Studies On Effects And Mechanism Of Puerarin On Diabetes Mellitus Induced Depression In Mice

The statistical data released by the International Diabetes Federation(IDF)in 2019 shows that there are 116.4 million patients with diabetes in China,which is becoming a major social problem threatening public health.Approximately 30%of patients with diabetes have depressive symptoms,that seriously impairs the quality of life and stable control of the disease,and the prognosis of patient gets worse and the mortality of patient increases.At present,there is no specific medicines for treating diabetes combined with depression effectively.The clinical treatment mainly adopts the combination of hypoglycemic medicines and antidepressants,thus it is difficult to control the comorbidity safely and effectively over the long haul.Long-term clinical practice has proved that Traditional Chinese Medicine(TCM)has unique advantages in the treatment of many chronic diseases.As a Chinese herb,Pueraria lobata and its relative compound decoctions have the significantly antidepressant effects,and are widely applied in treatments of depression after stroke,or cardiovascular disease combined with depression.Our previous study focused on the hypoglycemic mechanism of puerarin which is the active ingredient of Pueraria lobata.However,the potential effect of puerarin on depression symptoms caused by type 2 diabetes and the underlying mechanism are still largely unknown.In the present study,effects of puerarin on alleviating depression-related symptoms were evaluated in a type 2 diabetic(T2D)mouse model by behavioral analysis,cell and molecular biology experiments and other technique.The mechanism of puerarin in regulating hippocampal neuroplasticity of T2D mice was clarified further.Moreover,impacts of metformin and fluoxetine on hyperglycemia and depression of T2D mice were simultaneously investigated to compare with effects of puerarin,which would provide experimental evidence for the potential application of puerarin in treating diabetes combined with depression.The main experimental results are as follows:(1)Db/db diabetic mice and high-fat diet(HFD)induced diabetic mice were applied in our experiments to investigate the depression-related symptoms and pathological changes.The depression-like behaviors of db/db mice and HFD mice were observed and confirmed via analysis of the open field test,sucrose preference test,tail suspension test and forced swimming test.Using ELISA assay,it was found that 5-hydroxytryptamine(5-HT)in the serum of diabetic mice was decreased compared to control mice,while levels of corticosterone(CORT),IL-1β and IL-6 were significantly increased.Moreover,western blotting(WB)tests showed that levels of p-ERK,p-CREB,and BDNF in the hippocampus of diabetic mice were decreased compared to control mice,which suggested that hippocampal neuroplasticity was impaired.Meanwhile,we observed the significant down-regulation in expressions of GLP-1R and VDR in the hippocampus of diabetic mice compared to control.(2)To further evaluate effects and mechanism of puerarin on depression-related symptoms and pathological changes in HFD diabetic mice,behavioral analysis,ELISA,cell culture,TUNEL staining and WB were performed.The results showed that puerarin effectively relieved the depression-like behaviors of HFD mice,down-regulated levels of CORT and IL-1β in serum,up-regulated 5-HT respectively.Simultaneously,the expressions of GLP-1R and VDR in the hippocampus of HFD mice were enhanced by puerarin,and p-AKT,p-β-catenin,p-ERK,p-CREB,BDNF,p-TrkB,NCAM2 which are involved in hippocampal neuroplasticity were promoted subsequently.Notably,puerarin can effectively protect the survial of mouse hippocampal neuron cell line HT-22 cells and suppress the apoptosis of HT22 cells treated with high glucose and corticosterone.Furthermore,puerarin reduced the fasting blood glucose of HFD mice,improved oral glucose tolerance test(OGTT),promoted activations of AMPK and AKT in the liver of HFD mice to ameliorate insulin resistance.Metformin presented strong antidiabetic effects on HFD mice.However,the antidepressant effect of metformin is not as significant as puerarin.Fluoxetine relieved depression-related symptoms of HFD mice,but has no effects on glucose lowering.These findings indicate that puerarin has unique advantages in the treatment of comorbidity of diabetes and depression.(3)Here we noticed that the expression of VDR in the hippocampus of T2D mice down-regulated significantly.Our previous experiments also demonstrated that VDR knockout mice showed depressive disorder and hippocampal neuroplasticity impaired.In order to further clarify the relevance of VDR and depressive disorder,we performed RNA sequencing analysis using the hippocampus of VDR knockout mice and wild type mice.The differential matrix of mRNA,lncRNA,and circRNA were obtained,and related genes and signal pathways were analyzed and discovered.The data suggest that VDR knockout would significantly affect multiple pathways such as "long-term depression","neurotrophic pathway","insulin resistance",and "PPAR signaling".Transcripts related to depression,such as PPP2R1B,ARAF,PLA2G5,and transcripts involved in glucose metabolism including HK1,CaCNA1c and MAPK3 are all changed significantly.Notably,genes regulated by differential circRNA are closely related to synaptic formation and development,suggesting that VDR may regulate neuronal synaptic functions through the circRNA.Summary:Our previous study reported that puerarin could promote β-cell functions by activating the GLP-1R pathway.The present study demonstrated that puerarin could ameliorate depressive symptoms in HFD diabetic mice,and clarified that puerarin protected hippocampal neuroplasticity via up-regulating GLP-1R expression and activating downstream signaling including Akt,β-catenin,ERK,CREB,BDNF and TrkB.Type 2 diabetes and depression are chronic diseases and required long-term medication.Therefore,Pueraria lobata,used as medicine and food,has unique advantages in the treatment of comorbidities of diabetes and depression.Our study provides experimental evidence for the potential application of Pueraria lobata in treating comorbidities of diabetes and depression.Simultaneously,the transcriptome sequencing and analysis of the hippocampus of VDR knockout mice would contribute to elucidate the role of VDR in regulating glucose homeostasis and depression.