Study On The Effect And Mechanism Of Danggui Liuhuang Decoction On Type 2 Diabetic Cognitive Dysfunction Mice Based On IRS1/PI3K/Akt Signaling Pathway

Objective1.To clarify the pathological mechanism of cognitive dysfunction in type 2 diabetes in terms of traditional Chinese and Western medicine through theoretical research,and preliminarily explore the theoretical basis for Danggui Liuhuang Decoction in treating diabetic cognitive dysfunction.2.By probed the effect of cognitive function,pathological changes of hippocampal neurons,influence of glucolipid metabolism（fasting blood glucose,fasting serum insulin,HOMA-IR,liver oil red O staining）,oxidative stress indicators（SOD,MDA,GSH-Px,CAT）,inflammatory factors（TNF-α,IL-6,IL-8）and IRS1/IPI3K/AKT signaling pathway protein expression level in hippocampus of Diabetic cognitive dysfunction mice,to explore the effect and mechanism of Danggui Liuhuang Decoction on Diabetic cognitive dysfunction.Methods1.Theoretical researchSummarized the etiology and pathogens of DCD by sorting out related discussion and studies in Huangdi Neijing,ancient classics and modern literature.Analysed Danggui Liuhuang Decoction from the perspective of tradition Chinese and Western medicine respectively to clarify the theoretical and material basis of its treatment of DCD.Explored the regulation effect of IRS1/PI3K/AKT signaling path way on diabetes and cognitive function,and clarified the mechanism of Danggui Liuhuang Decoction in treating Diabetic cognitive dysfunction based on this pathway.2.Experimental study40 male C57 BL/6J mice were randomly divided into 6 mice as the normal Control group（Control）and fed with ordinary diet.The other mice were fed a high-fat diet（D12492）for 40 days,then intraperitoneally injected streptozotocin（STZ）（30mg/kg）to build type 2diabetes mellitus（T2DM）model.Y maze test（alternation and novel arm entry）was performed on the successful T2 DM mice on weekends 0,4,6,8 and 10 to evaluate the cognitive function and verify the DCD model.type 2 diabetic mice were placed from the end of arm no.1,observed and recorded the sequence and frequency of mice entering each arm within 8min.Alternation was defined in which the mouse entered the three arms in the order of arm 1,2 and 3 or arm 1,3 and 2,calculate the alternation rate.Closed No.3 arm and the mice were allowed to explore freely in no.1 and No.2 arms for 5min.One hour later,arm no.3 was opened and the mice were allowed to move freely between arm No.1,2 and3 for 5min.Observed and recorded the frequency of entering each arm,and caculated the exploration rate of novel arm.The mice with cognitive decline were set to the next period of the experiment.DCD mice were randomly divided into 4 groups: model group（T2DM）,Danggui Liuhuang Low dose group（DLD-L）,Danggui Liuhuang high dose group（DLD-H）and Donepezil group（DNPZ）,with 6 mice in each group.The mice in T2 DM model group and normal control group were given equal volume of normal saline intragastric administration for 35 d.As revealed by the place and spatial probe tests in Morris water maze to observe the spatial learning and memory ability of mice.The mice enter the water from the same entry point in one quadrant each time,and the mice were put into the water facing the pool wall.The instrument automatically recorded the road map of the mice searching for and climbing up the platform,recorded the time from the mice entering the water to finding and standing on the underwater hidden platform（escape the incubation period）and the total swimming distance.Every four quadrants of training is regarded as a cycle.If the mice did not find the platform in 90 s,it needs to be led to the platform.At this time,the incubation period is 90 s,and the mice is allowed to stay on the platform for 10 s before being put back into the cage.In this way,the four quadrants of the experiment were repeated for a total of 5 days of training,namely four cycles.The selection of quadrants and the training sequence of mice in each experimental group should be randomly determined or crossed.Five days after the end of positioning navigation experiment,removed the underwater platform,and four quadrants were repeated as one cycle test according to the method of positioning navigation experiment.The mice entered the water from the entry point of one quadrant every time,recorded the swimming distance,path and time of the mice in the four quadrants of the