| Parkinson 's disease(PD)is a neurodegenerative disease common in middle-aged and elderly people.The pathological feature is the selective loss of dopaminergic neurons in the substantia nigra(SN),which reduces the dopamine(DA)content in the striatum,and the presence of Lewy bodies(LBs),which alpha-synuclein(?-syn)is the main component in the remaining neurons.The pathogenesis of PD includes inflammatory response,oxidative stress,abnormal nigral iron deposition,abnormal protein aggregation and mitochondrial dysfunction.Recent studies have shown that gut microbiota is closely related to the pathogenesis of PD.Sampson et al reported that gut microbiota are required for motor deficits,microglia activation,and ?-syn pathology in ?-syn overexpressed mice.Colonization of ?-syn-overexpressing mice with microbiota from PD patients enhances physical impairments compared to microbiota transplants from healthy human donors.The gut microbiota is inseparable from the health of host.In most cases,the number and types of gut microbiota are in a relatively balanced state at different times,which can protect the host from pathogens.However,the composition of bacteria is affecting by many factors,especially dietary ingredients containing metal ions.The diet structure determines the microenvironment of gut microbiota,which in turn affects the diversity of microorganisms.Iron is one of the vital factors for bacterial growth.When the body iron uptake overload,it will destroy the balance of gut microbiota,which is more conducive to the growth of pathogenic intestinal bacteria and inhibits the growth of intestinal beneficial barrier bacteria.In addition,iron levels are directly relating to the virulence of many causative agent and conditioned pathogen.People who eat iron-rich foods have a 1.7-fold increased risk of PD.Animal research found that excessive uptake of exogenous iron in newborn mice will cause the degeneration of dopaminergic neurons.Our previous study showed that iron content in the SN increased and tyrosine hydroxylase-immunoreactive(TH-ir)cells decreased,when adult male mice were gaven a high-iron diet.Although gastrointestinal administration of iron can cause iron deposition and dopaminergic neurons loss in the SN of mice,the exact mechanism is still unclear.Based on the above research background,we propose the hypothesis: the increased iron content and dopaminergic neurons damage in the SN may be related with the gut microbiota imbalance caused by the high-iron diet.Therefore,to observe the effect of high-iron diet on iron content and dopaminergic neurons in the SN and mice gut microbiota,8-week-old male C57BL/6 mice fed with 3% carbonyl iron powder were used in the experiment.Bacterial 16 S r DNA sequence amplification and Mi Seq high-throughput sequencing technology were used to detect the impact of high-iron diet on gut microbiota.GC-MS technology was used to detect fecal SCFAs content.Immunohistochemistry staining,Perls' staining,immunofluorescence staining,western blot were used to observe the changes in the number of TH-ir neurons,the number of iron-positive cells,the expression of microglia marker Iba1 and the expression of TH protein in SN.ELISA was used to observe serum and fecal bacterial LPS content.In order to investigate whether dopaminergic neurons damage and iron deposition in the SN are related with the dysregulation of gut microbiota in mice,we used fecal microbiota transplantation(FMT)to transplant fecal suspension of high-iron diet mice(donor mice)into antibiotic-pretreated mice(recipient mice).Immunohistochemistry and Perls' staining were used to observe the changes in the number of TH-ir neurons and the iron staining positive cells in the SN.Results as follow:1.Effect of high-iron diet on gut microbiota in mice(1)Alpha diversity analysis.The H index increased in the high-iron diet mice of 1 month and 3 months groups,compared to the normal diet group(***P<0.001,*P<0.05,n=8-10),and the D index decreased significantly in the high-iron diet mice of 1 month and 3 months(***P< 0.001,*P<0.05,n=8-10),compared to the normal diet group,which indicating that the high-iron diet for 1 and 3 months has a high bacterial diversity.No significant change of H index and D index were found in high-iron diet mice of 2 weeks group(P>0.05,n=10).(2)Beta diversity analysis based on OTU.Samples in the high-iron diet group and the normal diet group distributed in different area,and the two groups are significantly separated.The samples aggregated significantly in the high-iron diet mice of 1 month and 3 months groups.However,the samples did not show obvious aggregation in the high-iron diet mice of 2 weeks group.(3)Species abundance difference analysis based on phylum level.Compared to the normal diet group,Proteobacteria(*P<0.05,n=10),Tenericutes and Verrucomicrobia(**P<0.01,n=10)species abundance increased significantly in high-iron diet for 2 weeks group.Compared to the normal diet group,Bacteroidetes species abundance significantly decreased(***P<0.001,n=10);species abundance of Firmicutes,Proteobacteria,Tenericutes(**P<0.01,n=10),Cyanobacteria,Verrucomicrobia(*P<0.05,n=10)and Actinobacteria(***P<0.001,n=10)significantly increased in the high-iron diet for 1month group.Compared to the normal diet group,the abundance of Firmicutes significantly decreased(*P<0.05,n=8),species abundance of Bacteroidetes and Verrucobacteria(*P<0.05,n=8)Cyanobacteria(**P<0.01,n=8)increased significantly in the high-iron diet for 3 months group.