| Purpose: In this study,we established a db/db mouse model and a high-glucose treated HT22 cell model,observed the effects of YTK on behavioral function,neuron and synaptic injury of db/db mice,observed the effects of YTK on iron metabolism,oxidative stress/lipid peroxidation and ferroptosis in db/db mouse model and high-glucose induced HT22 cells.The purpose of the present study was to discuss the improvement of YTK on cognitive impairment and ferroptosis of hippocampal neurons in T2 DM and the mechanism,so as to provide a theoretical basis for clinical application of YKT.Material and method:Article 1: Improvement of cognitive impairment of db/db mice and repair of damage of hippocampal neurons and synaptic plasticity by YKTThirty SPF male db/db mice were randomized into 3 groups: model group(db/db group),Western Medicine(Liraglutide)and YTK group;another 10 db/m mice were control(db/m group).Mouse model was constructed on Week 10 of feeding.Mice in YTK group were administrated with YTK decoction 30 g/kg/day,gavage,qd.Mice in LIRA group were administrated with Liraglutide 200 μg/kg,ip,qd.Mice in db/m group and db/db group were administrated with distilled water of equivalent volume of YTK decoction.Model construction lasted for 5 weeks.First,fasting glucose was determined in mice;Glucose metabolism,insulin reserve and liver gluconeogenesis were observed by tests of glucose tolerance,insulin tolerance and pyruvate tolerance;Serum levels of TG,TC and LDL-C were determined by biochemistry tests;Serum levels of INS and Hb A1 C were determined by ELISA;Behavior test was performed by Morris water maze,Y maze and open field tests;Synaptic integrity of neurons was evaluated by transmission electron microscopy;Neuronal morphology was observed by FJC staining and Nissl staining;The expression of Neu N and MAP2 in hippocampus was detected by immunofluorescence;The expression of synapseassociated proteins in hippocampus,including PSD95,SYN and BDNF was detected by Western blot;Whether YTK could improve cognitive impairment and damage of hippocampal neurons and synaptic plasticity in db/db mice was confirmed through these tests.Article 2: Improvement of ferroptosis of hippocampal neurons in db/db mice by YKTAssignment and route of administration were the same as those in Article 1.Mitochondrial function of hippocampal neurons was observed by immunostaining of both Cox1 and Cox4;Mitochondrial structure of hippocampal neurons was observed by transmission electron microscopy;The level of GSH and activity of GSH-Px in serum and hippocampal tissue were detected by biochemistry and ELISA;The expression of ferroptosisassociated proteins,including GPX4,SLC7A11 and ACSL4,in hippocampal neurons was detected by Western blot and immunofluorescence.Whether YTK could inhibit ferroptosis of hippocampal neurons in db/db mice was confirmed through these tests.Article 3: The mechanism underlining improvement of iron overload and oxidative stress in hippocampal neurons in db/db mice by YKTAssignment and route of administration were the same as those in Article 1.The levels of SOD,CAT and MDA in serum and hippocampal tissues were detected by biochemistry analysis;ROS content in hippocampus was detected by DHE staining;The expression of NOX2,NOX4 and SOD2 in hippocampus was detected by Western blot;The expression of 4-HNE in hippocampus was detected by Western blot and immunohistochemistry;Iron content in serum and hippocampal tissues was determined by serum iron and tissue iron kit;The distribution of iron in CA1,CA3 and DG regions in hippocampal tissues was observed by Prussian blue dyeing;The expression of ferritins(FTL and FTH),iron-transport-associated proteins(Tf R1、DMT1、FPN1),mitochondrial ferritin Mtft and mitochondrial ferroportin Mfrn2 in hippocampus was detected by Western blot;The expression of Tf R1 and FPN1 m RNA was detected by q-PCR.The effect of YTK on intracerebral iron overload and oxidative stress in hippocampal neurons in db/db mice and the underlining mechanism were confirmed through these tests.Article 4: The mechanism underlining the inhibition of iron overload,oxidative stress and ferroptosis in HT22 hippocampal