Ginsenoside Rg1 Alleviates Aβ Deposition And Neuronal Damage By Inhibiting NLRP1 Inflammasome Activation In APP/PSl Mice

Neuro-inflammation and Oxidative Stress（OS）play a pivotal role in Alzheimer’s disease（AD）.NADPH oxidase 2（NOX2）is an important enzyme that is responsible for ROS generation in many neurodegenerative diseases.The production of pro-inflammasome molecules in neurons is connected with the nucleotide-binding oligomerization domain（NOD）-like receptor protein 1（NLRP1）inflammasome.However,it is still unclear whether inhibition of NOX2 and NLRP1 inflammasome decreases amyloid-beta（Aβ）generation and deposition in APP/PS1 mice.Ginsenoside Rg1（Rg1）is an active component in ginseng,and maybe a potential agent for neurodegenerative diseases.In this study,we investigated the effects and mechanisms of Rg1 treatment on cognitive performance,neuronal damage,Aβdeposition,and NOX2-NLRP1 inflammasome activation in APP/PS1 mice.ObjectiveTo observe the effect of Rg1 on the learning and memory impairment and to explore the protective effect of Rg1 and its possible mechanism in APP/PS1 mice.To observe knockdown NLRP1 whether can achieve the same protective effect as Rg1.Methods1.A total of 72 male 6-month-old（M）WT and APP/PS1 male mice were divided into6 groups.They are the normal age control（WT-9M）group,pre-treatment control（APP/PS1-6M）group,model（APP/PS1-9M）group,Rg1 5 mg/kg,10 mg/kg group,positive drug Apocynin 50 mg/kg group.Except for the APP/PS1-6M group,they were given intragastric administration or saline for 12 weeks（dose 0.1ml/10g body weight）.Each group of mice was raised regularly,and they were free to eat and drink.Observe the abnormal activities of mice by open field tests,buried food tests and Morris water maze;observe the neuronal damage changes in mouse cortex and hippocampus by HE staining and Nissl staining;DHE fluorescent probe to detect the ROS generation in mice hippocampus and cortex;β-galactosidase kit to detect the aging degree of mice brain;immunofluorescence single staining to detect the production of PSD95 and Aβ_1-42 in mice cortex,hippocampus CA1 and CA3 areas;Thioflavin-S staining detects the Aβin the mice cortex and hippocampus;Western Blot detects the aging indicators（β-Gal）,synaptic function indicators（PSD95）,Aβindicators(APP,BACE,CTF-β,NCSTN,Aβ_1-42,TAU,p-TAU),OS indicators（p47phox,p22phox,NOX2）and inflammation indicators（NLRP1,Nf-k B,P-Nf-k B,IL-1β,ASC,Caspase-1）protein expression in the mice hippocampus and cortex;q-PCR detects OS indicators（p47phox,p22phox,NOX2）,inflammation indicators（NLRP1,IL-1β,ASC,Caspase-1）and Aβindicators（APP,BACE,NCSTN）m RNA expression.2.Divided into 3 groups,a total of 24 male 6M APP/PS1 mice,which are the normal saline（NS）control group,the lentivirus（LV）-scramble control group,and the LV-NLRP1-si RNA treatment group.Inject NS,control lentivirus or NLRP1-si RNA lentivirus（2μl each）stereotactically into the left lateral ventricle（coordinates:0.6mm after bregma;lateral:-1.5 mm;ventral:1.7 mm）.Live transfection was maintained for 12 weeks after injection of lentivirus.Each group of mice was raised regularly,and they were free to eat and drink.Observe the abnormal activities of mice by open field tests,buried food tests and Morris water maze;The transfer of lentivirus in mice brain was observed by frozen section staining;HE staining and Nissl staining to observe the change in mice cortex and hippocampus neuronal damage;Detect Aβ_1-42 in mice brian by immunofluorescence single staining;Detect aging indicators（β-Gal）,synaptic function indicators（PSD95）,Aβindicators(APP,BACE,CTF-β,NCSTN,Aβ_1-42,TAU,p-TAU)and inflammation indicators（NLRP1,Nf-k B,p-Nf）-k B,IL-1β,ASC,Caspase-1)protein expression changes in mice hippocampus and cortex by Western Blot;Detect inflammation indicators（NLRP1,IL-1β,ASC,Caspase-1）and Aβindicators（APP,BACE,NCSTN）m RNA expression by q-PCR.Results:（1）a.OFT:Group compared with control,the model mice show obviously abnormal activity.Group compared with model,the treatment group mice significantly reduced abnormal activity;b.BFT:Group compared with two