(4)Species abundance difference analysis based on genus level.Compared to the normal diet,the abundance of Bacteroides,Parabacteroides,Alloprevotella,Alistipes,Escherichia,Parasutterella and Erysipelatoclostridium increased significantly(***P<0.001,**P<0.01,*P<0.01,n=10)in high-iron diet for 2 weeks group,while Pseudomonas abundance significantly decreased(*P<0.05,n=10)in high-iron diet for2 weeks group.Compared to the normal diet group,the abundance of Bacteroides,Parabacteroides,Escherichia,Intestinimonas,Ruminococcus,Ruminiclostridium,Enterorhabdus,Lactococcus,Roseburia,Pseudomonas and Blautia significantly increased(***P<0.001,**P<0.01,*P<0.01,n=10)in the high-iron diet for 1 month group,while the abundance of Rikenella was significantly decreased in the high-iron diet for 1 month group(*P<0.05,n=10).Compared to the normal diet group,Bacteroides,Parabacteroides,Allo Prevotella,Escherichia,Intestinimonas,Erysipelatoclostridium,Oscillibacter,Lactococcus and Blautia species abundance significantly increased(***P<0.001,**P<0.01,*P<0.01,n=8)in the high-iron diet for 3 month group,while Lactobacillus,Pseudomonas and Allobaculum abundance decreased significantly(***P<0.001,**P<0.01,*P<0.01,n=8)in the high-iron diet for 3 month group.2.Compared to the normal diet group,the content of acetic acidwas significantly decreased(*P<0.05,n=10),while the content of hexanoic acid and isovaleric acid were significantly increased(**P<0.01,n=10)in the high-iron diet for 2 weeks group.Compared to the normal diet group,the content of valeric acid(**P<0.01,n=10)and hexanoic acid(*P<0.05,n=10)was significantly decreased,the content of isobutyric acid(***P<0.001,n=10)significantly increased in the high-iron diet for 1 month group.Compared to the normal diet group,the content of acetic acid and butyric acid were significantly decreased(*P<0.05,n=8)in the high-iron diet for 3 month group.3.LPS content in serum of mice increased significantly in high-iron diet mice of 1month and 3months group compared to the normal diet group(***P<0.001,n=6).No changes were observed in LPS content in feces of mice from high-iron diet group compared to the normal diet group(P>0.05,n=6).4.The numbers of TH-ir neurons decreased significantly in the SN of high-iron diet mice of 3months group compared to the normal diet group(**P<0.01,n=6);no changes were observed in high-iron diet mice of 2weeks and 1month groups compared to the normal diet group(P>0.05,n=6).5.TH protein expression in the SN of the high-iron diet mice of 3months group decreased significantly compared to the normal diet group(*P<0.05,n=6);no changes were observed in the high-iron diet mice of 2weeks and 1month groups compared to the normal diet group(P>0.05,n=6).6.The numbers of iron positive staining cells increased significantly in the SN of high-iron diet mice of 1month and 3 months groups compared to the normal diet group(*P<0.05,**P<0.01,n=6);no changes were observed in high-iron diet mice of2 weeks group compared to the the normal diet group(P>0.05,n=6).7.The numbers of microglia marker Iba1 positive staining cells increased significantly in the SN of high-iron diet mice of 3months group compared to the normal diet group(*P< 0.05,n=5);no changes were observed in high-iron diet mice of 2weeks and1 month groups compared to the normal diet group(P>0.05,n=5).8.The numbers of TH-ir neurons in the SN of the fecal microbiota transplantation group and the high-iron diet group significantly decreased compared to the normal diet group(*P<0.05,n=5);no significant change was found in the antibiotic pretreatment group compared to the normal diet group(P>0.05,n=5).The numbers of TH-ir neurons in the fecal microbiota transplantation group and the high-iron diet group significantly decreased compared to the antibiotic pretreatment group(##P<0.01,n=5).No significant change was observed in the fecal microbiota transplantation group compared to the high-iron diet group(P>0.05,n=5).9.The numbers of iron staining positive cells in the SN of the fecal microbiota transplantation group and the high-iron diet group increased significantly compared to the normal diet group(*P<0.05,**P<0.01,n=5);no significant change was found in the antibiotic pretreatment group compared to the normal diet group(P>0.05,n=5).The numbers of iron-staining positive cells in the fecal microbiota transplantation group and the high-iron diet group significantly increased compared to the antibiotic pretreatment group(#P<0.05,##P<0.01,n=5).No significant change was observed in the fecal microbiota transplantation group compared to the high-iron diet group(P>0.05,n=5).The above results indicate that the high-iron diet in mice for 1 month and 3 months can cause the gut microbiota dysbiosis,leading to an increase in the diversity of gut microbiota,changes in the abundance ratio of the dominant bacteria Bacteroides and Firmicutes,decreased contents of the beneficial gut microbiota metabolites(acetic acid and butyric acid).High-iron diet in mice can induce the reduction of dopaminergic neurons,iron deposition and microglia activation in the SN,cause increased level of serum bacterial LPS.Therefore,the iron deposition and the reduction of dopaminergic neurons in the SN caused by the high-iron diet may be related to the the gut microbiota dysbiosis. |
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