The Effect And Mechanism Of Ligustilide In The Prevention And Treatment Of Cognitive Impairment In Hypercholesterolemia Model Mice

ObjectiveAiming at the cognitive impairment caused by hypercholesterolemia,this study investigated whether ligustilide（LIG）could restored cholesterol homeostasis in the brain by acting on cholesterol-related metabolic enzymes to remove excess 27-hydroxycholesterol（27-OHC）.On the other hand,to investigate whether LIG could increase the glucose uptake by acting on neuronal glucose transporter 4（GLUT4）,thereby improving neuronal activity,reducing neuroinflammatory damage,oxidative stress,and improving cognitive function.Thereby preventing cognitive dysfunction caused by hypercholesterolemia.Methods1.The model mice with cognitive impairment caused by hypercholesterolemia.2-month-old male C57BL/6 mice were selected and divided into a normal group and a model group,the normal group was fed with ordinary feed and the model group was fed with high fat and high cholesterol feed for 8 months to create a hypercholesterolemia model.The behavioral tests of open field,novel object recognition and Y-maze were used to detect the cognitive function of model mice,and the blood lipid level of mice was detected by TC,TG,HDL-C,LDL-C kits.The model mice with hypercholesterolemia and cognitive impairment was established successfully.2.The effect of LIG on learning and memory in model miceThe selected model mice were divided into five groups:model group,model+LIG low dose group,model+LIG medium dose group,model+LIG high dose group,and model+positive drug group（simvastatin group）,with 10 mice in each group.The normal group and the model group were given an equal volume of 3%Tween-80 PBS buffer solution by gavage.The low,medium and high dose LIG groups were given LIG 10 mg/kg,LIG 20mg/kg and LIG 40 mg/kg solution by gavage,respectively.The positive drug group was given simvastatin 50 mg/kg by gavage once a day for 10 weeks.The next day after administration,the cognitive function of mice were tested using various behavioral-methods such as novel object recognition,Morris water maze and Y-maze experiments.3.The effects of LIG on brain glucose metabolism,serum cholesterol level,related cholesterol metabolic enzymes,oxidative stress and neuroinflammation in hypercholesterolemic mouse model.The glucose uptake rate in the hippocampus and cortex of mice were measured using micro-PET;the day after the end of the experiment,the pathological morphology of neurons was observed using HE staining.Blood lipid indicators（TC,TG,HDL-C,LDL-C）and cholesterol-related metabolic enzymes were detected using a kit and time-PCR methods,respectively.Western blotting was used to detect the levels of glucose-related transporters.Time-PCR method and kit were used to detect inflammatory factors（IL-6,TNF-α）and oxidative stress indicators（SOD,GSH,H₂O₂）,respectively.ResultsExperiment 1.1.The high-cholesterol diet created hypercholesterolemia and cognitive impairment model mice:Through the detection of blood lipid levels of mice,it could be found that high-cholesterol diet had a great impact on the blood lipid levels of mice,and the results showed that the TC,TG and LDL-C levels of modular mice increased significantly（P<0.05）,while HDL-C levels decreased significantly（P<0.05）.Through the detection of open-field,new objects and Y maze behavioral experiments,it was found that in the open-field experiment,the mice that built the module had less time to explore the central area of the new environment,lower degree of autonomy,and higher exploration of the new environment of the mice in the normal group.In the new object recognition experiment,the mice who built the module spent longer on the old objects and explored the new objects for a shorter time than the normal group,indicating that the high-cholesterol diet reduced the memory ability of the mice and made them more insensitive to new objects.In the Y-maze experiment,the correct alternation rate（P<0.05）in the three arms of the mice was less,indicating that their degree of exploration of new environments and spatial memory were poorer.The result indicates that the mold was successful.Experiment 2.1.The effect of LIG on cognitive function of model mice:Through the use of behavioral tests（Morris water maze,new object recognition and Y maze）on each group of mice,it was found that LIG could improve the memory impairment of hypercholesterolemia model mice.In the Morris water maze positioning cruise experiment,it was seen that the positive drug group and the L,M and H dose groups of LIG could shorten the evasion latency of the model mice,and the M and H dose groups were better than the positive drug group.By observing the number of platform crossings in each group,it can be seen that the LIG administration group was significantly higher than that of the model group.It was reflected that LIG can improve the spatial learning and memory ability of model mice.In the new object recognition experiment,it was found that the LIG administration group could improve the exploration time of mice on new objects,and the H-dose group was better than the M dose group and L dose group.Experiments showed that LIG could improve the learning and memory ability of model mice.The results of Y-maze experiment showed that the correct alternation rate of mice was significantly improved in the LIG administration group,and the results were better than those in the normal control group.The results suggested that LIG could improve the short-term recognition memory ability of new