| BackgroundIn recent years,the prevalence of diabetes in China has increased dramatically,according to the International Diabetes Federation’s November 2021 update:the number of adults with diabetes worldwide has reached 537 million,accounting for 10.5%of the total global population;the number of Chinese with diabetes has increased from 90 million to 140 million between 2011-2021.Diabetes can lead to mood disorders in addition to physical symptoms,with depression being one of the more common mood disorders,with depressed mood and lack of pleasure as the main symptoms,along with slow activity,loss of appetite and feelings of helplessness.The risk of depression is relatively higher in people with diabetes,with studies showing that more than 30%of people with diabetes are diagnosed with depression;the tendency to depression is three times higher in people with diabetes than in the general population.Although there was no exact records of diabetes mellitus combined with depression in the ancient texts of traditional medical classics,there are scattered records of related clinical manifestations in the ancient books of past dynasties.Diabetes mellitus is assigned to consumptive thirst in the traditional Chinese medicine(TCM).The symptoms of depression are rather complex and have many similarities with clinical manifestations such as lily disease,visceral agitation and depression syndrome in TCM.Modern research has demonstrated that liver-related pathological changes caused by various stresses,including the state of persistent hyperglycemia,may be related to functional changes of in neuro-endocrine-immune network.There is close relation between the function of the liver and the excitatory or inhibitory state of the cerebral cortex,and the sympathetic nerve predominantly autonomic function.At present,symptomatic treatment of hypoglycemic with antidepressant is commonly used treatment methods.There are disadvantages of frequent recurrence and serious adverse reflects.Brain-derived neurotrophic factor(BDNF)is a kind of nutritional factor related to nerve,immunity and metabolism.It provides necessary nutritional support for the survival of and normal physiological function of neurons.BDNF and its cognate receptors tropomyosin receptor kinase B(TrkB)not only play an important role in neuronal survival,differentiation and plasticity,but also regulate energy and glucose homeostasis by affecting of the central nervous system.Research shows that vagus nerve stimulation(VNS)can increase hippocampal BDNF levels and thus activate neuroprotective pathways to alleviate depressive symptoms,as well as regulate glucolipid metabolism and improve insulin resistance.However,it has surgical complications such as susceptibility to infection,dyspnea and hoarseness.Neuroanatomical studies manifest that the auricular concha,where auricular acupoints such as heart(CO15),liver(CO12)and spleen(CO13)locate,is the only body surface with vagal afferent fibers are distributed.The research on transcutaneous auricular vagus nerve stimulation(taVNS)conducted by our group over the years has fully confirmed the efficacy of taVNS based on VNS technology and auricular acupoint theory of TCM in treating diabetes and depression.VNS can improve depression-like behavior in diabetic rats while regulating glucolipid metabolism.The activation of vagus nerve improves liver metabolic function through the hepatic branch of vagus nerve while inhibiting apoptosis.However,the exact biological mechanism is not yet clear.The hippocampus is an important brain region for regulating emotion and also for glycolipid metabolism.The liver is also one of the peripheral target organs that maintain metabolism and mood regulation.Thus,it can be concluded that vagus nerve regulates the function of liver-brain axis through BDNF-TrkB pathway to achieve hypoglycemic and antidepressive effects.Therefore,the modeling method,high-fat diet combined with intraperitoneal injection of streptozotocin(STZ),was used to make the diabetic rats in this study.An implantable physiological signal telemetry system invented by American data sciences international(DSI)was used to detect the effect of taVNS on instant blood glucose once every 30s in diabetic rats to achieve real-time in vivo monitoring.The effect mechanism of taVNS in regulating glucolipid metabolism and antidepression will be investigated in diabetic rats by using K252a,the TrkB receptor specific blocker,and exploring the effect of taVNS on the behavior,biochemical indicators related to glucolipid metabolism,related proteins of BDNF-TrkB pathway in the hippocampus and liver and hippocampal neuronal apoptosis.Aim(1)To explore effect of taVNS on real-time blood glucose