Abstract:

Objective To investigate whether orexin A （orexin-A）， orexin receptor 1 （OX1R） and orexin receptor 2 （OX2R） are involved in iron death and lipid peroxidation regulation in chronically unpredictable mild stress （CUMS） depressed rats. Methods Forty rats were randomly divided into a normal group （NC group）， a modeling group （Mod group）， an exogenous Orexin-A group （Orexin-A group，）， and an OX1R/OX2R blocker group （TCS1102 group）， with 10 rats in each group. After modeling， behavioral changes were observed using the absent field test （OFT）， sugar-water preference test （SPT） and forced swimming test （FST）， action potential （PA） and resting membrane potential （Vm） were detected by diaphragm-clamp technique， Orexin-A/OX1R/OX2R protein expression in orbital frontal cortex （OFC） tissues was detected by protein immunoblotting （WB） method， RT-PCR The mRNA expression of glutathione peroxidase 4 （GPX4）， long chain acyl coenzyme A synthase 4 （ACSL4） and cysteine/glutamate transporter light chain （SLC7A11） were detected by RT-PCR method， and the intensity of the expression of rat glial fibrillary acidic protein （GFAP） and lipid peroxidation product 4-hydroxynonenal （4-HNE） was labeled by immunofluorescence. Results Compared with the NC group， there were significant differences in OFT， SPT and FST behavioral in the Mod group （P < 0.01）， with lower number of PA issuance （P < 0.001）， higher Vm （P < 0.01）， and higher expression of Orexin-A/OX1R/OX2R proteins （P < 0.01， P < 0.001， and P < 0.001）. GPX4/SLC7A11 mRNA expression was decreased （P < 0.01）， ACSL4 mRNA expression was elevated （P < 0.01）， and the fluorescence intensity expression of both GFAP and 4-HNE was elevated （P < 0.001）； the number of PA issuance was decreased in the Orexin-A group compared to the Mod group （P < 0.05）， and the Orexin-A/OX1R/ OX2R protein expression was elevated （P < 0.05， P < 0.01）， GPX4/SLC7A11 mRNA expression was decreased （P < 0.05）， ACSL4 mRNA expression was elevated （P < 0.05）， and the fluorescence intensity of GFAP and 4-HNE expression was elevated （P < 0.05， P < 0.01）； the TCS1102 group had higher expression of GFAP and 4-HNE in the behavioral， Orexin-A/OX1R/OX2R protein expression， PA and Vm， GPX4/SLC7A11/ACSL4 mRNA， and GFAP and 4-HNE fluorescence intensity expression showed a reversed trend. Conclusions Orexin-A/OX1R/OX2R is involved in the regulation of iron death and lipid peroxidation in CUMS depressed rats， and the mechanism may be that Orexin-A enhances the excitability of OFC neurons by activating the OX1R/OX2R signaling pathway， up-regulates the expression of the key factor of iron death， ACSL4/4-HNE， and decreases the expression of GPX4/SLC7A11， which promotes lipid peroxidation and iron death.

Key words: orexin, depression, iron death, lipid peroxidation, orbitofrontal cortex