Delayed recanalization reduced neuronal apoptosis and neurological deficits by enhancing liver-derived trefoil factor 3-mediated neuroprotection via LINGO2/EGFR/Src signaling pathway after middle cerebral artery occlusion in rats.

Delayed recanalization at days or weeks beyond the therapeutic window was shown to improve functional outcomes in acute ischemic stroke (AIS) patients. However, the underlying mechanisms remain unclear. Previous preclinical study reported that trefoil factor 3 (TFF3) was secreted by liver after cerebral ischemia and acted a distant neuroprotective factor. Here, we investigated the liver-derived TFF3-mediated neuroprotective mechanism enhanced by delayed recanalization after AIS. A total of 327 male Sprague-Dawley rats and the model of middle cerebral artery occlusion (MCAO) with permanent occlusion (pMCAO) or with delayed recanalization at 3 d post-occlusion (rMCAO) were used. Partial hepatectomy was performed within 5 min after MCAO. Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 (LINGO2) siRNA was administered intracerebroventricularly at 48 h after MCAO. Recombinant rat TFF3 (rr-TFF3, 30 µg/Kg) or recombinant rat epidermal growth factor (rr-EGF, 100 µg/Kg) was administered intranasally at 1 h after recanalization, and EGFR inhibitor Gefitinib (75 mg/Kg) was administered intranasally at 30 min before recanalization. The evaluation of outcomes included neurobehavior, ELISA, western blot and immunofluorescence staining. TFF3 in hepatocytes and serum were upregulated in a similar time-dependent manner after MCAO. Compared to pMCAO, delayed recanalization increased brain TFF3 levels and attenuated brain damage with the reduction in neuronal apoptosis, infarct volume and neurological deficits. Partial hepatectomy reduced TFF3 levels in serum and ipsilateral brain hemisphere, and abolished the benefits of delayed recanalization on neuronal apoptosis and neurobehavioral deficits in rMCAO rats. Intranasal rrTFF3 treatment reversed the changes associated with partial hepatectomy. Delayed recanalization after MCAO increased the co-immunoprecipitation of TFF3 and LINGO2, as well as expressions of p-EGFR, p-Src and Bcl-2 in the brain. LINGO2 siRNA knockdown or EGFR inhibitor reversed the effects of delayed recanalization on apoptosis and brain expressions of LINGO2, p-EGFR, p-Src and Bcl-2 in rMCAO rats. EGFR activator abolished the deleterious effects of LINGO2 siRNA. In conclusion, our investigation demonstrated for the first time that delayed recanalization may enhance the entry of liver-derived TFF3 into ischemic brain upon restoring blood flow after MCAO, which attenuated neuronal apoptosis and neurological deficits at least in part via activating LINGO2/EGFR/Src pathway.

Modulation of the Intestinal Barrier Integrity and Repair by Microbiota Extracellular Vesicles through the Differential Regulation of Trefoil Factor 3 in LS174T Goblet Cells.

Trefoil factor 3 (TFF3) plays a key role in the maintenance and repair of intestinal mucosa. TFF3 expression is upregulated by the microbiota through TLR2. At the posttranscriptional level, TFF3 is downregulated by miR-7-5p. Reduced TFF3 levels have been detected in the damaged tissue of IBD patients. Here, we investigate the regulation of TFF3 expression by microbiota extracellular vesicles (EVs) in LS174T goblet cells using RT-qPCR and inhibitors of the TLR2 or PI3K pathways. To evaluate the subsequent impact on epithelial barrier function, conditioned media from control and vesicle-stimulated LS174T cells were used to treat Caco-2 monolayers. The barrier-strengthening effects were evaluated by analysing the expression and subcellular distribution of tight junction proteins, and the repairing effects were assessed using wound-healing assays. The results showed a differential regulation of TFF3 in LS174T via EVs from the probiotic EcN and the commensal ECOR12. EcN EVs activated the TFF3 production through TLR2 and downregulated miR7-5-p through PI3K. Consistently, high levels of secreted TFF3 reinforced the tight junctions and stimulated wound healing in the Caco-2 cells. ECOR12 EVs did not cause these effects. TFF3 is a potential therapeutic target in IBD. This study contributes to understanding the molecular players (microbiota EVs) connecting gut microbes to health and may help in designing better nutritional interventions based on microbiota bioactive compounds.