in diabetic model rats;(2)To investigate effects of taVNS on the behavior and glucolipid metabolism of in diabetic model rats;(3)To elucidate the effect mechanism of taVNS on ameliorating depression-like behavior and disorder of glucolipid metabolism in diabetic rats from the perspective of the mediation of BDNF-TrkB pathway in the hippocampus and liver.MethodsStudy 1 The effects of taVNS on real-time blood glucose in diabetics ratsTwelve Sprague-Dawley(SD)rats were randomly divided into control group(group C),a model group(group M),and taVNS group(group T).After 4 weeks of high-fat diet,rats in Group M and Group T were fed with high-fat diet and intraperitoneally injected with 1%STZ according to the proportion of 35 mg/kg(dissolved into 0.1 mol/L with citrate buffer,pH=4.4)to keep the rats in a continuous hyperglycemic state.Fasting blood glucose(FBG)≥16.7 mmol/L was used as the standard to determine whether the model was successful.Rats in Group C were given a normal diet.All rats were given adequate diet and kept in natural day/night cycle.Group M and Group T:After successful modeling,taVNS was given under the condition of inhaled isoflurane.Under 2%isoflurane inhalation anesthesia,Han’s electroacupuncture apparatus was used to connect the two positive and negative poles of the electrodes which can be fixed with self-absorbed conductive magnets to noninvasively fixed onto the anterior and posterior of the auricular concha.The selected electrical stimulation site is the distribution area of the auricular vagus nerve.Stimulation parameters are dense-disperse wave,2/15 Hz and 2mA.The duration is 30 min each time,once a day for 7 days.After modeling,rats in Group M were given free food and water without intervention.An implantable physiological signal telemetry system invented by American data sciences international(DSI)was used to detect blood glucose and temperature at different time points under the state of being awake and free to move.Study 2 The effects of taVNS on depression-like behavior and glucolipid metabolism in diabetic ratsSixty healthy adult male SD rats of SPF class with a body mass of 200±20g were randomly divided into groups C,M,T,isoflurane control(IOS,group I)and TrkB receptor-specific blocker control(K252a,group K)after one week of adaptive feeding,with 12 rats in each group.The modeling method and criteria were the same as in Study 1.Every two weeks,the scores of open field experiment and the immobility time of forced swimming were recorded to evaluate the depression-like behavior.In the sixth week,rats in groups T and K were intervened for 21 consecutive days.Rats in Group C and M were not given taVNS intervention.Rats in Group K was injected intraperitoneally with K252a(25 ug/kg),the TrkB-specific blocker 30 min before taVNS.Rats in Group I were grasped simultaneously with group T under the anesthesia condition of inhaled,but not given taVNS intervention.Body mass and FBG were measured weekly.Open field experiment and forced swimming experiment were performed every two weeks.Caudal vein blood was collected before and after modeling respectively to measure serum insulin levels of all rats.Blood was collected from the abdominal aorta.Enzyme linked immunosorbent assay(Elisa)technique was applied to measure triglyceride(TG),serum total cholesterol(TC),low density lipoprotein(LDL-C)and glycosylated hemoglobin(HbA1C).Hematoxylin-eosin staining(HE)was used to observe the morphological changes of pancreatic tissue.Study 3 BDNF-TrkB pathway mediates the effect mechanism of taVNS to improve glucolipid metabolism and depression-like behavior in diabetic rats Western blot(WB)was applied to measure the protein expression of BDNF,TrkB,PI3K,AKT and mTOR in the hippocampus and liver of diabetic rats.HE staining was employed to observe morphological changes of hippocampal tissue.TUNEL method was used to investigate the apoptosis of hippocampal neurons in CA1 region.ResultsStudy 1 Effects of taVNS on real-time blood glucose with diabetes mellitus model rats Diurnal variation of blood glucose(1)Diurnal fluctuation of blood glucose:Compared with group C,the rats in group M had greater diurnal fluctuation of blood glucose;The diurnal fluctuation of rats in Group T tended to be similar to that of Group C after taVNS.(2)Diurnal fluctuation of temperature:Compared with group C,rats in Group M had greater diurnal fluctuation in temperature(P<0.05).The diurnal fluctuation of rats in Group T was significantly reduced compared with group M after taVNS(P<0.05).(3)Correlation analysis between temperature and blood glucose of rats in rats of all groups:There was a positive correlation between the temperature and blood glucose in all groups(Group C,r=0.219;Group M:r=0.097;Group T,r=0.359).In other words,temperature increases with the increase of blood glucose.