| Objective Intestinal flora can affect cognitive function through gut brain axis.In the process of aging,intestinal flora imbalance will produce a large number of pro-inflammatory neurotoxic substances lipopolysaccharide(LPS).LPS in the gut can enter the brain through the physiological barrier,induce neuroinflammatory reaction,and eventually lead to cognitive dysfunction.Therefore,it may be the key to alleviate neuroinflammation and cognitive impairment to take active measures to regulate intestinal flora in the process of aging.Therefore,this experiment takes D-galactose-induced aging model rats as the research object,starting from the relationship between LPS,the metabolite of intestinal flora,and neuroinflammation,to explore the regulation of pre electroacupuncture on the target intestinal flora,the regulation of intestinal mucosal barrier and blood-brain barrier,and the effect on systemic inflammation and neuroinflammation,so as to further clarify that pre electroacupuncture can regulate intestinal flora Objective to explore the possible mechanism of preventing and treating cognitive impairment related to aging.Method Sixty 3-month-old male Sprague Dawley rats were randomly divided into normal group,model group,Baihui group,Zusanli group and Baihui + Zusanli group,with 12 rats in each group.Except for the normal group,D-galactose(120mg / kg / day)was injected intraperitoneally for 8 weeks.Rats in Baihui group,Zusanli group and Baihui + Zusanli group were intervened by Electroacupuncture at Baihui,Zusanli,Baihui and Zusanli from the first day of modeling.Baihui Point was punctured 3-5mm forward,Zusanli point was punctured 3-5mm straight,and hans-200 a electroacupuncture therapeutic instrument was connected.Electroacupuncture treatment parameters: frequency 50 Hz,current 1 m A,time 20 min,once a day,a total of 8 weeks.The normal group and the model group received the same gripping and binding fixation when other groups intervened.Open field test and Morris water maze test were performed after the intervention.Then serum,fresh hippocampal tissue,intestinal mucosa tissue and perfusion were collected and the indexes were detected.The thickness of postsynaptic dense zone,the width of synaptic gap and the morphological changes of microglia in hippocampal CA1 area were observed by transmission electron microscope;the changes of intestinal microflora were detected by RT-PCR;the morphological changes of ileal mucosa were observed by HE staining;the expression of ZO-1 protein in colonic tissue,ZO-1,LPS,TLR4 and p-nf-κ B in hippocampal tissue were detected by Western blot The protein expression levels of p65,TNF-α,IL-6 and IL-1 β were detected;the serum levels of Dao,S100 β,LPS,TNF-α and IL-1 β were detected by ELISA;the expression of Iba-1 in hippocampal CA1 area was detected by immunofluorescence.Result 1.Pre electroacupuncture can improve the cognitive impairment ofD-galactose-induced aging model rats,and the protective effect of "sample matching points" pre electroacupuncture is stronger than that of single point.(1)Results of open field experiment: compared with the normal group,the movement distance,standing times and central area residence time of the model group were significantly decreased(P < 0.01);compared with the model group,the movement distance,standing times and central area residence time of the pre EA group were significantly increased(P < 0.01),indicating that pre EA can improve the depression of aging rats.In addition,compared with Baihui + Zusanli group,the movement distance,standing times and central area residence time of Baihui group and Zusanli group were significantly decreased(P < 0.01),indicating that "specimen matching points" is better in improving depression of aging model rats.(2)Morris water maze test results: compared with the normal group,the escape latency of the model group was significantly longer(P < 0.01);compared with the model group,the escape latency of the pre acupuncture group was significantly shorter(P < 0.01).Compared with Baihui + Zusanli group,the escape latency of Baihui group and Zusanli group was significantly longer(P < 0.01).In the space exploration experiment,compared with the normal group,the exploration time of the model group in the original platform quadrant was significantly reduced(P < 0.01);compared with the model group,the exploration time of the pre EA group in the original platform quadrant was significantly prolonged(P < 0.01);Compared with the Baihui + Zusanli group,the exploration time of Baihui group and Zusanli group in the original platform quadrant was significantly shorter(P < 0.05,P < 0.01).The "specimen matching points" pre EA was better than single point in improving learning and memory of aging model rats. 2.Pre EA can improve the synaptic damage and inhibit the activation of microglia in hippocampal CA1 area of aging model rats induced by D-galactose,and the protective effect of "sample matching points" is stronger than that of single point.1.Results show: there was no significant difference in the