Neuroprotective Effect And Molecular Mechanism Of An Engineered Strain Of Clostridium Butyricum-pMTL007-GLP-1 On Parkinson’s Disease Mice

Background and purpose:Parkinson’s disease(PD)is one of the most frequent chronic neurodegenerative disorders,and its incidence continues to escalate globally with the rapid aging of the population.Nevertheless,there are currently no effective drugs that can prevent or arrest its progression,so broadening the spectrum of PD drugs is of great significance.GLP-1(Glucagon-like peptide 1),as a type of incretin,is now widely recognized to be a promising target for the treatment of neurodegenerative diseases.However,its therapeutic potential is limited due to the rapid degradation of GLP-1 by widespread dipeptidyl peptidase-IV(DPP-IV)after entering the blood circulation,with a half-life of merely 1-2 minutes.Despite the development of GLP-1 analogs to overcome this defect,the exorbitant price and long-term injection requirements have wrought a significant impact on patients’financial burden and quality of life.With the application of gene editing technology in the pharmaceutical sector,engineered bacteria are increasingly becoming a promising modality of disease treatment,offering novel avenues for addressing this conundrum.On the basis,this study constructed a strain of Clostridium butyricum(C.butyricum)that can continuously express GLP-1,aiming to investigate the neuroprotective mechanism of C.butyricum-p MTL007-GLP-1 on PD mice models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),which provides an alternative therapeutic modality for PD.Methods:1.Construction of Clostridium butyricum-p MTL007-GLP-1 engineering strain:Firstly,integrate the GLP-1 gene into the p MTL007 plasmid,and then conjugate the recombinant plasmid GLP-1 into the genome of the C.butyricum host strain to construct a genome-integrated butyric acid C.butyricum-p MTL007-GLP-1engineering bacteria.2.Function verification of C.butyricum-p MTL007-GLP-1 engineering strain in vitro:GLP-1 secretion quantity of C.butyricum-p MTL007-GLP-1 is detected by ELISA.The probiotic properties of engineered bacteria were evaluated by growth curve,Bile salt and acid resistance test.3.Establishment and treatment of PD mouse model:Forty C57BL/6 eight-week-old male mice were separated into the control group(C group),model group(M group),model group+C.butyricum(CB group),model group+liraglutide group(L group),model group+C.butyricum-p MTL007-GLP-1 group(CBG group),with eight male mice in each group.The C group was given continuous gavage of saline gelatin for 14 days.Except for the C group,the remaining mice were given 20 mg/kg MPTP intraperitoneally for seven days to establish a PD mouse model.Then,the M group received 100μL of saline gelatin treatment gavage for seven days;the L group was given liraglutide at 0.4 mg/kg per day intraperitoneally for seven days;the CB group received 100μL of 10~8 CFU/m L C.butyricum gavage for seven days;CBG group received 10~8 CFU/m L C.butyricum-p MTL007-GLP-1 gavage for seven days.4.Mechanism analysis:The motor ability was assessed using behavioral assays.The pathological alteration was determined using immunohistochemistry and ultrastructural morphology.The expression of PD-related proteins,mitochondrial autophagy pathway-related proteins,and intestinal barrier proteins(ZO-1,Occludin)were detected by Western-blot.Effect of engineered bacteria on intestinal microbiome was detected by high-throughput sequencing.The levels of oxidative stress in mouse brain tissue and serum were measured via the kit.Results:1.Clostridium butyricum-GLP-1 has good probiotic properties in vitro:(1)ELISA results showed that the expression level of GLP-1 in C.butyricum-p MTL007-GLP-1 was 97.97 pg/m L(300 m L system);(2)Growth curve experiments indicated that no statistically significant differences in growth properties were observed between C.butyricum-PMTL007-GLP-1 and C.butyricum;(3)Acid resistance and bile salt resistance experiments showed that C.butyricum-p MTL007-GLP-1 could maintain a viable bacterial count higher than 10~6CFU/m L in the p H range of 3 to 7;under the condition of 0.5%bile salt,the number of viable bacteria remained to keep it above 10~7 CFU/m L,indicating that C.butyricum-p MTL007-GLP-1 has good acid and bile salt tolerance.2.Effect of C.butyricum-p MTL007-GLP-1 on PD:(1)C.butyricum-p MTL007-GLP-1 could improve the motor coordination and exploration ability of PD mice(P<0.01);(2)C.butyricum-p MTL007-GLP-1 could up-regulate PD characteristic proteinTH and DAT expression,and down-regulate the expression ofα-synuclein(α-syn)(P<0.01);(3)C.butyricum-p MTL007-GLP-1 promoted mitophagy signaling pathway proteins Beclin-1,PINK1,Parkin,Atg7,LC3B II and LAMP-1 expression(P<0.01),and suppressed p62 expression(P<0.05),to eliminate abnormal mitochondria;(4)C.butyricum-p MTL007-GLP-1 could restore the reduction of GSH-Px andSOD activities in serum and brain tissue of PD mice(P<0.01),and inhibit MDA activity(P<0.01);(5)C.butyricum-p MTL007-GLP-1 improved the diversity and richness of gut microbiota in PD mice,and reduced the relative abundance of PD-associated pathogenic bacteria Bifidobacterium(P<0.05).In addition,C.butyricum-p MTL007-GLP-1 increased the expression of intestinal barrier proteins(ZO-1,Occludin)(P<0.01).Conclusion:In this study,an engineered strain,C.butyricum-p MTL007-GLP-1,was constructed to continuously express GLP-1.In vitro experiments indicated that the engineered bacteria were well tolerant of acids and bile salts,which ensured their survival in the host gastrointestinal tract.In animal experiments,C.butyricum-p MTL007-GLP-1 improved motor dysfunction and neuropathological changes in PD,this neuroprotective effect may be attributed to the promotion of PINK1/Parkin-mediated mitochondrial autophagy and the attenuation of oxidative stress.In addition,C.butyricum-p MTL007-GLP-1 administration increased gut microbiota diversity in PD mice,thereby maintaining intestinal barrier function.