The Regulated Effects Of ?7nAchR On Behavior Of Chronic Restraint Stress Mice And Underlying Mechanism

First part: DMXBA prevents CRS-induced learning and memory impairment and depression like behaviorAims: To explore whether the learning and memory function and depression behavior was changed after chronic restraint stress injury, and to evaluate the effect of DMXBA on mice behavior.Methods: We used the chronic restraint stress model to simulate chronic stress,during the restraint stress 11 d to 21 d, mice were injected with,DMXBA(4mg/kg,i.p.)combinded with ?-BGT(1?g/kg, i.p.) or vehicle,until the end of the model, the spatial learning and memory was determined by the Morris water maze(MWM) test and the novel object recognition test(NOR), the depressive like behavior was determined using the sucrose preference test(SPT), forced swim test(FST) and tail suspension test(TST).Results:(1) In the Morris Water Maze test, the latency to find the platform was significantly longer in CRS mice compared with control group on days 3 and 4 of the training period, and CRS mice spent less time in target quadrant during test period. In the novel object recognition test, CRS mice showed the lower discrimination index.These results indicated that CRS induced learning and memory impairment.(2) In the sucrose preference test, CRS mice had the lower sucrose preference after chronic stress restraint, and the immobile time of CRS mice was significantly longer than control group. These results indicated that CRS induced depression like behavior.(3) CRS mice treated with DMXBA spent less time to find the platform and more time in target quadrant compared with CRS mice. DMXBA reversed the lower discrimination index after chronic stress restraint. And ?-BGT treatment can reverse the protective effects of DMXBA.(4) In the sucrose preference test, DMXBA reversed the lower sucrose preference after chronic stress restraint. In the forced swim test and the tail suspension test,DMXBA alleviated depressive-like behavior. And ?-BGT could reverse the therapeutic effects of DMXBA.Conclusions: DMXBA prevents CRS-induced learning and memory impairment and depression like behavior.Second part: DMXBA reverse CRS-induced neuron lossAims: To observe the morphology of neurons and dendritic spine density in hippocampus of mice after chronic restraint stress injury, and to evaluate the neuroprotection of DMXBA.Methods: Morphology of hippocampal neurons was examined by Nissal staining, and dendritic spine density was evaluated by Golgi silver staining.Results:(1) Nissl staining showed that neuronal damage in the hippocampus is characterized by shrunken cell bodies accompanied by shrunken and pyknotic nuclei. Significantly damaged neurons were observed in the hippocampus CA1 of CRS mice. DMXBA treatment alleviated CRS-induced neuronal damage in the CA1 region of hippocampus. And ?-BGT could inhibit the therapeutic effects of DMXBA.(2) CRS mice decreased the dendritic spine density of hippocampus CA1. DMXBA increased the dendritic spine density in the hippocampus CA1 of CRS mice. And?-BGT could reverse the therapeutic effects of DMXBA.Conclusions: Chronic treatment with DMXBA reverse CRS-induced the decrease in the dendritic spine density of hippocampus CA1 and neuron loss.Third Part: DMXBA regulate CRS-induced CNS inflammation and underlying mechanismAims: To observe the TLR4/NF-?B pro-inflammation and Ach anti-inflammation signaling pathway proteins expression pattern and the relationship with downstream inflammatory cytokines level. To explore the effect of DMXBA against inflammation in hippocampus induced by chronic restraint stress.Methods: Pro-inflammatory factor of TNF-? and IL-1? in the hippocampal homogenat were detected by the ELISA kit. ?7n Ach R,CHAT,Ach E,STAT3,TLR4,NF-?B,GFAP and Iba-1 protein expression were also measured.Results:(1) The expression of TNF-?, IL-1?, Iba-1, GFAP, TLR4 and My D88 of CRS mice increased signifigantly, while the expression of NF-?B of CRS mice decreased.DMXBA depressed TLR4/NF-?B signaling pathway in the hippocampus of CRS mice and decreased pro-inflammatory factor express in the hippocampal homogenat of CRS mice. DMXBA inhibited microglia and astrocyte activation in the hippocampus of CRS mice. And ?-BGT could reverse the effects of DMXBA.(2) The expression of STAT3 of CRS mice decreased significantly, and the expression of CHAT, ?7n Ach R increased dramatically, but level of ACh E in hippocampus had no difference between all groups. DMXBA treatment reversed the CRS-induced elevation of Ach E, but have no effect on the expression of STAT3, CHAT and?7n Ach R.Conclusions: DMXBA depressed TLR4/NF-?B signaling pathway in the hippocampus of CRS mice, activated Ach signaling pathways meanwhile.Fourth part: The effect of DMXBA on the amount of Treg cell of CRS miceAims: To observe the discipline of Treg-cell in spleen and hippocampus of CRS mice,to detect the leve of TNF-? and IL-1? in the peripheral blood and explore the effect of DMXBA on Treg-cell and pro-inflammatory factor.Methods: The amount of Treg cell in spleen was examined by flow cytometry; and the expression of Treg cell marker Foxp3+ in hippocampus was examined by Western blot. Pro-inflammatory factor of TNF-? and IL-1? in the peripheral blood were detected by ELISA.Results: The amount of Treg cell in spleen and hippocampus of CRS mice was decreased significantly, DMXBA treatment upregulated Treg-cell in the spleen and hippocampus of CRS mice. And ?-BGT can reverse the effects of DMXBA in hippocampus. DMXBA treatment have no effect on the expression of TNF-? and IL-1?.Conclusions: DMXBA treatment reverse the CRS-induced decline of Treg.