| Background and objective: Parkinson’s disease(PD)is the second most common neurodegenerative disease in the world,affecting about 1% of people over the age of60.The pathological hallmarks of PD are progressive death of dopaminergic neurons and pathological aggregation of α-Synuclein(α-Syn)to form Lewy bodies(LBs)or Lewy neuritis(LNs)in substantia nigra(SN)and striatum.The main clinical manifestations of PD are resting tremor,rigidity,bradykinesia and postural disability.Some non-motor symptoms including anxiety,depression,constipation,and rapid eye movement sleep behavior disorder(RBD)can also occur with disease progression.Diabetes mellitus(DM)is the most common chronic metabolic disease in the world,and its high prevalence and high disability and mortality rates resulting from numerous complications become a serious burden on society.Studies have found that PD and DM share common causes,and both diseases are caused by genetic factors and environmental factors.Moreover,PD and DM have similar pathophysiological changes,namely amyloid misfolding and aggregation,progressive cell death,and chronic inflammation.Based on this,we intend to study the association between DM and the risk and progression of PD,and screen out anti-diabetic drugs that can reduce the risk of PD.Then,the screened anti-diabetic drugs were used to verify their protective effect in animal and cell models of PD and explore their mechanism of regulating neuroinflammation,providing theoretical and experimental basis for the prevention and treatment of PD.Methods: This study is divided into three parts.In the first part,we searched Pub Med,Web of Science,Scopus,Embase and Cochrane Library for all literatures related to PD and DM,and then used literature management software Endnote X9 for screening.Finally,Stata SE 12.0 software was used for meta-analysis to quantify the association between DM and the occurrence and progression of PD,and to screen out anti-diabetic drugs that can reduce the risk of PD for subsequent experiments.In the second part,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)was used to construct the subacute PD mouse model.Behavioral testing methods such as open field test,rotarod test,pole test,inverted screen test,hindlimb clasping test and gait analysis were used to verify the effectiveness of the screened anti-diabetes drugs.Then,immunohistochemistry,immunofluorescence,western blot,real-time quantitative polymerase chain reaction(RT-q PCR),enzyme-linked immunosorbent assay(Elisa)was used to explore the mechanism of screened anti-diabetes drugs,especially the effects on neuroinflammation.In the third part,1-methyl-4-phenylpyridinium(MPP(+))was used to construct PD cell model.Human neuroblastoma cells(SH-SY5 Y cells)and mouse microglia cells(BV2 cells)were treated with MPP(+).Cell viability assay(CCK8 method),flow cytometry and other methods were used to observe the effects of the screened anti-diabetes drugs on cell viability,apoptosis and reactive oxygen species(ROS)of SH-SY5 Y cells and BV2 cells.Results: 1.In case-control studies,DM was not associated with the risk of PD,and the combined effect size was OR 0.82(95%CI 0.64-1.04).In cohort studies,DM patients had an increased risk of PD,and the probability of PD in DM patients was 1.32 times higher than that in non-DM patients,with a combined effect size of HR 1.32(95% CI1.22-1.43).Prediabetic status also increased the risk of PD,with a combined effect size of HR 1.04(95% CI 1.02-1.07).2.DM can accelerate the progression of PD,including motor symptom progression and cognitive decline.Movement Disorder SocietyUnified Parkinson Disease Rating Scale Part Ⅲ(MDS-UPDRS Part Ⅲ)of PD patients with DM increased by 0.3 points,and the combined effect size was SMD 0.30(95%CI0.06-0.54).The Montreal Cognitive Assessment(Mo CA)of PD patients with DM decreased by 0.47 points,and the combined effect size was