EGCG Reduces Microglial Inflammation And Neurotoxicity By Inhibiting Classical And Non-classical NLRP3 Inflammatory Body Activation

Objective:Alzheimer disease(AD)is the most common neurodegenerative disease,which is mainly manifested by progressive cognitive impairment and memory loss,which seriously endangers the physical and mental health and quality of life of middle-aged and elderly people.Alzheimer’s disease(AD)is also a progressive brain degenerative disease based on irreversible dementia.Its clinical feature is gradual impairment of cognitive function.Due to the chronic development of AD,dementia-type neurocalcification symptoms always occur in neurodegenerative lesions and are clinically identified as mild cognitive impairment(MCI).The rapid increase in population and the aging of the population have led to a sharp increase in the population of AD and MCI,which has placed a heavy burden on families and society,and has attracted widespread attention from the government and the medical community.Although cognitive impairment can be diagnosed through cognitive screening and neuropsychological assessment,patients should take soft preventive measures or early intervention.Therefore,early diagnosis and intervention are essential for the early diagnosis and treatment of MCI.Nucleotide binding oligomerization domain-like receptor protein 3(NLRP3)inflammatory body-mediated inflammatory response and MG activation play important roles in the pathogenesis of AD.There is growing evidence that elderly microglia exhibit an increased proinflammatory phenotype,and activated microglia play a central role in neuroinflammation in AD.Epigallocatechin gallate((-)-epigallocatechin-3-gallate,EGCG)is a major polyphenol component of green tea,which has significant anti-inflammatory,anti-viral,anti-oxidant,anti-cancer,anti-microbial infection and Neuroprotective effects.Previous research by the research team showed that EGCG can reduce Aβ deposition in the brain of APP / PS1 transgenic mice by regulating the balance of NGF-TrkA / p75 NTR signal pathway,thereby inhibiting neuronal apoptosis and exerting neuroprotective effects.Recent studies have shown that the activation of Cysteine-containing aspartic protease 11(caspase-11)can also cause the activation of the NLRP3-ASCcaspase1 pathway.This special activation pathway is called the "atypical NLRP3 inflammatory corpuscle activation pathway." The mature release of 1β and IL-18 mediates the inflammatory response.Recent studies have shown that the innate immune response,especially the immune response that activates inflammatory bodies,is involved in stimulating Aβ release and may contribute to the occurrence and development of AD.Therefore,this study explored the role and mechanism of epigallocatechin gallate(EGCG)in neuroprotective functions from the perspective of classical and non-classical NLRP3 inflammatory body activation.Methods: LPS + Aβ-induced BV2 cells and rat primary microglia were used to establish the MG activation model,and the blank group,LPS + Aβ group,and LPS + Aβ + EGCG group were established.1)Western blot and ELISA were used to detect the expression of Iba-1 in BV2 and rat primary microglia and the transcription,expression and secretion of related inflammatory factors IL-1β and IL-18 by EGCG.2)SH-SY5 Y was cultured with BV2 cells or rat primary microglia induced by LPS + Aβ,and the survival rate of SH-SY5 Y cells was detected by flow cytometry and CCK8.3)Western blot and immunofluorescence staining were used to detect the colocalization of caspase-1 and Iba-1,caspase-1 activity,caspase-1 p20,caspase-11 p26,and NLRP3 protein expression levels in BV2 cells.4)Caspase-1 specific inhibitor Z-YVAD-FMK was used to treat BV2 cells induced by LPS + Aβ.Western blot was used to detect caspase-1 activity and the expression levels of IL-1β,IL-18 and Iba-1.5)LPK + Aβ-induced BV2 cells were treated with IKK inhibitor wedelelolactone.Western blot was used to detect TLR4,pIKK / IKK and p-NF-κB / NF-κB,caspase-11 p26 and caspase-1 p20,IL-1β,IL-18,Iba-1 protein expression level.Results: 1)EGCG can inhibit the transcription,expression and secretion of inflammatory factors IL-1β and IL-18 on the basis of reducing the expression of Iba-1 in BV2 cells induced by LPS + Aβ and rat primary microglia,suggesting that EGCG can inhibit the inflammatory response in MG.2)EGCG significantly increased the survival rate of BV2 induced by LPS + Aβ or SH-SY5 Y cells cultured from rat primary microglia,suggesting that EGCG can improve neuron survival by inhibiting the inflammatory response in MG.3)EGCG can significantly reduce the colocalization of caspase-1 and Iba-1,the activity of caspase-1 and the expression of caspase-1 p20 and caspase-11 p26 proteins in BV2 induced by LPS + Aβ and in rat primary microglia.It suggests that EGCG’s inhibition of MG activation is related to its inhibition of caspase-11 expression and NLRP3 inflammatory body-mediated inflammatory response.4)EGCG can significantly reduce the expression levels of TLR4,p IKK / IKK and p-NF-κB / NF-κB in LPS + Aβ-induced BV2 and rat primary microglia.It is suggested that EGCG’s inhibition of caspase-11 expression and NLRP3 inflammatory body-mediated inflammatory response are related to its inhibition of TLR4 / NF-κB pathway.5)Consistent with EGCG treatment,Z-YVAD-FMK treatment can reduce caspase-1 activity,and the expression levels of caspase-1,IL-1β,IL-18,and Iba-1 wedelelolactone can reduce TLR4 of BV2 cells induced by LPS + Aβ,The expression levels of pIKK / IKK and p-NF-κB / NF-κB,caspase-11 p26 and caspase-1 p20,IL-1β,IL-18 and Iba-1.Conclusion: Gallate catechin-3-gallate(EGCG)can exert neuroprotective effects by inhibiting LPS + Aβ-induced MG activation and inflammatory response.This effect may be related to the caspase-11 inflammatory bodies and NLRP3 inflammatory bodies mediated by the TLR4 / NF-κB pathway.Therefore,in-depth study of the related mechanisms of EGCG neuroprotective effect will help provide a new direction for the treatment of Alzheimer’s disease and lay a solid theoretical foundation for future clinical applications.