小补心汤对应激模型啮齿类动物的抗焦虑作用及可能机制OA

OBJECTIVE To investigate the anxiolytic effect of Xiaobuxin decoction(XBXT)in rodent models of chronic restraint stress(CRS)and empty bottle stress(EBS),and to explore its regu-latory mechanism on the hypothalamic-pituitary-adrenal(HPA)axis as well as its impact on the hippo-campal Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)inflammatory pathway in CRS model mice.METHODS Male C57BL/6J mice were used to establish a CRS model.The mice were randomly divided to six groups:the normal control group,CRS model group,buspirone 5 mg·kg-1 group,and XBXT treatment groups 0.45,0.9,and 1.8 g·kg-1.Except the normal control group,all the mice received 3 hours of restraint stress daily for 14 consecutive days.Male sprague-dawley(SD)rats were used to establish an EBS model.The rats were randomly divided into the normal control group,EBS model group,buspirone 2 mg·kg-1 group,and XBXT treatment groups 0.28,0.56 and 1.12 g·kg-1.Except the normal control group,all the rats received one week of drinking adaptation training,followed by daily placement of an empty bottle at a random time point.One hour prior to modeling,mice/rats in each drug-treated group were intragastrically(ig)given corresponding doses of drugs while those in the normal control and model groups were garaged with an equal volume of deionized water.After modeling,the open field test and elevated plus maze test were combined to evaluate the anxiolytic effect of XBXT.Following behavioral tests in mice,Nissl staining was performed to observe neuronal morphology.Immunofluorescence staining was used to detect the expressions of the microglial-specific marker ionized calcium-binding adapter molecule 1(IBA-1)and the neuronal activation marker c-Fos protein.Enzyme-linked immunosorbent assay(ELISA)was adopted to measure the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6 and IL-2 in mouse hippocampal tissue,as well as the con-tents of corticotropin-releasing hormone(CRH),adrenocorticotropic hormone(ACTH)and corticoste-rone(CORT)in the serum of mice.The protein expression levels of phosphorylated NF-κB(p-NF-κB),total NF-κB,TLR4,NOD-like receptor pyrin domain-containing 3(NLRP3)and brain-derived neuro-trophic factor(BDNF)in the hippocampus were determined using Western blotting.RESULTS Normal mice and rats were stable in behavior,but obvious anxiety-like phenotypes emerged in both CRS and EBS model animals.In CRS mice,the number of entries and the time spent in the center of the open field were reduced,and the amount of time spent in the open arms and the percentage of open-arm entries in the elevated plus maze dropped markedly.The same was true of EBS rats that spent less time in the center of the open field.Compared with the CRS model group,mice that had received buspi-rone or XBXT 0.9 and 1.8 g·kg-1 spent a longer time and made more entries into the center of the open field.The proportions of time spent in the open arms and entries into the elevated plus maze increased.Compared with EBS model group,rats given 0.56 and 1.12 g·kg-1 XBXT spent more time in the center of the open-field.In CRS model mice,a marked loss of Nissl bodies was observed in the hippocampus,along with increased IBA-1 fluorescence intensity and elevated c-Fos expressions.Hippocampal levels of TNF-α,IL-1β,IL-6 and IL-2 became substantially higher,so did serum concentrations of ACTH,CORT and CRH.Meanwhile,the decrease of hippocampal BDNF protein abundance was pronounced,whereas the protein levels of TLR4 and NLRP3,as well as NF-κB phosphorylation,were significantly enhanced.Compared with the CRS group,XBXT 1.8 g·kg-1 significantly increased the number of Nissl bodies,suppressed microglial activation and c-Fos expression,and reduced the levels of TNF-α,IL-1β,IL-6,IL-2,ACTH,CORT and CRH while upregulating BDNF expressions and inhibiting TLR4 and NLRP3 protein expressions as well as NF-κB phosphorylation.CONCLUSION XBXT exerts strong anxiolytic effects in both CRS mice and EBS rats.The underlying mechanism is closely associated with the modulation of HPA axis activity,the suppression of the TLR4/NF-κB signaling pathway,and the restora-tion of BDNF expressions.