SMD-0.47(95%CI-0.74--0.19).3.Glitazone(GTZ)reduced the risk of PD in DM patients by 0.75 times,with a combined effect size of HR 0.75(95%CI 0.60-0.94).Glucagon-like peptide-1 receptor agonists(GLP-1Ra)reduced the risk of PD in DM patients by 0.40 times,with a combined effect size of HR 0.4(95%CI 0.22-0.73).Dipeptidyl peptidase 4 inhibitors(DPP4i)reduced the risk of PD in DM patients by 0.59 times,with a combined effect size of HR 0.59(95%CI 0.42-0.83).Metformin did not reduce the risk of PD in DM patients,with a combined effect size of HR 1.24(95%CI 0.52-2.92).4.Exendin-4 and Linagliptin can increase the total movement distance and rearing times of mice in open field test and increase the latent time of mice in rotarod test.They can improve the score of mice in hindlimb clasping test and reduce the maximum variation of mice in gait analysis.5.Immunohistochemistry and western blot results showed that Exendin-4 and Linagliptin increased the expression of tyrosine hydroxylase(TH)and dopamine transporter(DAT),and increased the number of dopaminergic neurons in the SN of MPTP mice.6.Immunohistochemistry and immunofluorescence results showed that Exendin-4 and Linagliptin decreased the number of activated microglia(Iba1 labeled)and astrocytes(GFAP labeled),and increased the expression of M2 microglia marker CD206 in the SN of MPTP mice.7.RT-q PCR showed that Exendin-4 and Linagliptin decreased the m RNA expression of pro-inflammatory cytokines including interleukin-1β(IL-1β),tumor necrosing factor α(TNF-α)and M1 microglia marker inducible nitric oxide synthase(i NOS)and increased the m RNA expression of anti-inflammatory cytokines including interleukin-10(IL-10),transforming growth factor β(TGF-β)and M2 microglia marker arginase 1(Arg1)in the SN of MPTP mice.8.Western blot showed that Exendin-4 and Linagliptin inhibited the expression of NLRP3 inflammasome pathway related proteins such as NLRP3,Caspase-1 p20,IL-1β and GSDMD N-terminal in the SN of MPTP mice.9.Elisa showed that Exendin-4 and Linagliptin could reduce the level of lactate dehydrogenase(LDH)in serum of MPTP mice.10.Immunohistochemistry showed that Exendin-4 and Linagliptin increased the expression of colonic tight junction protein ZO-1 in MPTP mice.11.CCK8 assay and apoptosis flow cytometry showed that Exendin-4 and Linagliptin had limited protective effect on SH-SY5 Y cells,and only 100 n M Linagliptin could mildly inhibit the early apoptosis of SH-SY5 Y cells induced by MPP(+).12.The green fluorescence expression of ROS probes was observed under inverted fluorescence microscope,and it was found that Exendin-4 and Linagliptin could reduce the green fluorescence expression of ROS probes in MPP(+)induced BV2 cells.Quantitative flow cytometry analysis showed that Exendin-4 and Linagliptin could reduce ROS expression,which was consistent with the results observed by inverted fluorescence microscope.Conclusions: 1.Meta-analysis results showed that DM is a risk factor for PD,and can aggravate the progression of motor symptoms and cognitive decline of PD.Metaanalysis showed that GTZ,GLP-1Ra,and DPP4 i but not metformin can reduce the risk of PD in diabetic patients.2.Exendin-4 and Linagliptin can reverse behavioral dysfunction in MPTP-induced PD mice,including autonomous motor behavior,exploration behavior,motor coordination.Exendin-4 and Linagliptin reduced the death of dopaminergic neurons in the SN in MPTP-induced PD mice.Exendin-4 and Linagliptin inhibited neuroinflammation by inhibiting NLRP3 signaling and promoting polarization of microglia to M2 phenotype in the SN in MPTP-induced PD mice.Exendin-4 and Linagliptin can improve intestinal barrier function.3.Exendin-4 and Linagliptin cannot directly protect SH-SY5 Y cells from MPP(+)injury.However,Exendin-4 and Linagliptin significantly reduced MPP(+)-induced ROS production in BV2 cells. |
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