| Objective: Parkinson ’s disease(PD)is the second most common degenerative disease of the central nervous system worldwide after Alzheimer’ s disease,with a prevalence of about 2-3% in people ≥ 65 years of age,and its typical pathological features are a gradual decrease in dopaminergic neurons in the substantia nigra pars acta(SNpc),resulting in a decrease in dopamine(DA)released into the striatum(Str),ultimately leading to motor dysfunction,and some patients are also accompanied by non-motor symptoms such as cognitive dysfunction and dementia.Existing drugs can relieve the motor symptoms of PD patients,but long-term use can lead to irreversible dyskinesia and drug resistance,which brings limitations to the treatment of PD.Neuroinflammation is thought to play an important role in the pathogenesis of Parkinson ’s disease,while huperzine A(Hup A),as an acetylcholinesterase inhibitor that improves cognitive function,has been shown in some animal models to improve neurological disease by reducing neuroinflammation.Hup A has been found to reduce apoptosis of dopaminergic neurons in the substantia nigra(SN)of a mouse model of PD and improve memory function in clinical patients with PD,but it is unclear whether Hup A can improve PD-induced neuroinflammation and what is the specific molecular mechanism by which Hup A improves memory function in PD.In this study,we observed the effects of Hup A treatment on motor and cognitive-related behaviors in PD mice through a mouse model of acute PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),and then investigated whether Hup A could inhibit PD-induced neuroinflammation in the substantia nigra and striatum,and preliminarily explored the molecular mechanism by Hup A in improving cognitive function in PD according to the results of hippocampal transcriptomics in PD mice,providing a theoretical and experimental basis for exploring the clinical application of Hup A in the treatment of PD.Methods: 1.Healthy male C57BL/6 J mice were randomly divided into control group,MPTP model group(MPTP group),Hup A low dose group(MPTP+Hup A-L group)and Hup A high dose group(MPTP+Hup A-H group).Acute PD mouse models were established by intraperitoneal injection of MPTP on day 0.MPTP + Hup A-L group and MPTP + Hup A-H group were continuously intraperitoneally injected with Hup A injection at concentrations of 0.1 mg/kg and 0.2 mg/kg for 2 weeks,respectively,and the other two groups were intraperitoneally injected with the same amount of saline.Open field test,pole climbing test and rotarod test were used to detect the motor function of mice,water maze test and shuttle box test were used to detect the cognitive function of mice.Brain tissue samples were collected from mice on the 14 th day of the experiment.2.Immunofluorescence was used to detect the number of tyrosine hydroxylase positive(TH +)cells in SNpc,and real-time PCR(q PCR)was used to detect the m RNA expression of NF-κB p65,TNF-α,IL-6,IL-1β,IL-10,IL-4,and IL-17 A in the midbrain and striatum.3.The differentially expressed genes in the hippocampus of mice were screened by comparing the gene expression levels between MPTP group and MPTP+Hup A-H group using transcriptome high-throughput sequencing(RNA-seq)technique,and the differentially expressed genes were analyzed by GO functional analysis and KEGG pathway enrichment analysis,and some of the enriched differential genes were verified by q PCR experiments.Results: 1.Behavioral test results showed that the total distance,mean velocity and maximum velocity of mice activity in the open field test were reduced in the MPTP group compared with the control group(P < 0.01,P < 0.01,P < 0.01);the time to climb to the bottom was increased in the pole climbing test(P < 0.05);the latency to fall was reduced in the rotarod test(P < 0.01);the number of mice crossing the platform was reduced in the Morris water maze test(P < 0.05),and the time to reach the platform for the first time was increased(P < 0.01);the number of mice actively avoiding was reduced in the active and passive shuttle box