| Objectives:In this study,an animal model of cervical 5 unilateral clamp spinal cord injury and every-other-day fasting(EODF)intervention was used to observe the behavior of rats,spinal cord histopathology,and intestinal bacteria.The changes of metabolites of bacteria and tryptophan bacteria and the expression of inflammatory factors,preliminary exploration of the effect of dietary restriction therapy on the intestinal flora of rats with spinal cord injury on the motor function of the intestinal flora regulating tryptophan metabolism and secondary inflammation.Methods:144 male SD rats were randomly divided into 4 groups,each with 36 rats:previously every-other-day fasting(pre-EODF)group,post every-other-day fasting(pos-EODF)group,ad libitum(AL)group and sham group.The entire experimental period is 56 days,including 28 days before SCI and 28 days after surgery.The pre-EODF group received EODF intervention before and after the operation,that is,fasting and eating were alternated at 24 hour intervals during the experiment,and drinking water was not restricted;the pos-EODF group was free to take food before the operation and EODF intervention after the operation;the AL group and the sham group can eat and drink freely before and after the operation.Observe the rats’ diet,body weight and behavior,and collect feces before surgery.Rats were sacrificed 1 days,7 days and 28 days after surgery.Serum,feces and spinal cord tissues were collected.Cylinder vertical climbing test and Irvine-Beatties-Bresnahan score were used to evaluate the recovery of forelimb motor function.HE staining to observe the pathological changes of the spinal cord,enzyme-linked immunosorbent assay to detect the levels of TNF-α and IL-10 in the rat serum,16S rDNA amplicon sequencing technology to detect and analyze the changes of intestinal flora in each group of rats at various time points Characteristics and different species,liquid chromatography tandem mass spectrometry technology to detect the content of tryptophan bacterial metabolites in feces.results:1.General situationBefore SCI,the average daily food intake and body weight of rats in the pos-EODF group,AL group and sham group were small at each time point(p>0.05).The average daily food intake of the pre-EODF group was reduced by about 35%compared with the AL group.After SCI,the average food intake of rats in the AL group,pos-EODF group and pre-EODF group all decreased on the 7th day after surgery.Among them,pos-EODF and pre-EODF group decreased significantly(p<0.05).The average daily food intake of the pre-EODF group was about 65%of that of the AL group.The body weight of rats in each group decreased to varying degrees on the first day after SCI.The weight of rats in the 4 groups gradually increased from the first day after modeling,but the weight of the rats in the pre-EODF group and pos-EODF was lower than that of the AL group at all time points,and the pre-EODF group and the AL group were significantly different(P<0.05).2.The effect of EODF intervention on the motor function of SCI ratsBefore SCI,there was no significant difference in the usage rate of the affected forelimb and IBB score in the cylinder vertical climbing test and the IBB score of the 4 groups of rats(P>0.05);1 days after SCI,except for the sham group of rats In addition,the use rate of forelimbs and IBB scores of the other three groups of rats were significantly reduced(P<0.0 5),and then gradually increased on 7,14 and 28 days,and the differences in different time points within each group were statistically significant.Significance(P<0.05);7 days and 14 days after SCI,there was no significant difference in the use rate of affected forelimbs and IBB scores between the AL group,pos-EODF and pre-EODF group(P>0.05);28 days after SCI,the pre-EODF group and pos-EODF’s ipsilateral forelimb usage and IBB score were significantly higher than the AL group(P<0.05).3.The effect of EODF intervention on histopathology in SCI ratsThe results of HE staining showed that 3 days after SCI,the spinal cord tissue of the sham group was normal,with clear boundaries between gray matter and white matter;the other three groups showed large spinal cord tissues on the injured side.Area of cavity,gray matter and white matter boundaries are unknown,spinal cord tissue structure is disordered,nerve fiber rupture and necrosis,myelin sheath loss,extensive hemorrhage,and a large number of glial cell proliferation and inflammatory cells Infiltration,especially in the AL group and pos-EODF group.At 7 and 28 days after surgery,the pathological conditions of spinal cord tissue in each group gradually improved.The morphology and structure of spinal cord in pos-EODF group and pre-EODF group were closer to those in the sham group.Smaller vacuoles were seen in the AL