Sulforaphane Reverses Aβ25-35 Fibre-induced Primary Pericytes Apoptosis By Inhibiting Autophagy And NLRP3 Inflammasome Activation

BackgroundCerebral amyloid Angiopathy(CAA)is one of the main types of cerebral small vessel disease(CSVD),involving capillaries known as capillary CAA,It is mainly caused by deposition of amyloid-β protein(Aβ).Pericytes are cells in the vascular wall,which play A key role in regulating the clearance of Aβ in the cerebral lymphatic system.Pericytes can cause Aβ clearance disorder in the brain when Pericytes are damaged,resulting in Aβ deposition,which is A key mechanism of CAA.NLRP3 Inflammasome mediates inflammatory response and apoptosis,and has a close interaction with autophagy.When autophagy is inhibited,inflammatory cells can be overactivated,leading to strong inflammatory responses causing damage to the body,which plays an important role in the occurrence and development of cerebrovascular diseases,neurodegeneration and aging related diseases.Sulforaphane(SFN)is a kind of isothiocyanate,which has anti-inflammatory,antioxidant,free radical scavenging,inhibition of Tau protein hyperphosphorylation and other functions,and has a significant protective effect on neurodegenerative diseases.In summary,we hypothesized that autophagy and NLRP3 inflammosomes may be overactivated when perihemispheric cells are continuously stimulated by AP,leading to A series of inflammatory cascade reactions leading to perihemispheric cell death,resulting in the failure of Aβ to be effectively cleared in the brain,further promoting the occurrence of CAA.SFN may play a protective role in this mechanism by inhibiting autophagy and anti-inflammatory effects,so as to achieve the effect of protecting pericytes.ObjectiveThe purpose of this study was to discuss the toxicity of Aβ on primary pericytes,the role of autophagy and NLRP3 inflammasome in its pathological mechanism,and the protective effect of SFN.Methods1.Immunofluorescence identified the purity of primary pericytes.2.CCK-8 assay and LDH assay detected the cytotoxicity and toxicity of Aβ25-35 on primary pericytes.3.qPCR detected the RNA expression of NLRP3 inflammasome in primary pericytes.4.Western blot analysis detected the expression of NLRP3 and autophagy protein in primary pericytes.5.Flow cytometry detected the apoptosis of primary pericytes.Results1.Immunofluorescence identification showed that the purity of primary pericytes gradually increased with the increase of passage times.2.Immunofluorescence identification showed that primary pericyte cells on Transwell plate coated with rat tail collagen type I were more pure than those coated without collagen.3.CCk-8 results showed that Aβ25-35 had no significant effect on the activity of primary pericytes.LDH results showed that Aβ25-35 had a "Hormesis"effect on the activity of primary pericytes.4.CCk-8 results showed that SFN had a "Hormesis" effect on the activity of primary pericytes,that is,it was activated at low dose and inhibited at high dose.5.qPCR results showed that SFN could inhibit the RNA expression of NLRP3,ASC and GSDMD in primary pericytes stimulated by Aβ25-35,and WB showed that SFN could inhibit the protein expression of NLRP3 in primary pericytes stimulated by Aβ25-3 5.6.WB results showed that Aβ25-35 increased the expression of autophagy specific protein LC-3Ⅱ in primary pericytes in a time-dependent manner,and degraded with time.SFN reversed the increased expression of LC-3Ⅱ protein and the decrease of P62 protein in primary pericytes stimulated by Aβ25-35.7.Flow cytometry results showed that AP25-35 increased the apoptosis rate of primary pericytes,while SFN inhibited it.Conclusions1.Aβ25-35 has a "Hormesis" effect on the toxicity of primary pericytes.SFN also has a "Hormesis" effect on primary pericytes,that is,a dual effect.2.Aβ25-35 can stimulate the activation of NLRP3 inflammasome in primary pericytes and enhance autophagy activity in A time-dependent manner.SFN can reverse the activation of NLRP3 inflammasome induced by Aβ25-35 and then mediated autophagy.SFN can reverse Aβ25-35 induced primary pericytes apoptosis by inhibiting autophagy and NLRP3 inflammasome activation.