Exploring The Therapeutic Effect Of Mesenchymal Stem Cell-derived Exosomes In Alzheimer’s Disease Based On Gut Microbiota And Its Related Metabolites

Background:Alzheimer’s disease（AD）is a widespread degenerative nervous system disease.Alzheimer’s disease usually starts slowly or insidiously and typically presents with memory loss and cognitive impairment.Currently,some approved drugs can reduce AD symptoms and improve patients’quality of life,but cannot stop the progression of the disease.Therefore,finding new therapeutic strategies for AD is a top priority.There is growing evidence that the gut microbiota is closely associated with AD,and that microbiota disorders can increase gut permeability,promote systemic inflammation and immune responses,and increase blood-brain barrier permeability,which in turn leads to neuroinflammation and amyloid plaque deposition and induces AD.According to the report that mesenchymal stem cell-derived exosomes（MSCs-exo）have similar properties to those of mesenchymal stem cells（MSCs）.MSCs have immunomodulatory and regenerative repair potential,and are more stable and easy to preserve.MSCs-exo have been found to promote the degradation of amyloid plaques,modulate the immune response,protect neurology,promote axonal growth,and improve cognitive impairment,but the exact mechanism is not clear.Objective:Since the gut microbiota is closely related to the development of AD,we investigated whether the gut microbiota of AD disorders affects the therapeutic effect of MSCs-exo.Methods:MSCs were first cultured for 3-6 generations,then MSCs-exo were extracted using ultracentrifugation,and the size,morphology and surface marker proteins of exosomes were identified by NTA,TEM and Western Blot.In vitro experiments:SH-SY5Y cells were treated with Aβ_1-40 and co-incubated with Aβ_1-40-treated SH-SY5Y cells after Di O-labeled MSCs-exo,and the uptake ability of MSCs-exo by SH-SY5Y cells was analyzed;the effect of MSCs-exo on proliferation and migration of the Aβ_1-40-treated SH-SY5Y cells was detected by CCK-8 assay and scratch assay.The effect of MSCs-exo on the proliferation and migration of the Aβ_1-40-treated SH-SY5Y cells was examined by CCK-8 assay and scratch assay.The effect of MSCs-exo on the Aβcontent of Aβ_1-40-treated SH-SY5Y cells was detected by cellular immunofluorescence.In vivo experiments:A small amount of mouse tail tissue was taken at 1 month of age to identify and screen 5×FAD mice.Then,4-month-old C57 and 5×FAD female mice were selected for the experiment.One group of 5×FAD mice was intravenously injected with PBS（5×FAD group）,and the other group was intravenously injected with MSCs-exo（1.0×109 in 100μL PBS）three times at 1-month intervals（MSCs-exo group）.We co-housed PBS-treated 5×FAD mice and MSCs-exo-treated 5×FAD mice（co-housed group）to test the effect of the disordered gut microbiota in AD on MSCs-exo treatment.To deplete the gut microbiota in AD,5×FAD mice received broad-spectrum antibiotic cocktails（Abx,vancomycin（0.5 mg/m L）,neomycin（1mg/m L）,metronidazole（1 mg/m L）,and ampicillin（1 mg/m L））in drinking water for 1week and was intravenously injected MSCs-exo（MSCs-exo+Abx group）.The cognitive abilities of the mice were examined in the contextual fear-conditioning and Morris water maze;the neuropathy was examined by HE staining,NISSL staining,Aβimmunohistochemical staining,GFAP and Thio S immunofluorescence double staining;and the gut microbiota and its related metabolites were examined by collecting feces from the mice.Results:The results showed that MSCs-exo had a protective effect on Aβ_1-40-treated SH-SY5Y cells.5×FAD mice had dysregulated intestinal flora and its associated metabolites,and MSCs-exo improved cognitive impairment and neuropathology in 5×FAD mice,but AD-disordered intestinal flora could eliminate the therapeutic effect of MSCs-exo.However,Abx treatment enhanced the improvement of cognitive impairment in5×FAD mice by MSCs-exo.HE staining,NISSL staining and immunohistochemical staining further confirmed that Abx treatment enhanced the repair of neuropathy in5×FAD mice by MSCs-exo.Conclusion:We found that the AD gut microbiota can eliminate the therapeutic effect of MSCs-exo,while the therapeutic effect can be enhanced by modulating the disordered gut microbiota and its associated metabolites with antibiotics.For patients with disordered gut microbiota or those who do not respond to MSCs-exo treatment,gut microbiota modulation may help achieve better therapeutic outcomes,providing an experimental basis for clinical treatment of AD.