Study On The Effects And Mechanisms Of Hydrogen Rich Saline On Brain Iron Metabolism And Ferroptosis With Chronic Cerebral Hypoperfusion/Ischemia Rats

BackgroundLong-term chronic cerebral hypoperfusion or ischemia（CCH）can lead to severe brain injury,which is one of the important causes of death and disability worldwide^[1].At present,although there is a great deal of preclinical evidence for neuroprotection of ischemic stroke,the neuroprotection of CCH is still a difficult problem,and the clinical treatment strategy is very limited.In recent years,studies have found that^[2,3]either acute or chronic cerebral ischemia can lead to abnormal brain iron metabolism or imbalance of iron homeostasis,and further lead to brain iron deposition and iron overload.Clinical studies also found that^[4-6]serum iron and brain iron deposition were significantly increased in patients with ischemic stroke,but the specific mechanism is unclear.Iron is an essential metal trace element for human body,which plays an important role in maintaining the normal structure and function of the nervous system.At the same time,iron is also an effective catalyst for oxidative stress,and the homeostasis imbalance of iron metabolism and it is an important factor leading to oxidative stress and lipid peroxidation damage.Oxidative stress and lipid peroxidation caused by low cerebral blood flow perfusion and increased reactive oxygen species（ROS）caused by abnormal iron metabolism,It plays an important role in the mechanism of CCH injury.Ferroptosis（FPT）)is a new type of non-apoptotic regulatory cell death discovered in recent years,which is often accompanied by a large amount of iron accumulation and lipid peroxidation in the process of cell death,which depends on iron.Ferroptosis inducing factor can directly or indirectly affect glutathione peroxidase（GSH-Px）through different pathways,which leads to the decrease of cell antioxidant ability,the destruction of intracellular redox balance,the accumulation of lipid reactive oxygen species（L-ROS）,and finally leads to cell oxidative death^[7,8].Research findings that^[9-11]there is a strong similarity in the pathogenesis between ferroptosis and many nervous system diseases such as stroke,brain trauma and I/R injury.Imbalance of iron homeostasis,oxidative stress and oxidative glutamate toxicity all have important effects on it,Therefore,FPT may play an important regulatory role in the occurrence and development of the above nervous system diseases.Activating or blocking the FPT pathway will slow down the progression of these diseases,which provides a promising treatment strategy for many nervous system diseases,and has become the key points and focus of research on the treatment and prognosis of related diseases.Although the understanding and research on Ferroptosis have been deepened gradually in recent years,the role and specific mechanism of FPT in CCH injury are not completely clear.CCH can lead to oxidative stress injury caused by iron overload and lipid peroxidation in nerve cells,The iron overload and accumulation of L-ROS caused by lipid peroxidation are the key factors in the occurrence of cell ferroptosis^[8].Therefore,in-depth study of the role of ferroptosis in CCH injury and inhibition of ferroptosis after CCH in various ways may be an important potential therapeutic target for improving CCH injury,This provides a new idea for clinical prevention and treatment of CCH injury and a series of nervous system diseases caused by it.In recent years,many scholars at home and abroad have confirmed that hydrogen can play the role of anti-oxidation and anti-inflammation by up-regulating the biological activities of a variety of antioxidant,such as SOD,CAT,GSH,GSH-Px and so on,Thus it has a definite brain protective effect on cerebral ischemic and hypoxic injury and various neurodegenerative diseases caused by different causes.but the specific mechanism is not clear^[12-20].The effects of hydrogen on brain iron metabolism after cerebral ischemia,especially on brain iron metabolism and Ferroptosis after CCH have not been reported in detail.Therefore,in this study,the rat model of CCH injury in vivo was established by using the modified bilateral common carotid artery permanent occlusion（2-VO）method,to observe the neuroprotective effect of hydrogen rich saline（HRS）on CCH injury and Ferroptosis induced by CCH injury in rats,and to explore the roles and possible mechanisms of hydrogen rich saline on iron metabolism homeostasis,System Xc-/GSH/GPX4 signal axis and GPX4 in regulating Ferroptosis