The relationship between dysbiosis and central nervous diseases has been proved in the last 10 years. Microbial alterations cause increased intestinal permeability, and the penetration of bacterial fragment and toxins induces local and systemic inflammatory processes, affecting distant organs, including the brain. Therefore, the integrity of the intestinal epithelial barrier plays a central role in the microbiota–gut–brain axis. In this review, we discuss recent findings on zonulin, an important tight junction regulator of intestinal epithelial cells, which is assumed to play a key role in maintaining of the blood–brain barrier function. In addition to focusing on the effect of microbiome on intestinal zonulin release, we also summarize potential pharmaceutical approaches to modulate zonulin-associated pathways with larazotide acetate and other zonulin receptor agonists or antagonists. The present review also addresses the emerging issues, including the use of misleading nomenclature or the unsolved questions about the exact protein sequence of zonulin.

Tissue fibrosis is characterized by chronic fibroblast activation and consequently excessive accumulation of collagen-rich extracellular matrix. In vitro microplate-based assays are essential to investigate the underlying mechanism and the effect of antifibrotic drugs. In this study, in the absence of a gold-standard method, we optimized a simple, cost-effective, Sirius Red-based colorimetric measurement to determine the collagen production of fibroblasts grown on 96-well tissue culture plates. Based on our findings, the use of a serum-free medium is recommended to avoid aspecific signals, while ascorbate supplementation increases the collagen production of fibroblasts. The cell-associated collagens can be quantified by Sirius Red staining in acidic conditions followed by alkaline elution. Immature collagens can be precipitated from the culture medium by acidic Sirius Red solution, and after subsequent centrifugation and washing steps, their amount can be also measured. Increased attention has been paid to optimizing the assay procedure, including incubation time, temperature, and solution concentrations. The resulting assay shows high linearity and sensitivity and could serve as a useful tool in fibrosis-related basic research as well as in preclinical drug screening.

It is increasingly known that Parkinson’s (PD) and Alzheimer’s (AD) diseases occur more frequently in patients with inflammatory gastrointestinal diseases including inflammatory bowel (IBD) or celiac disease, indicating a pathological link between them. Although epidemiological observations suggest the existence of the gut-brain axis (GBA) involving systemic inflammatory and neural pathways, little is known about the exact molecular mechanisms. Parkinson’s disease 7 (PARK7/DJ-1) is a multifunctional protein whose protective role has been widely demonstrated in neurodegenerative diseases, including PD, AD, or ischemic stroke. Recent studies also revealed the importance of PARK7/DJ-1 in the maintenance of the gut microbiome and also in the regulation of intestinal inflammation. All these findings suggest that PARK7/DJ-1 may be a link and also a potential therapeutic target in gut and brain diseases. In this review, therefore, we discuss our current knowledge about PARK7/DJ-1 in the context of GBA diseases.

Coeliac disease (CD) is a chronic, immune-mediated small intestinal enteropathy, accompanied with gluten-triggered oxidative damage of duodenal mucosa. Previously, our research group reported an increased mucosal level of the antioxidant protein Parkinson’s disease 7 (PARK7) in children with CD. In the present study, we investigated the role of increased PARK7 level on the epithelial cell and mucosal integrity of the small intestine. The presence of PARK7 was investigated using immunofluorescent staining on duodenal mucosa of children with CD and on FHs74Int duodenal epithelial cells. To investigate the role of oxidative stress, FHs74Int cells were treated with H₂O₂ in the absence or presence of Comp23, a PARK7-binding compound. Intracellular accumulation of reactive oxygen species (ROS) was determined by DCFDA-based assay. Cell viability was measured by MTT, LDH, and Annexin V apoptosis assays. Disruption of cytoskeleton and cell adhesion was investigated by immunofluorescence staining and by real-time RT PCR. Effect of PARK7 on mucosal permeability was investigated ex vivo using intestinal sacs derived from control and Comp-23-pretreated mice. Comp23 treatment reduced the H₂O₂-induced intracellular accumulation of ROS, thus preserving the integrity of the cytoskeleton and also the viability of the FHs74Int cells. Accordingly, Comp23 treatment increased the expression of antioxidants (NRF2, TRX1, GCLC, HMOX1, NQO1), cell-cycle regulators (TP53, CDKN1A, PCNA, BCL2, BAX), and cell adhesion molecules (ZO1, CDH1, VCL, ITGB5) of H₂O₂-treated cells. Pretreatment with Comp23 considerably decreased the small intestinal permeability. In this study, we demonstrate that PARK7-binding Comp23 reduces the oxidative damage of duodenal epithelial cells, via increased expression of NRF2- and P53-regulated genes. Our results suggest that PARK7 plays a significant role in the maintenance of mucosal integrity in CD.

Recent studies draw attention to how excessive salt (NaCl) intake induces fibrotic alterations in the peritoneum through sodium accumulation and osmotic events. The aim of our study was to better understand the underlying mechanisms. The effects of additional NaCl were investigated on human primary mesothelial cells (HPMC), human primary peritoneal fibroblasts (HPF), endothelial cells (HUVEC), immune cells (PBMC), as well as ex vivo on peritoneal tissue samples. Our results showed that a high-salt environment and the consequently increased osmolarity increase the production of inflammatory cytokines, profibrotic growth factors, and components of the renin–angiotensin–aldosterone system, including IL1B, IL6, MCP1, TGFB1, PDGFB, CTGF, Renin and Ace both in vitro and ex vivo. We also demonstrated that high salt induces mesenchymal transition by decreasing the expression of epithelial marker CDH1 and increasing the expression of mesenchymal marker ACTA2 and SNAIL1 in HPMCs, HUVECs and peritoneal samples. Furthermore, high salt increased extracellular matrix production in HPFs. We demonstrated that excess Na⁺ and the consequently increased osmolarity induce a comprehensive profibrotic response in the peritoneal cells, thereby facilitating the development of peritoneal fibrosis.

Among patients on peritoneal dialysis (PD), 50–80% will develop peritoneal fibrosis, and 0.5–4.4% will develop life-threatening encapsulating peritoneal sclerosis (EPS). Here, we investigated the role of extracellular vesicles (EVs) on the TGF-β- and PDGF-B-driven processes of peritoneal fibrosis. EVs were isolated from the peritoneal dialysis effluent (PDE) of children receiving continuous ambulatory PD. The impact of PDE-EVs on the epithelial–mesenchymal transition (EMT) and collagen production of the peritoneal mesothelial cells and fibroblasts were investigated in vitro and in vivo in the chlorhexidine digluconate (CG)-induced mice model of peritoneal fibrosis. PDE-EVs showed spherical morphology in the 100 nm size range, and their spectral features, CD63, and annexin positivity were characteristic of EVs. PDE-EVs penetrated into the peritoneal mesothelial cells and fibroblasts and reduced their PDE- or PDGF-B-induced proliferation. Furthermore, PDE-EVs inhibited the PDE- or TGF-β-induced EMT and collagen production of the investigated cell types. PDE-EVs contributed to the mesothelial layer integrity and decreased the submesothelial thickening of CG-treated mice. We demonstrated that PDE-EVs significantly inhibit the PDGF-B- or TGF-β-induced fibrotic processes in vitro and in vivo, suggesting that EVs may contribute to new therapeutic strategies to treat peritoneal fibrosis and other fibroproliferative diseases.