ABSTRACT
ObjectiveTo evaluate the intervention effect of dihydroartemisinin （DHA） on hippocampal nerve injury in L5 spinal nerve ligation （SNL） model and tumor necrosis factor-α （TNF-α） hippocampal continuous injection model. In primary cultured microglia-hippocampal neurons， the regulatory pattern of DHA on microglia-hippocampal neuronal interactions was confirmed. MethodThe experimental animals were divided into Sham group， SNL group， and DHA group （16 mg·kg-1）， with 3 mice in each group. The hippocampal CA3 glutamatergic neurons were labeled with adeno-associated virus ［Calmodulin-dependent protein kinase Ⅱ（CaMKⅡ） dTomato AAV］， and their contributions to the hippocampal CA1， prefrontal cortex （Frc）， anterior cortex （ACC）， projections of nucleus accumbens （Nac）， and Basolateral Amygdala （BLA） were traced by immunofluorescence staining. The experimental animals were divided into a Sham group， a TNF-α hippocampus continuous injection model group， DHA-L， DHA-M， and DHA-H groups （4， 8， 16 mg·kg-1）， and pregabalin group （25 mg·kg-1）， with 4 mice in each group. The morphology of pyramidal neurons in the hippocampal CA1 and CA3 regions was counted by Golgi staining. The continuous activation of hippocampal primary neurons and microglia was induced， DHA intervention was given by co-culture， and the cell soma area and the expression of postsynaptic density protein 95 （PSD95） inside and outside the primary and secondary dendritic spines of neurons were counted by immunofluorescence. ResultCompared with the Sham group， the projection of CA3 glutamatergic neurons to CA1 region， Frc， and ACC in the SNL group was significantly reduced （P<0.01）， while the projection to Nac and BLA was significantly increased （P<0.01）. As compared with the SNL group， the projection of hippocampal CA3 glutamatergic neurons to CA1 region， Frc， and ACC was significantly increased in the DHA group （P<0.01）， while the projection to Nac and BLA was significantly reduced （P<0.01）. Golgi staining results showed that as compared with the Sham group， the density of dendritic spines and the number of dendritic branches in the CA1 and CA3 pyramidal neurons in the TNF-α hippocampal continuous injection model group were significantly reduced （P<0.01）. As compared with the TNF-α hippocampal continuous injection model， the density of dendritic spines and the number of dendritic branches in hippocampal CA1 and CA3 pyramidal neurons in the DHA-M and DHA-H groups were significantly increased （P<0.05， P<0.01）. Compared with DHA-M group， the total dendrite length of CA1 pyramidal neurons in hippocampus in DHA-H group was significantly increased （P<0.01）， while the total dendrite length of CA1 neurons and the total dendrite base length of CA3 neurons in DHA-L group was significantly decreased （P<0.01）. Compared with the blank control group， the cell soma area of the glycine group and glutamate group increased significantly （P<0.01）. As compared with the glycine group and glutamate group， the cell area of the glycine + glutamate group was significantly increased （P<0.01）， and as compared with the glutamate group， the cell soma area of the glutamate + DHA group was significantly reduced （P<0.01）. As compared with the glycine acid + glutamate group， the cell soma area of the glycine + glutamate + DHA group was significantly reduced （P<0.01）， and as compared with the glutamate + DHA group， the cell soma area of the glycine + glutamate + DHA group was also significantly reduced （P<0.05）. Compared with the blank control group， the cell soma area of the glutamate group was significantly increased （P<0.01）. As compared with the glutamate group， the cell soma area of the glutamate + DHA-L， glutamate + DHA-M， and glutamate + DHA-H groups was significantly reduced （P<0.01）. As compared with the blank control group， the expression of the resting primary microglia + glycine group in primary and secondary dendritic internal and external postsynaptic density protein 95 （PSD95） was significantly increased （P<0.01）. As compared with the resting primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons of the activated primary microglia + glycine group was significantly reduced （P<0.01）. As compared with the activated primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons in the activated primary microglia + glycine + DHA group was significantly increased （P<0.01）. As compared with the activated primary microglia + DHA group， the expression of PSD95 in the primary and secondary dendritic spines and outside neurons in the activated primary microglia + glycine + DHA group was significantly increased （P<0.01）. ConclusionDHA has a significant repair effect on vertebral neuronal damage caused by hippocampal microglia and TNF-α overexpression in NP pathology， and this repair is closely related to the dual inhibition of neuronal-microglia by DHA.
ABSTRACT
ObjectiveTo establish a neuroinflammation-based obesity and depression comorbidity （COM） model in mice and explore the pharmacodynamics and preliminary pharmacological mechanism of tripterine on COM mice. MethodC57BL/6J mice were randomly divided into a normal group （Chow）， a diet-induced obesity group （DIO）， and a COM group. The mice in the COM group were fed on a high-fat diet and chronically stressed with moist litter for 12 weeks to establish the COM model. C57BL/6J mice were randomly divided into a Chow group， a COM group， and a tumor necrosis factor-α（TNF-α） knock-down group. In the TNF-α knock-down group， TNF-α shRNA adeno-associated virus was injected into the amygdala through brain stereotaxis， and the expression of TNF-α in the amygdala was down-regulated. C57BL/6J mice were randomly divided into a Chow group， a DIO group， a DIO + low-dose tripterine group （0.5 mg·kg-1）， a DIO + high-dose tripterine group （1.0 mg·kg-1）， a COM group， a COM + low-dose tripterine group （0.5 mg·kg-1）， and a COM + high-dose tripterine group （1.0 mg·kg-1）. The body weight， food intake， glucose tolerance， white/brown fat ratio， serum total cholesterol （TC）， triglyceride （TG）， and high-/low-density lipoprotein cholesterol （HDL-C and LDL-C） content were recorded， and obesity of mice in each group was evaluated. Forced swimming test （FST）， tail suspension test （TST）， and open field test were used to evaluate the degree of depression of mice in each group. Immunofluorescence staining was used to detect the protein expression levels of neuropeptide Y， tryptophan hydroxylase 2 （TPH2）， and brain-derived neurotrophic factor （BDNF） in various brain nuclei of mice. Correlation analysis was used to detect the correlation of obesity and depression indexes. ResultThe comparison of the Chow group and the DIO group indicated that COM mice showed obesity and depression. To be specific， obesity was manifested as increased body weight and food intake （P<0.05， P<0.01）， as well as increased NPY expression in the central amygdala， and depression was manifested as prolonged immobility time in FST and TST （P<0.01）， and reduced TPH2-positive 5-hydroxytryptamine neurons in the dorsal raphe nucleus （DRN） and basolateral nucleus of the amygdala （BLA）. The down-regulation of TNF-α protein in BLA of COM mice shortened the immobility time in FST and TST （P<0.05， P<0.01）， increased TPH2/BDNF-positive neurons in BLA， and showed no significant changes in obesity. In DIO mice， the administration of 0.5 mg·kg-1 tripterine for 9 days significantly decreased the 60 min blood glucose in glucose tolerance （P<0.01） and food intake （P<0.05）. In COM mice， 1.0 mg·kg-1 tripterine was administered for 14 days to significantly decrease 30 min blood glucose in glucose tolerance （P<0.01）， and food intake （P<0.05）， and immobility time in TST （P<0.01）， increase TPH2-BDNF double-labeled cells in BLA and DRN， and reduce the area of TMEM119-stained cells. ConclusionThe model of obesity and depression comorbidity can be properly induced in mice under the condition of dual stress of energy environment. Tripterine can effectively interfere with obesity-depression comorbidity， and its mechanism may be related to the inhibition of central nervous system inflammation.