| Objective:Neuroinflammation promotes the development of various neurodegenerative diseases.Microglial activation is one of the basic characteristics of neuroinflammation.Previous studies have shown that Roflupram(ROF)reduces the release of inflammatory factors.However,its underlying mechanism is not yet clear.This study aimed to study the inhibitory effect and mechanism of ROF on lipopolysaccharide(LPS)-induced microglial activationMethods:(1)BV-2 microglial cells were activated by LPS,the levels of IL-6 and TNF-? were detected by ELISA kits,the content of NO was detected by a Greiss kit,and the expression of iNOS and Iba1 were detected by Western blot,immunofluorescence for Iba1 was used to evaluate the inhibitory effect of ROF on microglial activation.(2)Western blot was used to detect the effects of ROF on the AMP-dependent protein kinase(AMPK)phosphorylation expression level(p-AMPK)and Sirtuin 1(SIRT1)protein expression in BV-2 cells.Pretreatment with AMPK inhibitor Compound C(CC)and SIRT1 inhibitor EX527 were performed to evaluate whether they can block the drug effect.Cells were pretreated with 10 ?M ROF with or without CC or EX527,and Western blot was used to detect the effects of ROF on p-AMPK and SIRT1 expression under LPS stimulation(3)Cells were pretreated with 10 ?M ROF with or without EX527,the contents of IL-6 and TNF-? were detected by ELISA kits,the content of NO was detected by the Greiss kit,and the expression of iNOS and Iba1 were detected by Western blot,which was to evaluate whether the inhibition of SIRT1 could block the effect of ROF.(4)Mice were administered intragastrically with ROF for 7 days and then injected intraperitoneally with LPS.The mice were sacrificed and the hippocampus and cortex were isolated.ELISA was used to detect the IL-6 and TNF-? levels in the hippocampus and cortex of mice to evaluate the anti-inflammatory effect of ROF.(5)Mice were administered intragastrically with ROF with or without EX527 for seven days and then injected intraperitoneally with LPS.New object recognition experiments were performed in mice,SIRT1 expression was detected by Western blot,inflammatory factor content in mouse hippocampal and cortex was detected by ELISA,and Iba1 expression was verified by Western blot and immunofluorescence.Results:(1)ROF(10 ?M)significantly reduced the levels of IL-6 and TNF-? in the supernatant of LPS-treated BV-2 cells(P<0.01),and reduced the content of NO(P<0.05)and the protein expression levels of iNOS and Iba1 in the cells(P<0.05)(2)ROF increased the expression of p-AMPK and SIRT1 in a time-and dose-dependent manner and ROF(10 ?M)increased the expression of p-AMPK and SIRT1 proteins(P<0.05)in LPS-treated BV-2 cells,and the inhibitors CC and EX527 reversed the drug effect.(3)Cells were pretreated with 10 ?M ROF with or without EX527.Compared with the group of ROF+LPS,EX527 pretreatment can reverse the drug effect,which reduce the expression level of SIRT1 protein,increase the release of IL-6 and TNF-?in the supernatant(P<0.01),and increase the NO content(P<0.05),iNOS and Iba1 protein expression were also increase in cells(P<0.01).(4)ROF reduced TNF-? content in the hippocampus and cortex of mice(P<0.05),decreases IL-6 content in the hippocampus of mice(P<0.05),and also reduces IL-6 content in cortex,but there is no statistics difference.(5)Compared with the control group,the weight of the mice in each group of LPS intraperitoneal injection decreased(P<0.05).ROF decreased the levels of IL-6 and TNF-? in the hippocampus and cortex of mice(P<0.05),reduced the expression level of Iba1(P<0.05)and increased the new object recognition index of LPS-challenged mice(P<0.01),Increase the expression level of SIRT1 in hippocampal cortex(P<0.01).Conclusion:ROF may inhibit the release of inflammatory factors in LPS-stimulated microglial cells and reduce BV-2 cell activation through AMPK/SIRT1 signal pathway.ROF reduces the levels of inflammatory factors in the hippocampal and cortex of LPS-challenged mice and the Anti-inflammatory effects which may depend on SIRT1. |
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