| BackgroundParkinson's Disease(PD)is a common degenerative disease of the central nervous system.The main motor symptoms of clinical patients include bradykinesia,rigidity,posture instability and resting tremor,accompanied by non-motor symptoms such as abnormal sleep behavior,depression,constipation,and olfactory loss.The main pathological manifestation of PD is the degeneration and death of dopaminergic neurons in substantia nigra par compacta(SNpc),accompanied by a biochemical change in the reduction of dopamine transmitters in the striatum.In recent years,the role of microglia-mediated neuroinflammatory responses in the pathogenesis of PD has been increasingly valued by researchers.Studies have confirmed that dopaminergic neurons are susceptible to inflammatory reactions.The autopsy results and PET imaging results of PD patients show that there are a large number of reactive microglia in the substantia nigra,suggesting that the pathogenesis and inflammatory response of PD are closely related.Therefore,the protection of DA neurons of anti-inflammatory drug by inhibiting the inflammatory responses,will postpone the progress of PD and become a new strategy for PD treatment.Ginsenoside Rbl(GRb1),the major components of ginseng,exhibits a wide range of neurotrophic and neuroprotective effects in the CNS.GRb1 has been reported to protect neurons from the injuries induced by amyloid ?-peptide and alpha-synuclein.Further more,Hashimoto reported that GRb1 could protect against apoptosis of dopaminergic neurons intoxicated by 1-methyl-4-phenylpyridinium-iodide(MPP+).These studies indicated that GRb1 might be a potent neuroprotectant for the neurodegenerative disorders including PD.Recently,several studies demonstrated that the neuroprotective effects of GRb1 might be partly attributed to its anti-inflammatory effects.In vitro studies have indicated that GRb1 could alleviate lipopolysaccharide(LPS)-induced inflammatory reaction in N9 microglia,RAW264.7 macrophages,EOC20 microglia and BV2 microglial cells.In in vivo study,Lee JS et al.have reported GRb1 could inhibit the microglial activation and proin-flammatory factors expression obviously in both cortex and hippocampus induced by systemic LPS treatment in mice.However,in Parkinson's disease model whether GRb1 can protect substantia nigra dopaminergic neurons by inhibiting the inflammatory response of microglia has not yet been reported.Therefore,in the first part of this study,LPS was injected into the substantia nigra of one side of Wistar male rats to prepare an inflammation model of PD rats to explore whether GRb1 can protect dopaminergic neurons through its anti-inflammatory effects at the overall level.In addition,the target of GRb1's anti-inflammatory effects is currently unclear.Insulin-like growth factor-I receptor(IGF-1R)is widely expressed on neurons and glial cells in the cerebral cortex,hippocampus,midbrain and other brain regions.Studies have shown that in the developmental stage of the central nervous system,the signal pathway mediated by IGF-1R not only promotes nerve growth and development,cell proliferation and differentiation,but also participates in inhibiting nerve inflammation and repairing nerve regeneration.And studies have shown that ginsenoside Rgl,which has a similar chemical structure to GRb1,can inhibit the inflammatory response of BV2 microglia through IGF-1R.In view of the above research background,we wondered that whether GRb1 can also inhibit the inflammatory response of glial cells through IGF-1R?In the second part of this study,BV2 cells were treated with LPS to establish an inflammatory cellular model.We explored the role of IGF-1R in the anti-inflammatory mechanism of GRb1 in in vitro study.This study aims to provide useful ideas for the treatment of Parkinson's disease and to discover prodrugs with potential clinical value for Parkinson's disease.MethodsPart I Experimental study of ginsenoside Rbl inhibiting lipopolysaccharide-induced inflammatory response in PD rats and protecting substantia nigra dopaminergic neurons Wistar male rats were divided into four groups:(1)control group,(2)GRbl group,(3)LPS model group,(4)GRb1+LPS group.A PD rat inflammatory model was established by unilateral injection of LPS into the substantia nigra.Apomorphine was injected intraperitoneally and the total number of revolutions of the rat was recorded 5 minutes after the injection.High performance liquid chromatography(HPLC)was used to detect the content of dopamine(DA)and its metabolites homovanicillic acid(HVA)and dihydroxyphenylacetic acid(DOPAC)in rat striatum(Str);Immunohistochemical experiments were performed to observe the damage of dopaminergic neurons and the activation of microglia in the compact part of the substantia nigra;Enzyme-linked immunosorbent assay was used to detect the changes in the content of TNF-? and IL-1?in the substantia nigra;Western blot was used to detect the changes in the expression of TH protein and Iba protein in the substantia nigra;the expression of COX-2,iNOS and the expression of I?B,phosphorylated I?B,IKK p65 and phosphorylated IKK p65 in the inflammation-related NF-?B signaling pathway.Part II The inhibitory effect of ginsenoside RbI on the inflammatory response of BV2 microglia induced by lipopolysaccharide through IGF-1RBV2 microglia was cultured conventionally.When the cell confluence reached 80%?90%,BV2 cells were