of the biochemical reactions of biomacromolecules, plays a crucial role in modulating the responses of cytomechanical proteins. LLPS leads to the formation of highly concentrated liquid-phase condensates through multivalent interactions between biomacromolecules, thereby regulating a series of intracellular life activities. It has been reported that a variety of cytomechanical proteins respond to external mechanical signals through LLPS, which in turn affects biological behaviors such as cell growth, proliferation, spreading, migration, and apoptosis. Herein, we introduced the mechanisms of cytomechanics and LLPS. In addition, we presented the latest findings on cytomechanical protein phase separation, covering such issues as the regulation of focal adhesion maturation and mechanical signal transduction by LIM domain-containing protein 1 (LIMD1) phase separation, the regulation of intercellular tight junctions by zonula occludens (ZO) phase separation, and the regulation of cell proliferation and apoptosis by cytomechanical protein phase separation of the Hippo signaling pathway. The proposition of LLPS provides an explanation for the formation mechanism of intracellular membraneless organelles and supplies new approaches to understanding the biological functions of intracellular physiology or pathology. However, the molecular mechanisms by which LLPS drives focal adhesions and cell-edge dynamics are still not fully understood. It is not clear whether LLPS under in vitro conditions can occur under physiological conditions of organisms. There are still difficulties to be overcome in using LLPS to explain the interactions of multiple intracellular molecules. Researchers should pursue answers to these questions in the future.

Graphene oxide nanosheets enhancing the photothermal response of a cellular barrier exposed to near infrared light

AbstractRecent developments in targeted photothermal therapy focus on integrating near‐infrared (NIR) light and biocompatible nanostructures. It is yet unclear if graphene oxide nanosheets (GO) can assist in the photothermal response of cellular barriers under NIR irradiation. Herein, this study investigates the photothermal response of a cellular barrier treated with different concentrations of GO under the irradiation of NIR light. The synthesized GO nanosheets show good stability in aqueous media. No toxic effects are imposed onto human umbilical vein endothelial cells (HUVECs) when the cells are treated with GO up to 20 μg/mL for 1 day. In addition, the in vitro cellular barrier model with intercellular tight junctions is formed by using HUVECs, which have a high transepithelial electrical resistance (TEER) value of 89.3 Ω · cm2. Under the irradiation of NIR light (980 nm), the photothermal response on the cellular barrier is enhanced by introducing GO, which has been verified by the decrease of TEER value and the increase in the transport of dextran. The results indicate that the thermal response of a cellular barrier exposed to NIR light is non‐linearly enhanced with increasing concentration of GO.

Fecal microbiota transplantation repairs intestinal permeability and regulates the expression of 5-HT to influence alcohol-induced depression-like behaviors in C57BL/6J mice.

The epidemic of alcohol abuse affects millions of people worldwide. Relevant evidence supports the notion that the gut microbiota (GM) plays a crucial role in central nervous system (CNS) function, and its composition undergoes changes following alcohol consumption. Therefore, the purpose of this study was to investigate the effect of reconstructing the gut microbiota by fecal microbiota transplantation (FMT) on alcohol dependence. Here, we established an alcohol dependence model with C57BL/6J mice and proved that FMT treatment improved anxiety-like behavior and alcohol-seeking behavior in alcohol-dependent mice. Additionally, we found that the expression of the intestinal intercellular tight junction structure proteins ZO-1 and occludin was significantly increased after FMT. FMT repaired intestinal permeability in alcohol-dependent mice and decreased the levels of lipopolysaccharide (LPS) and proinflammatory factors. Moreover, the serotonin (5-hydroxytryptamine, 5-HT) content was significantly increased in alcohol-dependent mouse intestinal and brain tissues after receiving the fecal microbiome from healthy mice. 16S rRNA sequencing demonstrated that FMT markedly reshaped the composition of the gut microbiota and elicited changes in the intestinal barrier and 5-HT levels. Collectively, our results revealed that FMT has a palliative effect on alcohol dependence and explored the underlying mechanisms, which provides new strategies for the treatment of alcohol dependence.

The Relationship Between Zonulin and Asthma: A Mouse Model Study

Background and Objectives: Zonulin is a human protein that regulates intercellular tight junctions and increases the permeability of the intestinal epithelium. In light of the increasing focus on zonulin’s role in numerous chronic inflammatory diseases, this study aimed to investigate whether differences exist in serum zonulin levels and bronchial epithelium zonulin expression in vivo between asthma and normal groups, using a mouse model.Methods: Sixteen mice were utilized in this study, divided evenly between the normal and asthma groups. Serum zonulin levels, the expression of zonulin antibody in the bronchial epithelium, and serum cytokine levels were evaluated in both groups. Enzyme-linked immunosorbent assay and RNA in situ hybridization were utilized for the analysis.Results: The asthma group exhibited significantly higher levels of serum zonulin. High zonulin antibody expression was also observed in the bronchial epithelium of the asthma group. Given that our mouse model demonstrated a significant difference in interleukin (IL)-4 and IL-6 between the normal and asthma groups, zonulin may be associated not only with type 2 responses but also with various subtypes of asthma. Further studies are required to investigate this relationship in greater detail.Conclusion: Zonulin may play a role in the complex pathophysiology of asthma and could serve as a biomarker in various asthma-related situations.