width of synaptic gap in hippocampal CA1 area among all groups.Compared with the normal group,the thickness of postsynaptic dense zone in hippocampal CA1 area of the model group was significantly decreased(P < 0.01);compared with the model group,pre EA intervention could increase the thickness of postsynaptic dense zone Compared with Baihui + Zusanli group,the thickness of postsynaptic dense zone in CA1 area of hippocampus in Baihui group and Zusanli group decreased significantly(P < 0.01).2.Morphological changes of microglia results: in the normal group,the boundary of cytoplasm and nucleus was clear,and no lysosome was found.In the model group,the cell body showed amoeba morphology,the boundary between nucleus and cytoplasm was unclear,and a large number of lysosomes were produced.After electroacupuncture intervention,the number of lysosomes decreased,the boundary between nucleus and cytoplasm was clear,and there was no obvious amebic degeneration.3.Pre EA can regulate the structure of intestinal flora,protect the barrier function of intestinal mucosa,reduce the entry of LPS into the blood,and alleviate systemic inflammation in aging model rats induced by D-galactose,and the protective effect of "sample matching acupoint" is better than that of single acupoint.(1)RT-PCR results of target intestinal flora: compared with the normal group,the relative gene expression of Lactobacillus and Bifidobacterium in the model group was significantly decreased,and the relative gene expression of Bacteroides fragilis and Escherichia coli was significantly increased(P < 0.01).Compared with the model group,the relative gene expression of Lactobacillus and Bifidobacterium in the pre EA group increased significantly,while that of Bacteroides fragilis and Escherichia coli decreased significantly(P < 0.01).Compared with Baihui + Zusanli group,the relative gene expression of Lactobacillus and Bifidobacterium in Baihui group and Zusanli group decreased significantly,while the relative gene expression of Bacteroides fragilis and Escherichia coli increased significantly(P < 0.01).It shows that pre EA can regulate intestinal flora,promote the growth of probiotics and inhibit inflammatory bacteria.(2)He staining results of colon tissue: in the normal control group,the intestinal mucosal epithelial cells were complete,the glands were arranged orderly and tightly,rich in goblet cells,no obvious inflammatory cell infiltration,no congestion and expansion of capillaries.In the model group,the intestinal mucosa was eroded,a large number of intestinal epithelial cells were destroyed,the glands were arranged disorderly,inflammatory cell infiltration was visible,and the capillaries were hyperemia and expansion.In each EA group,the epithelial cells of intestinal mucosa were relatively complete,the glands were arranged neatly and tightly,rich in goblet cells,only a small amount of inflammatory cells were seen in lamina propria,and a small amount of congestion and expansion of capillaries.(3)Results show: compared with normal group,the relative expression of ZO-1 protein in colon tissue of model group was significantly decreased(P < 0.01).Compared with the model group,the relative expression of ZO-1 protein was significantly increased in the pre EA group(P < 0.01).In addition,compared with Baihui + Zusanli group,the relative expression of ZO-1 protein in colon tissue of Baihui group and Zusanli group was significantly lower(P < 0.01). (4)Results show: compared with the normal group,the serum Dao content of the model group was significantly increased(P < 0.01);compared with the model group,the serum Dao content of the EA group was significantly decreased(P < 0.01);compared with the Baihui + Zusanli group,the serum Dao content of the Baihui group and the Zusanli group was significantly increased(P < 0.05,P < 0.01).It shows that electroacupuncture can improve the function of intestinal mucosal barrier,and "specimen matching points" pre electroacupuncture can protect intestinal mucosal barrier more effectively than Baihui or Zusanli.(5)Results: compared with the normal group,the contents of LPS,TNF-α and IL-1 β in the serum of the model group were significantly increased(P < 0.01);compared with the model group,the contents of LPS,TNF-α and IL-1 β in the serum of the EA group were significantly decreased(P < 0.01),indicating that pre EA can alleviate the peripheral inflammatory reaction.Compared with Baihui + Zusanli group,the levels of serum LPS,TNF-α and IL-1 β in Baihui group and Zusanli group were significantly increased(P < 0.05,P < 0.01).It shows that pre EA with "specimen matching points" is more effective in relieving peripheral inflammation than Baihui or Zusanli alone.4.Pre EA can protect the blood-brain barrier function of aging model rats induced by D-galactose,reduce the entry of LPS in peripheral blood into the brain,reduce the activation of microglia,alleviate the neuroinflammatory reaction by inhibiting TLR4 / NF-κ B signaling pathway,and the protective effect of "sample matching acupoint" is better than that of single acupoint.