| Objective The theoretical part of this paper summarizes the understanding of ancient and modern doctors of traditional Chinese medicine on xiaoke and yuzheng,and discusses the theoretical feasibility of treating Diabetes mellitus with Depression(DD)from nourishing Yin and tonifying kidney.The experiment part observes the changes of body weight,fasting blood glucose,depression-like behavior and myelin damage of DD model rats after different doses of Liuwei Dihuang Decoction,and the changes in the activity of key proteins in oxidative stress and Adenosine monophosphate activated protein kinase(AMPK)/protein kinase B(AKT)/Nuclear factor E2 related factor 2(Nrf2)signaling pathways,and changes in autophagy proteins and microglia phenotypes.Then we investigate the dual therapeutic effects of Liuwei Dihuang Decoction on improving blood glucose and depression-like behavior in DD rats,and the effects on promoting myelin sheath regeneration in DD rats and its underlying mechanisms.Methods1.Theoretical part Through consulting ancient medical books and searching modern databases,the understanding of ancient and modern doctors on the pathogenesis of xiaoke and yuzheng is combed,the feasibility of treating DD is discussed from the aspect of kidney deficiency,and the theoretical basis is provided for further exploring the mechanism of Treating DD by Liuwei Dihuang Decoction combined with the modern pharmacological results of Liuwei Dihuang Decoction.2.Experimental part Effect and mechanism of Liuwei Dihuang Decoction on improving depressive phenotype in DD model rats Experimental 1:SD rats were fed with high-fat diet and injected with low-dose Strep To Zotocin(STZ)into tail vein to prepare diabetes mellitus model.Chronic unpredictable mild stres(CUMS)was given for 28 days on this basis,and the DD model was verified by fasting blood glucose and tail suspension experiment.Then DD rats were randomly divided into 5 groups:model group,low-medium-high-dose Liuwei Dihuang Decoction group,fluoxetine group,10 in each group,and SD rats were set as normal group.The daily dose of Liuwei Dihuang Decoction low,medium and high dose group was 3.375/g/kg/d,6.75 g/kg/d,13.5g/kg/d,and the gavage dose of fluoxetine group was 10mg/kg/d.The normal group was given the same volume of physiological saline lavage,a total of 28 d,the same as model group.Body weight and fasting blood glucose were measured weekly.The open field test,tail suspension test and forced swimming test were used to evaluate the depressive behavior in rats after drug intervention.And then,blood and brain tissue samples were collected.Fasting blood glucose and insulin were measured,and insulin resistance index was calculated,Western blot was used to detect Myelin basic protein(MBP),myelin proteolipid protein(PLP)and Myelin oligodendrocyte glycoprotein(MOG)in ventral hippocampus(v HIP)and anterior cingulate cortex(ACC)tissues.The fluorescence expression level of myelin basic protein(MBP)in v HIP and ACC of rats in each group was detected by immunofluorescence method.Experimental 2: Animal grouping and intervention were the same as experiment 1.Malondialdehyde(MDA),reactive oxygen species(ROS)and 8-hydroxy-2 deoxyguanosine(8-Ohd G),superoxide dismutase(SOD)and Glutathione(GSH)in v HIP were measured by ELISA,and Western blot was used to detect phosphorylated adenosine 5'-monophosphate-activated protein kinase(P-AMPK)/AMPK,phosphorylated protein kinase B(P-Akt)/AKT and nuclear factor erythroid-2 related factor 2(Nrf2)in v HIP.Experiment 3:Animal grouping and intervention were the same as experiment1.The interleukin-1?(IL-1?),Tumor Necrosis factor-?(TNF-?)and the anti-inflammatory factor interleukin-4(IL-4),interleukin-10(IL-10)contentin v HIP and ACC was detected by ELISA.The expression levels of Beclin-1,Microtubule-associated protein 1 light chain 3(LC3-II),P62,inducible nitric oxide synthase(i NOS)and Arginase 1(Arg-1)in v HIP and ACC were detected by Western blot.Immunofluorescence staining was performed to observe the intensity of autophagy(co labeled by LC3 and Iba1)in v HIP and ACC.Results Experimental part Effect and mechanism of Liuwei Dihuang Decoction on improving depressive phenotype in DD model rats Experiment 1:1.General conditions:In terms of body weight,the model group had a decrease in body weight,and the difference was statistically significant compared with the normal group(P<0.01).The level of FBG in model group was significantly increased,and the difference was statistically significant compared with normal control group(P<0.01).The blood glucose of model group increased significantly with the extension of stress time.Behavioral results: Compared with the normal control group,the stationary time of rats in the model group was increased in the tail suspension experiment(P<0.01).2.Fasting blood glucose results in each group Compared with the control group,the level of FBG in model group was significantly higher(P<0.01),Compared with model group,FBG of rats in low,medium and high dose liuwei Dihuang Decoction group and fluoxetine group showed a significant downward trend,and the difference was statistically significant(P<0.01).3.Body weight change results Compared with the control group,the weight of rats in model group decreased significantly(P<0.01),Compared