Royal Jelly And 10-HDA Activate Autophagy Through The MTOR/ULK1 Pathway To Improve Cognitive Function In Diabetic Mice

Background and Objective:Type 2 diabetes and cognitive dysfunction are highly prevalent disorders worldwide.Type 2 diabetes increases the risk of developing cognitive dysfunction which will carry grave individual,societal and financial burdens.The mechanism and preventive measures of Type 2 diabetes and cognitive impairment are very important.10-hydroxydec-2-enoic acid(10-HDA),a unique component of royal jelly,has a variety of beneficial biological properties.The aim of this study was to investigate whether royal jelly and 10-HDA could improve cognitive function in diabetic mice by restoring autophagic flux in the hippocampus.Methods:Part Ⅰ:Fifty 6-8 week male C57BL/6J mice were randomly divided into five groups of 10 mice each:Normal control(Control,CON),High Fat Diet(HFD),Type 2 diabetes mice(DM),Royal Jelly intervention(DM+RJ)and 10-HDA intervention(DM+10HDA)groups.The mice were given royal jelly(400mg/kg)by gavage daily in the royal jelly group and 10-HDA(100mg/kg)by gavage daily in the 10-HDA intervention group for a total of 28 weeks.After 28 weeks,subjected to glucose tolerance test(OGTT)and Morris water maze test.Lipid-related indexes,and the concentration of 10-HDA cross blood-brain barrier in brain tissue were analyzed by HPLC-MS,the morphological structure of hippocampus was observed by HE staining.The expression levels of autophagy-related proteins and autophagy pathway proteins were detected by Western Blot.Part Ⅱ:Mouse hippocampal neuron HT22 cell was selected and cultured in high glucose.CCK-8 was be used to detect the effects of cell proliferation and toxicity,and 10-HDA intervention on cell viability.High glucose cell model was established and HT22 cells were divided into four groups:control group(Control,25mM D-glucose),high glucose group(HG,125mM D-glucose),10-HDA intervention group(HG+10-HDA,125mM D-glucose+1mM 10-HDA),inhibitor group(HG+10-HDA+3-MA,125mM D-glucose+lmM 10-HDA).Cell morphological changes were observed and the expression of autophagy-related proteins as well as autophagy pathway proteins were detected by Western Blot.Results:Part Ⅰ:①The diabetic mouse model was successfully established,and the fasting blood glucose level of mice was ≥11.1 mmol/L one week after STZ injection;②The body weight of mice in the DM,DM+RJ and DM+10-HDA groups showed a decreasing trend after STZ modeling;③The water intake of diabetic mice increased significantly compared with the CON and HFD groups(P<0.01);④Compared with the CON group,the FBG of diabetic mice increased significantly(P<0.01),10-HDA intervention significantly reduced FBG in diabetic mice compared with the DM group(P<0.05);⑤In the OGTT,the peak blood glucose level was the highest in DM-only mice,and the area under the AUC curve was significantly higher than that in the CON group(P<0.01).10-HDA intervention significantly improved the islet function of diabetic mice,with faster peak recovery and reduced area under the AUC curve;⑥The results of lipid content measurement showed that compared with the control group,the levels of TG,TC and LDL were increased in the DM group mice.There was a significant difference in TG(P<0.05)and a significant decrease in HDL levels.Both royal jelly and 10-HDA interventions reduced TG,TC and LDL levels and increased HDL levels in the diabetic mice,with royal jelly having a more significant effect(P<0.05);⑦During the localized navigation test,from day 1 to day 4,the escape latency of mice in the DM group was significantly longer than that of the CON group(P<0.05),while RJ and 10-HDA interventions significantly reduced the escape latency on day 4.In the spatial exploration test,compared to the CON group,the DM group showed a significant decrease in the dwell time and swimming distance in the target quadrant and the number of times crossing the quadrant where the original platform was located(P<0.05),which were improved to varying degrees by the RJ and 10-HDA interventions,with the 10-HDA intervention being the most significant(P<0.05),and the dwell time in the target quadrant and the number of times crossing the platform in the diabetic mice were significantly improved;⑧The results of HPLC-MS suggest that 10-HDA can cross the blood-brain barrier and is enriched in the brain and hippocampus,and the enrichment is stronger in the hippocampus;⑨HE staining showed that the hippocampal CA1,CA3 and DG areas of mice in the DM group showed obvious pathological changes,and the hippocampal neuronal cells in the DM+10-HDA intervention group were more closely arranged and tended to be in order,with an increase in the number of normal neurons and a reduction in nuclear fixation;⑩The expression of autophagy-related proteins was detected by Western Blot and found that compared with the CON group,the expression of Atg7,Atg16L1,Beclinl,Atg5,Atg12,Atg3,LC3Ⅱ/Ⅰ was significantly lower in the DM group,while the expression of p62 protein was significantly higher(P<0.05);compared with the DM group,the expression of Atg7,Atg16L1,Beclin1,Atg5,Atg12,Atg3,LC3Ⅱ/Ⅰ was significantly higher in the RJ and the 10-HDA intervention group,while the expression of p62 was reduced(P<0.05);11The expression of proteins related to the upstream pathway of autophagy was continued to be examined by Western blot,and the expression of ULK1 in the DM group mice was significantly lower than that in the CON group mice,while after the RJ and 10-HDA interventions,the ULK1 expression increased(P<0.05);on the other hand,the expression of mTOR,a target protein of rapamycin,increased in diabetic mice,and 10-HDA intervention inhibited the mTOR signaling pathway(P<0.05),the difference is statistically significant.Part Ⅱ:①The glucose CCK-8 assay showed that the cell survival rate decreased gradually with the increase of glucose concentration in a concentration-dependent manner,so the DMEM medium with 125mM glucose concentration was selected for 48h to construct the HT22 cell injury model;②The cells were treated with 0.1,0.2,0.4,0.6,1,2,4,and 6mM of 10-HDA for 48h,the survival rate decreased when the concentration of 10-HDA reached 2mM(P<0.01),so ImM was used as the administration concentration for the next experiment;③Compared with the control group,HT22 cells after 48h high glucose intervention,the number of cells was significantly reduced,the cell gap was widened,the arrangement was loosened and HT22 after 10-HDA intervention for 48h showed some degree of improvement in morphology,growth and arrangement distribution.However,after the addition of the autophagy inhibitor 3-MA,the number of cells was significantly reduced again and the degree of damage was increased;④Western blot showed that HG induced a decrease in the expression of Atg7 and LC3Ⅱ/Ⅰ,markers of autophagosome formation,and an increase in the expression of p62,marker of autophagic degradation(P<0.05).10-HDA intervention activated autophagy and increased intracellular Atg7 and LC3Ⅱ/Ⅰ.The autophagy inhibitor 3-MA decreased the expression of LC3-Ⅱ,resulting in an impaired protective effect of 10-HDA against high-glucose-induced injury(P<0.05);⑤ Compared with the control group,mTOR expression was elevated and ULK1 expression was reduced in the HG group(P<0.05),mTOR expression decreased after 10-HDA treatment.10-HDA and 3-MA co-treatment of cells decreased ULK1 expression in HT22 cells and attenuated the protective effect of 10-HDA.Conclusion:1.High-fat diet combined with streptozotocin injection successfully established a diabetic mouse model,which showed learning and memory impairment after 28 weeks.2.Royal jelly and 10-HDA could improve the learning and memory impairment of diabetic mice through activation of autophagy.3.10-HDA can protect high glucose-induced HT22 cells by modulating the mTOR/ULK1 pathway to activate autophagy,suggesting a potential role for 10-HDA in ameliorating cognitive impairment in diabetes.