A novel brain-derived peptide inhibits microglial pyroptosis through MBTPS1 in neonatal hypoxic-ischemic brain damage

This study aimed to identify whether a novel brain-derived peptide, hypoxic ischemic brain damage-associated peptide (HIBDAP), which was identified by our research group in previous studies through peptidome analysis, has a protective effect on the neonatal brain under hypoxic ischemia (HI), and to elucidate the underlying mechanism in a neonatal hypoxic-ischemic brain damage (HIBD) rat model.

The HIBDAP sequence was coupled with the cell-penetrating peptide TAT (YGRKKRRQRRR). Seven days after birth, neonatal rats were subjected to a sham operation or HI. The peptide or an equal volume of normal saline was injected into the left ventricular area with a stereotactic injector. The area of cerebral infarction was assessed via 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Behavioral tests, including the water maze test, suspension test, cliff escape test and step error test, were carried out at 21 days and 3 months after birth. Primary microglias were treated with different concentrations of TAT-HIBDAP. After different durations of oxygen-glucose deprivation (OGD), a Cell Counting Kit-8 (CCK-8) was used to detect the cell survival rate. To screen proteins that interact with this peptide, we labeled this peptide with biotin to perform pull-down and mass spectrometry assays. The mitochondrial membrane-bound transcription factor peptidase (MBTPS1) with the best binding effect of this peptide was selected, and its combination was further verified by immunofluorescence and pull-down. Lentiviral vectors were used to overexpress or knock down MBTPS1 in microglia. The pyroptosis rate was evaluated via a lactate dehydrogenase (LDH) release assay. The morphology of the pyroptotic cells was observed via electron microscopy. The expression of the NLRP-3 inflammasome and downstream inflammatory cytokines was detected via Western blotting. The expression levels of IL-18 and IL-1β in the cell culture supernatants were measured via enzyme-linked immunosorbent assay (ELISA). The average value used was n = 6, and every sample was analyzed three times.

A neonatal HIBD rat model in which the left ventricle was injected with TAT-HIBDAP resulted in reduced cerebral infarction size and improved motor, learning and memory-related abilities. HIBDAP suppressed microglial pyroptosis under OGD conditions. The direct relationship between HIBDAP and MBTPS1 was confirmed by pull-down and intracellular immunofluorescence colocalization. In microglia, HIBDAP down regulated the expression of MBTPS1 under OGD conditions. Knocking down MBTPS1 inhibited microglial pyroptosis, and overexpressing MBTPS1 promoted microglial pyroptosis. MBTPS1 overexpression attenuated the effects of HIBDAP on microglial pyroptosis under OGD conditions. The expression levels of NLRP-3, ASC, cleaved-Caspase-1, n-GSDMD, IL-18 and cleaved-IL-1β were significantly decreased in the peptide treatment group, and MBTPS1 overexpression increased their expressions.

HIBDAP inhibits microglial pyroptosis by combining with MBTPS1 to inhibit the expression of the NLRP-3/ASC/Caspase-1/GSDMD N-terminus, IL-1β and IL-18 and significantly improves motor, learning and memory-related abilities in neonatal HIBD rats.