Role of intestinal trefoil factor in protecting intestinal epithelial cells from burn-induced injury.

Although intestinal trefoil factor (ITF) can alleviate the burn-induced intestinal mucosa injury, the underlying mechanisms remains elusive. In this study, we investigated if ITF alters glutamine transport on the brush border membrane vesicles (BBMVs) of the intestines in Sprague-Dawley rats inflicted with 30% TBSA and the underlying mechanisms. We found that ITF significantly stimulated intestinal glutamine transport in burned rats. Mechanistically, ITF enhanced autophagy, reduces endoplasmic reticulum stress (ERS), and alleviates the impaired PDI, ASCT2, and B0AT1 in IECs and BBMVs after burn injury likely through AMPK activation. Therefore, ITF may protect intestinal epithelial cells from burn-induced injury through improving glutamine transport by alleviating ERS.

Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins.

Trefoil factors are effector molecules in gastrointestinal tract physiology. Each one improves healing of the gastrointestinal tract. Trefoil factors may be grouped into three classes: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Significant amounts of TFF3 are present in human breast milk. Previously, we have reported that trefoil factor 3 isolated from human breast milk produces down regulation of cytokines and promotes human beta defensins expression in intestinal epithelial cells. This study aimed to determine the molecular mechanism involved. Here we showed that the presence of TFF3 strongly correlated with protease activated receptors 2 (PAR-2) activation in human intestinal cells. Intracellular calcium ((Ca2+)i)mobilization was induced by the treatment with: 1) TFF3, 2) synthetic PAR-2 agonist peptide. The co-treatment with a synthetic PAR-2 antagonist peptide and TFF3 eliminates the latter's effect. Additionally, we demonstrated the existence of interactions among TFF3 and PAR-2 receptors through far Western blot and co-precipitation. Finally, down regulation of PAR-2 by siRNA resulted in a decrease of TFF3 induced intracellular (Ca2+)i mobilization, cytokine regulation and defensins expression. These findings suggest that TFF3 activates intestinal cells through PAR-2 (Fig. 4, Ref. 19).

mTOR signalling in the nucleus accumbens shell is critical for augmented effect of TFF3 on behavioural response to cocaine.

Neuropeptides play important roles in modulating the rewarding value of abused drugs. Trefoil factor 3 (TFF3) was recently reported to modulate withdrawal syndrome of morphine, but the effects of TFF3 on the cocaine-induced behavioral changes are still elusive. In the present study, cocaine-induced hyperlocomotion and conditioned place preference (CPP) rat paradigms were provided to investigate the role of TFF3 in the reward response to cocaine. High-performance liquid chromatography (HPLC) analysis was used to analyse the dopamine concentration. The results showed that systemic TFF3 administration (0.1 mg/kg i.p.) significantly augmented cocaine- induced hyperlocomotion and CPP formation, without any effects on locomotor activity and aversive or rewarding effects per se. TFF3 significantly augmented the increment of the dopamine concentration in the NAc and the activity of the mTOR signalling pathway induced by acute cocaine exposure (10 mg/kg, i.p.) in the NAc shell, but not the core. The Intra-NAc shell infusion of rapamycin blocked TFF3-induced hyperactivity in cocaine-treatment rats. These findings indicated that TFF3 could potentiate behavioural response to cocaine, which may be associated with regulating dopamine concentration. Furthermore, the findings indicated that mTOR signalling pathway in the NAc shell is important for TFF3-induced enhancement on the cocaine-induced behavioral changes.

Trefoil factor 3 shows anti-inflammatory effects on activated microglia.

Microglial cells are a major source of pro-inflammatory cytokines during central nervous system (CNS) inflammation. They can develop a pro-inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Shifting the phenotype from M1 to M2 might be an important mechanism to overcome CNS inflammation and to prevent or reduce neuronal damage. Here, we demonstrate that the anti-inflammatory protein trefoil factor 3 (TFF3) is secreted by astrocytes and that its transcription is significantly reduced after incubation with lipopolysaccharide (LPS). Moreover, we demonstrate that microglial cells cultured in the presence of TFF3 show reduced expression and secretion of pro-inflammatory cytokines after LPS stimulation.