neurons induced by high glucose by YKT(1)Part I consisted of 4 groups: Control(Con)group: treated by 25 m M glucose medium + 5% normal rat serum for 48 h;High-glucose(HG)group: treated by 50 m M glucose medium + 5% normal rat serum for 48 h;Ferroptosis inhibitor(Fer-I)group: treated by 50mM glucose medium + 5% normal rat serum + 10 μM Fer-I for 48 h;Yitangkang(YKT)group: treated by 50 m M glucose medium + 5% rat serum containing YTK for 48 h.The function and structure of mitochondria were observed by JC-1 staining and transmission electron microscopy;Lipid peroxidation and the expression of PSD95,ACSL4,GPX4 and SLC7A11 in HT22 cells were observed by C11 BODIPY 581/591 staining,Western blot and immunofluorescence.Whether high glucose induced ferroptosis in neurons and whether serum containing YTK could inhibit ferroptosis were confirmed in this part.(2)Part II consisted of 5 groups: Control(Con)group: treated by 25 m M glucose medium + 5% normal rat serum for 48 h;High-glucose(HG)group: treated by 50 m M glucose medium + 5% normal rat serum for 48 h;Iron chelating agent(DFO)group: treated by 50 m M glucose medium + 5% normal rat serum + 100 μM DFO for 48 h;Antioxidant(NAC)group: treated by 2 m M NAC for 4 h,then treated by 50 m M glucose medium + 5% normal rat serum for 48 h;Yitangkang(YKT)group: treated by 50 m M glucose medium + 5% rat serum containing YTK for 48 h.The function and structure of mitochondria were observed by JC-1 staining and transmission electron microscopy;Cellular oxidative stress,lipid peroxidation and the expression of SOD2 were observed by DCFH-DA staining,C11 BODIPY 581/591 staining and Western blot.Fe2+ level in HT22 cells was observed by calcein staining and Ferro Orange staining;The expression of iron-related proteins,including FTL,FTH,Tf R1,FPN1 and Mfrn2,were detected by Western blot.This part aimed to confirm whether high glucose induced iron overload and oxidative stress through relevant regulatory pathways,and further caused ferroptosis of neurons,and whether serum containing YTK could decrease iron overload and oxidative stress and further inhibit ferroptosis.(3)Part III consisted of 4 groups: Control(Con)group: treated by 25 m M glucose medium + 5% normal rat serum for 48 h;Ferroptosis agonist(RSL3)group: treated by 25 m M glucose medium + 5% normal rat serum + 0.25 μM RSL3 for 24 h;Ferroptosis inhibitor(RSL3+Fer-1)group: treated by 25 m M glucose medium + 5% normal rat serum + 0.25 μM RSL3 + 10 μM Fer-1 for 24 h;Yitangkang(RSL3+YKT)group: treated by 25 m M glucose medium + 0.25 μM RSL3 + 5% rat serum containing YTK for 24 h.The structure of mitochondria was observed by and transmission electron microscopy; Cellular lipid peroxidation was observed by C11 BODIPY 581/591 staining;The expression of ACSL4,GPX4 and SLC7A11 was detected by Western blot and immunofluorescence.This part aimed to confirm whether YTK could improve ferroptosis induced by RSL3 and demonstrate the direct effect of YTK on GPX4 and ferroptosis.Results:Article 1: Improvement of cognitive impairment of db/db mice and repair of damage of hippocampal neurons and synaptic plasticity by YKT1.Improvement of glycolipid level and insulin resistance by YKT:Fasting glucose was increased significantly(P<0.05),the AUCs of three resistance tests were increased significantly(P<0.05),the serum levels of INS and Hb A1 C were increased significantly(P<0.05)and the levels of TG,TC and LDL-C were increased significantly(P<0.05)in db/db group than those in db/m group;Fasting glucose decreased significantly(P<0.05),the AUCs of three resistance tests were decreased significantly(P<0.05),the serum levels of INS and Hb A1 C were significantly improved(P<0.05)and the levels of TG,TC and LDL-C were decreased significantly(P<0.05)in both LIRA group and YTK group than those in db/db group.These results suggested that YTK could decrease abnormal glycolipid level and improve insulin resistance in db/db mice.2.Improvement of behavior levels by YKT:In comparison with db/m group,the latency of escape in the water maze test of db/db mice was increased significantly in 1-5 days(P<0.05),the escape distance was increased in the fifth day,and the