Trimethylamine N-oxide Worsened Neuroinflammation On Parkinson’s Disease Model Mice By Activating Glial Cells

Background: Parkinson’s disease(PD),a neurodegenerative disease that still cannot be effectively treated,is mainly characterized by degeneration and death of dopaminergic neurons in the midbrain substantia nigra,which leads to a decrease in the level of the neurotransmitter dopamine in the striatum,resulting in a series of clinical symptoms.Its main clinical symptoms include: static tremor,motor retardation,myotonia as the typical manifestations and gastrointestinal dysfunction,cognitive disorders as the representative of non-motor symptoms.The pathogenesis and risk factors of PD are not fully understood.However,a large number of studies on the pathogenesis of PD have shown that gut microbiota and its metabolites can affect the central nervous system through the microbiomegut-brain axis,and thus participate in the pathogenesis of PD.Population studies have shown that gut microbiota disorders in PD patients are accompanied by changes in microbial metabolites.Animal experiments also found that fecal bacteria transplantation based on normal mouse gut microbiota or supplementation of short-chain fatty acids by gavage can play a neuroprotective role in various PD mouse models.In conclusion,gut microbiota and its metabolites may play an important role in the pathogenesis of PD.Trimethylamine N-oxide(TMAO)is an indirect metabolite of gut microbiota.Studies have shown that TMAO can cross the blood-brain barrier(BBB)and disrupt its integrity.Meanwhile,TMAO can induce neuroinflammation,damage mitochondrial function and increase the level of oxidative stress in the brain.Recent clinical studies have found that the plasma TMAO level of PD patients are significantly higher than that of healthy controls,and higher TMAO level is positively correlated with the severity and progression of PD.Interestingly,another study showed that the plasma TMAO level of PD patients were significantly lower than that of the control group,and a lower level of TMAO could be used as a biomarker for early PD.The above research results suggest that abnormal changes in TMAO level in vivo may be closely related to PD,but existing studies are very limited with different conclusions.Objective: Based on the above research background,we firstly increased the TMAO level in peripheral blood of mice by supplying 3%(w/v)TMAO into drinking water,and then constructed PD model mice by intraperitoneal injection of MPTP,so as to explore the potential effect of pre-existing higher circulating TMAO on PD model mice.In order to provide a new idea for clarifying the relationship between PD and TMAO.Methods: The 6-week-old male C57BL/6 mice were randomly divided into four groups:Control group,MPTP group,TMAO group and TMAO + MPTP group.The experiment lasted for 28 days.The Control and MPTP groups were fed conventionally throughout the experiment,and the TMAO and TMAO + MPTP groups were fed conventionally and supplemented with 3%(w/v)TMAO in water.On day 21 of the experiment,MPTP group and TMAO + MPTP group were injected intraperitoneally(20 mg/kg,4 times,every 2 hours)to construct acute animal models of Parkinson’s disease,and tissue samples were collected on day 28.The motor function of mice was evaluated by pole test and traction test.The serum TMAO level was detected by liquid chromatography-mass spectrometry(LC-MS).Immunofluorescence(IF),Western blotting and HPLC were used to detect the number of dopaminergic neurons in the SN,the level of tyrosine hydroxylase(TH)in the striatum,and the contents of neurotransmitters dopamine(DA)and its metabolites,respectively.Immunofluorescence(IF)was used to detect the activation of glial cells in striatum,substantia nigra and dentate gyrus of hippocampus.Tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the hippocampus were detected by enzyme-linked immunosorbent assay(ELISA).The polarization of hippocampal glial cells was evaluated by RT-qPCR.Results: The results showed that TMAO treatment significantly increased the serum TMAO level of mice.Although higher circulating TMAO did not have significant effects on the number of dopaminergic neurons in the substantia nigra,TH protein content in the striatum and DA level in PD mice,higher circulating TMAO significantly intensified DA metabolism.In addition,TMAO significantly increased the activation of microglia and astrocytes in the striatum and substantia nigra(SN).Meanwhile,immunofluorescence,ELISA and RT-qPCR results in the hippocampus also showed that higher circulating TMAO significantly promoted the activation of microglia and astrocytes in the hippocampal dentate gyrus(DG),and increased the levels of TNF-α and IL-1β.The mRNA levels of M1microglia-related markers(including CD16,CD32 and iNOS)and A2 astroglia-related markers(including S100a10,Ptx3 and Emp1)were up-regulated.Conclusion: In a word,the striatal tissue,substantia nigra tissue,and hippocampal tissue in the acute PD model mice with and without TMAO treatment were analyzed,we found that higher circulating TMAO exacerbates neuroinflammation in Parkinson’s disease model mice by inducing the activation of glial cells.Therefore,TMAO may be an important mediator of host-microbial interactions in PD and higher TMAO levels in the peripheral blood may be one of the risk factors for PD.