| Background:Type 2 diabetes mellitus(T2DM)is one of the most common and serious chronic metabolic diseases,which has a huge negative impact on the lives of people all over the world.Major Depressive Disorder(MDD),one of the mental disorders associated with diabetes,is characterized by loss of interest in daily activities,cognitive deficits and social dysfunction.In T2DM,the occurrence of depression is bidirectionally associated with it and may be related to a low-grade systemic inflammatory response,with diabetes-related inflammatory factors leading to depression and depression promoting the inflammatory response and thus lowering glucose tolerance and aggravating the abnormal blood glucose.Modern medicine believes that there is a common biological origin between diabetes and depression,especially the chronic cytokine-mediated inflammatory response caused by innate immune over-activation,and the treatment is mostly a combination of antidepressants and hypoglycemic drugs,but due to the interaction between drugs is prone to adverse reactions such as weight,body fat abnormalities.Traditional Chinese medicine(TCM)believes that diabetes mellitus accompanied by depression belongs to the category of "thirst and syndrome stagnation",which is mostly caused by blood stasis and liver depression due to the deficiency of both qi and yin,and treatment should be based on the patient’s specific evidence.Therefore,there is an urgent need for a safe and effective non-drug therapy for T2DM concomitant depression.Vagus nerve stimulation(VNS)has been approved by the Food and Drug Administration(FDA)for the treatment of depression since 2005.The vagus nerve is also involved in energy metabolism,food intake and blood glucose regulation,and plays an important role in controlling pancreatic hormone secretion by influencing glucagon and insulin secretion to regulate blood glucose.The auricular acupuncture is performed by stimulating disease response points on the surface of the auricle.The visceral representative area of the external auricular acupuncture points is located in the auricular nail region,where vagus nerve afferent fibers are distributed.Therefore,based on the combination of the TCM theory of auricular acupuncture and principles of modern neuroscience,our team innovatively proposed a new method for treating diseases-transcutaneous auricular vagus nerve stimulation(taVNS),taVNS has been found to have significant antidepressant and hypoglycemic effects in previous studies,so we believe that taVNS also has a certain alleviating effect on depressive-like behaviors associated with diabetic rats.Central inflammatory response is the key pathogenesis of diabetes mellitus with depression.Purinergic P2X7 receptor(P2X7R),a purinergic P2 receptor,plays an important role in the innate immune response by regulating the expression of pro-inflammatory cytokines in the Interleukin-1(IL-1)family.P2X7R can activate NOD-like receptor protein 3 inflammatory vesicles NOD-like receptor family pyrin domain-containing 3(NLRP3),causing IL-1βrelease induces depression-like behavior and insulin resistance in rats,NLRP3 inflammasomes and upstream P2X7R are biological substrates of diabetes and depression,and the P2X7R/NLRP3/IL-1β pathway is an important pathogenesis of diabetes associated depression.Therefore,this study used Zucker Diabetic Fatty(ZDF)rats as the research object to explore the effects of taVNS on P2X7R,NLRP3 and IL-1β neuroinflammatory markers of prefrontal cortex(PFC)microglia to further elucidate the central antidepressant mechanism of taVNS alleviating depression-like behavior in ZDF diabetic rats.Objectives:(1)To investigate the effect of taVNS on blood glucose in ZDF diabetic rats.(2)To investigate the effect of taVNS on depressive-like behavior in ZDF diabetic rats.(3)To investigate the effect of taVNS on the morphology of PFC microglia and the expression of P2X7R,NLRP3 and IL-1β proteins in ZDF diabetic rats,and to elucidate the central antidepressant mechanism of taVNS in ZDF diabetic rats.Methods:SPF-grade male 6-week-old ZDF rats(n=30)and Zucker Lean(ZL)rats of the same litter(n=10)with body masses of 150-180 g.After 1 week of adaptive feeding,10 ZL rats were fed normal chow as a blank group(ZL group),and 30 ZDF rats were randomly divided into a model group(ZDF group),a transcutaneous auricular vagus nerve stimulation group(ZDF+taVNS group)and a sham stimulation group(ZDF+sham-taVNS),the rats were housed in a single cage and fed with high fat diet Purina#5008(23.5%crude fat,6.5%crude protein).After successful modeling,the rats in the ZDF+taVNS group were subjected to taVNS intervention for 6 consecutive weeks starting from the 8th week:under isoflurane inhalation anesthesia at 2%concentration,the positive and negative self-absorbing conductive electromagnets were connected and fixed non-invasively to the bilateral auricular cavity of the rats from 14:00 to 16:00 pm daily,and electrical stimulation intervention was performed using a HANS electroacupuncture instrument with the following stimulation parameters:the intensity is 2 mA,the frequency is 2/15 Hz,dense wave,30 min once,once a day.The duration and location of the intervention in the ZDF+sham-taVNS group were the same as those in the ZDF+taVNS group,but the rats were not electrified.Body mass and fasting blood glucose(FBG)were measured weekly in each group to assess the hypoglycemic effect of taVNS.The behavioral changes of the rats were observed every 2 weeks in the open field test(OFT)and forced swimming test(FST)to observe the behavioral changes of ZDF rats to evaluate the preparation of diabetic model with depression and the antidepressant effect of taVNS.After the experiment,blood was collected from the abdominal aorta,and serum glucagon,glycosylated hemoglobin and insulin concentrations in each group of rats were detected by ELISA technique;the PFC of each group of rats was taken freshly,and the expression of P2X7R,NLRP3 and IL-1β in each group of rats was detected by Western Blot technique;The morphology of PFC microglia and the expression of P2X7R,NLRP3 and IL-1β were observed by immunofluorescence technique.Results:(1)Body mass:Rats in the ZDF group,ZDF+taVNS group,and ZDF+sham-taVNS group fed with high fat diet purina#5008 for 8 weeks always had higher body mass than rats in the ZL group(P<0.01);after the intervention from the 9th week,the body mass of rats in the ZDF group showed a decreasing trend,but was still higher than that in the ZL group(P<0.01);rats in the ZDF+taVNS group were higher than the ZDF group,but the difference was not statistically significant(P>0.05).