number of times of crossing the platform position was decreased significantly in 2 minutes(P<0.05),the percentage of time spent exploring the new arm in the Y maze test was decreased significantly(P<0.05),and the percentage of distance and time spent in the central area and the total distance and time in the open field test was decreased significantly(P<0.05);In comparison with db/db group,the escape time in the water maze test of mice in LIRA group and YTK group was decreased significantly(P<0.05),the escape distance was shortened on the fifth day,the number of times of crossing the circle position was increased significantly(P<0.05),and the percentage of exploring time in the new arm in the Y maze test was increased significantly(P<0.05),while the distance ratio and time ratio in the central area of mice in YTK group were increased significantly in the open field test(P<0.05),but there was no significant difference between LIRA group and db/db group.These results suggested that YTK could significantly improve the learning and memory function of db/db mice,relieve anxiety and improve cognitive dysfunction.3.Improvement of hippocampal neurons and synaptic plasticity by YKT:In comparison with db/m group,the number of FJC positive cells in the hippocampus of db/db mice increased significantly(P<0.05),the Nissl staining in the CA1,CA3 and dentate gyrus(DG)of the hippocampus became lighter,the number of Nissl bodies was decreased significantly(P<0.05),the positive areas of Neu N and MAP2 were decreased significantly(P<0.05),the expression of PSD95,SYN and BDNF decreased significantly(P<0.05),the width of synaptic space increased,and the dense substance between synapses decreased;In comparison with db/db group,the number of FJC positive cells in LIRA group and YTK group was significantly decreased(P<0.05),the number of Nissl corpuscles in each area of hippocampus was increased(P<0.05),the positive expression of MAP2 and Neu N was significantly increased(P<0.05),the expression of PSD95,SYN and BDNF was significantly increased(P<0.05),and the synaptic damage of hippocampal neurons was significantly improved.These results suggested that YTK could repair the damage of hippocampal neurons and synaptic plasticity in db/db mice and improve the expression of neurons and synaptic related functional proteins.Article 2: Improvement of ferroptosis of hippocampal neurons in db/db mice by YKT1.Improvement of mitochondrial function of hippocampal neurons by YTK:In comparison with db/m group,the fluorescence expression of Cox1 in db/db group decreased(P<0.05),and the fluorescence expression of Cox4 increased(P<0.05).Mitochondrial atrophy,mitochondrial membrane thickening,mitochondrial crista reduction,and partial mitochondrial membrane rupture were observed;In comparison with db/db group,Cox1 expression in LIRA group and YTK group increased(P<0.05),Cox4 expression decreased(P<0.05),mitochondrial structure improved,mitochondrial size restored,and mitochondrial cristae increased.These results suggested that YTK could improve the damage of mitochondrial function and structure of hippocampal neurons in db/db mice,and improve the pathological morphology of ferroptosis in neurons.2.Improvement of GSH level and GSH-Px activity in hippocampus:In comparison with db/m group,the GSH level and GSH-Px activity of db/db mice were significantly decreased(P<0.05);In comparison with db/db group,GSH and GSH-Px in LIRA group and YTK group were significantly improved(P<0.05).These results suggested that YTK could effectively increase GSH level and GSH-Px activity in db/db mice.3.Regulation of expression of ferroptosis-assocaited regulatory proteins in hippocampal neurons by YTK:In comparison with db/m group,the expression of GPX4 and SLC7A11 protein in hippocampus of db/db group was significantly decreased(P<0.05),while the expression of ACSL4 protein was significantly increased(P<0.05);In comparison with db/db group,the expression of GPX4 and SLC7A11 in LIRA group and YTK group was up-regulated(P<0.05),and the expression of ACSL4 was down-regulated(P<0.05).These results suggested that YTK could improve the expression of iron death related proteins