| Objective:Type 2 diabetes is a widespread disease in the world,which seriously affects the health of patients and the economic development of society.With the continuous deepening of diabetes research,more and more scholars have found that diabetic patients are at a higher risk of developing cognitive dysfunction.Central nervous system disease is one of the complications of diabetes.Due to its high incidence,it is necessary to study its pathogenesis and treatment strategies.Studies have shown that brain Aβ deposition is a typical pathological change in patients with diabetes-associated cognitive decline.Brain insulin resistance,blood-brain barrier changes,and abnormal intestinal flora are all closely related to the metabolism of central and peripheral Aβ.The surface of certain intestinal flora can produce amyloid fiber aggregates,which can participate in the formation of biofilms,promote the binding of bacteria and proteins to produce complexes to further regulate signal pathways or produce pro-inflammatory cytokines to cause neuronal apoptosis.The previous research showed that the ZiBu PiYin recipe can reduce the expression of Aβ in hippocampal neurons,improve insulin resistance,reduce neuronal degeneration,protect mitochondria,improve intestinal flora,and slow down the cognitive function of rats with diabetes-associated cognitive decline,it has obvious effect in slowing down the occurrence and development of diabetes-associated cognitive decline.However,it is still unclear whether it changes the central Aβ metabolism by regulating peripheral metabolism and intestinal flora.Therefore,this study explored the effects of ZBPYR on the production,transport and clearance of Aβ,as well as the changes in the structure of the blood-brain barrier in ZDF rats with Diabetes-associated cognitive decline.In addition,we analyzed the relationship between intestinal flora and Aβ metabolism to provide a research basis for diabetes-associated cognitive decline based on "treatment from the spleen".Methods:We selected male 5-week-old Zuker diabetes fatty(ZDF,fa/fa)rats and their littermate control male Lean Zuker rats(LZ,fa/+)housed in a specific pathogen-free(SPF)animal experiment center,they were randomly divided into normal group(L),diabetes model group(ZDF),diabetes model+high dose ZBPYR group(HZ),diabetes model+medium dose ZBPYR group(MZ),diabetes model+low dose ZBPYR group(LZ).The normal group was fed with ordinary feed,other groups were fed with Labdiet 5008 induction feed.HZ,MZ,LZ groups were given high,medium and low doses of ZBPYR by oral gavage per day,and the control group was orally administered an equal dose of ultrapure water instead of ZBPYR.The experiment was divided into two phases.The rats were in the type 2 diabetes stage from 5 weeks to 9 weeks,and in the diabetic cognitive dysfunction stage from 9 weeks to 15 weeks.Random blood glucose(RBG),body weight,and abdominal circumference were measured weekly.When the rats were 9 weeks old,We performed oral glucose tolerance test(OGTT),insulin tolerance taet(ITT)and Fasting serum insulin(FSI)to observe the diabetes status of the rats in each group,and observed the Aβ deposition of brain tissue and pancreatic tissue at the stage of type 2 diabetes by immunohistochemical experiments.At 15 weeks of age,we used the Novel object recognition(NOR)and the Morris water maze(MWM)experiment to observe the changes in cognitive function of ZDF rats with Diabetes-associated cognitive decline.At the same time,we collected all rat feces for 16S rRNA sequencing to observe the changes of intestinal flora.We used ELISA and immunohistochemical experiment to observe the changes of β-amyloid(Aβ)protein deposition in central hippocampus,cortex and peripheral plasma.Western blotting was used to observe Aβ production-related proteins(APP),β-site APP cleaving enzyme 1(BACE 1),transport-related protein Low-density lipopro-tein receptor-related protein 1(LRP 1),receptor for advanced glycation end products(RAGE),and removal of related proteins insulin degrading enzyme(IDE),neprilysin(NEP)expression changes of ZDF rats with Diabetes-associated cognitive decline.The transmission electron microscope(TEM)was used to observe the structural changes of the blood brain barrier(BBB).Result:1.ZBPYR can improve the symptoms of hyperglycemia and cognitive dysfunction in spontaneously obese ZDF rats.ZDF rats have the characteristics of type 2 diabetes at 9 weeks of age,such as increased random blood glucose,increased diet and water intake,and insulin resistance.At 15 weeks of age,they have cognitive dysfunction and reduced learning and memory capabilities.ZBPYR can reduce random blood glucose,reduce the amount of food and drink,improve insulin resistance,and improve learning and memory ability.Among them,high doses of ZBPYR have the best effect.2.ZBPYR can improve Aβ metabolism in ZDF rats.By comparing the deposition of Aβ in central brain tissue and peripheral pancreatic tissue,we found that there was a small amount of Aβ deposition in hippocampus,cortex and pancreatic tissue in model group at 9 weeks of age,and Aβ increased significantly at 15 weeks of age.ZBPYR could reduce the deposition of Aβ in the cytoplasm of neurons in hippocampus,cortex and pancreatic tissue,and reduce the level of Aβ in plasma of 15 weeks old rats.By observed the expression of Aβ metabolism related proteins at 15 weeks of age,it was found that the expression of Aβ production related proteins APP and BACE1 in hippocampus and cortex of model group was significantly increased;the expression of Aβ transport related protein RAGE was increased,the expression of LRP1 was decreased;the expression of Aβ clearance related proteins IDE and NEP in hippocampus,cortex,liver and kidney were decreased,while ZBPYR could improve the expression of central and peripheral Aβ metabolism related proteins expression.3.ZBPYR can regulate the changes of intestinal flora in ZDF rats.The results showed that ZBPYR could increase the diversity and abundance of intestinal flora,and adjust the structure and composition of intestinal flora in ZDF rats.At the phylum level,we found that ZBPYR could down regulate the abundance of Firmicutes,Proteobacteria and up regulate the abundance of Bacteroidetes in ZDF rats.At genus level,ZBPYR could up regulate the abundances of Clostridium,Pseudomonas and Streptococcus,and decrease the abundances of coprococcus,Allobaculum and Blautia.Correlation analysis showed that coprococcus was positively correlated with APP,BACE1 and RAGE,and negatively correlated with LRP1,IDE and NEP,while the correlation analysis of Streptococcus was opposite to Coprococcus.Conclusion:ZBPYR can improve the structure of blood-brain barrier,reduce the deposition of Aβ in central and peripheral tissues,and slow down the occurrence and development of diabetic cognitive impairment by improving the expression of Aβ production,transport and clearance related molecules in ZDF rats.In addition,ZBPYR can improve the diversity and composition of intestinal flora,down regulate the abundance of Coprococcus,which is closely related to Aβproduction,and up regulate the abundance of Streptococcus,which is closely related to Aβclearance.This research improved the mechanism of ZBPYR in the prevention and treatment of diabetes-associated cognitive decline from the perspective of Aβ metabolism,and screened out the different bacteria closely related to Aβ metabolism,suggesting that the way of ZBPYR in improving diabetic cognitive dysfunction may be closely related to the change of intestinal flora. |
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