| ObjectiveBased on the theory of microbiota-brain-gut axis,this paper provides a new idea and experimental basis for the development and treatment of Neuropathic pain(NP)from the perspective of "if there is no general pain,pain will lead to no pain".Firstly,the ligation standard suitable for the preparation of Chronic constriction injury(CCI)model of silk thread was explored by a semi-quantitative way.Secondly,a stable and reliable animal model was used to explore the possible mechanism of the microbiota-brain-gut axis in neuropathic pain.Finally,the regulation of emodin on the microbiota-brain-gut axis was investigated after the microbe and metabolites that may be involved in the formation of neuropathic pain were obtained by high-throughput sequencing technology.Methods1.Sevevty-four male SD rats were selected and divided into Sham group(SG,n=12),Loose group(LG,n=13),Medium group(n=11,MG),and Tight group(n=11,TG),whose ligation criteria were selected based on a summary of the literature.To assess constriction under different criteria,we calculated the total radial strain(?TR%)of ligated nerves from digital photographs.The mechanical withdrawal thresholds(MWT),thermal withdrawal latency(TWL)were observed in rats of different groups to assess the state of model.Changes of myelin sheath were detected by pathological staining and immunohistochemistry.2.Sixteen male SD rats were divided into Sham group(Sham,n=8)and CCI model group(CCI,n=8)randomly.After aptive raising,the rats were operated to CCI model according to the standards of MG and measured the MWT and TWL.Using 16 S rDNA amplification sequencing technique,the feces of rats were tested to observe the abundance of intestinal flora,the LC-MS/MS untargeted metabolomics to detect metabolite changes in the serum and spinal cord of each rats.Besides,IPA analysis on the changed serum metabolites to establish network interactions.Correlation analysis was carried out between the behavioral results and the significantly different flora,and between different intestinal flora and significantly different metabolites in the serum,respectively,to obtain the key flora and metabolites.3.Twenty four male SD rats were divided into Sham group(Sham,n=8),CCI group(CCI,n=8),and Emodin group(Emodin,n=8)randomly.CCI model was prepared according to the standard of group M after adaptive feeding.The Emodin group was given intragastric administration of emodin(50mg/kg)from the first day after surgery,once a day for 15 days.After measurements of MWT,TWL and sciatic nerve index(SFI),qPCR was used to detect differential intestinal flora in faeces of rats.LC-MS/MS non-targeted metabolomics was used to detect the changes of metabolites in the spinal cord and serum of rats in each group and the metabolite pathways were screened according to correlation analysis.The changes of metabolites and related indexes were detected by ELISA and immunofluorescence techniques.Results1.The median ? R values in the Loose,Medium and Tight groups were 13.6%,15.2%and 21.7%,respectively.Ligated groups had lower MWT than SG(P<0.05)and the TWL of rats in the LG approached to rats with SG(P>0.05),while that of the TG was higher than Medium group 14 days after surgery(P<0.05).The expression MPZ in MG and TG was significantly lower than that in SG(P<0.05),and LG was significantly higher than MG(P<0.05).MPZ levels decreased in the sciatic nerves as the degree of constriction increased,but myelin basic protein of the Medium group was lowest abundant in the spinal cords of all rats.2.There was no significant difference in the MWT and TWL between the Sham group and CCI group before surgery.Compared with the Sham group,the MWT and TWL in CCI group began to decrease significantly 3 days after surgery(P<0.05).And that remained at a low level until 15 days after surgery,which showed a significant difference compared with the Sham group(P<0.05).Besides,we found that the composition of the gut microbiota was different between rats with CCI operation.The abundances of Helicobacter,Phascolarctobacterium,Christensenella,Blautia,Streptococcus,Rothia and Lactobacillus were significantly increased(P<0.05),whereas the abundances of Ignatzschineria,Butyricimonas,Escherichia,AF12,and Corynebacterium were significantly decreased(P<0.05).Additionally,72 significantly differentially expressed serum metabolites and 17 significantly differentially expressed spinal cord metabolites were identified between the CCI rats and the Sham