induced by CCH injury in rats.Part one:Protective effect of hydrogen rich saline on CCH injury in rats in vivoObjective:To explore whether hydrogen rich saline has neuroprotective effect on CCH injury in rats in vivo.Methods:The rat model of CCH injury was established by modified 2-VO method.Thirty-six healthy adult male Wistar rats were randomly divided into three groups（n=twelve）:sham operation group（Sham group）,chronic cerebral ischemia group（CCH group）and saturated hydrogen rich saline group（Hydrogen group）.Observe and record the general changes of rats in each group during the making of the model;Morris water maze（MWM）experiment was used to evaluate the spatial learning and memory ability of rats,Nissl staining,immunohistochemistry and immunofluorescen-ce staining were used to detect the pathological changes of brain tissue and the expression of Neu N in hippocampus CA1 area after CCH injury in rats;The activity of SOD,GSH,GSH-Px and the content of MDA,4-HNE in brain tissues were detected respectively by colorimetry and ELISA.Results:1.The study found that during the making of the model,the activity of rats in Sham group was basically normal and there was not obvious restriction,the spontaneous activity,activity ability,food intake and drinking water of rats in CCH group and Hydrogen group decreased significantly after operation.A total of five rats died in the experiment,There was no death in Sham group,but three rats died in CCH group（three/twelve）,two rats died in Hydrogen group（two/twelve）.2.Compared with Sham group,the expression of Neu N positive nerve cells in hippocampal CA1region decreased significantly after CCH injury in rats（P<0.005）;The contents of MDA and 4-HNE,the metabolites of lipid peroxidation in brain tissue,were significantly increased,the activities of antioxidant such as SOD,GSH and GSH-Px decreased significantly（P<0.005 or P<0.001）;The escape latency of rats was significantly prolonged in MWM of Place navigation test,and in the Spatial probe test,the number of times of the target quadrant crossing the platform decreased significantly（P<0.01）;Compared with CCH group,In Hydrogen group,the expression of Neu N positive nerve cells in hippocampal CA1 region increased significantly after treatment with saturated hydrogen-rich saline（HRS）（P<0.005）;The activities of SOD,GSH,GSH-Px and other antioxidant increased significantly,The contents of MDA and 4-HNE,the metabolites of lipid peroxidation,were significantly decreased（P<0.001）;The escape latency of Place navigation test in rats was significantly prolonged,and the number of times of crossing the target quadrant platform in the Spatial probe test was increased significantly（P<0.001）.Conclusion:HRS can decrease the content of lipid peroxidation metabolites MDA and 4-HNE in nerve cells by increasing the activities of antioxidant such as SOD,GSH and GSH-Px,inhibit oxidative stress and lipid peroxidation damage after CCH,and then improve the pathomor-phological changes of brain tissue after CCH,increase the expression of neuronal marker Neu N,significantly improve the spatial learning and memory ability of injured rats,and have a definite protective effect on CCH injury.Part two:The role of Ferroptosis in CCH injury in rats and the effects of hydrogen rich saline on iron metabolism and Ferroptosis in brain of rats with CCHObjective:To determine the role of Ferroptosis in CCH injury in rats.To explore whether the effect of HRS on Ferroptosis induced by CCH injury in rats is related to maintenance of cellular iron homeostasis by regulating the expression of iron metabolism-related proteins in cells.Methods:The rat model of CCH injury was established by modified 2-VO method.Thirty two healthy adult male Wistar rats were randomly divided into four groups（n=eight）:Sham operation group（Sham group）,Chronic cerebral ischemia group（CCH group）,Saturated hydrogen-rich saline group（Hydrogen group）,Ferrostatin-1group（ferroptosis inhibitor group）.Transmission electron microscope（TEM）was used to observe the ultrastructural changes of mitochondria in hippocampal CA1region of neurons.Detection of iron content in cerebral cortex by tissue iron test kit,Detection of ROS content of nerve cells in brain tissue by flow cytometry,The expressions of iron metabolism related proteins TF,TFR1,DMT1,IREB2,FTL and FTH1 were detected by Western Blot.Results:1.Through TEM observation,it was found that,in Sham group,the morphology of mitochondria in hippocampal neurons of rats was normal,the structure of the mitochondrial membrane was intact and there was