DDEL-04. MODULATION OF THE BLOOD-TUMOR BARRIER FOR IMPROVED DRUG DELIVERY IN GLIOBLASTOMA

Abstract Effective drug delivery remains a major challenge in glioblastoma (GBM) treatment. Specialized intercellular endothelial cell tight junctions at the blood-brain and blood-tumor barriers (BBB/BTB) represent obstacles to therapeutic efficacy by limiting intratumoral drug uptake. To understand the factors at the BTB that limit drug accessibility we performed bioinformatics analysis of TCGA data which suggested enrichment of the tight junction protein VE-cadherin (CDH5). CDH5 expression at the BTB was confirmed by immunostaining of patient GBM specimens and spatial transcriptomics using the 10X Visium platform. We found that a broadly selective protein kinase inhibitor, 6-bromoindirubin-3’-acetoxime (BIA), potently down-regulated CDH5 in cultured human cerebrovascular endothelial cells (HCMEC/D3) at the transcriptional level and this correlated with decreased trans-endothelial electrical resistance (TEER) measurement. In vivo administration of BIA (20 mg/kg IP) to mice bearing intracranial GBM xenografts showed striking downregulation of CDH5 in tumor associated endothelium. Furthermore, increased BTB permeability was shown using fluorescent tracers as well as DCE-MRI, which demonstrated elevated Ktrans values 24 hours after BIA administration. Integrated phospho-proteomic and transcriptomic analysis of BIA treated HCMEC/D3 cells support a mechanism involving p38 MAP kinase regulation of Ets-family transcription factors that drive CDH5 expression. At least a 20-fold increase in intra-tumoral accumulation of cisplatin and temozolomide was observed using G9-pCDH GBM xenografts when administered 24 h after BIA treatment. No increased drug uptake was seen in healthy brain tissue, indicating specific targeting of tumor vasculature. Efficacy studies showed extended survival when using cisplatin in combination with BIA (median 27d, p=0.0016) when compared to cisplatin (18.5d), BIA (21.5d) and control (18d). Further efficacy studies are underway. These data therefore identify the GBM-associated vasculature as a target for small molecule inhibition of tight junction formation to enhance intratumoral drug uptake. Ongoing studies are investigating the potential of this approach with a range of agents.

Ultrasound mediated gold nanoclusters-capped gas vesicles for enhanced fluorescence imaging

The intercellular tight junction inhibits tumor imaging efficiency of nanomaterials, and enhanced cellular drug delivery with efficient detection is an important tool for tumor diagnosis. Herein, we fabricate fluorescence gold nanoclusters (Au NCs) decorated gas vesicles (GV-Au) for ultrasound (US)-mediated enhanced cellular delivery and imaging, in which GVs are living cell derived protein bubbles. GV-Au is rod-shaped sack-like structure around 230 nm, and displays improved stability and fluorescence ability compared with free Au NCs. Flow cytometry assay confirms the intracellular localization of Au NCs and GV-Au with a respective 2.20-fold enhanced cellular uptake post US treatment. Confocal images reveal the efficient cellular uptake of GV-Au under US impact, indicating that GV-Au is suitable for cellular and in vivo fluorescence imaging. Our strategy provides a new idea for efficient fluorescence imaging by penetrating cell membranes at the presence of US treatment.

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

Gasdermin A Is Required for Epidermal Cornification during Skin Barrier Regeneration and in an Atopic Dermatitis-Like Model

