The Role And Mechanism Of PGAM5 And ASK1 After Subarachnoid Hemorrhage In Rats

PURPOSE : Subarachnoid hemorrhage(SAH)is an acute cerebrovascular disease with a high mortality rate and complex pathological changes,and surviving patients are usually left with cognitive and motor deficits,etc.After SAH,severe mitochondrial dysfunction occurs,leading to oxidative stress damage and neuronal apoptosis,causing irreversible brain damage.Therefore,reducing oxidative stress levels and neuronal apoptosis are important therapeutic strategies to reduce brain injury after SAH and improve the prognosis of SAH.Phosphoglycerate mutase family member 5(PGAM5),a serine/threonine phosphatase localized in mitochondria,plays a key role in regulating mitochondria-mediated injury.Apoptosis signal-regulating kinase 1(ASK1),a member of the mitogen-activated protein kinase family,can be activated by a variety of cellular stresses and plays an important role in oxidative stress and apoptosis.The aim of this study was to evaluate the role of PGAM5 and ASK1 in early brain injury after SAH and to investigate their possible mechanisms.MATERIALS AND METHODS: The male Sprague-Dawley(SD)rat subarachnoid hemorrhage model was established by intravascular puncture.Protein immunoblotting(Western blot,WB)was used to detect the changes of PGAM5 and ASK1 contents in the sham-operated(Sham)group,3 h,6 h,12 h,24 h and 72 h after SAH in rats,and mitochondria were isolated to detect the changes of PGAM5 and ASK1 contents in mitochondrial lysate and de-mitochondrial cytoplasmic matrix,respectively.Silencing of PGAM5 expression by Silencer? PGAM5 siRNA injection in the lateral ventricle 48 h before SAH modeling,and the effect of silencing PGAM5 on short-term neurological function in rats24 h after SAH modeling was assessed by modified Garcia score and balance beam test;silencing PGAM5 was assessed by Rotarod rotating bar assay and Morris water maze assay on long-term neurological function after SAH modeling in rats.At 24 h after SAH modeling,the levels of oxidative stress damage protein 4-HNE and oxidative stress protective protein HO-1 were measured using dihydroethidium(DHE)staining and WB to assess the alteration of silencing PGAM5 on oxidative stress levels after SAH in rats;the levels of pro-apoptotic protein Bax and apoptotic protective protein Bcl-2 were measured using TUNEL staining and WB to assess the effect of silencing PGAM5 on long-term neurological function after SAH modeling in rats.The levels of pro-apoptotic protein Bax and apoptosis-protective protein Bcl-2 were measured by TUNEL staining and WB to assess the alteration of silencing PGAM5 on apoptosis after SAH in rats.The direct interaction between PGAM5 and ASK1 in the de-mitochondrial cytoplasmic matrix was detected by immunoprecipitation,and the phosphorylation activation of ASK1,P38 and JNK after PGAM5 silencing was detected by WB.On the other hand,immunofluorescence(IF)was used to observe the coexpression of ASK1 with the neuronal marker protein Neu N,microglia marker protein Iba-1 and astrocyte marker protein GFAP.The ASK1 inhibitor NQDI-1 was injected through the lateral ventricle 1 h after SAH modeling to assess the effects of NQDI-1 on short-term neurological function and long-term neurological function after SAH modeling in rats.ASK1 was silenced by Silencer? ASK1 siRNA injection via the lateral ventricle 48 h prior to SAH modeling,and p-ASK1,ASK1,p-P38,P38,p-JNK,and JNK inhibitor SP600125 were measured by intraperitoneal injection of P38 inhibitor BMS-582949 or JNK inhibitor SP600125 1 h after SAH modeling,using WB at 24 h after SAH,JNK,4-HNE,HO-1,Bcl-2,Bax protein content changes to explore the possible mechanism of the neuroprotective effect of NQDI-1.RESULTS: The content of PGAM5 at 30 kDa in mitochondria did not change significantly after SAH modeling in rats,whereas the content of PGAM5 at 55 kDa in the de-mitochondrial cytoplasmic matrix increased significantly.Silencer? PGAM5 siRNA significantly reduced PGAM5 content in both mitochondrial and de-mitochondrial cytoplasmic matrices,and improved both short-term and long-term neurological function after SAH modeling in rats.short-term neurological function and long-term neurological function in rats after SAH modeling.Further,PGAM5 directly bound to ASK1 and mediated oxidative stress and neuronal apoptosis after SAH modeling,at least partially through the ASK1-P38/JNK pathway.On the other hand,endogenous ASK1 levels were elevated after SAH modeling and peaked at 24 h.The ASK1 inhibitor NQDI-1 improved short-term neurological function and longterm neurological function after SAH modeling in rats and reduced the level of oxidative stress,neuronal degeneration,and neuronal apoptosis after SAH modeling.The use of the P38 inhibitor BMS-582949 or the JNK inhibitor SP600125 resulted in decreased levels of p-P38 or p-JNK,respectively,and decreased 4-HNE and Bax protein levels and increased HO-1 and Bcl-2 levels,but neither inhibitor had an effect on p-ASK1 or ASK1 levels.CONCLUSION: The amount of mitochondrial protein PGAM5 in the cytoplasmic matrix increased over time after SAH modeling in rats and its molecular weight was 55 kDa.55 kDa PGAM5 in the cytoplasmic matrix directly bound to ASK1 and mediated oxidative stress and neuronal apoptosis after SAH modeling,at least in part through the ASK1-P38/JNK pathway.Silencing PGAM5 improved short-term neurological function and long-term neurological function after SAH modeling in rats.NQDI-1 inhibited ASK1 phosphorylation and attenuated oxidative stress,reduced neuronal degeneration and neuronal apoptosis at least partially through the P38 and JNK signaling pathways,and improved short-and long-term neurological function and exerted neuroprotective effects after SAH modeling in rats.PGAM5 and ASK1 are promising and viable therapeutic targets after SAH.Figures 33,Table 16,References 135...