In this study, we presented evidence that the permeability of the brain, lung, and intestinal barriers increased after stroke, and that GRb1 could effectively mitigate the stroke-induced damage to the three barriers. We also demonstrated that GRb1 exerted these effects through the PPARγ/NF-κB signaling pathway, at least partly.
An increasing number of studies have shown that ischemic stroke causes a series of complications, including pneumonia and damage to the intestinal mucosa. For instance, focal ischemic stroke has been associated with brain–lung crosstalk, leading to increased pulmonary damage and permeability of the pulmonary capillary membrane. Additionally, intestinal mucosal barrier disturbance can be identified even in the early stages of cerebral ischemic stress. Necrosis occurs at the tip of the villi and the intestinal mucosa is damaged in the early stages of ischemic stroke, leading to gastrointestinal kinetic changes.
In our study, we found that both the pulmonary and intestinal barriers were simultaneously damaged in an experimental model of focal ischemic stroke. Several studies have demonstrated that GRb1 can be useful as a therapy for cardiovascular diseases, and exerts beneficial effects on the intestinal mucosal barrier and lung inflammation. However, its protective effects on brain/lung/intestinal barrier damage after ischemic stroke remain unclear.
In the present study, EB leakage was used to evaluate the integrity of the barriers of the three organs (brain, lung, and intestine). EB has a very high affinity for serum albumin, which cannot cross the blood barrier. However, when a blood vessel is compromised, albumin, with bound EB, leaks out. Our findings demonstrated that treatment with GRb1 reduced EB leakage and ameliorated histopathological damage in the brain, lung, and intestine after cerebral I/R (Figs 1C, 3A, and 5A).
In the brain, a physical paracellular barrier is created by the presence of TJs between endothelial cells. TJs are the luminal-most cellular junctions and are composed of claudins, occludins, and zona occludens. TJs are more often studied in terms of their role in the BBB, where they have been found to induce high transendothelial electrical resistance and reduce paracellular diffusion to a greater extent than adhesion junctions. In our study, the expression of ZO-1 and occludin in brain endothelial cells was significantly higher in the GRb1 group than in the Model group (Fig. 2).
In the lung, junction protein levels were reduced in mice with cerebral I/R. Adherens junctions play a major role in the regulation of pulmonary barrier permeability, while the transmembrane protein VE-cadherin is a central player in adherens junction formation and the regulation of pulmonary barrier integrity. Moreover, lung barrier injury has been associated with decreased mRNA and protein levels of claudin-5. In our study, the expression of VE-cadherin and claudin-5 in the lung was significantly increased in the GRb1 group compared with that in the Model group (Fig. 4A, B).
Patients with severe brain injury have been reported to experience reduced gastrointestinal motility. In our study, GRb1 administration led to a significant increase in mesenteric blood flow (Fig. 5D), indicating its potential efficacy for treating brain injury-induced gastrointestinal motility. Laboratory and clinical evidence have both indicated that the intestinal TJ-mediated barrier plays a critical role in the pathogenesis of intestinal and systemic diseases. TJs encircle the apical ends of the lateral membranes of epithelial cells and determine the selectivity of their paracellular permeability to solutes[48,49]. In the current study, we showed that the levels of ZO-1 and occludin were significantly decreased after cerebral I/R, but increased after treatment with GRb1 or Eda. This indicated that GRb1 can help maintain TJ-mediated barrier integrity following stroke-induced barrier disruption (Fig. 6A, B). Combined, these findings implied that GRb1 could effectively mitigate brain, pulmonary and intestinal barrier dysfunction in mice with cerebral I/R.
