Fig 2: EcN‐GLP‐1 improved the neuropathologic damages in 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) mice. (a) EcN‐GLP‐1 inhibited the microglia activation (Iba1), astrocyte activation (GFAP), and reduced the synaptic dysfunction (α‐syn) in the substantia nigra of PD mice. Representative results of immunohistochemical analysis (IHC) of the Iba1‐positive microglia, GFAP‐positive astrocytes, and the expression of α‐syn. Magnification ×400, Scale bar = 50 μm. (b) EcN‐GLP‐1 alleviated the reduction of tyrosine hydroxylase (TH)‐positive dopaminergic neuron numbers in the substantia nigra of PD mice. Representative results of immunofluorescence analysis (IF) of the TH‐positive cell numbers. Magnification ×200, scale bar = 100 μm. Quantification analysis of the content of Iba1 (c), GFAP (d), α‐Syn (e), and TH (f), which were related to the results of IHC and IF. C, The normal control group; M, the MPTP‐induced PD model group; ME, the EcN‐GLP‐1 treatment group; MG, the exenatide treatment group; MN, the EcN treatment group. Three mice of each group were randomly selected for analyzed, and data were presented as means ± SD. The significance of data was performed by Tukey's multiple Comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference (p > 0.05)

Fig 3: Schematic diagram of the underlying mechanisms of EcN‐GLP‐1 in improving Parkinson's disease (PD). EcN‐GLP‐1 exerted neuroprotective effects against neurodegeneration and intestinal dysbiosis in 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP)‐induced PD mice via delivering GLP‐1 and modulating gut microbiota. GLP‐1, glucagon‐like peptide‐1; EcN‐GLP‐1, the engineered Escherichia coli Nissle 1917 that GLP‐1 cluster genes has been recombinantly integrated into its chromosome

Fig 4: EcN‐GLP‐1 suppressed the neuroinflammation induced by 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) in Parkinson's disease (PD) mice. (a) Effects of EcN‐GLP‐1 on the expression of p‐AKT/AKT, p‐IκB‐α, TLR‐4, and p‐p65/p65 in the substantia nigra of MPTP‐induced PD mice. (b) EcN‐GLP‐1 reduced the pro‐inflammatory cytokines of TNF‐α, IL‐1β, and IL‐6 at gene level. C, The normal control group; M, the MPTP‐induced PD model group; ME, the EcN‐GLP‐1 treatment group; MG, the exenatide treatment group; MN, the EcN treatment group. Three mice were randomly selected for analyzed, and data were presented as means ± SD. Tukey's multiple Comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference (p > 0.05)

Fig 5: Expression of GLP‐1 in the engineered probiotic strain and evaluation its probiotic characteristics. (a) ELISA analysis of EcN‐GLP‐1 expressing and secreting GLP‐1. (b) Growth curves of EcN‐GLP‐1 and EcN. (c) Acid tolerance capacity of EcN‐GLP‐1 and EcN. (d) Bile salt resistance of EcN‐GLP‐1 and EcN. (e) Antioxidant property of EcN‐GLP‐1 and EcN. (f) Antibacterial activity of EcN‐GLP‐1 and EcN. (g) Representative diagram of EcN‐GLP‐1 and EcN adhesion CT26 cells. (h) The CFU of adherent bacteria per 100 CT26 cells. Means ± SD was calculated from three independent experiments. Compared with EcN, *p < 0.05, no asterisk indicates no significant difference (p > 0.05)