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.

Intestinal Trefoil Factor 3 Alleviates the Intestinal Barrier Function Through Reducing the Expression of TLR4 in Rats with Nonalcoholic Steatohepatitis.

BACKGROUND: Previous studies have reported that nonalcoholic steatohepatitis (NASH) is relevant to intestinal mucosal barrier dysfunction. AIM OF THE STUDY: To investigate the effects of intestinal trefoil factor 3 (TFF3) on intestinal barrier function and endotoxin/toll-like receptor 4(TLR4) expression in NASH rats. METHODS: Sixty NASH rats were divided into control, NASH and NASH-TFF3 treated group. Intestinal permeability, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), endotoxin (ET), diamine oxidase (DAO) and liver index were examined. HE and PAS staining were performed to observe the histopathology of liver and terminal ileum. Expression of TFF3 and occludin were detected by immunohistochemical staining. mRNA and protein expression of TLR4, nuclear factor-κB (NF-κB), Mucin-2(Muc2) were detected by RT-qPCR and Western Blot. Interleukin (IL) -1ß and IL-10 levels in the ileum were measured by ELISA. RESULTS: In NASH group, levels of AST, ALT, ET, DAO, NAS, liver index and intestinal permeability were higher while occludin expressions were lower than control and NASH-TFF3 treated groups (p <0.05). Histopathology examination showed pathological damages of liver and ileum were alleviated in NASH-TFF3 treated group. NASH-TFF3 treated group had decreased expression levels of TLR4 and NF-κB and increased expression levels of Muc2 than NASH group. Besides, NASH group showed increased IL-1ß and IL-10 levels compared with control group. NASH-TFF3 treated group showed decreased IL-1ß level however increased IL-10 level compared with NASH group. CONCLUSION: Recombinant human TFF3 (rhTFF3) can reduce the expression of TLR4, reduce intestinal permeability, alleviate liver damage and thus may play a therapeutic role in the treatment of NASH rats.