Effects Of Sigma-1 Receptor Agonist PRE-084 On Secondary Brain Injury Following Intracerebral Hemorrhagel Hemorrhage

Backgrounds AND purpose:Intracerebral hemorrhage(ICH)is one of the most devastating disease with high mortality and disability rate.Long-term intensive cares are often needed for those who survived from ICH,which results a great burden to the family and society.Secondary injuries following ICH are closely related to the prognosis of the patients.Excessive neuroinflammation has been proved to play a key role in ICH-induced brain damages,and therapies that target on attenuating neuroinflammation have become a hit in ICH related studies.Accumulating evidence indicated that astrocytes exert important influences on neuroinflammation after brain injuries.However,much is unknown about the role of astrocytes in regulating neuroinflammation after ICH to date.Considering that astrocytes are the most abundant glia cells in the central nervous system,deep and further studies on the mechanisms of astrocytic regulation of neuroinflammation following ICH may shed light on future preclinical studies.Sigma-1 receptor(Sig1R)has been proved to be neuroprotective in ischemic stroke and neurodegenerations.As a highly selective agonist of Sig1 R,PRE-084 alleviates mitochondrial dysfunctions and inflammatory responses under various kind of stress.Whether Sig1 R or PRE-084 affects the neurological dysfunctions induced by ICH remains undetermined.Our study shows that Sig1 R highly co-locates with GFAP,a biomarker of astrocyte,both before and after ICH induction.Astrocytic Sig1 R knockdown mouse model and primary astrocyte culture were established to evaluate the effect of PRE-084 on brain injuries following ICH and explore the underlying mechanisms.Materials and methods:In the first section of this study,collagenase VII was used to induce ICH in C57BL/6mice.PRE-084 were administered via i.p.injection at different dosages 1 hour after ICH induction,and neurological performances were evaluated 7 days after ICH to determine the best dosage for further studies.Western blotting(WB)was used to detect the expression of Sig1 R at different times after surgery,and immunofluorescence(IF)was used to determine the cellular localization of Sig1 R.Mice model of astrocytic specific Sig1 R knockdown was established and used to evaluate the degree of neurobehavior dysfunction and tissue damage.In the second section of this study,ELISA kits were used to measure the level of TNF-α and IL-1β in the basal ganglia.Oxyhemoglobin(Oxy Hb)was used to establish in vitro ICH models in primary microglia and astrocyte cultures.ELISA kits and q RT-PCR method were utilized to determine the level of TNF-α and IL-1β.Mitochondrial functions were evaluated by flow cytometry analysis of Mito-tracker Red and DCFH staining and measurement of cellular ATP level.Protein levels of NLRX1,IκB-α and NF-κb under different treatment were detected by WB.Small interfering RNA was introduced to primary astrocyte culture to knockdown NLRX1.Astrocytes in ICH models were collected via fluorescence activated cell sorting 7 days after surgery,and the changes of NLRX1/IκB-α/NF-κb signaling pathway were detected by WB.Results: 1.Neurological deficits were significantly alleviated after 7 days treatment of PRE-084.2.Sig1 R mainly expressed on astrocytes.Astrocytic Sig1 R knockdown aggravated neurological dysfunctions after ICH in mice and diminished the protective effects of PRE-084 against ICH.3.PRE-084 protected mitochondrial functions and decreased the level of ROS,TNF-α and IL-1β.4.Activation of astrocytic Sig1 R by PRE-084 attenuated neuroinflammation both in vivo and in vitro via NLRX1/IκB-α/NF-κb signaling pathway.Conclusions: PRE-084 alleviated neuroinflammation,tissue damage and neurological deficits after ICH through activating astrocytic Sig1 R and the following NLRX1/IκB-α/NF-κb signaling pathway.PRE-084 could be a potential therapeutic drug in treating ICH in the future.