with model group,low,medium and high-dose group and fluoxetine group could significantly improve the trend of weight loss in rats(P<0.01).4.The behavioral test Compared with the control group,the central area movement time of rats in the model group decreased in the open field experiment(P<0.01),the stationary time increased and swimming time decreased in suspended tail test and forced swimming(P<0.01);Compared with model group,the low,medium and high-dose group and fluoxetine group significantly increased the movement time of central region in open field experiment(P<0.01,P<0.05),the immobility time was shortened and swimming time was increased in suspended tail test and forced swimming(P<0.01)Compared with fluoxetine group,there was no statistical significance in the medium-high dose group(P>0.05).5.Western blot results Compared with control group,the expression of myelin related protein(MBP PLP MOG)in v HIP and ACC in model group decreased(P<0.01);Compared with model group,the expression of MBP,PLP and MOG protein in v HIP and ACC in medium and high dose group increased(P<0.05,P<0.01).there was no significant difference between the fluoxetine group and the medium and high dose group(P>0.05).6.Immunofluorescence results Compared with the control group,the average fluorescence intensity of MBP in v HIP and ACC in model group was significantly decreased(P<0.01);and the average fluorescence intensity of MBP in the low,medium and high dose groups increased significantly(P<0.05,P<0.01).Experiment 21.Western blot results Compared with the control group,the expression level of P-AMPK p-Akt Nrf2 protein in v HIP in model group decreased(P<0.01);Compared with model group,the expression level of P-AMPK p-Akt Nrf2 protein in v HIP in low-dose group was slightly increased without significant difference(P>0.05),the expression of p-AMPK,p-Akt and Nrf2 protein in v HIP increased in medium and high dose group and fluoxetine group(P<0.01).2.ELISA test results Compared with the control group,the content of SOD and GSH in v HIP in model group decreased(P<0.01),the content of MDA,ROS and 8-OHDG increased(P<0.01);Compared with model group,the content of MDA,ROS and 8-OHDG in v HIP in medium and high dose group decreased(P<0.01),the content of SOD and GSH increased(P<0.05,P<0.01);Compared with fluoxetine group,the effect of high dose on oxidative stress index of v HIP was more significant(P< 0.05).Experiment 3:1.Western blot results Compared with control group,the expression of i NOS protein in v HIP and ACC in model group increased(P<0.01),the expression of Arg1 protein increased slightly(P<0.05);Compared with model group,the expression of i NOS protein decreased and the expression of Arg1 protein increased in medium and high dose group(P<0.05,P<0.01),there was no significant difference between middle and high dose groups(P> 0.05).Compared with control group,the expression of beclin-1 and LC3-II/LC3-I protein in v HIP and ACC in model group decreased significantly,and the expression level of P62 protein increased(P<0.01);Compared with model group,There was no significant difference of the expression of beclin-1,LC3-II/LC3-? protein in v HIP and ACC in low-dose group(P>0.05),the expression of beclin-1 and LC3-II/LC3-I protein in v HIP and ACC in medium and high dose group increased and the expression of P62 protein decreased(P<0.05,P>0.01),there was no significant difference between the medium and high dose groups(P>0.05);There was no significant difference between fluoxetine group and medium and high dose group(P>0.05).2.ELISA test results Compared with control group,the content of il-1? and TNF-? in v HIP and ACC in model group increased significantly(P< 0.01),the content of il-4and IL-10 increased in different degrees(P<0.01,P<0.05);Compared with the model group,there was no significant difference in the content of inflammatory factors in v HIP and ACC in low-dose group(P>0.05),the content of IL-1? and TNF-? in medium and high dose group decreased(P<0.05),the content of IL-4 and IL-10 Increased(P<0.01).There was no statistically significant difference between the medium and high dose groups(P>0.05).3.Immunofluorescence results Compared with the control group,the number of LC3+Iba1+immunofluorescence co-staining cells in v HIP and ACC in model group decreased(P<0.01);Compared with model group,LC3+Iba1+ cell number increased in v HIP and ACC in medium and high dose group(P<0.05);compared with fluoxetine group,there was no significant difference in the high-dose group(P>0.05).Conclusion1.Liuwei dihuang decoction improves the sugar metabolism,At the same time can reduce the myelin damage,improve behavior of depression,may be a better treatment options in patients with DD.This study further confirmed the myelin damage play an important role on the mechanism of the DD,may provide more intervention targets and treatment strategies for the treatment of DD and mechanism research.2.Liuwei Dihuang Decoction up-regulated the expression of Nrf2 protein through AMPK/Akt pathway,inhibited oxidative stress and alleviated myelin damage.3.Liuwei Dihuang Decoction promotes M2 polarization of microglia cells by enhancing autophagy and ultimately alleviates myelin damage. |
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