Andrographolide Regulates The Mechanism Of LRP1-mediated PPARγ/NF-κB Pathway And Autophagy-lysosome Pathway For Neuroprotective Effects

Objective: Alzheimer’s disease(AD)is one of the serious neurodegenerative diseases that plague the lives of elderly people.Its main pathological features are plaques formed by extracellular β-amyloid(Aβ)deposits and neurofibrillary tangles formed by highly phosphorylated Tau.Low density lipoprotein receptor associated protein 1(LRP1)is a multifunctional scavenger and signal transduction receptor that is highly expressed in neurons,microglia and astrocytes.Andrographolide(Andro)is a diterpene lactone extracted from Andrographis paniculata(Burm.f)Nees,family Sirtugaceae,which has antiinflammatory,antioxidant and therapeutic effects on cardiovascular and cerebrovascular diseases.In this paper we investigated the ameliorative effect of Andrographis paniculata lactones on the production of inflammation and autophagy in AD and the related mechanisms.Methods: In vitro AD cell model was established by Aβ induction in BV-2 microglia,and cell viability was measured by MTT after treatment with andrographolide,and intracellular LRP1,peroxisome proliferator-activated receptorγ(PPARγ)and peroxisome proliferatoractivated receptorγ(PPARγ)were detected by Western blot and RT-PCR,respectively.receptorγ(PPARγ),nuclear factor B p65(Nuclear factor kappa-B p65 nucleotidebingding,NF-κB(P65)),intracellular autophagy-associated(p62,LC3II/LC3Ⅱ),endocytosis-associated(Rab5,Rab7)and lysosome-associated membrane protein 1(LAMP1)protein and m RNA expression levels,Western blot and RT-PCR for proinflammatory factors(IL-1β,IL-6,TNF-α),EMSA for NF-κB binding ability to proteins,immunofluorescence for changes in cellular LRP1 localization,and dual fluorescent m RFPe GFP-LC3 system was used to detect the fusion of autophagosomes with lysosomes.Fivemonth-old β-amyloid precursor protein/progerin-1/tau protein(APP/PS-1/tau)triple transgenic mice(3x Tg)male mice were selected in vivo,AAV virus was injected into the lateral ventricle of the brain and administered intraperitoneally for 3 months,the spatial memory ability of mice was tested by Morris water maze,kits were used to detect the expression of inflammatory factors IL-1β,IL-6,TNF-α and SOD activity,HE staining to detect the cell status in the hippocampus of mice,Elisa kit to detect the level of Aβ in the brain,Western blot to detect the expression of p62,LC3II/LC3Ⅱ,Rab5,Rab7,LAMP1,NF-κB(P65),LRP1,PPARγ protein,immunofluorescence to detect the brain Aβ,LRP1,P62 fluorescence intensity.Results: In the Aβ-induced BV-2 microglia AD cell model,1.MTT method results revealed that 6 μM Aβ cells survived up to 47.48±4.108%,and co-treatment of 6 μM Aβ and 10 μM andrographolide was able to rescue the cell damage caused by Aβ.2.Andrographolide was able to increase LRP1 as well as PPARγ m RNA and protein expression,and decrease NF-κB(P65)expression and decreased IL-1β,IL-6 and TNF-α expression.3.Andrographolide not only increased LRP1 expression but also promoted LRP1 nucleation.4.Reducing LRP1 and PPARγ expression followed by co-treatment with Aβ and andrographolide showed a further decrease in LRP1 and PPARγ expression and increased expression of proinflammatory inflammatory factors while NF-κB(P65)expression increased.5.Andrographolide reduced the expression of autophagy factors(p62,LC3-Ⅱ/LC3-Ⅱ)and increased the expression of autophagy factors after reducing LRP1 expression.6.Lysosomal factors(Rab5,Rab7,ULK1,Beclin1,LAMP1)expression decreased after treatment with andrographolide and increased after reducing LRP1 expression.7.The fusion of autophagosomes with lysosomes was detected by the dual fluorescence m RFPe GFP-LC3 system,which showed that autophagy increased and fusion increased after the reduction of LRP1 expression by andrographolide treatment.In the 3x Tg mouse model,1.Morris water maze experiments showed that Andrographolide improved memory impairment in mice.2.Histopathological sections of mouse hippocampus showed that Andrographolide reduced the formation of solidified atrophic cells in the hippocampal region and promoted the compact arrangement of cells.3.expression,while increasing LRP1,PPARγ protein expression and decreasing NF-κB,p-NF-κB and p-PPARγ expression.The co-treatment group of AAV-LRP1 and andrographolide further decreased P62,LAMP1,LC3II/LC3 Ⅱprotein expression,while increasing LRP1,PPARγ protein expression and decreasing p PPARγ,NF-κB and p-NF-κB expression.4.Immunofluorescence results further demonstrated that andrographolide reduced Aβ plaque production,increased LRP1 expression,P62 expression,decreased NF-κB expression and reduced NF-κB entry.5.After LRP1 expression in the brain of overexpressing mice,PPARγ expression increased,pPPARγ,NF-κB and p-NF-κB expression was decreased,and inflammatory factor expression was reduced.After treatment with andrographolide LRP1 and PPARγ expression further increased,p-PPARγ,NF-κB,p-NF-κB and inflammatory factors expression decreased.6.After overexpression of LRP1,autophagy factor(p62,LC3-Ⅱ/LC3-Ⅱ)expression decreased,lysosomal factors(Rab5,Rab7,ULK1,Beclin1.LAMP1)expression was reduced and further reduced by Andrographis paniculata lactone treatment.Conclusions: Andrographolide was able to improve spatial memory capacity and inflammation as well as autophagy and Aβ-induced BV-2 cell damage in memory-impaired mice by modulating the LRP1-PPARγ signaling pathway and inhibiting NF-κB.Furthermore,when knocking down the expression of LRP1 or PPARγ in cells,the original therapeutic effect of Andro was observed to be diminished and NF-B activity was increased.On the other hand upregulation of LRP1 expression in the brain of 3x Tg mice resulted in a further decrease in NF-κB(P65)expression in the brain,an attenuated inflammatory response and attenuated autophagy.the LRP1-PPARγ pathway has a significant inhibitory effect on cellular inflammation and attenuated autophagy.