Anti-TXNIP monoclonal antibody Made to order

Background: The endoplasmic reticulum (ER) is responsible for the control of correct protein folding and protein function which is crucial for cell survival. However, under pathological conditions, such as hypoxia-ischemia (HI), there is an accumulation of unfolded proteins thereby triggering the unfolded protein response (UPR) and causing ER stress which is associated with activation of several stress sensor signaling pathways, one of them being the inositol requiring enzyme-1 alpha (IRE1 alpha) signaling pathway. The UPR is regarded as a potential contributor to neuronal cell death and inflammation after HI. In the present study, we sought to investigate whether microRNA-17 (miR-17), a potential IRE1 alpha ribonuclease (RNase) substrate, arbitrates downregulation of thioredoxin-interacting protein (TXNIP) and consequent NLRP3 inflammasome activation in the immature brain after HI injury and whether inhibition of IRE1 alpha may attenuate inflammation via miR-17/TXNIP regulation. Methods: Postnatal day 10 rat pups (n = 287) were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O-2). STF-083010, an IRE1 alpha RNase inhibitor, was intranasally delivered at 1 h post-HI or followed by an additional one administration per day for 2 days. MiR-17-5p mimic or anti-miR-17-5p inhibitor was injected intracerebroventricularly at 48 h before HI. Infarct volume and body weight were used to evaluate the short-term effects while brain weight, gross and microscopic brain tissue morphologies, and neurobehavioral tests were conducted for the long-term evaluation. Western blots, immunofluorescence staining, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and co-immunoprecipitation (Co-IP) were used for mechanism studies. Results: Endogenous phosphorylated IRE1 alpha expression was significantly increased after HI. Intranasal administration of STF-083010 alleviated brain injury and improved neurological behavior. MiR-17-5p expression was reduced after HI, and this decrease was attenuated by STF-083010 treatment. MiR-17-5p mimic administration ameliorated TXNIP expression, NLRP3 inflammasome activation, caspase-1 cleavage, and IL-1 beta production, as well as brain infarct volume. Conversely, anti-miR-17-5p inhibitor reversed IRE1 alpha inhibition-induced decrease in TXNIP expression and inflammasome activation, as well as exacerbated brain injury after HI. Conclusions: IRE1 alpha-induced UPR pathway may contribute to inflammatory activation and brain injury following neonatal HI. IRE1 alpha activation, through decay of miR-17-5p, elevated TXNIP expression to activate NLRP3 inflammasome and aggravated brain damage.

Activation of peroxisome proliferator activated receptor beta/delta (PPAR-beta/delta), a nuclear receptor acting as a transcription factor, was shown to be protective in various models of neurological diseases. However, there is no information about the role of PPAR-beta/delta as well as its molecular mechanisms in neonatal hypoxia-ischemia (HI). In the present study, we hypothesized that PPAR-beta/delta agonist GW0742 can activate miR-17-5p, consequently inhibiting TXNIP and ASK1/p38 pathway leading to attenuation of apoptosis. Ten-day-old rat pups were subjected to right common carotid artery ligation followed by 2.5 h hypoxia. GW0742 was administered intranasally 1 and 24 h post HI. PPAR-13/8 receptor antagonist GSK3787 was administered intranasally 1 h before and 24 h after HI, antimir-17-5p and TXNIP CRISPR activation plasmid were administered intracerebroventricularly 24 and 48 h before HI, respectively. Brain infarct area measurement, neurological function tests, western blot, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), Fluoro-Jade C and immunofluorescence staining were conducted. GW0742 reduced brain infarct area, brain atrophy, apoptosis, and improved neurological function at 72 h and 4 weeks post HI. Furthermore, GW0742 treatment increased PPAR-beta/delta nuclear expression and miR-17-5p level and reduced TXNIP in ipsilateral hemisphere after HI, resulting in inhibition of ASK1/p38 pathway and attenuation of apoptosis. Inhibition of PPAR-13/8 receptor and miR-17-5p and activation of TXNIP reversed the protective effects. For the first time, we provide evidence that intranasal administration of PPAR-beta/delta agonist GW0742 attenuated neuronal apoptosis at least in part via PPAR-beta/delta/miR17/TXNIP pathway. GW0742 could represent a therapeutic target for treatment of neonatal hypoxic ischemic encephalopathy (HIE).