Atopic dermatitis (AD) is featured with impaired skin barrier. The stratum corneum (SC) and the intercellular tight junctions (TJs) constitute the permeability barrier, which is essential to protect the host water loss and pathogen entrance. The epidermal barrier is constantly renewed by differentiating keratinocytes through cornification, during which autophagy contributes to organelles and nucleus elimination. The human GSDMA and its mouse homologs Gsdma1-3 are expressed in the suprabasal epidermis. Although a pyroptotic role for GSDMA/Gsdma1 in host defense against Streptococcus pyogenes has been reported, the physiological function of Gsdma1/a2/a3 in epidermal homeostasis remains elusive. Herein, through repeated epidermal barrier disruption, we found that TJ formation and SC maturation were defective in the Gsdma1/a3-deficient epidermis. Using comparative gene profiling analysis, mitochondrial respiration measurement, and in vivo tracing of mitophagy, our data indicate that Gsdma1/a3 activation leads to mitochondrial dysfunction and subsequently facilitates mitochondrial turnover and epidermal cornification. In calcipotriol (MC903)-induced AD-like animal model, we demonstrated that Gsdma1/a3-deficiency selectively enhanced the Th2 response. Remarkably, the GSDMA expression is reduced in the epidermis of patients with AD compared to normal individuals. Gsdma1/a3-deficiency might be involved in AD pathogenesis, likely through GSDMA-mediated epidermal differentiation and cornification.

Analysis of Communal Molecular Mechanism Between Chronic Obstructive Pulmonary Disease and Osteoporosis.

Chronic obstructive pulmonary disease (COPD) patients with osteoporosis (OP) usually experience more frequent exacerbations, worse quality of life, and heavier economic burden, however, few studies have investigated common molecular mechanisms of COPD and OP. To explore the relationship between COPD and OP through bioinformatics analysis. The miRNA microarray data of COPD and OP were retrieved from the Gene Expression Database (GEO), and the differentially expressed microRNAs (DEmiRNAs) were screened and the intersection was obtained. The Targetscan, miRDB, and miRWalk databases were used to predict the target genes of DEmiRNA, and the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the R package clusterProfiler, the STRING database was used to analyze the target protein-protein interaction network (PPI) and screens to determine the core modules and core genes. Two DEmiRNAs (miR-23a-5p, miR-194-3p) have been found in COPD and OP, which have predicted 76 and 114 target genes, respectively. GO functional annotations of miR-23a-5p were significantly enriched in CD40 signaling pathway, ubiquitin-conjugating enzyme activity, etc; KEGG pathways of miR-23a-5p were significantly enriched in ubiquitin-mediated proteolysis, folate biosynthesis, and regulation of actin cytoskeleton. GO function annotations of miR-194-3p were significantly enriched in T cell activation regulation, ubiquitin protein ligase activity, and DNA transcription factor binding; KEGG pathways of miR-194-3p were significantly enriched in cell adhesion molecules, intercellular tight junctions, and lysosomal pathway. PPI analysis found target coding proteins formed complex regulatory networks. Ten core genes (TP53, SRC, PXN, CHD4, SYK, TNRC6B, PML, KAT5, BRD1 and IGF2) were picked out among them, then we used the MCODE plugin found three core subnetworks. Two identical DEmiRNAs (miR-23a-5p, miR-194-3p) exist in the peripheral blood of COPD and OP patients, which are important biomarkers for COPD patients with OP and may represent novel targets for diagnosis and treatment of COPD patients with OP.

Effects of Volatile Anesthetics versus Ketamine on Blood-Brain Barrier Permeability via Lipid-Mediated Alterations of Endothelial Cell Membranes.

The purpose of this study was to investigate the effects of the volatile anesthetic agents isoflurane and sevoflurane, at clinically relevant concentrations, on the fluidity of lipid membranes and permeability of the blood-brain barrier (BBB). We analyzed the in vitro effects of isoflurane or ketamine using erythrocyte ghosts (sodium fluorescein permeability), monolayers of brain microvascular endothelial cells ([13C]sucrose and fluorescein permeability), or liposomes (fluorescence anisotropy). Additionally, we determined the effects of 30-minute exposure of mice to isoflurane on the brain tight junction proteins. Finally, we investigated in vivo brain uptake of [13C]mannitol and [13C]sucrose after intravenous administration in mice under anesthesia with isoflurane, sevoflurane, or ketamine/xylazine in addition to the awake condition. Isoflurane at 1-mM and 5-mM concentrations increased fluorescein efflux from the erythrocyte ghosts in a concentration-dependent manner. Similarly, in endothelial cell monolayers exposed to 3% (v/v) isoflurane, permeability coefficients rose by about 25% for fluorescein and 40% for [13C]sucrose, whereas transendothelial resistance and cell viability remained unaffected. Although isoflurane caused a significant decrease in liposomes anisotropy values, ketamine/xylazine did not show any effects. Brain uptake clearance (apparent Kin) of the passive permeability markers in vivo in mice approximately doubled under isoflurane or sevoflurane anesthesia compared with either ketamine/xylazine anesthesia or the awake condition. In vivo exposure of mice to isoflurane did not change any of the brain tight junction proteins. Our data support membrane permeabilization rather than loosening of intercellular tight junctions as an underlying mechanism for increased permeability of the endothelial cell monolayers and the BBB in vivo. SIGNIFICANCE STATEMENT: The blood-brain barrier controls the entry of endogenous substances and xenobiotics from the circulation into the central nervous system. Volatile anesthetic agents like isoflurane alter the lipid structure of cell membranes, transiently facilitating the brain uptake of otherwise poorly permeable, hydrophilic small molecules. Clinical implications may arise when potentially neurotoxic drugs gain enhanced access to the central nervous system under inhalational anesthetics.

