Effects Of Papaverine On The Activation Of Retinal Microglia And Its Possible Mechanism

Background and purposeOptic nerve damage can cause apoptosis and gliosis of RGC axons,,leading to RGC axon regeneration disorder,resulting in irreversible visual damage.Abnormally activated microglia release a large number of cytotoxic factors such as TNF-?,IL-1?,NO and ROS,causing secondary damage to RGC.Our study has found that papaverine inhibits activation of primary retinal microglia by inhibiting the release of inflammatory factors.cAMP is closely related to the development,survival and axonal regeneration of mammalian central neurons and plays a key role in many signaling pathways.Woo et al.found that increasing cAMP levels activates PKA,thereby inhibiting the release of the inflammatory mediator IL-1? by BV2 cells.The MAPK/Erk cascade is involved in the activation of microglia.Park et al.found that fucoidan attenuated expression of LPS-induced iNOS,COX-2,IL-1? and TNF-? in BV2 microglia by down-regulating MAPK(ERK,JNK,p38)and Akt pathways.PI3 K also plays an important role in cell growth,adhesion,differentiation and inflammatory responses.In order to investigate the mechanism of papaverine inhibiting the activation of retinal microglia,we analyzed the activation of cAMP/PKA,MEK/Erk,PI3K/Akt pathway.And the release levels of TNF-?,IL-1?,IL-10 in retina microglia in each pathway were analyzed.We futher explored the relationship between the various signaling pathways,providing theoretical basis and experimental basis for papaverine to inhibit microglia activation and protect the central nervous system.Materials and methodsMaterialsThe retinal primary microglia of newborn SD rats were isolated and purified,and a LPS-induced microglia activation model was established.MethodsTo observe the abnormal activation of retinal microglia,RT-PCR and ELISA were used to detect the transcription and protein expression of TNF-?,IL-1? and IL-10,respectively.The protein phosphorylation levels of the signaling pathways were detected by western blot,and the activation of each pathway could be observed.The cAMP inhibitor Rp-isomer,PKA inhibitor H89,MEK inhibitor U0126 and PI3 K inhibitor LY294002 were separately added to further investigate the role of each pathway in the inhibition of primary retinal microglial activation and the relationship between the signaling pathways.Rusults1.Papaverine inhibits transcription and expression of TNF-? and IL-1? and promotes transcription and expression of IL-102.Papaverine inhibits expression of TNF-? and IL-1? by activating cAMP/PKA pathwayPapaverine up-regulated cAMP content,and Rp-isomer and H89 added to block cAMP/PKA pathway,and the expression levels of TNF-? and IL-1? increase.3.Papaverine inhibits MEK/Erk signaling pathwayLPS upregulates phosphorylation of MEK and Erk,and these effects are blocked by papaverine.4.U0126 attenuates the transcription and protein expression of inflammatory factors TNF-? and IL-1?LPS increases TNF-? mRNA and IL-1? mRNA,up-regulates the protein expression of TNF-? and IL-1?,and addition of U0126 significantly blocks the above effects.5.U0126 increases transcription and expression of the anti-inflammatory factor IL-106.MEK/Erk pathway is partially regulated by cAMP/PKAThe addition of Rp-isomer and H89 blocks the cAMP/PKA pathway,and then the phosphorylation levels of MEK and Erk increase.7.Papaverine activates the PI3K/Akt pathway and has a tendency to inhibit microglia activationPapaverine up-regulates the phosphorylation levels of PI3 K and Akt,and LY294002 shows the tendancy to increase the expression of TNF-? and IL-1?.ConclusionPapaverine inhibits LPS-induced primary retinal microglial activation via the cAMP/PKA and MEK/Erk pathways,and the MEK/Erk pathway is partially regulated by the cAMP/PKA pathway.