Experimental Study On Every-other-day Fasting Regulating Phenotypic Polarization Of Microglia In Rats With Spinal Cord Injury By Affecting Gut Microbiota

Objective:The rat model of clamped C5 spinal cord injury was established,and the intervention was Every-other-day fasting(EODF).By evaluating the motor function of rats,detecting the related genes and proteins of microglia phenotypic polarization in spinal cord tissue,gut microbiota and the inflammatory factors in serum,to investigate the effect of EODF on gut microbiota and phenotypic polarization of microglia in rats with spinal cord injury.Methods:A total of 144 male SD rats were randomly divided into 4 groups:pre-EODF group,post-EODF group,AL group and Sham group,36 rats in each group.Before injury,the pre-EODF group was treated with EODF,that is,throughout the experiment,fasting and ad libitum were carried out alternately every 24 hours(no restriction on drinking water),the rats in the other three groups were ad libitum.After4 weeks,the spinal cord injury model was performed.In Sham group,only lamina was removed without spinal cord injury.After injury,pre-EODF group continued EODF intervention,post-EODF group started EODF intervention immediately,both AL and Sham group were ad libitum.The experiment lasted for 8 weeks.Before injury,1day,7 days,14 days,and 28 days after injury,the grooming test was used to evaluate the motor function of rats’forelimbs.Rats in each group were sacrificed at 3,7,28 days after injury.Observe the changes in body weight and food intake of rats before and after injury,use 16Sr DNA to detect the changes of gut microbiota,use RT-PCR and Western blot to detect the m RNA and protein relative expression of Arg-1,CD206,i NOS and CD86 in spinal cord tissue,respectively,use immunofluorescence staining to detect the phenotypic polarization of M1 and M2microglia in spinal cord tissue,enzyme-linked immunosorbent assay to detect the content of IL-4,IL-1β,BDNF and LPS in serum.Results:1.Food intake and body weight of rat(1)Food intake:28 days before injury,the average weekly food intake of rats in post-EODF group,AL group and Sham group was basically the same.In pre-EODF group,it was less than the other three groups,which was reduced by 34%.28 days after injury,the average weekly food intake of rats in pre-EODF group and post-EODF group was significantly lower than AL group,which was 60%and 67%of the AL group,respectively.(2)Body weight:At 7,14,21 days before injury,the body weight of rats in pre-EODF group was significantly lower than that in the other three groups(P<0.05).At 1,7,14,21,28 days after injury,the body weight of rats in pre-EODF group and post-EODF group was significantly lower than AL group(P<0.05).2.Effect of EODF on motor function in rats with spinal cord injuryThe grooming test scores of rats in each group were higher before injury,and there was no significant difference between groups(P>0.05).One day after injury,the grooming test scores of rats in each group decreased,and gradually increased with the passage of time.On the 1st,7th,14th and 28th day after injury,the grooming test score in AL group was lower than that in Sham group(P<0.05),in pre-EODF group and post-EODF group,it was higher than that in AL group 28 days after operation(P<0.05).3.Effect of EODF on the levels of serum IL-4,BDNF and IL-1βin rats with spinal cord injury(1)On the 7th day after injury,the serum IL-4 content of pre-EODF group was higher than that of AL group(P<0.05).On the 28th day,the serum IL-4 content of pre-EODF group and post-EODF group was higher than that of AL group(P<0.05).(2)On the 28th day after injury,the serum BDNF content of pre-EODF group was higher than that of AL group(P<0.05).(3)Compared with the AL group,the serum IL-1βcontent of pre-EODF group was significantly reduced at 3,7 and 28 days after injury(P<0.05),and the IL-1βcontent of post-EODF group was reduced at 28 days after injury(P<0.05).4.Effect of EODF on phenotypic polarization of microglia in spinal cord tissue of rats with spinal cord injury(1)The RT-PCR results showed that compared with AL group,the relative m RNA expression of Arg-1 in pre-EODF group and post-EODF group were increased at 3 days after injury(P<0.05).At 7 days,the relative m RNA expression of CD206obviously increased,and the i NOS m RNA decreased significantly in pre-EODF group(P<0.05),the i NOS m RNA in post-EODF group also decreased significantly.At 28days,the relative m RNA expression of CD206 and Arg-1 in pre-EODF group and post-EODF group were significantly increased,and the CD86 and i NOS m RNA was significantly reduced(P<0.05).