Effects And Mechanisms Of A20 O-GlcNAc Modification On The Regulation Of Inflammatory Responses In RASMCs

Objective:The present project aims to investigate the effect of A20 O-GlcNAc(O-linked-N-acetylglucosamine) on NF-κB signaling pathway and elucidate the effect of protein O-GlcNAcylation on A20 activation and its anti-inflammative action, finally identify the key target of vascular protective effect mediated by protein O-GlcNAc modification.Methods:(1) RASMCs were transfected with an empty adenovirus vectors(AD-NULL), adenovirus vectors carrying A20shRNA(AD-shA20-2) and adenovirus vectors carrying A20 overexpression gene(AD-A20-IRES), serum-starved cells were pretreated with Thiamet-G(150nmol/ml) for 1 hour and then stimulated with TNF-a(10ng/ml) for 6 hours, total RNA were extracted, inflammatory mediator mRNA expression was measured by real-time RT-PCR.(2) RASMCs were transfected with AD-NULL, AD-shA20-2 and AD-A20-IRES, serum-starved cells were pretreated with Thiamet-G(150nmol/ml) for 1 hour, then cells were seeded into upper chamber, TNF-α(10ng/ml) were added to the lower chamber and continued to incubate for 24 hours, cells were fixed, stained and rinsed, ten fields were randomly selected in each group and the number of migrated cells were counted.(3) RASMCs were transfected with AD-NULL, AD-shA20-2 and AD-A20-IRES, cells were seeded into 96-well plates, serum-starved cells were pretreated with Thiamet-G(150nmol/ml) for 1 hour, then incubated with 10% FBS for 24 hours, the cell proliferation were measured by using CCK8 kit.Results:(1) In RASMCs transfected with AD-NULL, Thiamet-G pretreatment rapidly increased the level of intracellular O-GlcNAc modification and significantly inhibited TNF-a-induced expression of inflammatory mediators; In RASMCs transfected with AD-shA20-2, A20 protein expression was significantly reduced, Thiamet-G pretreatment no longer was able to inhibit TNF-a-induced the expression of inflammatory mediators; In RASMCs transfected with AD-A20-IRES, A20 protein expression was significantly increased, Thiamet-G pretreatment still inhibited TNF-a-induced expression of inflammatory mediators.(2) In RASMCs transfected with AD-NULL, Thiamet-G pretreatment could significantly inhibit TNF-a-induced RASMCs migration; In RASMCs transfected with AD-shA20-2, Thiamet-G pretreatment failed to inhibit TNF-a-induced RASMCs migration; In RASMCs transfected with AD-A20-IRES, Thiamet-G pretreatment still inhibited TNF-a-induced RASMCs migration.(3) In RASMCs transfected with AD-NULL, Thiamet-G pretreatment significantly suppressed serum-induced RASMCs proliferation. In RASMCs transfected with AD-shA20-2, Thiamet-G pretreatment failed to suppress serum-induced RASMCs proliferation. In RASMCs transfected with AD-A20-IRES, Thiamet-G still suppressed serum-induced RASMCs proliferation.Conclusion:Rapid increase of the level of intracellular O-GlcNAc modification can inhibit TNF-a-induced expression of inflammatory mediators, cell migration and serum-induced cell proliferation in RASMCs. Scilencing A20 by infected recombinant adenovirus-carrying A20 shRNA can abolish the inhibitory effect of the fast O-GlcNAc modification on TNF-a-induced expression of inflammatory mediators and migration and serum-induced cell proliferation. Therefore, A20 is likely the key molecular target which mediates the vascular protective effects of rapid O-GlcNAc modification.