(1)Results: compared with the normal group,the relative expression of ZO-1 protein in the hippocampus of the model group was significantly decreased(P < 0.01);compared with the model group,the relative expression of ZO-1 protein in the hippocampus of the EA group was significantly increased(P < 0.01);compared with the Baihui + Zusanli group,the relative expression of ZO-1 protein in the ileum of the Baihui group and Zusanli group was significantly increased(P < 0.01)01).(2)Results: compared with the normal group,the content of serum S100-β in the model group was significantly increased(P < 0.01);compared with the model group,the content of serum S100-β in the electroacupuncture group was significantly decreased(P < 0.01),indicating that electroacupuncture can alleviate the damage of blood-brain barrier.Compared with Baihui + Zusanli group,the content of serum S100-β in Baihui group and Zusanli group was significantly increased(P < 0.01),indicating that the protective effect of "sample matching acupoint" on BBB was better than that of single acupoint.(3)Results: compared with the normal group,the relative expression of LPS in the hippocampus of the model group was significantly increased(P < 0.01).Compared with the model group,the relative expression of LPS in hippocampus of EA group was significantly decreased(P < 0.01).In addition,compared with Baihui + Zusanli group,the relative expression of LPS in hippocampus of Baihui group and Zusanli group was significantly increased(P < 0.01).It shows that EA can reduce the LPS entering the brain through blood-brain barrier,and the protective effect of "sample matching points" is better.(4)Compared with the normal group,the number of activated microglia and the expression of Iba-1 protein in the hippocampal CA1 area of the model group were significantly increased(P < 0.01).These results indicate that continuous intraperitoneal injection of D-galactose can induce the activation of microglia in rats.Compared with the model group,the number of microglia activated in hippocampal CA1 area and the expression of Iba-1 protein were significantly decreased in EA group(P < 0.01).These results indicate that EA can reduce the number of microglia activated in hippocampal CA1 area of aging rats.Compared with Baihui + Zusanli group,the number of activated microglia and the expression of Iba-1 protein in hippocampus of Baihui and Zusanli groups were significantly increased(P < 0.01),which indicated that "specimen matching acupoints" could reduce the activation of microglia more effectively.(5)Results: compared with the normal group,the expressions of TLR4 and p-NF-κ B in the hippocampus of the model group were significantly increased(P < 0.01).Compared with the model group,the expressions of TLR4 and p-nf-κ B in the hippocampus of EA group were significantly decreased(P < 0.01).Compared with the Baihui + Zusanli group,the expressions of TLR4 and p-nf-κ B in the hippocampus of Baihui group and Zusanli group were significantly increased(P < 0.01);compared with the normal group,the expressions of TNF-α,IL-6 and IL-1 β in the hippocampus of the model group were significantly increased(P < 0.01).Compared with the model group,the protein expressions of TNF-α,IL-6 and IL-1 β in hippocampus of EA group were significantly decreased(P < 0.01).Compared with Baihui + Zusanli group,the expressions of TNF-α,IL-6 and IL-1 β protein in hippocampus of Baihui group and Zusanli group were significantly increased(P < 0.05,P < 0.01).These results indicate that EA can alleviate the neuroinflammatory reaction through TLR4 / NF-κ B signaling pathway,and the protective effect of "sample matching points" is better than that of single point.Conclusion: 1.Pre electroacupuncture can improve the depression and spatial learning and memory ability of aging model rats induced by D-galactose,and the protective effect of "sample points" is better than that of single point. Alleviating synaptic dysfunction and neuroinflammation may be one of the important mechanisms of pre EA in alleviating cognitive impairment in aging rats.2.Pre EA can regulate the structure of intestinal flora in D-galactose-induced aging model rats,protect the barrier function of intestinal mucosa,reduce the entry of LPS into the blood,alleviate systemic inflammation,and the protective effect of "sample matching acupoint" is better than that of single acupoint.3.Pre electroacupuncture can protect the blood-brain barrier function of aging model rats induced by D-galactose,reduce the entry of LPS into the brain and the activation of microglia,so as to alleviate the neuroinflammatory reaction,and the protective effect of "sample matching acupoint" is better than that of single acupoint.It may be one of the important mechanisms of pre EA in preventing and treating cognitive impairment of aging model rats by inhibiting TLR4 / NF-κ B signaling pathway and reducing the synthesis and release of downstream inflammatory factors. |
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