| [objective]Alzheimer’s disease(Alzheimer’sdisease AD)is an age-related neurodegenerative disease,which is characterized by memory loss and cognitive impairment.Epidemiological and pharmacological studies have shown that there is a significant correlation between diabetes and cognitive impairment.Diabetes greatly increases the risk of AD.Some people even put forward the concept of "type 3 diabetes" and regard AD as type 3 diabetes.Abnormal glucose metabolism can cause insulin resistance,affect the transmission of insulin signal pathway,cause synaptic damage and even lead to neuronal dysfunction.As a key protein in insulin signaling pathway,abnormal function of GSK3 β will lead to insulin resistance.The late clinical features of AD include not only cognitive impairment,but also other emotion-related encephalopathy,such as depression,anxiety,fear and so on.In this experiment,the model of memory impairment caused by abnormal glucose metabolism was established,and the single medicine and the active ingredient of Gardenia jasminoides in Yueju pills and Yueju pills were selected to explore its protective effect on memory impairment and its mechanism.[Methods](1)Adult male db/db mice and ICR mice were used.ICR mice were injected with STZ through caudal vein and lateral ventricle to establish a mouse model of memory impairment caused by abnormal glucose metabolism.After modeling,ICR mice had similar behavioral and pathological characteristics to db/db mice,so we used the model established by intravenous injection of ICR mice for eight consecutive weeks.Fasting blood glucose and body weight were monitored every two weeks.At the eighth week,glucose tolerance was measured.Morris water maze was used to test the learning and memory function of mice.The expression levels of GSK3β,P-S9,P-S389,AMPK,P-AMPK and synaptic proteins NR2B and PSD95 were detected by Werstern Blot.(2)HT22 cells were cultured with high glucose to establish a high-glucose cell injury model,and gardenoside was used to protect the high-glucose cell injury model.Cell activity was detected by MTT and Werstern Blot to detect the expression levels of GSK3β,P-S9,P-S389,AMPK,P-AMPK and synaptic proteins such as NR2B and PSD95 in mouse brain hippocampal tissues,and the protective effects were detected.[Results](1)① ICR mice were injected with 200mg/kg STZ(STZ)through tail vein for 3 days,and their body weight began to decrease and their blood sugar increased.The general physiological state was observed,and it was found that "three more and one less" appeared,indicating the successful establishment of diabetes model.② Morris water maze test showed that the escape latency of STZ mice and db/db mice was significantly prolonged,and the spatial learning and memory ability of the mice was decreased.③Conditional fear test showed that the percentage of fear memory freeze time was increased and the fear memory was abnormally enhanced.④ There were no significant changes in the expression levels of GSK3β and AMPK in the hippocampal tissues of the two models,but the expression levels of P-S9,P-S389 and P-AMPK were significantly decreased.The expression levels of NR2B and PSD95 in db/db mice were decreased,while the expression levels of NR2B and PSD95 in STZ mice were decreased,and the expression levels of PSD95 remained unchanged.(2)① Yueju pill formula was intragastric administered to STZ mice,and it was found that Yueju pill formula had no significant change in blood glucose,significantly reduced the body weight of mice,and increased the mortality of mice.② Effectively shorten the escape latency of STZ mice and improve the spatial memory of mice.③Effectively reversed the percentage of rigidity time in STZ mice,and reduced the abnormal enhancement of fear memory.(3)①The single drugs of Gardenia Fructus,Atractylodes Atractylodes.Ligusticum chuanxiong and Fructus Atractylodes were intragastrically administered to STZ mice with 70%ethanol extract respectively.It was found that each single drug had no significant effect on the blood glucose of mice.Gardenia jasminoides,Atractylodes tractylodes and Ligusticum chuanxiong had no effect on the body weight of mice.② Morris water maze results showed that each single drug administration group could improve the spatial memory impairment of mice to a certain extent,but only the difference of gardenia was statistically significant.③ Similarly,the results of conditional fear showed that all single drug administration groups could reduce the enhancement of conditional fear memory in mice to a certain extent,but only the gardenia drug administration group showed statistical significance.(4)①Gardenia alcohol extract(0.6g/kg ZZL;1.2g/kg,ZZH),the main active ingredient of gardenia jasminoside(25mg/kg GEL;50mg/kg,GEH)were administered intragastrically to 200mg/kg STZ(model)mice through tail vein.It was found that the body weight of the mice could not be improved in each administration group,and the blood glucose in GEL group began to decrease at the sixth week with statistical significance.② The glucose tolerance,insulin and insulin resistance index increased in the model group.The results of glucose tolerance test showed that the blood glucose level at 120min in ZZL group was significantly decreased.The blood glucose values of Gel group at 0min,60min and 120min pairs were significantly lower than those of Model group.The area under the curve(AUC)was calculated,and it was found that the AUC of ZZL group and GEH group could be significantly reduced.