no thickening,and the mitochondrial crest was intact.In CCH group,Hippocampal neurons showed characteristic Ferroptosis features,such as significant reduction of mitochondrial volume,shrinking,thickening of mitochondrial membrane,decrease or even disappearance of mitochondrial crest,etc.The morphological changes of mitochondria in hippocampal neurons of Hydrogen group and Ferrostatin-1 group were mild,the structure of mitochondrial membrane was basically intact,the thickness of mitochondrial membrane was basically normal,and the mitochondrial crest was basically intact.2.Compared with Sham group,after CCH injury,the content of iron in brain cortex tissue were increased significantly,the content of ROS in neurons was increased significantly,the expression level of FTL and FTH1 protein were decreased significantly,and the expression level of TF,TFR1,DMT1 and IREB2protein were increased significantly（P<0.05 or P<0.01 or P<0.005 or P<0.001）;Compared with CCH group,the iron content in brain cortex tissue of rats in Hydrogen group and Ferrostatin-1 group were decreased in varying degrees,and the ROS content of neurons was decreased significantly.The protein expression level of FTL and FTH1 were increased significantly,while the protein expression level of TF,TFR1,DMT1 and IREB2 were decreased significantly in rat brain（P<0.05 or P<0.01or P<0.005 or P<0.001）.There was no significant difference in the content of iron in brain cortex tissue and the content of ROS in neurons between Hydrogen group and Ferrostatin-1 group,but the regulatory effects on the expression of iron metabolism-related proteins were slightly different.Conclusion:Ferroptosis is an important form of neuron death after CCH injury,and HRS has a significant inhibitory effect on Ferroptosis after CCH.The mechanism of HRS inhibit Ferroptosis after CCH may be through down-regulating iron transport or iron regulation related proteins TF,TFR1,DMT1,IREB2 and up-regulating the expression of iron metabolism related proteins such as iron storage-related white FTL and FTH1 in rat brain tissue after CCH,reduce iron overload in brain cortex tissue,maintain the iron homeostasis of neurons after CCH,and then reduce cellular toxic ROS accumulation,enhance cellular antioxidant capacity,and finally inhibit the Ferroptosis and play a neuroprotective role.Part three:The effects and mechanisms of System Xc-/GSH/GPX4 signal axis and GPX4 in hydrogen rich saline regulating Ferroptosis induced by CCH injury in ratsObjective:Separate or combined application HRS and System Xc-subunit SLC7A11inhibitor Erastin and GPX4 inhibitor RSL3,to explore the effects and mechanisms of System Xc-/GSH/GPX4 signal axis and GPX4 in HRS regulating Ferroptosis induced by CCH injury in rats,and to determine whether HRS can increase the synthesis of GSH and further improve the expression and activity of GPX4 protein by up-regulating the activity of SLC7A11,or directly up-regulating and improving the expression and activity of GPX4 protein,finally inhibit or scavenge toxic ROS and in turn inhibits Ferroptosis after CCH.Methods:Part Ⅰ:The rat model of CCH injury was established by modified 2-VO method.Forty healthy adult male Wistar rats were randomly divided into five groups by random number table method,with eight rats in each group（n=eight）:Sham operation group（Sham group）,Chronic cerebral ischemia group（CCH group）,Saturated hydrogen-rich saline group（Hydrogen group）,SLC7A11 subunit inhibitor group（Erastin group）,SLC7A11 inhibitor+saturated hydrogen-rich saline group（Erastin+Hydrogen group）.MWM experiment was used to evaluate the spatial learning and memory ability of rats;Observation of ultrastructural changes of neuronal mitochondria in hippocampal CA1 region by TEM;The contents of GSH,GSH-Px,MDA and 4-HNE in brain cortex tissue were detected by colorimetry and Elisa respectively,Western Blot technique was used to detect the protein expression of ferroptosis-related markers PTGS2,IREB2,SLC7A11 and GPX4.Part Ⅱ:The rat model of CCH injury was established by modified 2-VO method.Thirty healthy adult male Wistar rats were randomly divided into five groups by random number table method,with six rats in each group（n=six）:Sham operation group（Sham group）,Chronic cerebral ischemia group（CCH group）,Saturated hydrogen-rich saline group（Hydrogen group）,GPX4 inhibitor group（RSL3 group）,GPX4 inhibitor+saturated hydrogen-rich saline group（RSL3+Hydrogen group）.MWM experiment was used to evaluate the spatial learning and memory ability of rats;Detection of ROS content of nerve cells in brain