Functional Study Of GDF15 In LPS-induced Inflammation Model And Rotenone-induced Parkinson’s Disease Model

BackgroundParkinson disease(PD)is a common degenerative disease of the nervous system in the middle-aged and elderly.It has characteristic motor symptoms,including static tremor,bradykinesia,muscle rigidity and postural balance disorder.The pathophysiology of Parkinson’s disease is characterized by selective loss of dopaminergic neurons in the substantia nigra and striatum,which is associated with a complex interplay of abnormal αsynuclein deposition,mitochondrial dysfunction,lysosomal or vesicular transport disorders,and neuroinflammation.It has been reported that neuroinflammation plays a crucial role in the pathogenesis of Parkinson’s disease in both patients and animal models.During the autopsy of the patients,it was found the microglia activation,the infiltration of T lymphocytes and the release of all kinds of proinflammatory factors in the substantia nigra,Parkinson’s disease patients imaging studies confirmed that the reducing of dopaminergic neurons also along with the increasing of the microglia activation,at the same time it was found the increasing of the proinflammatory factor in the blood and cerebrospinal fluid of patients with Parkinson’s disease.Microglia is the main effector cell in the process of neuroinflammation and its function is similar to that of macrophages in the peripheral system.After activation,microglia transformed from a branching resting state to an amoeba-like activated state and produced a large number of pro-inflammatory mediators.These pro-inflammatory mediators can cause damage to surrounding dopaminergic neurons and release damage-related molecular patterns,which can further activate more microglia to produce more pro-inflammatory mediators,ultimately leading to the degeneration and death of dopaminergic neurons.Therefore,intervention of neuroinflammatory response may be a potential treatment for PD.Growth differentiation Factor 15(GDF15)is a member of the transforming growth factor-β(TGF-β)superfamily.GDF15 has a broad range of anti-inflammatory and immunosuppressive effects,which have been demonstrated in disease models of myocardial infarction,atherosclerosis,and rheumatoid disease.GDF15 is widely distributed in central and peripheral nervous system,such as choroid plexus,cortex,hippocampus,striatum,pons and medulla oblongata,etc.It has been found that GDF15 increases in peripheral blood of PD patients and is related to the course of PD.Therefore,we speculated that GDF15 might play a neuroprotective role in PD by inhibiting microglia-mediated neuroinflammatory response.Methods1.BV2 cells were cultured in vitro and stimulated with LPS at different concentrations.After receiving the cells,the mRNA expression of TNF-α,IL-1β,iNOS and COX-2 were detected by real-time RT-PCR.2.BV2 cells were cultured in vitro and treated with exogenous GDF15 and LPS stimulation.The mRNA expression of TNF-α,IL-1β,iNOS and COX-2 were detected by real time RT-PCR.The protein expression of STAT3,P-STAT3,P65,P-P65,ERK1/2 and PERK1/2 was observed by Western blot.The morphological changes of microglia were observed under microscope.Intra-nuclear metastasis of P65 was observed by immunofluorescence staining.3.LPS-induced neuroinflammation model was established in GDF15 knockout mice,and the mRNA levels of TNF-α,IL-1β,iNOS and COX-2 in substantia nigra and striatum were detected by real-time RT-PCR.Western blot was used to observe the protein expression of STAT3,P-STAT3,P65,P-P65,ERK1/2 and P-ERK1/2 in substantia nigra and striatum.4.BV2 cells were cultured in vitro and stimulated with rotenone at different concentrations,and the effect of rotenone on BV2 cell survival was observed by CCK8.5.BV2 cells were cultured in vitro and treated with exogenous GDF15 and rotenone stimulation.The mRNA expression of TNF-α,IL-1β were detected by real-time RT-PCR.Western blot was used to observe the protein expression of P65,P-P65,p38 and p-p38.6.Rotenone-induced Parkinson’s disease model was constructed in GDF15 knockout mice,and the changes of body weight in each group were recorded during modeling,and behavioral tests such as rotarod test,pole test,grasping experiment and gait analysis experiment were performed.The mRNA expression of TNF-α,IL-1βin substantia nigra and striatum were detected by real-time RT-PCR.Western blot was used to observe the protein expression of P65,P-P65,p38 and p-p38 in substantia nigra and striatum.Results1.GDF15 inhibited LPS-induced neuroinflammation in BV2 cells,decreased the expressions of TNF-α,IL-1β,iNOS and COX-2,inhibited the phosphorylation of STAT3,P65,ERK1/2,and reduced the nuclear metastasis of P65.2.In LPS-induced mouse model,GDF15 knockout resulted in increasing expression of TNF-α,IL-1β iNOS and COX-2,and increasing phosphorylation of STAT3,P65,ERK1/2.3.GDF15 inhibited rotenone-induced inflammation in BV2 cells,decreased the expression of TNF-α,IL-1β,and inhibited the phosphorylation of P65 and p3 8 protein.4.In rotenone-induced mouse model,GDF15 knockout mice lost weight earlier than wild-type mice during intraperitoneal injection;Compared with the wild-type mice rotenone injection group,the duration time on the rotating rod was significantly shortened in the GDF15 knockout mice rotenone injection group.GDF15 knockout resulted in increasing expression of TNF-α,IL-1β,and increasing phosphorylation of P65 and p38.ConclusionGDF15 may inhibit inflammation through the STAT3/P65/ERK signaling pathway in LPS-induced neuroinflammatory models.In rotenone-induced Parkinson’s disease models,it may play a protective role in Parkinson’s disease by inhibiting inflammation through the P65/P38 signaling pathway.