RNA interference silencing P2X7 receptor restrained inflammatory factors released from the microglia / 中华老年医学杂志

ABSTRACT

Objective To observe the effect of silencing P2X7 receptor (P2X7R) by RNA interference on microglial releasing interleukin 1β (IL-1β),tumor necrosis factor-α (TNF-α) and nitric oxide (NO).Methods The small interfering RNA (siRNA) targeting P2X7R gene was observed.The primary microglial cells activated by amyloid-β (Aβ-42) were infected with the lipofectaminesiRNA.The groups were designated as Aβ-1-42,Aβ1-42/siNC,Aβ1-42/siP2X7R and blank control groups according to the different stimulus.After RNA interference for 48 hours,the microglial cells were collected.The survival rate of microglia was detected by CCK-8.The levels of P2X7R mRNA and protein were detected by Real-time PCR and Western blotting respectively.The microglial morphology and the P2X7R immune response were observed by immunocytochemistry staining.Then levels of IL-1β,TNF-α and NO in the supernatantwere measured.Results The Aβ1-42 and siRNA had no effects on the survival rate of microglia.After RNA interference silencing P2X7R,the expression levels of P2X7R mRNA and protein in Aβ1-42/siP2X7R group were decreased significantly,and in this group,the microglial activity and P2X7R immune response were all reduced.In Aβ1-42,Aβ1-42/siNC,Aβ1-42 /siP2X7R and blank control groups,the supernatant levels of IL-1β were (52.54±3.21) μg L,(54.94±2.54) μg/L,(28.70±3.58) μg/L,(24.55±4.34) μg/L,respectively and the supernatant levels of TNF-α were (64.40±4.80) μg/L,(60.94±1.63) μg/L,(25.69±3.13) μg/l,(19.21±1.97)μg/L,respectively.As compared with Aβ1-42 and Aβ-42/siNC groups,the levels of IL 1β and TNF-α in Aβ1-42 /siP2X7R group was decreased significantly (P＜ 0.05),and IL-1β level had no significant difference between Aβ1-42/siP2X7R and blank control groups.The same result was observed in the level of NOin the supernatant.The levels of NO were (1.33±0.19) μmol/L,(4.49±0.28) μmol/L,(4.78±0.33) μmol/L and (1.07±0.36) μmol/L in Aβ1-42/siP2X7R,Aβ1-42,Aβ1-42 /siNC and blank control groups respectively.Conclusions The silencing expression of P2X7 R by RNA interference effectively decreases the levels of IL-1β,TNF-α and NO released by microglia.P2X7 R can be used as an effective therapeutic target for RNA interference treatment of Alzheimer's disease.