The Molecular Regulation Mechanism Of β-arrestin In The Apoptosis And TLR4-mediated Innate Immune Response

Objective:To detect the effect of β-arrestins on SD-induced apoptosis and signaling pathway in MEFsMethods:WT, β-arrestinl KO and β-arrestin2KO MEFs were cultured in DMEM medium without serum for12h、24h and48h, Apoptotic cells were determined by TUNEL assay. β-arrestin2full-length plasmid and control vector were transfected into β-arrestin2KO MEFs and then detected the cell apoptosis. Caspase-3, ERK1/2, p38MAPKs and Akt phosphory-lation were determined in WT,β-arrestinl and2KO MEFs following SD in a different time point by Westernblot.Results:1. Significantly greater cell apoptosis was observed when β-arrestinl or β-arrestin2deficient MEFs were exposed to SD than WT MEFs were exposed to SD. Moreover, transfection with β-arrestin2full-length plasmid strongly rescued the number of apoptosis in a deficiency of β-arrestin2MEFs.2. The level of Caspase-3activation was significantly higher in the absence of β-arrestinl or2than in WT cells following SD treatment for12and24h.3. The levels of phospho-ERK1/2and phospho-p38MAPKs were significantly higher in β-arrestinl or2KO MEFs than in WT MEFs. While the levels of phospho-Akt in β-arrestinl or2KO MEFs were significantly lower compared to in WT cells.Conclusions:1. β-arrestinl and β-arrestin2significantly inhibit MEFs apoptosis following SD 2.β-Arrestinl and β-arrestin2prevents cell apoptosis through pro-apoptotic ERKl/2and p38MAPKs and anti-apoptotic Akt pathways Objective:To detect the effect of β-arrestins on LPS、IL-1β-mediated the production of the cytokine and signaling pathway in MEFs and HEK293cells.Methods:RT-PCR analysis of relative IL-6、IL-8and TNF-a mRNA after LPS stimulation in WT and p-arrestinl/2DKO MEFs. p-arrestinl/2DKO MEFs were transfected with β-arrestinsl or β-arrestins2plasmid. Forty-eight hours posttransfection, cells were treated with or without LPS, mRNA and the concentration of IL-6、IL-8and TNF-a in culture supernatants were quantified by RT-PCR and ELISA, ERK1/2, p38MAPKs and Akt phosphorylation were determined by Westernblot. Dual-Luciferase reporter assay showing IL-1β-induced activation of transcription factor NF-κB and AP-1in HEK293cells.Results:1. Significantly increased production of IL-6、IL-8and TNF-a were dectected in β-arrestinl/2DKO MEFs than WT MEFs after LPS stimulation. Moreover, transfection with β-arrestinl or β-arrestin2full-length plasmid strongly reduced the producrion of IL-6、 IL-8and TNF-a in a deficiency of β-arrestinl/2MEFs.2. The levels of phospho-ERK1/2and phospho-p38MAPKs were significantly higher in β-arrestinl/2DKO MEFs than in WT MEFs after LPS stimulation. While transfection with β-arrestinl or β-arrestin2full-length plasmid strongly decreased the activation of ERK1/2and p38MAPKs in β-arrestinl/2DKO MEFs compared to in WT cells.3. IL-1β stimulation increased NF-κB and AP-1reported activity in HEK293cell-whereas transfection with β-arrestinl or β-arrestin2plasmid inhibit this increase.1 contrast, knockdown of β-arrestinl or β-arrestin2by siRNA increased the IL-1β-stimulated NF-κB and AP-1actication.Conclusions:1.β-arrestinl and β-arrestin2significantly inhibit the production of cytokine following LPS stimulation and negative regulate TLR4-mediated innate immune response.2. β-Arrestinl and β-arrestin2negative regulate TLR4-mediated innate immune response through ERK1/2and p38MAPKs signaling pathways3. β-Arrestinl and β-arrestin2inhibits IL-1β-induced NF-κB and AP-1activation. Objective:To determine the protection effect and possible mechanism of β-arrestin2in TLR4-mediated murine liver ischemia-reperfusion injury.Methods:C57BL/6β-arrestin2WT、KO male mice were randomly divided into four groups: control group (WT, β-arrestin2KO), experimental group (IR+WT, IR+β-arrestin2KO). Hepatic partial warm ischemia was performed for60minutes. After6h reperfusion, liver function, the degree of apoptosis, cytokine and the activation of TLR4、Akt/GSK3β, Bcl-2family, MAPKs, NF-κB were assessed. The survival rate was also investigated.Results:1. Serum ALT, AST, apoptotic cells and the production of cytokine were significantly higher in the I/R+P-arrestin2KO groups compared with those in WT group at6h after reperfusion. The survival rates of mice in I/R+P-arrestin2KO group significantly lower than in WT group.2. The activation of Akt/GSK, Bcl-2significantly lower in I/R+β-arrestin2K group than in WT group. In contrast the levels of phospho-ERK1/2, phosphc p38MAPKs and NF-κB were significantly higher in I/R+β-arrestin2KO group than in WT group.Conclusions:β-arrestin2can negative regulate TLR4signaling and attenuate hepatocellular injury after hepatic warm I/R injury through Akt/GSK3β、 MAPKs and NF-κB signaling pathway.