The Mechanism Of Nec-1 Inhibiting RIPK1 To Regulate Neuroinflammation In MPTP-induced Parkinson’s Disease Mouse Model

Background: Parkinson’s disease(PD)is the second most common neurodegenerative disease after Alzheimer’s disease.The main pathogenic factors of PD include selective loss of dopamine neurons in the substantia nigra,α-synuclein aggregation,and neuroinflammation,which can cause motor dysfunctions such as tremors,muscle rigidity,bradykinesia,and gait instability,as well as non-motor dysfunctions such as constipation,depression,anxiety,and cognitive impairment.Currently,the treatment methods for PD mainly include medication and surgery.However,medication treatment has many side effects,while surgical treatment,which is expensive,may require a long recovery period,and has poor long-term effectiveness.Although there are various treatment options for PD,there is no universally recognized completely effective treatment method.Recent studies have found that receptor-interacting protein kinase 1(RIPK1)is an initiator of necroptosis and a key upstream regulator of cell death and inflammation.It is a potential therapeutic target in neurodegenerative diseases and inhibiting RIPK1 expression has a neuroprotective effect on neuroinflammation.Currently,RIPK1 is mainly used in basic research on cell apoptosis and necrosis.In addition,RIPK1 has been shown to play an important role in neuroinflammation in neurodegenerative diseases such as Alzheimer’s,stroke,and multiple sclerosis.Recent studies have shown that RIPK1 plays an important role in the pathogenesis of PD by participating in the pathways of cell death and inflammation.However,the detailed mechanisms by which RIPK1 regulates neuroinflammation and glial cells in PD models and participates in disease progression have not been clearly demonstrated.Objectives: RIPK1 has been shown to participate in the regulation of inflammation in various diseases in domestic and foreign research.Our previous study showed that RIPK1 is upregulated in the brain tissue of PD model mice,but its role in the progression of PD is not clear.Therefore,RIPK1 inhibitor Necrostatin-1(Nec-1)was utilized in the current study in order to explor the mechanism of RIPK1 in the glial cell-induced neuroinflammation in PD.Methods: In vivo experiment: 7-week-old male C57BL/6J mice were used to establish a Parkinson’s disease(PD)model by injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)into the abdominal cavity four times at a dose of 20 mg/kg and a volume of 0.1m L/10 g every 2 hours,12 hours after the first injection of Nec-1(at a dose of 1.65 mg/kg and a volume of 0.1 m L/10g).From day 1 to day 6 after modeling,Nec-1 was injected into the abdominal cavity once a day.On day 7 after modeling,the mice were subjected to pole and open field tests to evaluate the effect of RIPK1 inhibition on motor dysfunction and anxiety-like behavior in PD mice.Real-time quantitative polymerase chain reaction(RT-q PCR)and Western blotting(WB)were used to detect the gene and protein expression levels of RIPK1 in the striatum of mice to evaluate the inhibitory effect of Nec-1 on RIPK1.WB and immunofluorescence staining(IF)were used to detect the expression of tyrosine hydroxylase(TH)and the number of dopamine neurons in the substantia nigra of mice to evaluate the effect of RIPK1 inhibition on dopamine neurons.WB and IF were used to detect the distribution and activation of microglia and astrocytes in the striatum of mice,and RT-q PCR was used to detect the transcription levels of inflammatory cytokines,chemokines,and A1/A2 subtype markers of astrocyte neurotoxicity and neuroprotection in the striatum of mice to evaluate the effect of RIPK1 inhibition on glial cell polarization and neuroinflammation.In vitro experiment: Based on the results of animal experiments,we further focused on the effect of RIPK1 on astrocyte polarization.The RIPK1 inhibitor Nec-1(50 μM pretreatment for 30 min and treatment for 24 h)was used as a therapeutic intervention,and lipopolysaccharide(LPS)was used to stimulate the mouse astrocyte cell line(MA cells)to establish a neuroinflammatory cell model(treated with 0.1 μg/m L LPS for24 h).The MTT assay was used to determine the safe concentration of LPS,and WB was used to detect the expression of RIPK1 to determine the final working concentration of LPS and Nec-1.RT-q PCR was used to detect the transcription levels of chemokines,inflammatory cytokines,and A1/A2 subtype markers of astrocytes to evaluate the effect of RIPK1 inhibition on LPS-induced astrocyte inflammation.Results: In vivo experiments have shown that administering Nec-1 can suppress the expression of RIPK1 in the striatum of mice.The research results indicate that inhibiting RIPK1 can reduce the time spent by PD mice in climbing the pole,increase the number of grid crossings,average speed,and total distance traveled in the open field test,thus improving their motor dysfunction and anxiety-like behavior.Inhibiting RIPK1 can also increase the expression of TH in the striatum of PD mice and the number of dopamine neurons in the substantia nigra compacta,improving the dysfunction of the dopaminergic system in PD mice.Inhibiting RIPK1 can also reduce the activation of astrocytes in the striatum of PD mice.Furthermore,it was found that it can reduce the m RNA levels of C-C motif chemokine ligand 2(CCL2),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),complement factor B(CFB),and histocompatibility 2,T region locus 23(H2-T23)in the astrocytes of PD mice,indicating that inhibiting RIPK1 can reduce the polarization of astrocytes to the neurotoxic A1 type,improving the inflammatory state of astrocyte-mediated neuroinflammation.In vitro experiments have also confirmed that Nec-1 can inhibit the expression of RIPK1 in MA cells.Inhibiting RIPK1 can reduce the production of CCL2 and the A1 type complement marker C3 induced by LPS,that is,reduce the polarization of MA cells to A1 type.Conclusions:1.Nec-1 can inhibit the expression of RIPK1 in the striatum of mice and MA cells.2.Inhibition of RIPK1 can improve the motor function impairment and anxiety-like behavior in PD mice.3.Inhibition of RIPK1 can reduce the death of dopaminergic neurons in PD mice.4.Inhibition of RIPK1 can reduce the activation of striatal astrocytes and the inflammatory response in PD mice.5.Inhibition of RIPK1 can reduce the LPS-induced A1-type polarization of astrocytes and improve the inflammatory response.Our study suggests that RIPK1 could be an important drug therapy target for neuroinflammatory disease like PD.