GPX4,SLC7A11 and ACSL4 in the hippocampus of db/db mice.Article 3: The mechanism underlining improvement of iron overload and oxidative stress in hippocampal neurons in db/db mice by YKT1.YTK improved oxidative stress of hippocampal neurons:In comparison with db/m group,the content of SOD in serum and hippocampus of db/db group was significantly decreased(P<0.05),the level of ROS in DHE staining was significantly increased(P<0.05),the expression of NOX2 and NOX4 in hippocampus was significantly increased,and the expression of SOD2 was decreased(P<0.05);In comparison with db/db group,the level of SOD in LIRA group and YTK group increased(P<0.05),the level of ROS decreased significantly(P<0.05),the expression of NOX2 and NOX4 in hippocampus decreased,and the expression of SOD2 increased(P<0.05).These results suggested that YTK could improve oxidative stress in hippocampus of db/db mice.2.YTK improved lipid peroxidation of hippocampal neurons:In comparison with db/m group,the level of MDA in serum and hippocampus of db/db group increased significantly(P<0.05),and the expression of 4-HNE increased significantly(P<0.05);In comparison with db/db group,the MDA level in LIRA group and YTK group decreased(P<0.05),and the expression of 4-HNE protein in hippocampus decreased(P<0.05).These results suggested that YTK could reduce the accumulation of lipid peroxide and lipid peroxidation in hippocampus of db/db mice.3.YTK improved iron overload in hippocampus:In comparison with db/m group,the content of iron in serum and hippocampus of mice in db/db group increased significantly(P<0.05),and the area of Fe positive areas in CA1,CA3 and DG regions of hippocampus increased significantly(P<0.05);In comparison with db/db group,the iron content in serum and hippocampus of mice in LIRA group and YTK group was significantly lower than that in db/db group(P<0.05),and the area of Fe positive areas in CA1,CA3 and DG regions of hippocampus was decreased(P<0.05).These results suggested that YTK could effectively reduce the iron overload in the hippocampus of db/db mice.4.YTK regulated iron-related proteins in hippocampus:In comparison with db/m group,the expression of iron storage protein FTH in the hippocampus of db/db mice decreased(P<0.05),the expression of iron-related transporters Tf R1 and DMT1 increased significantly(P<0.05),the expression of FPN1 decreased(P<0.05),the expression of Tf R1 m RNA increased significantly,the expression of FPN1 m RNA decreased(P<0.05),the expression of mitochondrial iron storage protein Mtft decreased(P<0.05),and the expression of mitochondrial iron transport protein Mfrn2 increased significantly(P<0.05);In comparison with db/db group,the expression of FTH in hippocampus of LIRA group and YTK group increased(P<0.05),the expression of Tf R1 and DMT1 decreased(P<0.05),the expression of FPN1 increased significantly(P<0.05),the expression of Tf R1 m RNA in hippocampus decreased,the expression of FPN1 m RNA increased(P<0.05),the expression of Mtft increased(P<0.05),and the expression of Mfrn2 decreased(P<0.05).These results suggested that YTK could up-regulate the expression of iron storage protein FTH,down-regulate the expression of iron transfer protein Tf R1 and DMT1,up-regulate the expression of iron transfer protein FPN1,and up-regulate the expression of mitochondrial iron storage protein Mtft in the hippocampus,and down-regulate the expression of mitochondrial iron transport protein Mfrn2.Article 4: The mechanism underlining the inhibition of iron overload,oxidative stress and ferroptosis in HT22 hippocampal neurons induced by high glucose by YKT1.YTK improved ferroptosis of HT22 cells induced by high glucose:In comparison with Con group,the ratio of JC-1 Aggregation/JC-1 Monomer in HG group was decreased significantly(P<0.05),mitochondrial structure shrank and mitochondrial membrane thickened,C11 BODIPY 581/591 oxidation/reduction ratio was significantly increased(P<0.05)and the expression of ACSL4 was up-regulated,the expression of PSD95,GPX4 and SLC7A11 was down-regulated(P<0.05);In comparison with HG group,the ratio of JC-1 Aggregation/JC-1 Monomer in both Fer-I group and YTK group was decreased