(4)The effect of taVNS on real-time blood glucose in rats:TaVNS caused an increasing trend of random blood glucose in all groups.The random blood glucose of rats in Group M decreased to the lowest value at the time,25 min after the end of taVNS on Day 1 and Day 4,and 40 min after the end of taVNS on Day 7.Then it had an increasing trend,increased and kept in a steady state at the time,60 min after the end of taVNS.The final level were significantly lower than that before taVNS on Day 1,4 and 7(all P<0.05),respectively.The final level on Day 7 were significantly lower than the initial level on,Day 1(P<0.05).After continuous stimulation for 7 days,there was no statistical difference between random blood glucose in rats of Group C compared with that of the initial level(P<0.05).The random blood glucose in Group M rats was significantly lower than that of the initial random blood glucose(P<0.01).Study 2 The effects of taVNS on depression-like behavior and glucolipid metabolism in diabetic ratsGeneral:Rats in Group C were active,responsive,well-proportioned,with a continuous increase in body mass,healthy and lustrous hair,normal stool and urine,and transparent corneas.Rats in group M showed obvious hyperphagia,polydipsia and polyuria.The growth rate of body mass was significantly reduced compared with Group C.It manifested typical symptoms of diabetes,"three more and one less".With the persistence of hyperglycemia,the rats gradually became thin,less active,depressed,unresponsive,with dull,yellowing and shedding fur,and some rats had loose stools after blood collection.There was little change in the comparison between group I and K and group M.Body mass:There was no statistical difference in body mass between the groups before modeling(P>0.05);body mass of rats fed with high-fat diet increased faster and showed statistical difference at 3w(P<0.01).There was a significant decrease in body mass at 7w(P<0.05).After taVNS intervention,the decrease in body mass of rats in group T was alleviated compared to group M.The effect of taVNS could be blocked by blocking agents.(1)Open-field experiment:Before modeling,there was no statistical difference between the horizontal and vertical movement scores of the rats in the open-field experiment(P>0.05),and the groups were comparable;at the beginning of the 4th w,the horizontal and vertical movement scores of the rats in the open-field experiment were significantly lower in the M group compared with the C group(P<0.001),indicating that the rats in the M group had reduced exploratory ability and showed anxiety and depression-like behavior.At the 3rd w of taVNS intervention,the horizontal and vertical locomotion scores of rats in group T increased significantly compared with group M(P<0.01);the horizontal and vertical locomotion scores of rats in group K decreased significantly compared with group T(P<0.01).(2)Forced swimming experiment:Before modeling,there was no statistical difference(P>0.05)in the resting time of each group of rats in the forced swimming experiment,and the groups were comparable;at the beginning of 4w,compared with group C,the resting time of forced swimming in group M and group T increased significantly(P<0.01),indicating that the rats in group M showed depression-like behavior;after 3w of taVNS intervention,compared with group M,the resting time of forced swimming in group T decreased significantly(P<0.01);compared with group T,group K increased significantly(P<0.01).After 3w of taVNS intervention,the forced swimming resting time was significantly reduced in the T group compared with the M group(P<0.01);compared with the T group,the forced swimming resting time was significantly increased in the K group(P<0.01).(3)Fasting blood glucose:Compared with group C,the FBG of rats in group M was significantly higher from 5w(P<0.001);after taVNS intervention,the blood glucose of group T was significantly lower at 7w compared with group M(P<0.001).Blood glucose was significantly higher in rats in group K compared with group T(P<0.01).(4)Serum INS,TG,TC and LDL-C contents:there was no statistical difference in serum INS,TG,TC and LDL-C contents among the rats in each group before modeling(P>0.05),and the groups were comparable;at the beginning of the 4w,compared with group C,the serum INS contents of rats in groups M and T increased significantly(P<0.001),and this experiment indicated that rats in group M showed insulin After 3w of taVNS intervention,serum INS,TG,TC and LDLC levels decreased significantly in group T compared with group M(P<0.001),while serum INS,TG,TC and LDL-C levels increased significantly in group K compared with group T(P<0.001).