pretreated with ginsenoside Rbl(1,10,20,50 ?M))for 1 h and added LPS to act together for 6 h.Quantitative PCR was used to detect the expression of inflammatory factors TNF-? and IL-1? mRNA to determine the optimal concentration of ginsenoside Rb1.In the presence or absence of JB-1(1 ?g/ml),BV2 cells were pretreated with ginsenoside Rbl(10 ?M)for 1 h and then added LPS to act together for 0.5 h,6 h or 24 h.Fluorescence quantitative PCR and Western blot were used to detect changes in inflammatory factor gene and protein expression and changes in the phosphorylation level of proteins related to the NF-?B signaling pathway to explore the mechanism of GRbl's anti-inflammatory effects through IGF-1R.ResultsPart I Experimental study of ginsenoside Rbl inhibiting lipopolysaccharide-induced inflammatory response in PD rats and protecting substantia nigra dopaminergic neurons1.In the LPS group,the number of rotations of rats induced by APO was significantly higher than that of the Control group(P<0.001).The number of rotations in the GRb1 protection group(20 mg/kg,intraperitoneal injection)was significantly lower than that of the LPS group(P<0.001)·Rats in the GRb1 single-use group had no obvious rotational behavior.2.The HPLC results displayed that the contents of DA and its metabolites HVA and DOPAC in the injured striatum of LPS group were significantly lower than those in Control group(P<0.001).Compared with LPS group,the contents of DA,HVA and DOPAC in Str injured side of GRb1 group were significantly increased and there was a significant difference(P<0.01,P<0.05,P<0.05).There was no significant difference in the content of the above three substances in the GRb1 single-use group compared with the Control group(P>0.05).3.The injection of LPS on the substantia nigra side resulted in a significant decrease in the number of TH-positive neurons in the substantia nigra of the injured side compared with the uninjured side.At the same time,the TH protein content of the injured side was also significantly lower than that in the Control group(P<0.001).The number of TH-positive neurons and the content of TH protein in the substantia nigra injury side of the rats in GRb1 treatment group were significantly higher than those in the LPS group(P<0.001).There was no difference in the number of TH-positive neurons and TH protein content in the GRb1 single-agent group compared with the Control group(P>0.05).At the same time,the microglia on the substantia nigra injury side of the LPS group showed hyperactivity,and the number was significantly increased compared with the Control group(P<0.001).GRb1 administration can significantly reduce the number of activated microglia and the activation degree of the injured side in the substantia nigra(P<0.01).GRbl alone has no obvious activation effect on microglia(P>0.05).4.The Enzyme-linked immunosorbent assay results showed that the levels of TNF-? and IL-1? in the substantia nigra of the LPS group were significantly higher than those in the Control group(P<0·001).GRb1 significantly inhibited the expression of TNF-? and IL-1? in the LPS group(P<0.01).There was no significant difference in the levels of TNF-? and IL-1? in the GRb1 group compared with the Control group(P>0.05).5.Compared with the control group,the expression of iNOS and COX-2 protein in the LPS group was significantly increased(P<0.001).GRb1 significantly decreased the expression of iNOS and COX-2 protein in the LPS group(P<0.05).The phosphorylation levels of I?B and IKK protein in the substantia nigra of the LPS group were significantly higher than those in the Control group(P<0.001).The GRb1 treatment significantly inhibited the increase of phosphorylation levels of the two proteins(P<0.05).Part II The inhibitory effect of ginsenoside Rbl on the inflammatory response of BV2 microglia induced by lipopolysaccharide through IGF-1R1.The mRNA expression levels of TNF-? and IL-1? in the LPS group were significantly increased(P<0.01,P<0.001).Different concentrations of GRb1(1 10,20,50 ?M)can reduce the increase of TNFa and IL-1? mRNA levels induced by LPS and 10 ?M GRb1 has the most significant inhibitory effect(P<0.05,P<0.01).In subsequent experiments,10?M GRb1 was used to study the mechanism.2.GRb1 could significantly inhibit the increase of TNF-? and IL-1? expression levels induced by LPS at the gene level(P<0.01),and JB-1 could block the protective effects of GRb1(P<0.05).3.GRb1 could significantly inhibit the increase of COX-2 and iNOS expression induced by LPS at the protein level(P<0.01),and JB-1 could block the protective effects of GRb1(P<0.05).4.The phosphorylation levels of I?B and IKK protein in the LPS group was also significantly increased(P<0.01);GRb1 could significantly inhibit the phosphorylation levels of I?B and IKK protein induced by LPS(P<0.01);JB-1 could block this protective effect of GRb1(P<0.05,P<0.01).ConclusionGRb1 exerts protective effects on dopaminergic neurons by inhibiting neuroinflammation characterized by excessive activation of microglia.The possible mechanism is that GRbl reduces the release of pro-inflammatory mediators caused by the activation of NF-?B signaling pathway through IGF-1R and inhibits the inflammatory response of glial cells.These data provide important experimental basis for GRb1 in the prospective treatment of Parkinson's disease(PD). |
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