The Ultrastructure of Hepatic Stellate Cell-Macrophage Intercellular Crosstalk as a New Morphological Insight into Phenomenon of Fibrogenesis in Pediatric Autoimmune Hepatitis.

Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4-17 with clinic-pathologically diagnosed untreated AIH. Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis.

The role of Nrf2 in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated MMP9

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.

Morphological and Functional Effects of Ultrasound on Blood–Brain Barrier Transitory Opening: An In Vitro Study on Rat Brain Endothelial Cells

With the recent advances in medicine, human life expectancy is increasing; however, the extra years of life are not necessarily spent in good health or free from disability, resulting in a significantly higher incidence of age-associated pathologies. Among these disorders, neurodegenerative diseases have a significant impact. To this end, the presence of the protective blood-brain barrier (BBB) represents a formidable obstacle to the delivery of therapeutics. Thus, this makes it imperative to define strategies to bypass the BBB in order to successfully target the brain with the appropriate drugs. It has been demonstrated that targeting the BBB by ultrasound (US) can transiently make this anatomical barrier permeable and in so doing, allow the delivery of therapeutics. Thus, our aim was to carry out an in depth in vitro molecular and morphological study on the effects of US treatment on the BBB. The rat brain endothelial (RBE4) cell line was challenged with exposure to 12 MHz diagnostic US treatment for 10, 20, and 30 min. Cell viability assays, Western blotting analysis on the endoplasmic reticulum (ER), and oxidative stress marker evaluation were then performed, along with cytological and immunofluorescence staining, in order to evaluate the effects of US on the intercellular spaces and tight junction distribution of the brain endothelial cells. We observed that the US treatment exerted no toxic effects on either RBE4 cell viability or the upregulation/dislocation of the ER and oxidative stress marker (GRP78 and cytochrome C, respectively). Further, we observed that the application of US induced an increase in the intercellular spaces, as shown by Papanicolaou staining, mainly due to the altered distribution of the tight junction protein zonula occludens-1 (ZO-1). This latter US-dependent effect was transient and disappeared 20 min after the removal of the stimulus. In conclusion, our results show that US induces a transient alteration of the BBB, without altering the intracellular signaling pathways such as the ER and oxidative stress that could potentially be toxic for endothelial cells. These results suggested that US treatment could represent a potential strategy for improving drug delivery to the brain.

Experimental Study on the Treatment of Ulcerative Proctitis in Rats by Combining Baitouweng Decoction with Mesalamine Retention Enema

To study the effect and mechanism of Baitouweng decoction, mesalamine and its combination with an enema in the treatment of ulcerative proctitis in rats is the objective of the study. The rats were divided into seven groups: Control (Normal), model (ulcerative proctitis), mesalamine, Baitouweng decoction, lowdose Baitouweng decoction combined with mesalamine, medium-dose Baitouweng decoction combined with mesalamine, high-dose Baitouweng decoction combined with mesalamine group. Except for the control group, all rats were induced with a 5 % acetic acid solution for ulcerative proctitis. The severity of ulcerative proctitis was measured by the disease activity index score; morphological changes of rectal tissue were observed by hematoxylin and eosin staining; expression of cytokines tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interferon‐gamma, interleukin-10 messenger ribonucleic acid was detected by reverse transcription-polymerase chain reaction; Western blot analysis of signaling pathway proteins nuclear factor kappa B p65 and phosphorylated-nuclear factor kappa B p65 and the expression of tight junction proteins claudin-1, occludin and zonula occludens-1 was detected by Western blot and immunohistochemistry. The results revealed that the disease activity index scores of rats in each dosing group were significantly lower than those in the ulcerative proctitis group. After treatment, the expression of pro-inflammatory factors tumor necrosis factor alpha, interleukin-1 beta, interleukin-6 and interferon‐gamma was inhibited and the expression of the anti-inflammatory factor interleukin-10 was up-regulated. In conclusion, Baitouweng decoction combined with mesalamine had a synergistic effect on the treatment of ulcerative proctitis. The mechanism of action may be to regulate the synthesis and release of inflammatory factors by inhibiting the over-activation of nuclear factor kappa B p65, reducing the damage of pro-inflammatory factors to rectal mucosa and repairing the intercellular tight junction structure, thus improving the intestinal barrier and reducing the rectal inflammatory response.