(5)GHbA1C:GHbAlC levels were significantly increased in rats in group M compared with group C(P<0.01);after 3w of taVNS intervention,GHbA1C levels were significantly decreased in group T compared with group M(P<0.01).Compared with the T group,GHbA1C was significantly increased in the K group rats(P<0.05).(6)Morphological changes of the pancreas:the islets of rats in group C were round or oval cell clusters with intact,regular structure and clear edges,and the pancreatic islets were rich in β-cells,uniform in size,full and closely arranged;the pancreatic cell clusters of rats in group M were irregular in shape,the edges of the islets were unclear and disordered in structure,and the cytoplasmic staining of the islets was light or vacuolated;compared with rats in group M,the pancreatic tissue morphology and structure of rats in group T were improved;compared with rats in group T,the pancreatic tissue morphology and structure of rats in group K were not significantly changed.Study 3 BDNF-TrkB pathway mediates the effect mechanism of taVNS to improve glucolipid metabolism and depression-like behavior in rats with diabetes mellitus model(1)Protein expression of BDNF,TrkB,PI3K,AKT,mTOR in hippocampus and liver:compared with group C,the protein expression of PI3K,AKT,mTOR in BDNF-TrkB pathway as well as downstream pathway was decreased in hippocampus of rats in group M(P<0.05).Compared with the M group,the expression of PI3K,AKT,and mTOR protein in the BDNF-TrkB pathway as well as the downstream pathway was significantly higher in the T group rats(P<0.01),while the expression of BDNF,PI3K,AKT,and mTOR protein was significantly lower in the I group(P<0.05),and there was no statistical difference in TrkB protein expression;compared with the T group,the expression of BDNF,TrkB,PI3K,AKT,and mTOR proteins were significantly lower in the K group compared with the T group(P<0.05).(2)Morphological changes of hippocampal neurons:In group C,hippocampal neurons in CA1 area were regular in morphology,arranged in a tight and orderly manner,with clear cellular structure and uniform chromatin,and normal neuronal gaps,with round or oval nuclei.In group M,hippocampal neurons were swollen,with nuclear consolidation,strong cytoplasmic staining,vacuolated structure,irregular arrangement,disorganized hierarchy,and extended neuronal gaps.Compared with the group M,the number of hippocampal vacuolated cells in the group T was significantly reduced and the arrangement was slightly more regular,while the hippocampal neurons in the group I were more disorganized and arranged more loosely.Compared with the group T,the hippocampal neurons in the group K were less regular in shape and slightly disorganized in arrangement.(3)Hippocampal neuron apoptosis rate:Compared with group C,the hippocampal neuron apoptosis rate of rats in group M increased significantly(P<0.01);compared with group M,the hippocampal neuron apoptosis rate of rats in group T showed an increasing trend,but there was no statistical difference(P>0.01);compared with group T,the hippocampal neuron apoptosis rate of rats in group K showed an increasing trend,but there was no statistical difference(P>0.01).ConclusionsStudy 1 Effects of taVNS on real-time blood glucose in diabetic ratsHyperglycemic state increased circadian blood glucose and body temperature fluctuations in model rats,while taVNS ameliorated physiological rhythm disturbances and significantly reduced random blood glucose in diabetic model rats.Study 2 Effects of taVNS on depression-like behavior and glucolipid metabolism in diabetic ratsDiabetic model rats prepared by high-fat diet combined with intraperitoneal injection of STZ can spontaneously develop depression-like behaviors.taVNS exerts hypoglycemic effects by regulating glucolipid metabolism in diabetic model rats,while improving depression-like behaviors in diabetic rats.And these effects of taVNS can be blocked by K252a,a specific blocker of TrkB.Study 3 BDNF-TrkB pathway mediates the effect mechanism of taVNS to improve glucolipid metabolism and depression-like behavior in diabetic ratsThe BDNF-TrkB pathway and the PI3K,AKT and mTOR proteins of the downstream pathway were significantly reduced in diabetic model rats.taVNS stimulation significantly upregulated the protein expression of BDNF,TrkB,PI3K,AKT and mTOR in hippocampus and liver of diabetic model rats,and then inhibited hippocampal neuronal apoptosis,while the specific blockade of TrkB could inhibit the above effects,suggesting that hippocampal and liver BDNF-TrkB signaling pathway may be one of the mechanisms of the hypoglycemic and antidepressant effects of taVNS. |