pool in 90 s.Based on this,the percentage of the data in the quadrant of the platform in the total distance and total time and the frequency of crossing the platform were calculated to reflect the spatial memory ability of mice to the location of the platform,so as to judge the memory storage,extraction and reprodu-ction ability of the diabetic mice.HE staining was used for pathological analysis of hippocampal tissues.Detected fasting blood glucose by automatic biochemical analyzer,determined FINS content by ELISA,and calculated insulin resistance evaluation index.Observed the changes of liver fat in Diabetic cognitive dysfunction mice by Liver oil red O staining.The contents of SOD,MDA,GSH-Px and CAT in hippocampus were determined by UV spectrophotometry.ELISA to detected the levels of TNF-α,IL-6and IL-8 in hippocampus.Western blot（WB）to detect the protein expression levels of IRS1,P-IRS1,PI3 K,P-PI3 K,AKT and P-Akt in hippocampal tissues.Results1.Theoretical research resultsThe internal heat making Five Zang Organs Weak was the pathogenesis of Quench thirst;Stagnant heat injures Yin,leading to the deficiency of vital essence,then occured the insufficiency of Medullary sea and Brain dystrophy,which was the primary source of dementia.The syndrome of quenching thirst was either due to diet,or indulgence in sexual activities,or cold and heat evil.The disease could be divided into Fire thirst,Hot thirst,Yin thirst,Stagnant thirst and Deficiency thirst.The position of the disease was different from upper-jiao,middle-jiao and lower-jiao.The disease of dementia was because of liver depression,or stomach failure,or phlegm,or blood stasis,ultimately due to the insufficiency of cerebrospinal,disturbing the mentality.DCD was based on Yin deficiency with internal heat,may company with phlegm evil,or blood stasis,or toxin.the cardinal treatment principle was nourish ing Yin and Clearing Heat,discriminating brain consciousness,Dredging cerebral vessels,combined with Invigorating Qi,nourish ing blood,removing blood stasis,removing dampness and detoxify ing.The characteristics of nourishing Yin and clearing heat of Danggui Liuhuang Decoction,has the pharmacological effects of resisting insulin resistance,regulating glucose and lipid metabolism,anti-inflammation and anti-oxidatio.integrated the regulation of IRS1/PI3K/AKT signaling pathway on diabetic cogni tive dysfunction,is the basis of treatment on DCD based on this pathway.2.Experimental resultsGeneral condition of mice: after feeding high fat diet for 40 days,the weight of mice increased significantly and formed obesity.After intraperitoneal injection of STZ,the mice took more food and urine volume increased.The mice were subsequently fed with highfat diet,and the weight gain was not significant or even reduced.After modeling,the mice had loosen hair,lethargy and slow action.No mouse death occurred during modeling,and 4 mice with substandard blood glucose were excluded.Mice in control group were in good condition,the spirit,activities,eating and drinking were fine.The Y maze experiment showed that the alternation rate and the proportion of novel arm entry began to decrease in T2 DM model group at week 6,and the difference was statistically significant compared with that at modeling time.At the 10 th week,the rate of alternation and novel arm entry decreased significantly,which was significant-ly different from that at modeling time.The results indicated that cognitive impairment began to occur in T2 DM mice at the 6th week,indicating successful modeling of DCD,which was consistent with relevant literature reports.2.1 Morris water maze: The trajectory of mice in the control group was clear and short,while the trajectory of mice in the T2 DM group was chaotic and long.Compared with the control group,the track of mice in DLD-L group was clearer and their route was shortened,while the route of mice in DLD-H group was significantly clearer and shortened.Compared with the control group,the incubation period of escape in T2 DM group was significantly prolonged（P <0.01）.Compared with T2 DM group,escape latency of DLD group was shortened,and the high dose group was better than the low dose group（P <0.05）.Compared with the control group,the frequency of crossing platform in T2 DM group was significantly reduced（P <0.01）.Compared with T2 DM group,the frequency of crossing platform in DLD group was increased,and the high dose