③ Serum insulin content in model group was significantly decreased,and there was no significant effect on serum insulin content of mice in each administration group;Compared with the control group,the insulin resistance index of model group was significantly increased,and the insulin resistance index of mice in GEL and GEH groups was significantly decreased.④ The escape latency of the model group was significantly prolonged,and the spatial learning and memory function was impaired.ZZH group and GEL group significantly shortened the escape latency,and improved spatial learning and memory.⑤ Both gardenia alcohol extract and gardenia glycoside could significantly reduce the percentage of mouse rigidity time,but only GEL and GEH showed significant differences.⑥ Liver function:Model group(AST/GOT)and(ALT/GPT)had no significant changes,but AKP was significantly increased,gardenia alcohol extract and gardenia glycoside had a certain degree of decreased AKP content,only GEL group had statistical significance.Renal function:There was no significant change in CRE in the model group,but the BUN content was significantly increased,and the BUN content in the GEH group was significantly increased than that in the model group.The results showed that GEH had a certain damage effect on renal function.⑦ Western Blot results showed that Gel and GEH enhanced the expression of P-AMPK,P-S389,P-S9 in the hippocampus of mice injected with STZ tail vein.The expression level of synaptic protein PSD95 in the Model group was unchanged,and the expression level of NR2B was significantly decreased,while the expression level of NR2B was significantly increased in the GEL and GEH groups.(5)① Lateral ventricular injection of STZ by GEL and GEH was found to have no effect on blood glucose and body weight of mice after LV injection of 5mg/kg STZ(model).②The escape latency of mice in the model group was significantly prolonged and the spatial memory was impaired.Both GEL and GEH had a certain improvement effect on the spatial memory impairment of mice,but only the improvement effect in the GEL group was statistically significant.③ In contrast to the mice injected with 200mg/kg STZ by caudal vein,the percentage of rigor time of the lateral ventricle injected with 5mg/kg STZ decreased significantly and the fear memory was impaired.Both the GEH and GEH groups could increase the percentage of high rigor time to a certain extent and enhance the fear memory,but only the GEL group showed significant differences.④ Western Blot results showed that the levels of AMPK and GSK3β in the hippocampus of model mice were decreased,the expression of P-AMPK and P-S9 were significantly decreased,but the expression of P-S389 was significantly increased.Compared with the Model group,the expression of P-AMPK and p-S9 protein in GEL group was significantly increased,and the expression of P-S389 was decreased.Synaptic proteins:the expression levels of PSD95 and NR2B in the Model group remained unchanged.(6)①HT22 cells were cultured with different concentrations of high glucose.MTT results showed that high glucose had more inhibitory effect on cell activity at 48h than at 24h,and 35mM glucose had more inhibitory effect than 15mM and 25mM glucose.In the high-glucose group(15mM,25mM,35mM),the expression levels of total AMPK and GSK3β were not significantly changed,while the expression levels of P-AMPK and P-S9 were significantly decreased,and the expression levels of synaptic proteins NR2B and PSD95 were not changed.Therefore,HT22 was treated with 35mM glucose for 48h to simulate the damage state of diabetic hippocampal neurons.②:Gardenoside with different concentrations(1uM-2.5mM)was used to protect HT22 cells damaged by high glucose.MTT results showed that gardenoside with 0.5mM and ImM enhanced the activity of HT22 cells.The expression of P-AMPK and P-S9 was significantly enhanced after the intervention of gardenoside(0.2mM-2.5mM).[Conclusions](1)200mg/kg STZ injection through caudal vein induced abnormal peripheral glucose metabolism and 5mg/kg STZ injection through lateral ventricle induced abnormal central glucose metabolism,resulting in impaired spatial learning and memory,insulin resistance,P-AMPK,P-S9 in ICR mice;Abnormal fear memory(increased/decreased),abnormal P-S389 expression(decreased/increased);NR2B expression was decreased and synaptic homeostasis was disrupted.(2)Yueju pill and Gardenia jasminoides alcohol extract can effectively enhance the spatial memory impairment caused by abnormal peripheral glucose metabolism,and the abnormal enhancement of fear memory(3)Gardenoside can not only enhance the spatial memory damage caused by peripheral and central glucose metabolism abnormalities,but also regulate the fear memory caused by glucose metabolism abnormalities in two ways.In vivo animal experiment results showed that gardenoside can effectively enhance AMPK,GSK3β(Ser9)phosphorylation level,enhance the expression of P-AMPK,P-S9;The expression of bidirectional P-S389 was abnormal(increased/decreased).The expression of NR2B in synapses of mice with peripheral glucose metabolism was enhanced to maintain synaptic homeostasis.(4)HT22 cells cultured with high glucose produced neurotoxicity,inhibited cell activity,and impaired phosphorylation of AMPK and GSK3β(Ser9).The results of cell experiments in vitro showed that gardenoside could protect the high-glucose cell injury model and enhance the expression of P-AMPK and P-S9.(5)The protective effect of Yueju Pills on memory impairment in mice with abnormal glucose metabolism may be the pharmacological effect of gardenoside,the active component of Gardenia jasminoides. |
