Objective It is observe the role of receptor-interacting protein 1(RIP1)/RIP3/mixed lineage kinase do-main-like(MLKL)-mediated necroptosis in lipopolysaccharide(LPS)-induced damage in substantia nigra dopaminergic neurons of rats,and to explore the mechanism of action of ginsenoside Rb1 in protecting dopaminergic neurons.Methods Forty-eight SD rats were randomly divided into control group,Parkinson group,Rb1+Parkinson group and Nec-1+Parkin-son group,with 12 rats in each group.The rats of the control group were injected with 2 μL of saline in substantia nigra,followed by 14 consecutive days of intraperitoneal injection of saline(2 mL/kg);the rats of the Parkinson were injected with 5 μg(2 μL)of LPS in substantia nigra,followed by 14 consecutive days of intraperitoneal injection of saline(2 mL/kg);the rats of the Rb1+Parkinson group were treated with ginsenoside Rb1(20 mg/kg)by intraperitoneal injection for 3 days,and then injected with 5 μg of LPS in substantia nigra on the 4th day,then continuously treated with ginsenoside Rb1(20 mg/kg)by intraperitoneal injection for 14 days;the rats of the Nec-1+Parkinson group were injected with 5 μg of LPS in substantia nigra for 3 days,followed by intraperitoneal injection of ginsenoside Rb1(20 mg/kg)for 14 days.14 days af-ter LPS injection,the rats were given apomorphine(APO)by subcutaneous injection to observe their behavioral changes;the number of dopaminergic neurons in substantia nigra and the morphology of microglia were observed by immunohisto-chemistry;the contents of dopamine(DA)and its metabolites homovanillic acid(HVA)and dihydroxyphenylacetic acid(DOPAC)in striatum were dettected by HPLC;the mRNA expressions of interleukin-1β(IL-1β)and tumor necrosis fac-tor-α(TNF-α)in substantia nigra were detected by RT-PCR;the activity of superoxide dismutase(SOD)and content of malondialdehyde(MDA)in substantia nigra were detected by chemical assay kit;the expressions of tyrosine hydroxylase(TH),ionic calcium-binding adapter molecule 1(Iba-1)and necroptosis-associated proteins(RIP1,RIP3,MLKL)in substantia nigra were detected by immunoblotting.Results After subcutaneous injection of APO,obvious rotational behav-iors occurred in the rats of the Parkinson group,and the rotational behaviors of rats in both the Rb1+Parkinson group and Nec-1+Parkinson group were significantly improved compared with those in the Parkinson group.Compared with the con-trol group,there were a large loss of dopaminergic neurons and transitional activation of microglia in the substantia nigra on the injected side of rats in the Parkinson group;the contents of DA,DOPAC,and HVA in the striatum on the injected side were significantly reduced(all P<0.05);the SOD activity and relative expression of TH protein in the substantia nigra on the injected side were significantly reduced(all P<0.05),and the content of MDA and relative expressions of IL-1β,TNF-α mRNA,and relative expressions of Iba-1,RIP1,RIP3,MLKL proteins were significantly increased(all P<0.05).Compared with the Parkinson group,the dopaminergic neuron loss and microglia activation were attenuated in the substantia nigra on the injected side of the rats in the Rb1+Parkinson group and Nec-1+Parkinson group;the contents of DA,DOPAC,and HVA in the striatum on the injected side were significantly increased(all P<0.05);the relative ex-pression of TH protein in the substantia nigra on the injected side was significantly increased(all P<0.05),and relative expressions of IL-1β,TNF-α mRNA and Iba-1,RIP1,RIP3,MLKL proteins were all significantly decreased(all P<0.05).The SOD activity in the substantia nigra on the injected side of the rats in the Rb1+Parkinson group was signifi-cantly higher than that in the Parkinson group(P<0.05),and the MDA content was significantly lower than that in the Parkinson group(P<0.05),but the differences in SOD activity and MDA content in the Nec-1+Parkinson group were not statistically significant when compared with those in the Parkinson group(all P>0.05).Conclusion Necroptosis is in-volved in LPS-induced damage to rat substantia nigra dopaminergic neurons,and ginsenoside Rb1 can exert anti-inflamma-tory and neuroprotective effects via inhibiting the necroptosis pathway.
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