Effects Of Different Administration Sequence Of Lipopolysaccharide And Rotenone On Dopaminergic Neurons And Glial Cells In Rat’s Midbrain

Objective: The purpose of this study was to investigate the effects of different administration methods and sequences of lipopolysaccharide(LPS)and Rotenone(ROT)on dopaminergic(DA)neurons and glial cells in the midbrain of rats and related mechanisms.Methods: Healthy male Sprague-Dawley(SD)rats(180-220 g)were randomly divided into six groups with 12 rats in each group,control group,LPS group,ROT group,LPS and ROT(LPS + ROT)group,LPS was given first then ROT(LPS→ROT)group and ROT first then LPS(ROT→LPS)group.Rats in LPS group were injected intraperitoneally(1mg/kg/d)for 4 days continuously,and ROT group was injected with ROT(0.5 mg/kg/d)subcutaneously at the back of the neck for 4 weeks,6 times per week.The last two groups were given LPS or ROT separately according to the aforementioned method.After 4 weeks,ROT or LPS were given according to the aforementioned methods separately.Behavioral evaluation and index testing were performed uniformly after the end of the administration.The change of motor ability was evaluated by rotating rod experiment,and the change of exploration habit was observed by open field experiment.Immunohistochemistry and Western blot were used to detect proteins expression: tyrosine hydroxylase(TH),characteristic marker of DA neurons in rats midbrain substantia nigra(SN),microglia surface marker ionized calcium binding adaptor(Iba1)and astrocyte surface marker glial fibrillary acidic protein(GFAP).Then,the expressions of pro-/anti-inflammatory mediators secreted by microglia,astrocyte-related toxic mediators and neurotrophic factors in the midbrain of rats were detected by Western blot.In addition,Western blot and PCR were used to detect the expression of nuclear factor-E2-related factor 2(Nrf2)and its pathway related proteins in rats midbrain.Besides,the levels of lipid oxidation index malondialdehyde(MDA)and antioxidant enzyme superoxide dismutase(SOD)in the midbrain of rats were detected by the kit.The protein expressions of mitochondrial complex I NADH: Ubiquinone oxidoreductase core subunit S3(NDUFS3)and complex II succinate dehydrogenase complex flavoprotein subunit A(SDHA)were detected by Western blot.The expression of TH and NDUFS3 in rat midbrain substantia nigra was detected by double immunofluorescence assay.Results:(1)Rotating rod experiment showed that the time spent on the rod of rats in LPS and ROT groups was significantly shorter than that in the control group.Rats in the LPS +ROT group spent less time in the rod than those in the LPS group.The residence time of rats in LPS group and ROT group with different treatment sequence was significantly shorter than that in LPS + ROT group.The duration of ROT→LPS group was shorter than that of LPS→ROT group.There was no significant difference in the total moving distance.(2)Compared with the control group,the damage of DA neurons in ROT group was increased.Compared with the single administration group,DA neuron damage of rats in LPS +ROT,LPS → ROT and ROT → LPS groups increased successively.(3)Compared with control group,the expression of NDUFS3 decreased in ROT group.Compared with the single administration group,the expression of NDUFS3 in LPS +ROT,LPS→ROT and ROT → LPS groups decreased successively,while the expression of SDHA had no significant changed.In addition,the immunofluorescence double-labeling method showed that DA neuron damage increased while the expression of NDUFS3 gradually decreased.(4)Compared with the control group,activation of microglia and astrocytes in LPS or ROT,LPS+ROT,LPS→ROT and ROT→LPS groups increased successively,and anti-inflammatory factors(Arg1 and IL-10)and pro-inflammatory factors(TNF-α and IL-1β)secreted by microglia increased gradually.The protein levels of A1 phenotype C3 and LCN2 in astrocytes gradually increased,while the protein levels of BDNF and GDNF were gradually decreased.(5)Compared with the control group,MDA levels in LPS or ROT,LPS+ROT,LPS → ROT and ROT → LPS groups gradually increased,SOD levels gradually decreased,and m RNA and protein expression levels of Nrf2,HO1,NQO1 and Keap1 gradually increased.Conclusion: ROT-induced oxidative stress combined with LPS-induced neuroinflammation can aggravate the damage of DA neurons and glial cells activation in the midbrain of rats,especially upon the stress damage of DA neurons induced by ROT,the synergistic damage of LPS-induced neuroinflammation is more serious.