group,and the degree of pathological damage recovery was less.4.The effect of EODF intervention on the expression levels of serum TNF-α and IL-10 in SCI rats(1)3 days after operation,the relative level of serum TNF-α in the AL group was significantly higher than that of pos-EODF and the pre-EODF group(P<0.05),and the relative expression level of IL-10 in pos-EODF was significantly higher than that of the AL group(P<0.05);(2)After 7 days,the relative levels of serum TNF-α in each group showed an upward trend,and the AL group was significantly higher than the two intervention groups(P<0.05);(3)At 28 days after surgery,compared with 7 days after surgery,the relative levels of serum TNF-α in each group were significantly lower,and pos-EODF and the pre-EODF group were significantly lower than the AL group(P<0.05),Compared with 7 days after the operation,the relative expression level of IL-10 in pos-EODF and the pre-EODF group Significant increase(P<0.05).5.The effect of EODF intervention on the gut microbiota of SCI rats(1)DiversityThe results of Alpha diversity showed that compared with the sham group,the Chaol index,Shannon index and Simpson index of the AL group,pos-EODF and pre-EODF group decreased significantly(P<0.05)at 3 and 7 days after SCI.The Chao1 index and Simpson index of pos-EODF and the pre-EODF group were significantly increased compared with the AL group on the 28 days after SCI(P<0.05).Beta diversity showed that the gut microbiota communities of the 4 groups of rats were different at each time point.(2)Relative abundanceAt the phylum level:3 days after surgery,the relative abundance of Bacteroides in theAL group and pos-EODF was significantly lower than that in the sham group(P<0.05);the relative abundance of the phylum Firmicutes in pos-EODF was significantly higher than that in the sham group The AL group and the pre-EODF group(P<0.05);the relative abundance of Proteobacteria in the AL group and pos-EODF was significantly higher than that of the sham group(P<0.05).At 7 days postoperatively,the relative abundance level of Actinomycota in pos-EODF was lower than that in the pre-EODF group(P<0.05).At 28 days after operation,compared with the sham group,the relative abundance of Bacteroides in the AL group was significantly increased,and the relative abundance of Firmicutes was significantly decreased(P<0.05);the pre-EODF group was relatively abundant in Actinobacteria The degree was higher than that of the AL group(P<0.05).At the species level:3 days after surgery,the relative abundance of Lactobacillus murine in the sham group was significantly higher than that in the AL group and the pre-EODF group(P<0.05);the pre-EODF group was in the relative abundance of L.gasseri,L.reuteri and L.acidophilus was significantly higher than that of the AL group(P<0.05);the relative level of Klebsiella variicola in the pre-EODF group was significantly lower than that of the AL group(P<0.05).At 7 days after operation,the relative abundance of L.murine in the intestinal tract of rats in pos-EODF and the pre-EODF group was significantly higher than that in the AL group and the sham group(P<0.05);The relative abundance was significantly lower than that of the sham group(P<0.05);the relative abundance of L.acidophilus in the pre-EODF group and the AL group was higher than that of pos-EODF and the sham group(P<0.05).6.The effect of EODF intervention on tryptophan in SCI ratsAt 3 days after SCI,the concentration of tryptophan,indole acetic acid and indole-3-aldehyd in the sham group was higher than that of the other three groups(P<0.05);7 days after SCI,the pre-EODF group The level of indole acetic acid was higher than that of pos-EODF,(P<0.05);28 days after SCI,the levels of indole acetic acid and indole-3-aldehyde in the pre-EODF group were higher than compared with the AL group(P<0.05).From 3 days to 28 days after SCI,the concentration of tryptophan and its bacterial metabolites in the feces of rats in each group gradually increased.the levels of indole acetic acid in the AL group and the pre-EODF group were significant increased at 28 days after SCI(P<0.05).Conclusions:EODF improved the diversity of gut microbiota after SCI,increased the relative abundance of beneficial bacteria such as L.murine,L.gasseri,L.reuteri and L.acidophilus,and reduced the relative abundance of pathogenic bacteria of Klebsiella variicola,which in turn increased the relative expression level of tryptophan metabolites,indicates that dietary restriction therapy every other day reduces the secondary inflammatory response and pathological damage in the rat model of cervical spinal cord injury and improves motor function The mechanism may be related to the effect of gut microbiota and tryptophan metabolism. |
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