significantly(P<0.05),mitochondrial structure improved and size recovered,C11 BODIPY 581/591 oxidation/reduction ratio was significantly decreased(P<0.05))and the expression of ACSL4 was down-regulated,the expression of PSD95,GPX4 and SLC7A11 was up-regulated(P<0.05).These results suggested that YTK could improve ferroptosis of HT22 cells induced by high glucose.2.YTK improved oxidative stress and iron overload of HT22 cells induced by high glucose:In comparison with Con group,ROS level and BODIPY 581/591 oxidation/reduction ratio in HG group were significantly higher(P<0.05),the expression of SOD2 was decreased significantly(P<0.05);In comparison with HG group,ROS levels and BODIPY 581/591 oxidation/reduction ratios in DFO group,NAC group and YTK group were significantly increased(P<0.05),the expression of SOD2 was increased significantly(P<0.05).These results suggested that YTK could reduce oxidative stress and lipid peroxidation level of HT22 cells induced by high glucose.3.YTK improved iron overload induced by high glucose and regulated related proteins in HT22 cells:In comparison with Con group,the positive areas of calcein staining and Ferro Orange staining in HG group were increased significantly,the level of Fe2+ was increased(P<0.05),the expression of Tf R1 and Mfrn2 was increased significantly,and the expression of FTH and FPN1 was decreased significantly(P<0.05);In comparison with HG group,the positive areas of calcein staining and Ferro Orange staining in both DFO group and YTK group were increased significantly,the level of Fe2+ was increased(P<0.05),the expression of Tf R1 and Mfrn2 was increased significantly,the expression of FTH and FPN1 was increased significantly(P<0.05).These results suggested that YTK could reduce the iron overload of HT22 cells induced by high glucose by up-regulating the expression of ferritin FTH,downregulating the expression of cytoplasmic and mitochondrial iron transferrin Tf R1 and Mfrn2,and up-regulating the expression of transferrin FPN1.4.The effect of YTK on ferroptosis of HT22 cells induced by RSL3:In comparison with Con group,the mitochondrial structure of RSL3 group was shrunk,the mitochondrial membrane was thickened,some mitochondria were broken,and the typical expression of ferroptosis occurred.C11 BODIPY 581/591 oxidation/reduction ratio was significantly increased(P<0.05),and the level of lipid peroxidation was increased.At the same time,the expression of ACSL4 was up-regulated,and the expression of GPX4 and SLC7A11 protein was down-regulated(P<0.05);In comparison with RSL3 group,the structure of mitochondria in Fer-I group was improved,the size of mitochondria was restored,BODIPY 581/591 oxidation/reduction ratio was decreased(P<0.05),the expression of ACSL4 was decreased,and the expression of GPX4 and SLC7A11 protein was increased(P<0.05);These results suggested that the regulation of underexpressed GPX4 and ferroptosis induced by RSL3 by YTK was limited.Conclusion: 1.YTK could improve cognitive impairment and repair neuronal and synaptic plasticity damage caused by T2 DM.2.YTK could repair the damage of mitochondrial structure and function,up-regulate the expression of GPX4 and SLC7A11 protein,down-regulate the expression of ACSL4 protein,and improve the ferroptosis of hippocampal neurons induced by T2 DM.3.YTK may improve the iron overload of hippocampal neurons induced by T2 DM by upregulating the expression of ferritin FTH,down-regulating the expression of transferrin Tf R1 and DMT1,up-regulating the expression of transferrin FPN1,and up-regulating the expression of mitochondrial ferritin Mtft in hippocampus,and down-regulating the expression of mitochondrial transferrin Mfrn2.4.YTK may improve the oxidative stress and lipid peroxidation of hippocampal neurons induced by T2 DM by reducing the levels of ROS and lipid oxides and increasing the activity of antioxidants.5.YTK may alleviate iron overload by regulating iron metabolism-related proteins,reduce oxidative stress and lipid peroxidation,thus improving the ferroptosis of hippocampal neurons induced by T2 DM and recovering cognitive function. |
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