(2)FBG:Before modeling,there was no difference in FBG among the rats in each group(P>0.05).After feeding with high-fat diet Purina#5008 for 8 weeks,the FBG of rats in ZDF group was always higher than that of rats in ZL group during the period(P<0.01);after the intervention,the FBG of rats in ZDF+taVNS group started to decrease from the week 9 and was significantly lower than that of ZDF group from week 12 to week 14(P<0.01);compared with the ZDF+taVNS group,the FBG of ZDF+sham-taVNS rats first decreased and then increased,and was significantly higher than that of the ZDF+taVNS group at week 10-14(P<0.01).(3)The concentration of plasma glucagon,insulin and glycated hemoglobin concentrations:After the experiment,blood was collected from the abdominal aorta of rats,and the serum glucagon,insulin and glycated hemoglobin concentrations of rats in each group were detected by ELISA.Glucagon and glycated hemoglobin concentrations of rats in ZDF group were significantly higher than those in ZL group(P<0.01).After the taVNS intervention,the concentrations of glucagon and glycated hemoglobin were significantly lower in the ZDF+taVNS group compared with the ZDF group(P<0.01),while the concentrations of glucagon and glycated hemoglobin in the ZDF+sham-taVNS group were significantly higher than those in the ZDF+taVNS group(P<0.01).On the contrary,the insulin concentration of rats in the ZDF group was significantly lower than that in the ZL group(P<0.01).After the intervention,compared with the ZDF group,the insulin concentration of rats in the ZDF+taVNS group was significantly higher(P<0.01),while the insulin concentration of rats in the ZDF+sham-taVNS group was significantly lower than that in the ZDF+taVNS group(P<0.01).(4)OFT:Before modeling,there was no difference between the horizontal and vertical movement scores of rats in each group(P>0.05).After feeding with high fat diet Purina#5008,the horizontal and vertical locomotion scores of rats in ZDF group,ZDF+taVNS group and ZDF+sham-taVNS group were decreased.From week 8 to 14,the horizontal locomotion scores of rats in the ZDF group were always significantly lower than those in the ZL group(P<0.01);after the intervention,at week 14,the horizontal locomotion scores of rats in the ZDF+taVNS group were higher than those in the ZDF group(P<0.01),and the horizontal locomotion scores of rats in the ZDF+sham-taVNS group were significantly lower than those in the ZDF+taVNS group(P<0.01).The vertical locomotion score of rats in the ZDF group was always significantly lower than that in the ZL group from week 4 to 14(P<0.01);after the intervention,at week 14,the vertical locomotion score of rats in the ZDF+taVNS group was higher than that in the ZDF group(P<0.01),and the vertical locomotion score of rats in the ZDF+sham-taVNS group was significantly lower than that in the ZDF+taVNS group(P<0.01).(5)FST:Before modeling,there was no statistical difference in the forced swimming immobility time of rats in each group(P>0.05).After feeding with high fat diet Purina#5008,the immobility time of rats in ZDF group,ZDF+taVNS group and ZDF+sham-taVNS group was prolonged.The taVNS intervention was started at week 8,and the immobility time was significantly reduced in the ZDF+taVNS group compared with the ZDF group from week 10 to 14(P<0.01),and decreased in the ZDF+sham-taVNS group,but the FST immobility time was significantly higher in the ZDF+taVNS group at week 12-14(P<0.01).(6)Microglia morphology in PFC:At the end of the experiment,the brain was taken by perfusion,sectioned,and the morphological changes of microglia stained by Iba-1 and the expression of P2X7R,NLRP3 and IL-1β in the PFC of rats in each group were observed by immunofluorescence technique.The results showed that the microglia in the ZL group were fewer in number and in the resting state,mainly in the form of smaller cytosol and elongated branches;compared with the ZL group,the microglia in the ZDF group were more in number and in the activated state,mainly in the form of shorter and thicker cytosolic branches;compared with the ZDF group,the microglia in the ZDF+taVNS group changed from the activated state to the resting state;while the microglia in the ZDF+sham-taVNS group were also more in the activated state.(7)The protein expression of P2X7R,NLRP3,IL-1β in PFC:After the experiment,the PFC of rats were freshly taken and snap frozen in liquid nitrogen,and then placed in-80℃refrigerator for examination.The protein expression of P2X7R,NLRP3 and IL-1β in PFC of rats in each group was detected by Western Blot.Compared with the ZL group,the expression of P2X7R,NLRP3,and IL-1β proteins were significantly increased in the PFC of ZDF rats(P<0.01).After taVNS intervention,P2X7R,NLRP3,and IL-1β protein expressions were significantly decreased in the PFC of rats in the ZDF+taVNS group compared with the ZDF group(P<0.01).Compared with the ZDF+taVNS group,P2X7R,NLRP3,and IL-1β protein expressions in the PFC of rats in the ZDF+sham-taVNS group were significantly higher(P<0.01).Conclusions:(1)taVNS may exert hypoglycemic effect by regulating serum insulin and glucagon concentration.(2)taVNS has central antidepressant effect and can relieve depressive-like behavior of ZDF diabetic rats induced by high-fat diet.P2X7R/NLRP3/IL-1β inflammatory signaling pathway in PFC may be one of the mechanisms of taVNS central antidepressant effect. |
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