The Role of Src Kinase in the Caspase-1 Pathway After Hypoxia in the Brain of Newborn Piglets

Abstract

Hypoxia induces a cerebral inflammatory response, which contributes to brain injury. Inflammasomes are complex intracellular molecular structures that initiate the inflammatory cascade. Caspase-1 and interleukin 1-β (IL-1β), have been established as markers of inflammasome activation. Src kinase, a cytosolic non-receptor protein tyrosine kinase, is linked to cell proliferation and differentiation and is up regulated during hypoxia. The role of Src kinase in the above pathway is not fully understood. The present study tests the hypothesis that inhibition of Src kinase, by a selective inhibitor, PP2, will prevent the activation of caspase-1 and production of IL-1β acutely, as well as at 1 and 15 days after hypoxia in the cerebral cortex of the newborn piglet. Piglets were divided into: Normoxia (Nx), Hypoxia acute (Hx), Hypoxia-day 1 (Hx-day 1), and Hypoxia day 15 (Hx-day 15). Piglets pretreated with Src kinase inhibitor, PP2, 1 mg/kg IV, 30 min prior to hypoxia were divided into: Hypoxia acute (Hx + PP2), 1 day (Hx + PP2-day 1), and day 15 (Hx + PP2-day 15). Hypoxia was induced by exposing the piglets to an FiO2 of 0.07 for 1 hour. Caspase-1 activity and expression were determined with spectrophotometry and Western blot respectively, while IL-1β levels were measured by solid phase ELISA. Caspase-1 activation was achieved immediately (within 1 h) after hypoxia and persisted for 15 days. IL-1β level was also increased after hypoxia reaching a maximum level at 24 h following hypoxia and returned to baseline by 15 days. Administration of PP2 attenuated the activity acutely, but not the expression of the caspase-1. IL-1β level at 24 h after hypoxia returned to baseline in piglets that were pretreated with PP2. We provide evidence that inhibition of Src kinase in the acute phase after hypoxia involves changes in the production or processing of caspase-1 subunits. Our data suggest that Src kinase mediates hypoxia-induced caspase-1 activation in the cerebral cortex of newborn piglets. Inhibition of Src kinase may attenuate the neuroinflammatory response and could represent a potential target for neuroprotection after hypoxic injury.

Inflammasomes in livestock and wildlife: Insights into the intersection of pathogens and natural host species

“…In lactating sows, feed reduction was demonstrated to reduce levels of expression of NLRP3 , CASP1 , and IL1B in liver approximately two-fold, indicating potential for influences of environment on porcine inflammasomes (Gessner et al, 2015). Hypoxia in piglets leads to brain injury and is associated with caspase-1 activation and small increases (<2-fold) in IL-1β release in the cerebral cortex (Angelis et al, 2014). Additionally, activation of NLRP3 , ASC ( PYCARD ), and IL1B transcription in aortal tissue was observed in a porcine atherosclerosis model (Li et al, 2013); the authors indicate that this may be due to cholesterol crystals.…”

Effect of Concurrent Src Kinase Inhibition with Short-Duration Hypothermia on Ca²⁺/Calmodulin Kinase IV Activity and Neuropathology after Hypoxia-Ischemia in the Newborn Swine Brain

“…[22] Single protein inhibition with small molecules resulted in satisfactory in vivo results in the HI porcine model. [10,22] In order to ensure adequate energy failure after hypoxia we measured the high energy compounds in the groups and our hypoxic groups were very comparable in the ATP and PCr levels. HT or Src inhibition did not restore the energy failure but they were applied one hour following the hypoxia.…”

“…[9] We have previously shown that cerebral hypoxia results in increased activity of Src kinase and CaM kinase IV in neuronal nuclei of the cerebral cortex of newborn piglets. [10,11]…”

Effects of Src Kinase Inhibition on Expression of Protein Tyrosine Phosphatase 1B after Brain Hypoxia in a Piglet Animal Model

“…During this procedure, piglets that undergo hypoxia, become acidotic, and develop hypotension with mean arterial pressure at the limits of loss of autoregulation. This experimental model of combined brain hypoxia ischemia as well as the details in changes of blood pressure and acid-base status of the ventilated piglets is previously described [13]. …”