Mechanism Of The Engineered Probiotic Strain L.lactis MG1363-pMG36e-GLP-1 On Parkinson’s Disease Via Inhibiting Ferroptosis

Background and purpose:Parkinson’s disease(PD)is the second most common neurodegenerative diseases(NDDs)of central nervous system(CNS),and PD patients is expected to exceed 9.3million by 2030.At present,levodopa(L-DOPA)is commonly used in the treatment of PD,which has obvious effect on motor symptoms at the beginning,but the effect will decrease with the prolongation of use time.In addition,the movement complications and side effects will also increase with the progression of PD.PD has long course of disease,high disability rate,and lack of effective treatment,which brings great burden to patients,families and society.Therefore,exploring and clarifying the pathogenesis of PD,and specifically slowing down or preventing the pathological progression of PD is the foundation of treatment.The neuroprotective effect of glucagon-like peptide-1(GLP-1)and its analogues are receiving attention.However,the short half-life of GLP-1 mediated by transient enzymolysis from dipeptidyl peptidase IV(DPP-IV)restricted its clinical use.The previously constructed engineered probiotics Lactococcus lactis(L.lactis)MG1363-p MG36e-GLP-1 was confirmed to steadily secrete GLP-1 as well as exerting neuroprotective effect on MPTP-induced PD mice model by derepressing the death of Dopaminergic(DAergic)neurons and reducing α-syn aggregation to improve motor functions.However,the mechanism by which the engineered probiotics exert their neuroprotective effects is unknown.Herein,we continued to study the neuroprotective role of engineered probiotic strain L.lactis MG1363-p MG36e-GLP-1on MPTP-induced PD mice model by intragastric administration and determined the neuroprotection and neurotrophy mediated by suppression of DAergic neuronal ferroptosis.Methods:Fifty 3-month-old male C57BL/6 mice were randomly divided into 5 groups,including control group(C group),model group(M group),MPTP-induced Liraglutide treatment group(L group),MPTP-induced L.lactis treatment group(R group)and MPTP-induced L.Lactis MG1363-p MG36e-GLP-1 treatment group(RG group).Specific experimental methods are as follows:(1)The improvement of the engineered strains on behavioral disorders in PD mice was evaluated by open field test,pole test and hanging wire test;(2)The expression of TH,α-syn,BDNF and GDNF in PD mice was assessed by IHC staining,IF staining and Western blotting,to evaluate the survival of DAergic neurons,abnormal folding of aggregation and changes in neurotrophic factors after treatment with engineered strains;(3)The distribution of GLP-1R and the specific content of GLP-1 in the brain tissue were determined by ELISA and Western blotting;(4)The integrity of the blood brain barrier(BBB)and the intestinal barrier was determined by H&E staining and Western blotting;(5)The important role of ferroptosis was defined by TEM,Perls’ staining,IF staining and Western blotting in PD mice,and the main pathways of ferroptosis in brain tissue were further explored;(6)The change of oxidative stress level in serum and brain tissue was detected by DHE staining and oxidative stress factor measurement;(7)The composition and abundance of intestinal flora were detected by 16 S r DNA high-throughput sequencing technology in order to clarify the changes of intestinal flora during the occurrence and development of PD.Results:(1)L.lactis MG1363-p MG36e-GLP-1 could increase the expression of TH,DAT,BDNF and GDNF,and decrease the expression of α-syn,so as to further improve the exploration ability and motor function in MPTP-induced PD mice model;(2)L.lactis MG1363-p MG36e-GLP-1 could effectively improve the epithelial integrity and the expression of tight junction related proteins occludin and ZO-1 of intestinal barrier and BBB in PD mice,in addition,GLP-1 content in brain tissue was significantly increased;(3)L.lactis MG1363-p MG36e-GLP-1 could effectively increase DMT1 in the substantia nigra(SN)of PD mice,thus reducing the content of iron ions.Furthermore,ferroptosis was inhibited by activating Keap1-Nrf2-GPX4 signaling pathway and reducing oxidative stress level in PD mice,including up-regulating GSH-Px and SOD and down-regulating ROS and MDA;(4)L.lactis MG1363-p MG36e-GLP-1 could restore α-diversity,β-diversity and the abundance of intestinal flora to a certain extent in PD mice,especially increased the abundance of Akkermansia.Conclusion:In this study,we constructed the engineered strain L.lactis MG1363-p MG36e-GLP-1 based on L.lactis,and evaluated the therapeutic efficacy and potential mechanism of the engineered strain on the MPTP-induced PD mice model by various molecular biotechnology.The results of showed that the engineered strain L.lactis MG1363-p MG36e-GLP-1 can inhibit ferroptosis in the brain tissue of PD mice by activating Keap1-Nrf2-GPX4 signaling pathway and reducing oxidative stress levels,thus exerting neuroprotective role in PD.Furthermore,DAergic neurons death and synucleinopathy were further inhibited in PD mice,so as to improve the motor symptoms of PD mice.L.lactis MG1363-p MG36e-GLP-1 not only have the neuroprotective effect of GLP-1 protein,but also have a positive protective effect on human health due to the gastrointestinal probiotics of L.lactis itself.More importantly,bacteriological drugs can greatly reduce the cost of drugs and have a very broad utilization potentiality.