tissue by flow cytometry,RT-PCR and Western Blot techniques were used to detect the m RNA and the protein expression of ferroptosis-related markers PTGS2,IREB2 and GPX4.Results:Part Ⅰ results:1.TEM observation found that compared with Sham group,the mitochondrial ultrastructure of neurons in rat’s brain hippocampal CA1 region showed characteristic changes of ferroptosis,such as volume reduction,membrane thickening and mitochondrial crest reduction or disappearance in different degrees after CCH.Compared with CCH group,after treatment with HRS,rats in Hydrogen group could significantly improve the mitochondrial ultrastructural changes of neurons in rat’s brain hippocampal CA1 region after CCH,the characteristic changes of ferroptosis,the thickening of mitochondrial membrane and the reduction of mitochondrial crest were significantly alleviated;However,the characteristics of ferroptosis in the ultrastructural changes of mitochondria of neurons in rat’s brain hippocampal CA1 region in Erastin group were more typical,the volume of mitochondria decreased significantly,the membrane thickened obviously,and the cristae of mitochondria almost disappeared completely.The ultrastructural changes of mitochondria of neurons in rat’s brain hippocampal CA1 region in Erastin+Hydrogen group were lighter than those in Erastin group.2.Compared with Sham group,the escape latency of Place navigation test in CCH group was significantly prolonged and the number times of crossing the original platform position in the target quadrant in Spatial probe test were significantly reduced（P<0.001）;The contents of lipid peroxidation metabolites:MDA and 4-HNE in brain tissue were significantly increased,while the activities of antioxidant of GSH and GSH-Px were significantly decreased（P<0.005）;The protein expression of Ferroptosis specific marker factors:PTGS2 and IREB2 were significantly increased,while the protein expression of ferroptosis negative regulatory factors:SLC7A11 and GPX4 were significantly decreased（P<0.001）.Compared with CCH group,The escape latency of rats in Hydrogen group was significantly shortened after treatment with HRS and the number times of crossing the original platform position in the target quadrant were increased significantly in the experiment（P<0.05 or P<0.005 or P<0.001）;The activities of antioxidant of GSH and GSH-Px in brain tissue were significantly increased,The contents of MDA and 4-HNE,the metabolites of lipid peroxidation,were significantly decrease（P<0.05 or P<0.005）;The protein expression of ferroptosis specific marker factors PTGS2 and IREB2 were decreased significantly,However,the protein expressions of SLC7A11 and GPX4,the negative regulators of ferroptosis,were significantly increased（P<0.001）.Compared with CCH group,the escape latency of rats in Erastin group was significantly prolonged,And the number times of crossing the original platform position in the target quadrant were decreased significantly in the experiment（P<0.001）;The activities of antioxidant of GSH and GSH-Px in brain tissue were decreased significantly,The contents of MDA and 4-HNE,the metabolites of lipid peroxidation,were significantly increased（P<0.005）;The protein expression of Ferroptosis specific marker factors PTGS2 and IREB2 were increased significantly,However,the protein expressions of SLC7A11 and GPX4,the negative regulators of ferroptosis,were significantly decreased（P<0.001）.Compared with Erastin group,The escape latency of rats in Erastin+Hydrogen group was shortened,the number times of crossing the original platform position in the target quadrant were increased in the experiment（P<0.001）;The activities of antioxidant enzymes GSH and GSH-Px in brain tissue were increased,however,the contents of MDA and 4-HNE,the metabolites of lipid peroxidation,were decreased（P<0.005）;The protein expression of ferroptosis specific marker factors PTGS2 and IREB2 were decreased significantly,However,the expression of SLC7A11 and GPX4 protein,the negative regulators of ferroptosis,were increased significantly（P<0.001）.Part Ⅱ results:Compared with Sham group,In the MWM experiment of rats in CCH group,the escape latency of Place navigation test was obviously prolonged and the number times of crossing the original platform position in the target quadrant in Spatial probe test were decreased significantly（P<0.001）;The content of ROS in nerve cells of brain tissue was increased significantly after CCH,the expression of m RNA and protein of Ferroptosis specific marker factors PTGS2 and IREB2 were significantly increased,However,the