Study On The Protective Mechanism Of Naotai Fang On Ischemic Stroke Based On The HSF1/HSPB1 Pathway Regulating Cellular Iron Transport And Inhibiting Ferroptosi

Objective:1 To clarify the effects of different cerebral ischemia time on HSF1/HSPB1 pathway and cellular iron transport,reveal the correlation between HSF1/HSPB1 pathway regulation of cellular iron transport and neuronal damage in ischemic stroke.2 To clarify whether Naotaifang can reduce brain iron deposition,reactive oxygen deposition and lipid peroxide production by regulating the HSF1/HSPB1 pathway,thereby inhibiting neuronal ferroptosis in ischemic stroke.Methods:1 In the first part of the experiment:50 normal SD male rats were randomly divided into Sham operation group(Sham group),MCAO model 2h group(MCAO 2h group),MCAO model 6h group(MCAO 6h group),MCAO model 12h group(MCAO 12h group),MCAO model 24h group(MCAO 24h group),10 rats in each group,adaptive feeding for 3 days.The cerebral ischemia model was prepared by the middle cerebral artery occlusion(MCAO)thread embolization method,and the heads were decapitated at 2h,6h,12h,and 24h after the operation.HE staining to observe the morphology and damage;Prussian blue staining to observe the level of iron accumulation in neurons;Western blotting and immunohistochemical experiments to detect the protein expression levels of HSF1,HSPB1,TFR1,and FTH1.2 The second part of the experiment:68 normal SD male rats were randomly divided into sham operation group(Sham group),MCAO model group(MCAO group),MCAO model+deferiprone group(MCAO+DFP group),MCAO model+Naotaifang extract group(MCAO+NTE group),17 rats in each group,start to dosing after 3 days of adaptive feeding.The cerebral ischemia model was prepared by the middle cerebral artery occlusion(MCAO)thread embolization method,and the neurological function was scored and obtained 2 hours after the operation.TTC staining to detect cerebral infarct volume;HE staining to observe the morphology and damage;Nissl staining to observe the number of Nissl bodies;Prussian blue staining to observe the level of iron accumulation in neurons;Western blotting and immunohistochemical experiments to detect the protein expression levels of HSF1,HSPB1,TFR1,and FTH1;iron Ion detection kits,malondialdehyde detection kits and reactive oxygen detection kits detect brain iron content,malondialdehyde content and reactive oxygen content.Results:1 The first part is the experimental results1.1 Compared with the Sham group,the cell morphology and structure of the rats in the MCAO 2h group,MCAO 6h group,MCAO 12h group,and MCAO 24h group were destroyed,the number of iron-containing aggregate granule cells increased,And within 2-24h after MCAO,the damage to the brain tissue of SD rats gradually increased,and the level of iron accumulation in neurons gradually increased.1.2 Compared with the Sham group,the expression of HSPB1 and TFR1 protein in the MCAO+2h group increased(P<0.05 or P<0.01),the expression of FTH1 protein decreased(P<0.01),and the expression of HSF1 protein was not statistically significant(P>0.05);The expression of HSPBl,HSF1,and TFR1 protein increased in the MCAO+6h group,MCAO+12h group and MCAO+24h group(P<0.05 or P<0.01);the FTH1 protein expression increased in the MCAO+12h group(P<0.01).The FTH1 protein expression of rats in the MCAO+6h group and MCAO+24h group was not statistically significant(P>0.05 each).1.3 Compared with MCAO+12h group,HSPB1,HSF1,TFR1,FTH1 protein expression increased in MCAO+2h group and MCAO+6h group(P<0.05 or P<0.01);HSF1 and FTH1 protein expression in MCAO+24h group decreased(P<0.05 each),TFR1 protein expression increased(P<0.01),HSPB1 protein expression was not statistically significant(P>0.05).2 The second part is the experimental results2.1 Compared with the sham operation group,the cerebral infarction volume of the rats in the MCAO model group increased,and the neurological function score increased(P<0.01);the morphological structure of the cells in the cerebral ischemia area was destroyed,and the Nissl body was reduced;the number of iron-containing aggregate granule cells increased(cortical iron-containing aggregate particles The number of cells was significantly more than that of hippocampal CA2 area);brain iron content,malondialdehyde content and reactive oxygen content increased(P<0.01each);FTH1 protein expression was down-regulated(P<0.01),HSF1,HSPB1 and TFR1 protein expression was up-regulated(P<0.05 or P<0.01).2.2 Compared with the MCAO model group,the cerebral infarction volume of the rats in the MCAO model+Naotaifang extract group decreased,and the neurological function score decreased(P<0.01);Nissl bodies in cerebral ischemia area increased,and the degree of cell damage decreased;the number of iron-containing aggregate granule cells decreased;brain iron content and malondioxide Aldehyde content and reactive oxygen content decreased(P<0.05 or P<0.01);HSF1,HSPB1 and FTH1 protein expression were up-regulated(P<0.05 or P<0.01),and TFR1 protein expression was down-regulated(P<0.05).Conclusion:1 The injury of different cerebral ischemia time affects the HSF1/HSPB1 pathway and cellular iron transport.Cerebral ischemia 12h has the most obvious effect on the HSF1/HSPB1 pathway and cellular iron transport.HSF1/HSPB1 pathway and cellular iron transport may be the ischemic brain An important therapeutic target for neuronal damage in stroke.2 Naotaifang may increase the HSF1/HSPB1 pathway,inhibit the expression of TFR1 to reduce neuronal iron absorption,and increase the expression of FTH1 to increase the iron storage of ferritin,thereby regulating the homeostasis of iron metabolism,thereby inhibiting the passage of excess iron through fen.The reactive oxygen species produced by the sudden reaction and the subsequent lipid peroxidation products cause neuronal ferroptosis in ischemic stroke.