(2)The results of western blotting showed that compared with AL group,the relative protein expression of Arg-1 in pre-EODF group increased significantly at 3days after injury(P<0.05),and the CD206 protein in pre-EODF group and post-EODF group was relatively higher,the i NOS protein decreased at 7 days after injury(P<0.05).28 days after injury,the relative protein expression of CD206 and Arg-1 in pre-EODF and post-EODF groups increased significantly compared with AL group,and the level of CD86 protein decreased(P<0.05).(3)The results of immunofluorescence detection showed that on the 3rd day after injury,the proportion of M1(i NOS~+/Iba-1~+)microglia in AL group was higher than that in Sham group(P<0.05).On the 7th and 28th day after injury,the proportion of M2(CD206~+/Iba-1~+)microglia in pre-EODF group and post-EODF group was higher than that in AL group,while the proportion of M1(i NOS~+/Iba-1~+)microglia decreased in pre-EODF group and post-EODF group(P<0.05).5.Effect of EODF on gut microbiota of rats with spinal cord injury(1)Alpha diversity analysis results:3 days after injury,the Chao1 and Shannon indexes of AL group were lower than those of Sham group(P<0.05).7 days after injury,there was no significant difference in Alpha diversity index in each group(P>0.05).28 days after injury,compared with AL group,the Chao1 index of the pre-EODF group and post-EODF group was significantly increased(P<0.05),and there was no significant difference in Shannon index between groups(P>0.05).(2)Beta diversity analysis results:After SCI,the composition of gut microbiota of rats was different from that of the Sham group.EODF would affect the composition of gut microbiota,which was closer to the Sham group.(3)Comparison results of gut microbiota relative abundance:Family level:Compared with AL group,the relative abundance of Lachnospiraceae and Clostridiaceae＿1 in post-EODF group was significantly higher at 3 days after injury(P<0.05).At 7 days after injury,the Lactobacillus in pre-EODF group and post-EODF group were significantly higher,but the Clostridiales＿vadin BB60＿group decreased(P<0.05),Tannerellaceae and Marinifilaceae in pre-EODF group are lower(P<0.05).28 days after injury,the Ruminococcaceae in pre-EODF and post-EODF groups significantly increased(P<0.05),Rikenellaceae increased significantly in post-EODF group(P<0.05),Erysipelotrichaceae increased significantly in pre-EODF group(P<0.05).Genus level:Compared with AL group,the relative abundance of Escherichia-Shigella in pre-EODF group was lower at 3 days after injury(P<0.05),and the post-EODF group also decreased.The relative abundance of[Eubacterium]＿coprostanoligenes＿group in pre-EODF group and post-EODF group decreased,[Ruminococcus]＿gnavus＿group and Clostridium＿sensu＿stricto＿1increased in post-EODF group(P<0.05).The relative abundance of Lactobacillus in pre-EODF group and post-EODF group both increased significantly,and the Prevotellaceae＿UCG-001,Prevotellaceae＿NK3B31＿group also increased(P<0.05),Clostridium＿sensu＿stricto＿1 in post-EODF group was significantly reduced(P<0.05).After 28 days,Escherichia-Shigella in pre-EODF group was significantly reduced,and Ruminococcaceae＿UCG-014 was significantly increased(P<0.05),the pre-EODF group and post-EODF group Prevotella＿9 decreased,and[Eubacterium]＿coprostanoligenes＿group increased significantly(P<0.05).6.Effect of EODF on serum LPS content in rats with spinal cord injuryAt 3 days after injury,the serum LPS content in pre-EODF group was lower than that in AL group(P<0.05).At 7 days after injury,serum LPS content in AL group was significantly higher than that in pre-EODF group and Sham group(P<0.05).28 days after injury,the serum LPS content of rats in pre-EODF group and post-EODF group was significantly lower than that in AL group(P<0.05).Conclusion:EODF may increase the relative abundance of beneficial bacteria in the intestines(such as Lachnospiraceae,Lactobacillus,[Eubacterium]＿coprostanoligenes＿group,etc.)and reduce the relative abundance of pathogenic bacteria(Such as Enterobacteriaceae,Escherichia-Shigella,etc.)by affecting the disordered gut microbiota of SCI rats,reduce the production of LPS,thereby regulating the phenotypic polarization of microglia,increasing the expression of M2 microglia,and reducing the expression of M1 microglia.This may be one of the mechanisms by which EODF alleviates the inflammatory response and plays a neuroprotective role.