expression of m RNA and protein of GPX4,a negative regulator of ferroptosis,were decreased significantly（P<0.005 or P<0.001）.Compared with CCH group,the escape latency of rats in Hydrogen group was significantly shortened after being treated with HRS and the number times of crossing the original platform position in the target quadrant were increased significantly in the experiment（P<0.001）;The content of ROS in nerve cells of brain tissue was decreased significantly,the expression of m RNA and protein of Ferroptosis specific marker factors PTGS2 and IREB2 were decreased significantly,However,the expression of m RNA and protein of GPX4 were significantly increased（P<0.005 or P<0.001）.Compared with CCH group,the escape latency of rats in RSL3 group was significantly prolonged and the number times of crossing the original platform position in the target quadrant were decreased significantly in the experiment（P<0.05or P<0.005）;The content of ROS in nerve cells of brain tissue was increased significantly,The expression of m RNA and protein of Ferroptosis specific marker factors PTGS2 and IREB2 were significantly increased,However,the expression of m RNA and protein of GPX4,a negative regulator of Ferroptosis,were significantly decreased（P<0.005 or P<0.001）.Compared with RSL3 group,in the experiment,the escape latency of rats in RSL3+Hydrogen group was shortened,the number times of crossing the original platform position in the target quadrant were increased（P<0.001）;The content of ROS in nerve cells of brain tissue was decreased significantly,the expression of m RNA and protein of Ferroptosis specific marker factors PTGS2 and IREB2 were significantly decreased,However,the expression of m RNA and protein of GPX4,a negative regulator of ferroptosis,were significantly increased（P<0.005 or P<0.001）.Conclusion:HRS can up-regulate the activity of SLC7A11,the key element of System Xc-,and then activate the signal axis of System Xc-/GSH/GPX4,increase the cystine uptake of cells,and then increase the synthesis of GSH intracellular and further improve the expression and activity of GPX4 protein;In addition,HRS can also directly up-regulate and improve the expression and activity of GPX4 protein.The final results of the above effects are that HRS can reduce the degree of oxidative stress and lipid peroxidation damage after CCH by inhibiting the production of toxic ROS and accelerating the clearance of ROS in nerve cells after CCH,and then inhibit the occurence of ferroptosis and the expression of ferroptosis specific marker factors PTGS2 and IREB2 in neurons;Improve the neurobehavioral changes in rats after CCH;Moreover,HRS could partially reverse the biological effects of Erastin（the inhibitor of SLC7A11 of System Xc-subunit）and RSL3（the inhibitor of GPX4）induced Ferroptosis.Summary1.HRS has a clear protective effect on CCH injury in rats,It is possible that HRS can activate the activity of antioxidant such as SOD,GSH and GSH-Px and reduce the content of MDA,4-HNE and other lipid peroxidation products on antioxidant stress and lipid peroxidation.2.Ferroptosis is an important way of neuron death caused by CCH.The neurobehavioral changes induced by CCH,the reduction in the number and morphological changes of neurons in hippocampal CA1 region may be related to oxidative stress damage and FPT induced by CCH.HRS has obvious inhibitory effect on Ferroptosis after CCH injury.3.This study has been confirmed that HRS can decrease the content of iron in brain tissue after CCH by regulating the expression of iron metabolism related proteins such as TF/TFR1 and FTL,FTH1,Furthermore,it can reduce the production of toxic ROS and inhibit Ferroptosis after CCH injury.4.This study has been proved that HRS can up-regulate the activity of SLC7A11,the key element of System Xc-,and then activate the signal axis of System Xc-/GSH/GPX4,increase the cystine uptake of cells,and then increase the synthesis of GSH intracellular and further improve the expression and activity of GPX4 protein;In addition,HRS can also directly up-regulate and improve the expression and activity of GPX4 protein.The final results of the above effects are that HRS could reduce the degree of oxidative stress and lipid peroxidation damage after CCH by inhibiting the production of toxic ROS and accelerating the clearance of ROS in nerve cells after CCH,and then inhibit Ferroptosis after CCH injury.5.HRS could partially reverse the biological effects of Erastin（the inhibitor of SLC7A11 of System Xc-subunit）and RSL3（the inhibitor of GPX4）induced Ferroptosis.