group was better than the low dose group（P <0.05）.2.2 HE staining of hippocampal neurons: In the control group,the vertebral cells in the CA1 area of mice were compact and full in shape,with clear nucleoli and rare cell necrosis.In T2 DM group,the number of vertebral cells was reduced,and the arrangement was disorderly,with vacuol-like changes,a large number of nuclear pyknosis and increased cell necrosis.Compared with T2 DM group,the number of vertebral cells in DLD-L group was increased,arranged slightly orderly,the layers were clearer,the necrotic cells were reduced,and the pathological changes were improved compared with model group.The number of vertebral cells in DLD-H group was significantly increased,the arrangement was regular,the layers were obvious,the necrotic cells were significantly reduced,and the pathological changes were significantly improved compared with the model group.2.3 Glucolipid metabolism: Compared with control group,FBG,FINS and HOMA-IR were significantly increased in T2 DM group（P<0.01）.Compared with T2 DM group,the levels of FBG,FINS and HOMA-IR in DLD group were decreased（P <0.05）,and the high dose group was better than the low dose group（P <0.05）.Oil red O staining section of Liver of DCD mice showed that the structure of liver cells of control mice was intact without obvious lipidation and necrosis.Compared with the control group,the liver of T2 DM mice showed significant fat infiltration and bulla-like lipid droplets.Compared with T2 DM group,the steatosis of The DLD group was less,and the DLD-H group was more obvious.2.4 Oxidative stress: Compared with the control group,the contents of SOD,GSH-Px and CAT in T2 DM group were significantly decreased（P <0.01）;Compared with T2 DM group,the contents of SOD,GSH-Px and CAT in DLD group were increased（P <0.05）,and the high dose group was better than the low dose group（P <0.05）.Compared with control group,MDA content in T2 DM group was significantly increased（P <0.01）;Compared with T2 DM group,MDA content in DLD group was decreased（P <0.05）,and the high dose group was better than the low dose group（P <0.05）.2.5 Inflammatory factors: Compared with control group,the levels of TNF-α,IL-6 and IL-8 in T2 DM group were significantly increased（P <0.01）;Compared with T2 DM group,the levels of TNF-αand IL-6 in DLD group were decreased（P <0.05）,and the high dose group was better than the low dose group（P <0.05）.Compared with T2 DM group,IL-8 level in DLD group was decreased,but there was no significant difference（P >0.05）.2.6 IRS1/PI3K/AKT signaling pathway: Compared with the control group,the phosphorylation expressions of IRS1,PI3 K and AKT were significantly decreased in T2 DM group（P <0.01）.Compared with T2 DM group,phosphorylation expressions of IRS1,PI3 K and AKT were increased in DLD group（P <0.05）,and the high dose group was better than the low dose group（P <0.05）.Conclusions1.Yin deficiency and internal heat was the root of Diabetic cognitive dysfunction pathology in Traditional Chinese medicine.The characteristics and pharmacological effects of Danggui Liuhuang Decoction for nourishing Yin and clearing heat fitted the pathogenesis of DCD.2.A high-fat diet combined with intraperitoneal injection of STZ was used to build the diabetes model.Y-maze test verify the cognitive decline,and successfully replicated the DCD model.3.Danggui Liuhuang Decoction can significantly improve the learning and memory ability of DCD mice,alleviate pathological changes of hippocampal neurons and ameliorate cognitive dysfunction.4.Disorder of glucose and lipid metabolism is critical for diabetic cognitive dysfunction.Danggui Liuhuang Decoction could improve glucose and lipid metabolism,reduce insulin resistance and improve insulin sensitivity in DCD mice.5.Oxidative stress is an important mechanism of diabetes cognitive dysfunction.Danggui Liuhuang Decoction can effectively regulate oxidative stress and improve cognitive function.Inflammatory response is an important feature in the pathological process of diabetic cognitive dysfunction.Ddanggui liuhuang Decoction can effectively inhibit inflammation and improve cognitive function.6.IRS1/PI3K/AKT signaling pathway plays an important role in the regulation of the occurrence and development of DCD.Danggui Liuhuang Decoction can up-regulate the protein expression of IRS1/PI3K/AKT signaling pathway in hippocampus of DCD mice and improve cognitive function.