Neuroprotective Effects And Mechanisms Of PGC-1? On Intracranial Hemorrhage Through TOM70/MICU1 Pathway

BackgroundsAs a common type of stroke,intracranial hemorrhage is high in mortality as well as morbidity.It consists of 10-15%of all kinds of stroke worldwide.Besides the primary damage induced by blood accumulation after ICH onset,secondary brain injury has also been proven to cause neurological deficits and poor outcome.It has been reported that the fatality rate of ICH at 1 month is approximately 40%,which has not changed over the past twenty years.This high rate of mortality and morbidity likely results from a lack of effective treatment options to improve a patient's survival following ICH,since standard management is limited to primarily supportive therapies such as control of intracranial pressure,treatment of brain edema,and maintenance of hemodynamic stability.With an increasing elderly population,the incidence of ICH is expected to increase.Thus,understanding the mechanisms underlying ICH and identification of novel therapeutic targets are of paramount interest to researchers in the development of new medical therapies for ICH.Inflammation is an important host defense response to brain injury after ICH.When ICH occurs,blood components including RBCs,leukocytes,macrophages,and plasma proteins such as thrombin immediately enter the cerebral parenchyma.The inflammatory response begins immediately after the presence of blood components in the parenchyma,and is characterized by accumulation and activation of inflammatory cells.Increasing evidence has shown that inflammatory injury plays a critical role in ICH-induced secondary brain injury.Activation of innate immunity and inflammatory responses contributes to the pathogenesis of inflammatory injury after ICH.The activation of innate immunity following ICH results in microglial activation,perihematomal inflammation reactions,infiltration of blood-derived inflammatory cells,release of inflammatory cytokines such as TNF-?and IL-1?,and brain edema.Inflammatory responses following ICH aggravate ICH-induced brain injury,ultimately leading to tissue damage,BBB disruption,and massive brain cell death.Recently,drug treatment for ICH-induced secondary brain injury,especially the process of neuroinflammation is of great importance and beneficial to alleviate brain injury and improve neurological dysfunction.PGC-1?is a nuclear transcriptional co-activator of nuclear receptors and other transcription factors and is strongly expressed in brown adipose tissue,heart,skeletal muscle,kidneys,and the brain.PGC-1?is a key contributor to up-regulation of the antioxidant activities that take place in response to oxidative stress and it is also a strong activator of mitochondrial biogenesis.Previous studies showed that PGC-1?is playing an important role in several central nerve system diseases due to its anti-inflammatory effect.A recent study on ICH indicated that inhibition of PGC-1?aggravated ICH-induced brain injury and neurological deficits.The specific activator of PGC-1?was discovered recently years,and showed neurological protection in hypoxia-ischemia-induced injury through activating mitochondrial biogenesis.PGC-1?regulated the expression of mitochondrial Ca²⁺-associated protein TOM70 and MICU1,inhibiting reactive oxygen specials,upregulating inflammatory cytokines,eventually inhibiting inflammatory reaction.Thus,through autologous blood injection model,we investigate neuroprotective effect of PGC-1?in ICH-induce secondary brain injury and its possible role of TOM70/MICU1-associated mitochondria way in neuroinflammation and hope to provide a new treatment target.MethodPart 1Rats were randomly assigned into the following 4 groups,including sham,ICH,ICH+vehicle as well as ICH+ZLN005 group.The sham received equal saline injection instead of blood into basal ganglia.In our study,all rats were estimated at 72 hours after ICH.Behavior test,MRI,immunofluorescence staining,western blot,ROS assays were carried out in each group.Part 2Experiment 1:A division of seven groups was conducted,including sham,3 h,6 h,12 h,24 h,48 h and 72 h group.Western blot was carried out to detect the expression of TOM70 and MICU1.Double staining of TOM70 or MICU1 with ionized calcium-binding adaptor molecule 1?Iba-1?were operated 72 h after ICH.Experiment 2:Rats were randomly assigned into the following 4 groups,including sham,ICH,ICH+vehicle as well as ICH+ZLN005 group.The sham received equal saline injection instead of blood into basal ganglia.In our study,all rats were estimated at 72 hours after ICH.Western blot,ATP assays,transmission electron microscopy?TEM?were carried out in each group.Experiment 3:To further investigate the fundamental mechanisms of PGC-1?'s neuroprotective effects,a random assignment of five groups was performed,including ICH+vehicle,ICH+ZLN005,ICH+ZLN005+scramble si RNA,ICH+ZLN005+PGC-1?si RNA as well as ICH+ZLN005+TOM70 si RNA group.ROS assays,ATP assays,western blot analysis and double immunostaining were measured at 72 hours after ICHResultsPart 172 hours after the occurrence of ICH,we can see the increased expression of PGC-1?,decreasing of neurological scores of rats,brain swelling,reactive oxygen specials accumulation,the expression of proinflammatory inflammatory factors increased.And the proportion of M1 microglia/macrophages around the hematoma increased significantly.After intravenously administering ZLN005 at a dose of 2.5 mg/kg daily,the neurological scores of rats significantly improved.The overall content of PGC-1?was further increased,the content of reactive oxygen species was decreased and cerebral edema was alleviated.MRI scan also indicated that the lysis of hematoma,was reduced after ZLN005 treatment.ZLN005 also inhibited the expression of inflammatory factors TNF-?,IL-1?and IL-6,and up-regulated the expression of IL-10.At the same time,the proportion of M2 microglia/macrophages around the hematoma was increased,and the proportion of M1 microglia/macrophages was decreased.Part 2After ICH,TOM70 and MICU1 expression were significantly down-regulated and reached a minimum at 72 hours.TOM70 and MICU1 are highly expressed in microglia,and double staining of immunofluorescence suggests that both TOM70 and MICU1 are localized in the cytoplasm.At 72h after ICH,the expression of mitochondrial TOM70and MICU1 were decreased.TEM showed mitochondrial vacuolation and the inhibition of ATP level.Treated with ZLN0005 restored these effects.Either PGC-1?si RNA or TOM70 si RNA abolished the protective effect of ZLN005.ConclusionPart 1Activation of PGC-1?by ZLN005 improve brain edema,reduce ROS,alleviate hematoma resolution,promote microglia/macrophages transforming to M2 type,reduce inflammatory response after cerebral hemorrhage,and thereby improve neurological function in rats.Part 2The neuroprotective effect of PGC-1?is related to its activation of the TOM70/MICU1 mitochondrial pathway,which may reduce the production of reactive oxygen species by regulating the calcium ion homeostasis in the mitochondria,and promote the transformation of microglia types,thereby reducing ICH After the inflammatory response.