environment in mice with hypercholesterolemia models.Experiment 3.1.HE staining analysis was used to show that the structure of neurons in the hippocampal region of mice in the normal control group was normal,the number was large and the arrangement was clear.In the model group,the number of neurons in the hippocampus was reduced,the arrangement was scattered,the neurons were wrinkled,the morphology was irregular,some nucleoli were not obvious,and the nuclear membrane boundary was blurred.Compared with the model group,the LIG administration group had a larger number of neurons and a closer arrangement.2.The effect of LIG on cholesterol and the effect of related metabolic enzymes in mice:Compared with the normal group,the TC,TG,LDL-C in the model group were elevated（P<0.05）,and HDL-C decreased.Compared with the model group,the lipid levels in the dosing group were improved.The detection of relevant metabolic enzymes by realtime-PCR showed that in the cortex,the m RNA expression level of CYP7B1 enzyme in the model group was significantly reduced compared with the normal group,and the m RNA expression level of the positive drug group and the H-dose group of LIG were significantly increased compared with the model group.Compared with the normal group,the m RNA expression level of CYP46A1 enzyme in the model group was increased without significant difference,and compared with the model group,the m RNA expression level was increased in the positive administration group and the H-dose group of LIG.In the hippocampus,the expression level of CYP7B1 enzyme m RNA in the hippocampal region of mice in the model group was significantly reduced compared with the normal control group.Compared with the model group,the expression level of CYP7B1 enzyme m RNA in the active drug group and the M and H-dose group was significantly increased.For CYP46A1 enzyme,compared with the normal group,the m RNA expression level of model mice increased without significant difference.Compared with the model group,m RNA expression levels were significantly increased in the H-dose group（P<0.01）.Compared with the normal group,the m RNA expression level of CYP27A1 enzyme in liver tissues decreased significantly in the model group（P<0.01）,compared with the model group,the m RNA expression level in the active dosing group increased significantly,and the m RNA expression level in the medium and H dose groups of LIG increased（P<0.05）.3.The effect of LIG on brain glucose uptake rate in mice:Using micro-PET method to detect the rate of glucose uptake in the brain of mice,it could be found that comparing with the normal group,the uptake rate of the model group showed a decrease to different degrees,while the LIG administration group had different degrees of upregulation effect.The expression of GLUT4 and IRAP proteins in the hippocampus and cortex of mice were detected by western-blot method,and the relative expression of GLUT4 protein in the model group decreased（P<0.05）compared with the normal group,and the relative expression of GLUT4 protein in the positive group increased（P<0.05）compared with the model group,and the relative expression of proteins in the LIG group increased but there was no statistical difference.There was no significant difference between the expression of IRAP protein in the brain of mice in the normal control group and the model group and the administration groups.4.The effects of LIG on brain inflammatory factors,oxidative stress indicators and neuronal pathological morphology in mice:q-PCR was used to detect related inflammatory factors,and in cortical tissue.Comparing with the normal group,expression of m RNA of the inflammatory factors IL-6 and TNF-αin the cortical area of mice in the model group were increased（P<0.05 or P<0.01）,and compared with the model group,the m RNA levels in the drug group showed different degrees of decrease（P<0.05 or P<0.01）,and TNF-αhad a downward trend in the L-dose group without significant difference.In hippocampal tissues,compared with the normal group,the expression of inflammatory factors（IL-6 and TNF-α）’s m RNA level in the hippocampal region of mice in the model group was increased（p<0.05 or P<0.01）;Compared with the model group,the m RNA levels in the LIG administration group showed different degrees of decrease（P<0.05）.Compared with the normal group,the SOD viability and GSH content of mice in the model group were significantly reduced,and compared with the model group,the GSH level in the H-dose LIG administration group was significantly increased（P<0.01）.The rest of the drug group had some improvement,but there was no difference,and compared with the model group,the SOD level in the LIG administration groups were adjusted to a certain extent,but there was no statistical difference.Compared with the normal group,the H₂O₂ content in the model group was significantly increased（P<0.01）,and compared with the model group,the H₂O₂ content in the positive dosing group and the medium and high dose groups of LIG decreased to varying degrees（P<0.05 or P<0.01）.ConclusionThe long-term feeding of high-fat and high-cholesterol diets could cause cognitive dysfunction and hypercholesterolemia in mice.LIG could improve cognitive dysfunction by regulating the cholesterol metabolism and the glucose uptake in the brain.Besides,LIG could reduce the inflammation and oxidative stress in mouse brain cells.These may be related to the regulation of cholesterol metabolism enzymes（CYP46A1,CYP7B1 and CYP27A1）and the improvement of the glucose uptake in the brain of mice.