| BACKGROUND&OBJECTIVEPost-traumatic stress disorder(PTSD),a traumatic and stress-related disorder,is char-acterized by exposure to extreme traumatic events,recurrent and invasive traumatic memo-ries,avoidance of event-related stimuli,cognitive impairment,negative emotions,high arousal states,hypervigilance,clinical stress,and social damage.PTSD is a global health problem with serious negative social and personal consequences.The high prevalence of PTSD significantly increases the risk and burden of those suffered from mental disorders.Although,recent studies have identified the molecular biological,neurochemical,and genetic factors involved in the pathological process of PTSD,the strategy of prevention and treat-ment for PTSD are limited.Studies have suggested that the stress-induced activation of in-flammatory response is involved in the development and pathogenesis of PTSD.Data from clinical studies and experimental findings have verified the efficacy and safety of acupunc-ture in the treatment of PTSD.However,the exact mechanism remains unclear.Therefore,the rat model in the present study was established by exposure to Single-prolonged Stress(SPS)to simulate PTSD process.We hypothesize that the therapeutic effect of acupuncture is based on regulating the stress-induced activation of inflammatory response.The present study aims to elucidate the mechanism of acupuncture on regulating the hippocampal inflam-matory response in rats exposed to single-prolonged stress,which might further identify the mechanism of the acupuncture in the treatment of PTSD.This is the starting point of this experiment.METHODSForty male Sprague Dawley rats were randomly divided into four groups:blank group,model group,acupuncture group and sertraline group.Except the blank group,the rats in the other groups were exposed to single prolonged stress(SPS)method.And the establishment of rat model was completed within 1 day after adaptive feeding.The blank group and model group were exposed to no intervention and were fed normally.The rats in the acupuncture group received acupuncture intervention at the acupoints of "Baihui" and "Yintang" at 2:00 PM every day,once per day for 15 days.Sertraline suspension(0.2mg/ml)was given intra-gastric once a day(10mg/kg)for the rats in sertraline group,once per day for 15 days.The experimental procedures were as follows.(1)The effect of acupuncture on the behavioral changes of rats exposed to SPS.The rat model of PTSD was established by SPS methods.Body mass of rats were measured before modeling(day 0),on day 8 and after intervention(day 15).And the differences of body mass of each group were compared at three time points.The rats were exposed to elevated cross maze experiment before modeling(day 0)and after intervention(day 15).The present part aims to investigate the effect of acupuncture on the behavioral changes of rats exposed to SPS.(2)The effect of acupuncture on regulating the stress-induced inflammatory response mediated by microglia activation in the hippocampus in the SPS model rats.In this part,HE staining method was used to observe the basic pathological morphological changes in the hippocampus.And immunofluorescence staining method was used to observe the activation of hippocampal microglia.The present part aims to explore the effect of acupuncture on reg-ulating the activation of hippocampal microglia in the SPS rat model.(3)The effect of acupuncture on regulating the expression of serum IL-6 and IL-1β in SPS rat model.This part aims to observed the effect of acupuncture on regulating the expres-sion of serum IL-6 and IL-1β in rats exposed to SPS,and explored the mechanism of acu-puncture on regulating the stress-induced inflammatory response mediated by microglia ac-tivation in the hippocampus,which might further elucidate the potential mechanism of acu-puncture in the treatment of PTSD.RESULTS(1)The effect of acupuncture on the behavioral changes of rats exposed to SPS.The results of this part showed that SPS stress could induce a series of behaviors associated with PTSD,which were mainly shown as follows:①Before the experiment(day 0),there was no statistically significant difference in body mass among the rats(P>0.05),and the baseline level was consistent.Compared with the blank group,the body mass of rats in model group significantly decreased after exposing to SPS at day 8(P<0.01).There was no significant difference between blank group and model group at day 15(P>0.05).It indicated that the single prolonged stress induce obvious decrease of body mass of rats in model group at day 8,thus influence the growth and the overall situation of SPS rats.Compared with the model group,there was no difference between acupuncture group and model group at day 8 and day 15(P>0.05).So it was between sertraline group and model group at day 8 and day 15(P>0.05).It can be seen that SPS stress can induce a decrease in body mass in rats,which influ-ence the natural growth and overall situation of rats.②The results of the elevated cross maze experiment showed that SPS stress significantly reduced the percentage of time and the per-centage of times of the rats entering the elevated cross maze(P<0.01;P<0.01),which indi-cates that SPS induce PTSD-like anxiety behavior in rats.Notably,acupuncture intervention reversed the reduction in the percentage of time to arm opening(P<0.01),suggesting that acupuncture could effectively improve the SPS-induced anxiety behavior and exert therapeu-tic effect in PTSD rats.(2)The effect of acupuncture on regulating the stress-induced inflammatory response mediated by microglia activation in the hippocampus in the SPS model rats.①The results of HE staining showed that the hippocampal cells of the blank group were closely arranged and uniform,with clear outlines,blue nuclei,oval bodies and clear nucleoli.Compared with the blank group,the model group showed disordered cell arrangement,decreased cell number,enlarged cell space,blurred or disappeared cell structure,cell vacuoles,degeneration and necrosis of neuronal cells,nuclear shrinkage,accompanied by softening foci.However,acu-puncture reversed the pathological injury to some extent.The cells of the acupuncture group returned to orderly arrangement,the number of cells increased and the number of cell vacu-oles decreased.In the sertraline group,the number of cells increased and the number of ne-crotic cells decreased as well.②The results of the immunofluorescence staining showed that compared with the blank group,the morphology of hipocampal microglia cells in the model group was enlarged,the processes were thickened,and the activated microglia cells were significantly increased(P<0.01).Compared with the model group,the activated microglia of the acupuncture group were significantly reduced(P<0.01)and compared with the model group,the activated microglia in sertraline group was also significantly reduced(P<0.01),suggesting that both acupuncture and sertraline intervention effectively reversed the activated microglia of the hippocampus of the rats of SPS model.(3)Effect of acupuncture on regulating the expression of serum IL-6 and IL-1β in SPS rat model.①The results of the expression of serum IL-6 levels of the rats in each group showed no statistically significant difference in the present study(P>0.05).Our team will conduct further studies based on the present findings in the follow-up study.②Compared with the blank group,the expression of IL-1β in the model group was significantly increased(P<0.05).Compared with the model group,the expression of IL-1β in the acupuncture group was significantly decreased(P<0.05),and the expression of IL-1β in the sertraline group was significantly decreased as well(P<0.05).Accordingly,acupuncture can significantly reverse the increased expression of serum IL-1β induced by SPS stress,thus exhibiting an anti-PTSD effect.CONCLUSIONThe present study preliminarily indicated that single prolonged stress could induce the activation of hippocampal microglia,increase the expression of serum pro-inflammatory cy-tokine IL-1β,mediate the degeneration and necrosis of hippocampal neurons,nuclear shrink-age,accompanied by a softening lesion.Accordingly,it can be found that SPS plays an im-portant role in the pathogenesis of PTSD via inflammatory response mediated by activation of hippocampal microglia.Of note,acupuncture could reverse the pathological process of the inflammatory response mediated by the activation of microglia induced by SPS stress,which may be the underlying mechanism of acupuncture in the treatment of PTSD.Further studies will be conducted in the future based on the present study. |
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