NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

Abstract

Background: Neuroinflammation plays an important role in pathogenesis of germinal matrix hemorrhage (GMH). Neurotrophin-4 (NT-4) is a member of the neurotrophin family, and it interacts with the tyrosine kinase B (TrkB) receptor. It has been studied that NT-4 has neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4 and it’s high affinity receptor TrkB as well as its downstream mediator phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/Forkhead box protein O1 (FoxO1) following GMH in neonatal rats, with a specific focus on inflammation. Methods: GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U) in P7 rat pups. A total of 163 seven-day-old pups were used in this study. The recombinant human NT-4 was administered intranasally at 1 hour after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002 and FoxO1 activating CRISPR were administered intracerebroventricularly at 24 hours prior to NT-4 treatment to investigate the potential mechanism. Short-and-long-term neurobehavior assessments, immunofluorescence staining, Nissl’s staining and Western blot were performed. Results ： The expression of p-TrkB increased after GMH with a peak at day3. The TrkB receptor was expressed by neurons, microglia, and astrocytes. The administration of rh-NT-4 increased phosphorylation of TrkB, expression of PI3K, phosphorylation of Akt and decreased FoxO1, IL-1beta and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. The use of FoxO1 activation CRISPR increased the expression of IL-6, suggesting that FoxO1 might potentially induce pro-inflammatory factors. These results demonstrated that PI3K/Akt/FoxO1 may be the downstream pathway of TrkB phosphorylation. The rat pups treated with rh-NT-4 performed better than untreated animals both in short-and-long-term behavior test. Conclusion： These data showed that rh-NT-4 can reduce the expression of pro-inflammatory cytokines, improve neurological function, attenuate neuroinflammation and post-hemorrhagic hydrocephalus after GMH by promoting TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic target to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

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“…For example, TrkB selectively binds BDNF and NT4 (reF. 19 ), and TrkC has a high affinity for NT3 (reF. 20 ).…”

Exercise postconditioning reduces ischemic injury via suppression of cerebral gluconeogenesis in rats

“…In normal settings, AKT downregulates FoxO1 signaling by phosphorylation at three phosphorylation sites: Thr24, Ser256, and Ser319 (Zhang et al., 2018). During ischemia, phosphorylation of PI3K/Akt is inhibited causing decreased FoxO1 phosphorylation as well as increased nuclear translocation (He et al., 2019; Wang et al., 2020) and gluconeogenesis regulation (Pitaloka et al., 2019). Recent studies showed that mebhydrolin or puerarin ameliorated blood glucose homeostasis by suppressing hepatic gluconeogenesis via activation of the PI3K/AKT/FoxO1 pathway in T2DM mice (Liu et al., 2021; Zhao et al., 2021).…”

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“…The procedure for Nissl staining was the same as in previous studies [25]. Brain slices (10 mm thick) were dehydrated in 95% and 70% FLEX (ThermoFisher) for 1 min respectively, then rinsed with tap water and distilled water for 10 s. The brain slices were then stained in 0.5% cresyl violet (Sigma-Aldrich) for 1.5 min, followed by rinsing with distilled water for 10 s, and then put into the dehydration in 100% FLEX and xylene for 1 min (twice each).…”