There is clear evidence that human stroke causes multi-organ systemic disease through multiple mechanisms. After a stroke, the stimulation of the vagus nerve through nicotinic acetylcholine receptor α7 (nAChRα7) induces microglial activation, resulting in reduced alveolar macrophage phagocytic capability and leading to lung injury. Meanwhile, systemic inflammation consequent to stroke leads to an increase in the release of inflammatory mediators such as interleukin (IL)-6 and TNF-α, resulting in lung inflammation and alveolar-capillary injury. There exists bidirectional communication and interaction between the gut and the brain. This link is thought to be based on the brain–gut–microbiota axis, and involves the autonomic nervous system (ANS), the hypothalamic–pituitary–adrenal (HPA) axis, and the gastrointestinal tract. Moreover, inflammatory molecules and cellular debris, the so-called DAMPs, are released from damaged tissue after a stroke. The release of DAMPs and cytokines, as well as the activation of the vagus nerve, induces gut dysmotility, gut dysbiosis, and increased gut permeability, resulting in the translocation of intestinal bacteria.
PPARγ is an important member of the nuclear receptor superfamily and predominantly expressed in adipocytes. However, PPARγ is also expressed in vascular cells, and data generated have shown the great importance of this receptor in vascular barrier integrity . Studies have suggested that PPARγ activation can inhibit inflammation, thereby maintaining barrier integrity. PPARγ may reduce pro-inflammatory phenotypes via suppression of the AP-1 and NF-κB pathways. PPARγ agonists can also significantly reduce ischemia-triggered increases in cerebrovascular/BBB permeability in mice by suppression of proapoptotic microRNA-15a (miR-15a). Systematic administration of the PPARγ agonist pioglitazone was shown to ameliorate DSS-induced colitis by helping to repair the intestinal mucosal barrier. Moreover, pioglitazone could also inhibit ERK and NF-κB phosphorylation, as well as upregulate the expression of the tight junction proteins ZO-1 and claudin-5 in a PPARγ signaling-dependent manner.
Increasing evidences have shown that PPARγ plays a upstream signal of NF-κB in the anti-inflammatory process. The direct impact of PPARγ on NF-κB may be associated with its enzymatic properties. PPARγ is an E3 ubiquitin ligase, which cooperates with E2 UBCH3. PPARγ mediates the ubiquitination of Lys48 NF-κB p65, which leads to the proteolytic degradation of this NF-κB subunit. Recent studies have reported that GRb1 binds to the PPARγ ligand-binding domain as determined by surface plasmon resonance, which suggests that GRb1 may be able to activate PPARγ, while polTIRFM analysis also indicated that GRb1 could bind to PPARγ. GRb1 has also been shown to exert anticalcification effects through the PPARγ/Wnt/β-catenin axis and can promote the browning of 3T3-L1 adipocytes through the activation of PPARγ. Based on these observations, we assessed the protein levels of PPARγ and phospho-NF-κB p65 in MCAO/R mice treated with GRb1. We found that GRb1 treatment upregulated PPARγ levels and inhibited those of phospho-NF-κB p65, with a concomitant reduction in the concentrations of proinflammatory factors, thereby helping to maintain the integrity of the brain, lung, and intestinal barriers. Moreover, we also found that these effects were suppressed by cotreatment with the PPARγ antagonist GW9662.
The vascular barrier maintains vascular and tissue homeostasis and modulates many physiological processes. The integrity of vascular barriers can be disrupted by a variety of soluble permeability factors, and changes in barrier function can result in multi-organ damage during disease progression. Dysregulated vascular hyperpermeability can participate in the progression of many pathological states, such as rheumatoid arthritis, inflammatory bowel disease, asthma, acute lung injury, and cardiovascular diseases. PPARγ expression is highest in brown and white adipose tissues, the large intestine, and immune cells, but can also be found in various other tissues, including the muscle, pancreas, liver, small intestine, and kidney. Activators of PPARγ exert a broad spectrum of biological functions, such as regulating fatty acid metabolism, reducing inflammation, influencing immune cell balance, inhibiting apoptosis and oxidative stress, and improving endothelial function. Such pleiotropic activity makes them interesting therapeutic targets for the treatment of various conditions. Therefore, the protective effects elicited by GRb1 on barrier damage in multiple organs via PPARγ suggest that it may have potential as a therapeutic option for the treatment of vascular barrier-related diseases.