| Objective:To study the dynamic changes of the effects of LPS on PI3K-Ⅰ,PI3K-Ⅲ and their key molecules in signal transduction pathway in mouse macrophages and the effects of artesunate(AS).Methods:1.Effect of AS on release of proinflammatory cytokines induced by LPS1.1 Mouse macrophages were treated with LPS(100 ng/m L)for 2,4 and 8 hours to establish the model,determine the time of LPS treatment,and detect the level of pro-inflammatory cytokine TNF-α in the supernatant by ELISA.1.2 Autophagy inhibitor 3-MA(0.60 mg/m L)was pre incubated for 2 h,and the mouse macrophages were treated with LPS(100 ng/m L)for 4 h.The level of TNF-α in the supernatant was detected by ELISA.1.3 RAW264.7 cells were pretreated with different concentrations of AS(10,20,30μg/m L)for 2h,and then treated with LPS(100 ng/m L)for 4 h.The level of TNF-α in the supernatant was detected by ELISA.1.4 Mouse peritoneal macrophages(PMs)were pre incubated with AS(20 μg/m L)for 2 h,and then treated with LPS(100 ng/m L)for 4 h.The levels of proinflammatory cytokines(TNF-α,IL-6)in supernatant were detected by ELISA.1.5 RAW264.7 cells were pretreated with 3-MA(0.60 mg/m L)and AS(20 μg/m L)for 2 h,and then treated with LPS(100 ng/m L)for 4 h.The level of TNF-α in supernatant was detected by ELISA.2.Effect of LPS on the mRNA expression of PI3K-Ⅰ,PI3K-Ⅲ and their key molecules in signaling pathwayAfter PMs were treated with LPS(100 ng/m L)for different time(0,0.5,1,2,4,8,12,24 h),cell precipitates were collected and real-time quantitative polymerase chain reaction(RT-q PCR)was performed,RT-q PCR was used to detect the dynamic changes of PI3K-Ⅲ,TLR4,My D88,TRAF6,Beclin1,NF-κB p65,NF-κB p50,ATG16L1,p62,TNF-α,IL-6,IL-1β,IFN-γ,PI3K-Ⅰ,AKT and m TOR mRNA.3.Effect of LPS on phosphorylation of PI3K-Ⅰ and PI3K-Ⅲ and the intervention of AS3.1 After PMs were treated with LPS(100 ng/m L)for different time(0,0.25,0.5,1,2,4,8,12 h),the cell precipitates were collected,and the phosphorylation levels of PI3K-Ⅰ and PI3K-Ⅲ were detected by Western blotting.3.2 PMs were pretreated with AS(20 μg/m L)for 2 h,and treated with LPS(100ng/m L)for 0.5 h.The cell precipitates were collected and the phosphorylation level of PI3K-Ⅲ(Ser249)was detected by WB.Results:1.Inhibitory effect of artesunate on LPS induced release of proinflammatory cytokines1.1 LPS(100 ng/m L)could induce mouse macrophages to release a large amount of proinflammatory cytokine TNF-α.1.2 3-MA(0.60 mg/m L)could significantly inhibit the release of TNF-α induced by LPS.1.3 AS(20 μg/mL)could significantly inhibit the release of pro-inflammatory cytokines(TNF-α,IL-6)in PMs induced by LPS.1.4 Whether 3-MA and AS were given alone or combined with 3-MA and AS,LPS induced TNF-α release from RAW264.7 cells was significantly inhibited.2.Effect of LPS on the mRNA expression of PI3K-Ⅰ,PI3K-Ⅲ and their key molecules in signaling pathwayLPS could significantly up regulate the mRNA expression of TLR4/My D88/TRAF6/Beclin1/PI3K-Ⅲ signaling pathway,and down regulate the mRNA expression of PI3K-Ⅰ/AKT/m TOR signaling pathway.3.Effect of LPS on phosphorylation of PI3K-Ⅰ and PI3K-Ⅲ and the intervention of AS3.1 LPS could significantly increase the phosphorylation levels of PI3K-Ⅲ(Ser249)and PI3K-Ⅰ.3.2 LPS(100 ng/mL)could significantly increase the phosphorylation level of PI3K-Ⅲ.AS(20 μg/m L)could significantly inhibit the increase of phosphorylation level of PI3K-Ⅲ(Ser249)induced by LPS.Conclusions:LPS could significantly increase the mRNA expression of PI3K-Ⅲ and decrease the mRNA expression of PI3K-Ⅰ,which was closely related to LPS induced release of a large number of proinflammatory cytokines from PMs;And LPS could significantly increase the phosphorylation of PI3K-Ⅲ(Ser249)and PI3K-Ⅰ,which was closely related to LPS induced release of a large number of proinflammatory cytokines from PMs.AS has significant anti-inflammatory effect,and its molecular mechanism is closely related to the inhibition of PI3K-Ⅲ(Ser249)phosphorylation. |
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