test(P < 0.05),and there was no significant difference in the latency of active avoidance(P > 0.05).The above results indicate motor and cognitive dysfunction in the MPTP-induced acute PD mouse model.Compared with the MPTP group,the maximum velocity of mice in the open field test increased in the MPTP+Hup AL and MPTP+Hup A-H groups(P < 0.01,P < 0.05),and there was no significant difference in the total distance and mean velocity of mouse activity in the MPTP+Hup A-L group(P > 0.05,P > 0.05),but there was an increasing trend,and the total distance and mean velocity of mouse activity increased in the MPTP+Hup A-H group(P<0.05,P<0.01);the time to climb to the bottom decreased in the pole climbing test(P < 0.05,P = 0.0509);the latency of mouse falling increased in the rotarod test(P < 0.01,P < 0.01);there was no significant difference in the number of mouse crossing the platform in the Morris water maze test(P > 0.05,P > 0.05),but there was an increasing trend,and the time to reach the platform decreased for the first time(P < 0.01,P < 0.05);The latency of active avoidance was reduced(P < 0.01,P < 0.05);the above results showed that Hup A treatment could improve motor and cognitive function in PD mice.2.Immunofluorescence results showed that the number of TH + cells in the SNpc of mice in the MPTP group was decreased compared with the control group(P < 0.05);the number of TH + cells in the SNpc of mice in the MPTP + Hup A-L and MPTP + Hup A-H groups was increased compared with the MPTP group(P < 0.01,P < 0.05).q PCR results showed that compared with the control group,the m RNA expression of NF-κB p65,TNF-α,IL-6,and IL-17 A was increased(P < 0.01,P < 0.01,P < 0.01,P < 0.01),the m RNA expression of IL-10 and IL-4 was decreased(P < 0.05,P < 0.05),and IL-1β was not significantly different in the SN of mice in the MPTP group,but tended to increase;the m RNA expression of NF-κB p65 was decreased(P < 0.01,P < 0.01),the m RNA expression of TNF-α was decreased(P < 0.05,P < 0.01),the m RNA expression of IL-6 was decreased(P < 0.01,P < 0.01),the m RNA expression of IL-1β was decreased(P < 0.01,P < 0.01),and the m RNA expression of IL-17 A was decreased(P < 0.01,P < 0.01)in the MPTP+Hup A-L and MPTP+Hup A-H groups compared with the MPTP group.There was no significant difference in the m RNA expression of IL-10(P > 0.05,P > 0.05)or IL-4(P > 0.05,P > 0.05),but the m RNA expression of IL-10 and IL-4 tended to increase.3.Transcriptome results showed that 490 DEGs,including 75 up-regulated genes and 415 down-regulated genes,were identified in the damaged hippocampal tissues of the MPTP +Hup A-H group compared with the MPTP group by transcriptome sequencing experiments.GO results showed that tumor necrosis factor-mediated signaling pathways,positive regulation of inflammation and other biological processes associated with the inflammatory response were significantly enriched.KEGG results showed that pathways associated with inflammatory responses such as IL-17 signaling pathway,TNF signaling pathway and Chagas’ disease(American trypanosomiasis)were also significantly enriched.Integrating the above inflammation-related pathways,9 inflammation-related DEGs were obtained,from which 8 DEGs(CCL2,SERPINE1,NFKBIA,ACE,LCN2,FAS,KRT8,KRT18)were selected and validated by q PCR experiments,and it was found that the m RNA expression of these DEGs was increased in the MPTP group compared with the control group,and decreased in the MPTP+Hup A-L group and MPTP+Hup A-H group compared with the MPTP group.Conclusion: 1.Huperzine A can improve motor and cognitive function and reduce dopaminergic neuron death in substantia nigra of MPTP-induced acute PD mice.2.Huperzine A may improve the motor function of PD mice by regulating the expression of pro-inflammatory and anti-inflammatory gene m RNA in the midbrain and striatum of PD mice.3.Huperzine A may improve the cognitive function of PD mice by regulating the expression of m RNA of inflammatory response-related genes in the hippocampus of PD mice. |
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