rats.Correlation analysis suggesting that MWT and TWL were mainly positively correlated with Ignatzschineria,Butyricimonas,Escherichia,Corynebacterium and AF12.Streptococcus,Phascolarctobacterium,Helicobacter,Blautia,Rothia,Lactobacillus and Christensenella were negatively correlated with MWT and TWL.It can be found that Ignatzschineria,Butyricimonas and Streptococcus are associated with more metabolites in the serum.Some metabolites associated with these two bacteria had the same changes in the spinal cord and serum,such as N6,N6,N6-trimethyl-L-Lysine,3-methylhistidine,Allantoin,D-Quinovose and D(-)-beta-hydroxy butyric acid.3.There were no significant differences in MWT and TWL between Sham group,CCI group and Emodin group before operation(P>0.05),but the MWT and TWL of CCI and Emodin group began to decrease significantly compared with Sham group 3 days after operation(P<0.05).MWT of Emodin group was significantly higher than CCI group at 7days after surgery(P<0.05),TWL of Emodin group was significantly higher than CCI group at 11 days(P<0.05),until 15 days after surgery.But MWT and TWL of them were significantly lower than Sham group(P<0.05).qPCR results indicated that the expression of Ignatzschineria in model group was significantly lower than Sham group(P<0.05),and the level of Ignatzschineria was increased after emodin treatment(P<0.05).Compared with the Sham group,both Helicobacter and Streptococcus were increased in the CCI group(P<0.05),and were significantly decreased after emodin treatment(P<0.05).In serum,there were 29 metabolites significantly different between the CCI group and Emodin group,such as Choline,L-Arginine and Allantoin.In the spinal cord,there were 18 siginificant changed metabolites in emodin group,such as L-Histidine,L-Histidinol and L-Glutamine.Correlation analysis indicated significant correlation between changed metabolites,including L-histidine,pro-glu and Citrate,and changed microbiotas,including Ignatzschineria,Helicobacter and Streptococcus(P<0.05).ELISA results showed that the levels of Histamine and L-histidine were decreased in CCI group compared with Sham group(P<0.05),while the expression of IL-1?,TNF-? and IL-6 were increased(P<0.05).Compared with CCI group,the levels of Histamine and L-histidine in Emodin group were increased(P<0.05),and the expression levels of IL-1?,TNF-? and IL-6 were decreased(P<0.05).By immunofluorescence,the expression of astrocytes markers(GFAP)showed that the CCI group were higher than Sham group(P<0.05),while those in Emodin group were significantly lower than those CCI group(P<0.05).Conclusion1.The optimal degree of tightness for silk ligatures in this model is defined by a 14-15% reduction in the diameter of the sciatic nerves with a brief twitch of surrounding muscles.When the sciatic nerve is constricted approximately 21% or more,the entire hind limb will twitch,and the pain thresholds will begin to increase and fluctuate.2.CCI operation induced the decrease of MWT and TWL in rats.16 S rDNA analysis of the feces in each group showed that the intestinal flora structure of rats in the CCI model group was significantly changed compared with Sham group,such as Ignatzschineria,Butyricimonas,Streptococcus,Helicobacter and Phascolarctobacterium.And there were some changes in the metabolites in spinal cord and serum,such as D(-)-beta-hydroxy butyric acid,3-methylhistidine,2-hydroxy-butanoic acid,Anthranilic acid and Kynurenic acid.And differences of intestinal flora,metabolites and CCI has certain correlation between the behavior of rats,the changes of intestinal flora were associated with significant changes in the level of serum metabolites,which prompt the imbalance of intestinal flora is the regulation of metabolic disturbance under the background of neuropathic pain.This funding provides a new idea for the development mechanism of NP based on the microbiota-brain-gut axis.3.After emodin treatment,the gene expression of Ignatzschineria?Helicobacter?Streptococcus in Emodin group was changed,and the levels of metabolites such as Histamine,L-histidine,L-histidinol,L-glutamine and L-glutamate in the spinal cord were changed.Among them,emodin may adjust the histidine metabolism pathway to increase the level of Histamine and inhibit glial cells releasing inflammatory cytokines to improving hyperalgesia of NP rats.This found illustrates the emodin may be the analgesic of NP by the microbiota-brain-gut axis. |
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