The Role Of Altered Intestinal Flora In The Development Of Elderly Epilepsy Through LPS-Mediated Inflammatory Responses

BackgroundEpilepsy is the third most common neurological disorder in the elderly(> 65 years),after cerebrovascular disease and dementia.The etiology,clinical manifestations,and treatment of senile epilepsy are quite different from those of youth.Nearly half of the elderly patients with epilepsy have an unknown etiology.The occurrence of epilepsy is closely related to age.The increase in epileptic conditions such as imbalances of neurotransmitters,abnormal glial cell function,and the release of inflammatory mediators during aging promotes susceptibility to epilepsy.Gut microbes play an important role in the maintenance of endocrine,immune,and metabolic processes and have been shown to influence central nervous system development and disease through bidirectional regulation of the brain-gut axis.ObjectThe structure and changes in intestinal microbial composition of C57 mice at different months of age were investigated by 16 SrRNA high-throughput sequencing,and serum LPS levels were measured in each group.Small doses of broad-spectrum antibiotics altered the composition of intestinal flora in aged mice,while low concentrations of exogenous lipopolysaccharide endotoxin(LPS)were administered intraperitoneally to observe its alterations on cognitive function,seizure threshold,and spontaneous epileptiform discharges,and to detect the expression of serum LPS,associated inflammatory factors,hippocampal microglia activation,β-amyloid(Aβ)and microtubule-associated protein(Tau)to clarify whether the intervention of the intestinal flora exerted pro-inflammatory and antiepileptic effects by elevating and decreasing LPS.TLR4 signaling inhibitors were administered to determine whether TLR4 inhibition reduced spontaneous epileptiform discharges and increased seizure thresholds in aged rats and whether it reduced the associated inflammatory response and Tau and Aβ.MethodsRectal faces were collected from 3,13,and 20 month old C57 mice,10 from each group,and the structural composition and changes in α-diversity,β-diversity and intestinal flora of the three age groups were analyzed by 16 SrRNA high-throughput sequencing.Forty 13-month-old male C57 were randomly divided into a blank control group(CON),a TLR4 receptor inhibition group(TAK),a broad-spectrum antibiotic treatment group(ABX),and an antibiotic treatment plus LPS treatment group(ABX+LPS).The CON group was gavaged and drank distilled water without ABX;the TAK group was injected intraperitoneally with TAK-242,3 mg/kg,3 times a week;the ABX group was gavaged and drank distilled water dissolved with broad-spectrum antibiotics;and the ABX+LPS group was injected intraperitoneally with LPS,0.33mg/kg,once a day,on top of the ABX group.After 6 months,Morris water maze was performed to observe cognitive function,6-Hz seizure threshold,and EEG to monitor seizure susceptibility and cortical discharges,and serum and brain tissue specimens were collected at the end of the experiment.Serum LPS,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),and interleukin-(IL-10)were measured by enzyme-linked immunosorbent assay(ELISA),and TNF-α,IL-1β and IL-6 m RNA expression in hippocampal tissues were measured by quantitative reverse transcription-polymerase chain reaction(q RT-PCR).The levels of TNF-α,IL-1β and IL-6 m RNA in hippocampal tissues were measured by immunofluorescence technic.Results(1)The intestinal microorganisms of aged mice were significantly altered,showing a decrease in diversity and an increase in abundance of pathogenic bacteria such as Proteobacteria,Burkholderiales,and Prevotellaceae.Serum LPS levels were significantly different in aged,middle-aged,and young mice,with older mice having significantly higher serum LPS levels than young ones.(2)After ABX treatment,compared with the control group,serum LPS levels were significantly reduced,expression of the anti-inflammatory factor IL-10 was increased,and expression of the pro-inflammatory factors TNF-α,IL-1β and IL-6 was reduced,consistent with the q-PCR results.The ABX+LPS group showed an enhanced inflammatory response,serum LPS levels were increased compared with the ABX group to no difference from the CON group,microglia activation,Aβ and Tau protein expression increased,mice showed cognitive impairment,lower seizure threshold and increased cortical discharges,demonstrating that geriatric intestinal flora acted as a pro-inflammatory and pro-epileptic agent by elevating LPS.Blocking TLR4-related signaling pathways resulted in a significant reduction in inflammatory responses and cortical discharges,suggesting that intestinal LPS exerts a pro-epileptic effect through activation of TLR4.ConclusionsIn this study,we found that the intestinal microecology of aged mice was disturbed,with significant increases in Gram-negative bacteria such as Proteobacteria,Burkholderiales and Prevotellaceae,and that serum LPS levels were significantly higher in aged mice than in young adults.Activation of TLR4 by intestinal flora via LPS promoted an inflammatory response leading to microglia activation and Aβ and Tau protein deposition in the brain,cognitive impairment,decreased seizure threshold,and increased cortical discharge in mice.