control groups,the significant olfactory dysfunction was in the model group.In comparison with model,the group of treatment mice olfactory dysfunction shows alleviate;c.MWM:In comparison with two control groups,the model mice show significant lower spatial positioning memory and learning capabilities.In comparison with model,the group of treatment mice spatial positioning memory and learning ability shows alleviate;d.HE staining:Group compared with two control groups,the more neuronal damage caused by Aβshows in the model.Group compared with model,the neuronal damage caused by amyloid deposits was obviously decreased in treatment group mice hippocampus and cortex areas;e.Nissl staining:Group compared with two control groups,the more Nissl bodies lost,and the staining was reduced in the model group mice hippocampus CA1,CA3 and cortex areas.Group compared with model,the less Nissl bodies lost,and the staining was aggravated in treatment group mice hippocampus CA1,CA3 and cortex areas;f.β-galactosidase kit:In comparison with two control groups,the moreβ-Gal shows in the model.Group compared with model,the lessβ-Gal shows in treatment group mice;g.DHE fluorescent probe:Group compared with two control groups,the more ROS shows in model.In comparison with model,the less ROS was in treatment group mice;h.Thioflavin-S staining:Group compared with two control groups,the more amyloid plaques were in model group mice.Group compared with model,the less amyloid plaques were in treatment group mice;i.Immunofluorescence single staining:In comparison with two control groups,the less PSD95 and the more Aβ_1-42 expression shows in the model.In comparison with model,the more PSD95expressions and the less Aβ_1-42shows in treatment group mice;j.Western blot:In comparison with two control groups,except the expression of NCSTN protein keep balance,and the less PSD95,the other indiactors show more in the model.In comparison with model,except the expression of NCSTN protein keep balance,and the more PSD95,the other indiactors show less treatment mice;k.q-PCR:In comparison with two control groups,except the m RNA expression of NCSTN keep balance,the other indiactors show more in the model.In comparison with model,except the m RNA expression of NCSTN keep balance,the other indiactors show less in treatment mice.（2）a.OFT:Group compared with NS,there were no obvious abnormal activities changes in LV-scramble,and the obviously reduced abnormal activities were in LV-NLRP1-si RNA;b.BFT:Group compared with NS,the LV-scramble olfactory did not change significantly,and the olfactory dysfunction of the LV-NLRP1-si RNA was improved,but it was not statistically significant;c.WMW:Group compared with NS,the LV-scramble has no significant changes,while the LV-NLRP1-si RNA group mice have obviously improved spatial positioning memory and learning capabilities;d.HE staining:Group compared with NS,the LV-scramble has no significant changes.The neuronal damage caused by amyloid deposition was obviously improved in LV-NLRP1-si RNA;e.Nissl staining:Group compared with NS,the LV-scramble have no significant changes,while the fewer Nissl bodies were lost and the staining was aggravated in LV-NLRP1-si RNA;f.Immunofluorescence single staining:Group compared with NS,the LV-scramble has no significant changes,and the obvious decreased expression of Aβ_1-42 was in LV-NLRP1-si RNA;g.Western blot:Group compared with NS,the protein expression of LV-scramble has no significant changes.Except the NCSTN protein expression did not change significantly,and the PSD95protein expression was significantly increased,the other indicators decreased obviously in LV-NLRP1-si RNA;h.q-PCR:Group compared with NS,the m RNA expression of LV-scramble has no significant changes,and except the m RNA expression of NCSTN did not change significantly,the other indiactors were significantly reduced in LV-NLRP1-si RNA.ConclusionRg1 treatment could alleviate learning and memory impairment,neuronal damage,and reduce Aβgeneration and deposition by inhibiting NLRP1 inflammasome activation in APP/PS1 mice.