Saturday, June 3, 2023

Intestine and brain TLR-4 modulation following N-acetyl-cysteine treatment in NEC rodent model – Scientific Reports

In previous studies using this NEC model, we and others18,19 demonstrated features of NEC with severe bowel damage in pups, inflammatory changes, and increased oxidative stress accompanied by offspring brain injury. This model is based on insults that are recognized to contribute to the development of NEC in humans, including intestinal immaturity, hypoxia, and artificial hyperosmolar formula feeding. Dams are separated from the pups soon after birth to avoid breastfeeding, which is known to be protective against NEC.

In the current study, we have demonstrated that offspring with NEC had increased ileum TLR-4 protein levels as well as increased brain TLR-4 levels and decreased brain and ileum glutathione levels as compared to control. NAC administered to dams during pregnancy and/or to offspring under NEC conditions decreased offspring ileum and brain TLR-4 protein levels and increased brain and ileum glutathione levels as compared to NEC offspring.

In recent years exaggerated TLR-4 levels and increased activity in the immature intestine of preterm neonates has emerged as an inciting event in the pathophysiology of NEC. High TLR-4 activity in epithelial cells results in the initiation of an exaggerated immune response with increased production of pro-inflammatory cytokines and destruction of the mucosal barrier1. It has been demonstrated that TLR-4 contributes to bacterial translocation across the mucosal barrier, which facilitates the development of the severe illness observed during NEC2. The exact reason for increased expression of TLR-4 in enterocytes of infants with NEC has not been fully elucidated, though Soliman et al. suggested that platelet-activating factor was responsible for TLR-4 over-expression20.

Azjifurther demonstrated the importance of TLR-4 in the pathogenesis of NEC, showing that a specific deletion in the TLR4 locus in endothelial cells in a mouse model of NEC was associated with a significant reduction in NEC severity when compared to endothelial TLR-4 sufficient animals21.

Our novel finding that NAC administered to either dams or offspring decreased TLR-4 protein levels in offspring ileum is of great importance. These findings are consistent with reports of improved NEC features observed in previous studies following NAC treatment22,23.

We have recently demonstrated that NAC treatment was associated with decreased inflammatory response and attenuated activation of NFKB in a rodent NEC model. It is uncertain where NAC interferes in the cascade of the inflammatory response. Normally, following activation of TLR-4, a cascade of events occurs through the MyD88 dependent pathway which involves activation of the IRAK family of kinases, in which TAK1 activates the downstream kinase IKK in the final step, which in turn phosphorylates the NF-κB inhibitor IκBα, leading to ubiquitin-dependent IκBα degradation and NF-κB activation. This cascade results in transcription of inflammatory genes, including those encoding TNF-α, IL-1β, IL-6, IL-12p40, and cyclooxygenase24.

The findings of our current study might infer that the attenuation of the inflammatory response associated with NAC administration in NEC might be connected to modulation of the TLR-4 levels in the ileum and brain upstream of the cascade. A recent study by Niño et al. (2018) demonstrated a gut-brain signaling axis in a mouse model of NEC in which activation of intestinal TLR-4 signaling led to release of high-mobility group box 1 (HMGB1) protein in the intestine, which in turn, promoted activation of TLR-4 on brain microglial cells resulting in accumulation of reactive oxygen species, loss of oligodendrocyte premature cells, dysmyelination, and cognitive impairment. The authors further demonstrated the role of TLR-4 in mediating brain injury by generating mice lacking TLR-4 in microglia. The mice lacking microglial TLR-4 were protected from NEC associated brain injury. Further evidence for this intestinal-brain axis was generated by demonstration of reduced microglial activation with administration of intranasal anti-HMGB1 antibody to wild-type mice under NEC conditions9.

In the present study, we have demonstrated that under NEC conditions there is a significant increase in TLR-4 protein levels in the offspring brain. Our findings demonstrating reversal of NEC associated increase in intestinal and brain TLR-4 levels may explain the mechanism of NAC protective effect in both ileum and brain. Despite the data presented by Niño9 showing that NAC administration reduced microglial activation, they did not present any data on brain or gut TLR-4 levels in response to the treatment. Our data may explain the mechanisms behind the protective effects of NAC in both the ileum and brain.

We further demonstrated that the decrease in brain TLR-4 following NAC administration was associated with an increase in brain antioxidant glutathione levels, potentially enabling the brain to cope with increased oxidative stress and thus to amelioate brain injury. This finding is in accordance with the findings by Niño et al.9. Although we did not evaluate behavior in the current study, we demonstrated in a previous study on a hypoxia model (not NEC model), that NAC treatment significantly attenuated sensorimotor dysfunction in neonatal rats exposed to hypoxia13. In another study, we found that NAC protected the brain from injury as compared to LPS group, as demonstrated by MRI at 30 days of age25.

N-acetyl cysteine, a known anti-inflammatory and anti-oxidative agent, is considered safe for use during pregnancy (class B)23. Our data regarding litter size, offspring weight, and mortality support the safety of NAC administration. We did not analyze possible sex influences in our study as in previous research, despite finding differences in immunity parameters between the sexes and higher mortality in males, as we did not find any differences in gut structure, function, or NEC incidence between males and females26. NAC’s therapeutic properties stem from its action on the cystine-glutamate antiporter system and as an antioxidant to regulate the neuroinflammatory response27,28. Glutamate is implicated in fetal brain injury. Overexposure to glutamate and subsequent excess intracellular calcium influx, termed excitotoxicity, destroys neurons both in vivo and in vitro29. Thus, NAC may exert an additional therapeutic benefit through decreasing synaptic glutamate release, hence mitigating subsequent excitotoxic neurological damage27.

Interestingly, we have demonstrated that NAC was effective even when administered during pregnancy to dams long before exposing the offspring to NEC conditions. Our novel finding that maternal NAC administered long before delivery may protect the offspring from NEC associated changes in both intestine and brain is supported by a human study which has demonstrated rapid transfer of NAC from the mother to the fetus through the placenta, with umbilical cord concentrations frequently exceeding maternal concentrations30. Buhimshi et al. demonstrated in a maternal inflammation model in mice that maternal inflammation resulted in oxidative stress associated with maternal and fetal liver glutathione (GSH) precursor depletion, while maternal NAC restored both maternal and fetal oxidative balance and increased liver GSH levels in both dams and fetuses. Maternal NAC administration may be of benefit for newborns at increased risk of NEC, as it attenuates the inflammatory and oxidative responses. These novel findings, if confirmed by future studies, imply that administrating NAC to mothers in very early preterm birth (< 30 weeks) before delivery, may prevent NEC and improve the outcome of the newborns14.

The strength of our study is that we used an established rat model of NEC which replicates the key clinical features of NEC. This allowed us to assess the brain and intestine and to study the mechanisms mediating the development of NEC and NEC- associated brain injury. There are some limitations to our study. It is an animal study with a relatively small sample size and little clinically correlated data. Also, we studied the offspring at 5 days of age which might be too early a stage in brain development. Further studies should be performed at later stages with larger numbers.

In conclusion, we have demonstrated that prenatal and postnatal NAC could prevent an increase in TLR-4 protein levels in both ileum and brain in an established NEC model, while increasing brain glutathione levels. This study extends our understanding of the mechanisms associated with NEC injury and its prevention, and thus may help develop new strategies to cope with offspring injury associated with NEC.

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