| Objective: To observe the effects of "Tiaoshen Changzhi Method" acupuncture on the intestinal flora and cAMP/PKA signaling pathway related proteins in rats with Parkinson’s disease associated with depression,and to explore the possible mechanism of "Tiaoshen Changzhi Method" acupuncture in treating Parkinson’s disease associated with depression.Methods: 6-OHDA injection combined with dopamine the striatum CUMS prepared comorbid depression rat model of Parkinson’s disease,to set up the normal group,control group,model group,god chang chi group,the ordinary electric acupuncture group and western medicine group,the god chang chi group selection can be "hundreds of each side to the sun through","zhong wan","qi hai","shen men" on both sides,"Tai chong","San yin jiao" on both sides;In the ordinary acupuncture group,"Dance tremor control area","Yintang" and "Sanyinjiao" were selected.The western medicine control group was given fluoxetine and doxazide tablets by gavage.Apo induced behavior test,weight measurement,open field test and sucrose solution preference test were used to observe the behavior of rats in each group.16 Sr DNA high-throughput sequencing was performed to observe the composition of intestinal flora of various rats.The expression of BDNF in brain tissue,CCK and VIP in intestinal tissue were observed by immunohistochemistry.The contents of cAMP,CCK and VIP in brain tissue,brain tissue and serum were detected by ELISA.The expression of PKA and P-CREB protein in brain tissue was detected by Western-blot.The expression of BDNF mRNA and VIP mRNA in brain tissues were detected by RT-PCR.Results: 1.Behavioral test: the body weight growth rate,horizontal exercise number,vertical exercise times and sucrose solution preference rate of rats in model group were lower than those in normal group(P < 0.01);Western medicine group,common acupuncture group and Tiaoshen Changzhi group were significantly higher than the model group;Both the western medicine group and the tiaoshen changzhi group were significantly higher than the ordinary acupuncture group(P < 0.05),and there was no statistical significance between the western medicine group and the tiaoshen changzhi group(P > 0.05).2.High throughput sequencing results: Alpha diversity analysis and Beta diversity analysis showed that the Chao1 index,Observed species index,Shannon index and Simpson index of rats in each group were observed at the same sequencing depth,and the indexes of the western medicine group,the ordinary acupuncture group and the changshen changzhi group were higher than those of the model group,among which,the indexes of the changshen changzhi group were better than those of the ordinary acupuncture group and the western medicine group.Classification analysis showed that at the phylum level,the proportion of intestinal microorganism Bacteroidetes in the normal group and the sham group was higher than that in the model group,while the proportion of Firmicutes was lower than that in the model group.After the intervention of the Tiaoshen Changzhi group and the western medicine group,the proportion of Bacteroides increased significantly,the proportion of Firmicutes decreased,and the distribution was close to the normal group,followed by the ordinary acupuncture group.3.Comparison of cAMP/PKA signaling pathway related proteins in rat brain tissues : The mRNA protein expressions of BDNF,cAMP,PKA,p-CREB and BDNF in brain tissues of rats in Tiaoshen Changzhi group,ordinary acupuncture group and western medicine group were significantly higher than those in model group(P < 0.05).The levels of BDNF and BDNF mRNA protein in brain tissue of Tiaoshen Changzhi group were lower than those of western medicine group(P < 0.05),and higher than those of ordinary acupuncture group(P < 0.05).There were no significant differences in cAMP content,PKA and p-CREB protein expression between Tiaoshen Changzhi group and western medicine group(P > 0.05),and those were higher than those in ordinary acupuncture group(P < 0.05).4.Expression of CCK-8 and VIP in rat intestine : The expression levels of CCK-8,VIP and VIPmRNA in intestinal tissue,brain tissue and serum of rats in Tiaoshen Changzhi group,ordinary acupuncture group and western medicine group were higher than those in model group(P < 0.05).The mRNA protein levels of VIP and VIP in Tiaoshen Changzhi group were lower than those in western medicine group(P < 0.05),and higher than those in ordinary acupuncture group(P < 0.05).There was no significant difference in the contents of VIP in intestinal tissue and serum between Tiaoshen Changzhi group and western medicine group(P >0.05),and it was higher than that of ordinary acupuncture group(P < 0.05).The expression of CCK-8 in intestinal tissue,brain tissue and serum of Tiaoshen Changzhi group was higher than that of western medicine group and ordinary acupuncture group(P < 0.05).Conclusion: 1."Tiaoshen Changzhi Method" acupuncture can significantly improve the behavioral performance of Parkinson’s disease associated with depression.2.In the rats with Parkinson’s disease and depression,intestinal microbial richness decreased and microbial distribution was disordered.After acupuncture intervention,"Tiaoshen Changzhi Method" can restore intestinal microbial richness,promote the uniform distribution of intestinal microorganisms and restore the normal composition of intestinal microbial community.3."Tiaoshen Changzhi Method" acupuncture can increase the protein expression levels of cAMP,PKA,p-CREB,BDNF and so on in the brain tissue of rats,so as to have an impact on the cAMP/PKA signaling pathway and play an anti-PDD role.4.Acupuncture can improve the contents of brain-intestinal peptide CCK-8 and VIP in the brain,intestine and serum,activate the cAMP/PKA signaling pathway and regulate brainintestinal interaction,thus improving the depressive state associated with Parkinson’s disease. |
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