Effect And Mechanism Of Irisin In Improving Lung Injury On Sepsis By Promoting Anti-inflammatory Differentiation Of Macrophages

Objective:1.To observe whether exogenous administration of Irisin can promote M2-type polarization of macrophages and reduce lung injury in LPS-induced septic mice.2.To clarify the role of Irisin in the anti-inflammatory regulation of M2 macrophages.3.To investigate the potential molecular regulatory mechanism of Irisin-mediated macrophage anti-inflammatory response.Methods:1.Establishment of animal and cellular experimental models.24 male C57BL/6J mice were randomly divided into four groups,normal control group,irisin control group,LPS-exposed group and irisin-treated group.Mice in the normal control group were injected intraperitoneally with 10 m L/kg saline and 2,26 and 50 h later with saline(10m L/kg);mice in the irisin control group were injected intraperitoneally with saline(10m L/kg)and 2,26 and 50 h later with recombinant irisin(0.5 μg/kg/day);mice in the LPSexposed group were injected intraperitoneally with the mice in the LPS-exposed group were injected intraperitoneally with LPS(10 mg/kg)and intraperitoneally with saline(10m L/kg)after 2,26 and 50 h.The mice in the irisin-treated group were injected intraperitoneally with LPS(10 mg/kg)and intraperitoneally with recombinant irisin(0.5μg/kg/day)after 2,26 and 50 h,respectively.After 72 h of treatment in the above four groups,mice were anesthetized with isoflurane by inhalation,and lung tissues were collected from the eyes.After irisin treatment of macrophages,macrophage viability changes were detected by CCK-8 method,and macrophage apoptosis changes were detected by flow cytometry.The effect of Irisin on LPS-induced inflammatory response by promoting anti-inflammatory differentiation of macrophages was analyzed in vitro and in vivo.2.200 ng/m L irisin was used to treat RAW264.7 and BMDMs cells for 0,6,12,24 and 48 h.Western blot was used to detect PPAR-γ and Nrf2-dependent antioxidant enzyme protein expression.Immunofluorescence was used to detect PPAR-γ and Nrf2 expression and localization in macrophages.RT-q PCR was used to detect Nrf2-dependent antioxidant enzyme expression.ELISA for inflammatory factor content,enzymatic activity of SOD2 and CAT and GSH content,and flow cytometry for ROS levels.The mechanism of action of irisin in regulating anti-inflammatory and antioxidant responses of macrophages through activation of PPAR-γ and Nrf2 was investigated in vivo using PPAR-γ inhibitor T0070907,small interfering PPAR-γ and Nrf2 inhibitor ML385 intervention.3.200 ng/m L Irisin was used to treat RAW264.7 and BMDMs cells for 0,6,12,24 and 48 h.STAT6 and p-STAT6 protein expression were detected by Western blot.STAT6 expression and localization were detected by immunofluorescence in macrophages.STAT6 and inflammatory factor m RNA expression were detected by RT-q PCR,and inflammatory factors were detected by ELISA.Irisin regulated anti-inflammatory differentiation of M2 macrophages through phosphorylation of STAT6 using STAT6 inhibitor AS1517499 or knockdown of STAT6.Dual luciferase reporter genes were used to detect the binding site of STAT6 to PPAR-γ/Nrf2,and chromatin immunoprecipitation was used to detect the extent of STAT6 binding to PPAR-γ/Nrf2.4.RNA-seq sequencing was performed to analyze the differentially expressed genes of the JAK family in Irisin-acting RAW264.7 cells to find the key regulators of antiinflammatory differentiation of macrophages.Western blot was performed to detect the expression of JAK2,p-JAK2 and STAT6,p-STAT6 proteins.RT-q PCR was performed to detect PPAR-γ/Nrf2,inflammatory factor and antioxidant enzyme m RNA expression,and ELISA for inflammatory factors.Changes in macrophage M2 type markers were observed using JAK2 inhibitor FLLL32,integrin αVβ5 inhibitor Cilengitide or knockdown of integrin αVβ5.Immunoprecipitation was analyzed to observe if JAK2 and integrin αVβ5 interacted with each other.Results:1.Irisin ameliorates LPS-induced inflammatory response by promoting the antiinflammatory differentiation of macrophages.In vivo study showed that Irisin did not cause lung toxicity,but it significantly reduced lung injury and inflammatory cell infiltration in LPS-induced septic mice,reduced the levels of TNF-α and IL-6 in serum and lung tissues,and increased the levels of IL-10 and Arg-1.The number of F4/80+CD11c+(M1)macrophages in lung tissue was decreased,and the number of F4/80+CD206+(M2)macrophages was increased.200 ng/m L Irisin increased the expression and secretion of M2-type macrophage markers such as IL-10,Arg-1 and CD206 in RAW64.7 and BMDMs at 0,6,12,24 and 48 h,and inhibited the LPS-induced expression and secretion of pro-inflammatory factors.It promotes the expression and production of IL-10 and Arg-1.2.Irisin promotes macrophage anti-inflammatory differentiation by activating PPAR-γ and Nrf2 signaling.Treatment with Irisin enhanced the m RNA and phosphorylation of PPAR-γ in macrophages,and promoted its nuclear translocation.PPAR-γ specific antagonist T0070907 attenuates Irisin-induced phosphorylation of PPAR-γ and inhibits expression and production of Arg-1 and IL-10 in RAW264.7.Moreover,knockdown of PPAR-γ by specific si RNA significantly suppressed Irisininduced m RNA expression of PPAR-γ,Arg-1,IL-10 and CD206.On the other hand,Irisin increased Nrf2 phosphorylation and nuclear translocation,enhanced the m RNA and protein expression of Nrf2 and its downstream antioxidant enzymes,and promoted SOD2 and CAT enzyme activities and GSH content in RAW264.7.Moreover,Irisin treatment attenuated LPS-induced ROS increase in RAW264.7 cells.The Nrf2-specific antagonist ML385 also inhibited Irisin-mediated Arg-1 and IL-10 expression and production.3.Irisin promotes PPAR-γ and Nrf2 signaling activation by regulating STAT6 phosphorylation.In response to Irisin,STAT6 was rapidly phosphorylated and increased in nuclear translocation.STAT6 inhibitor AS1517499 pretreatment blocked Irisinmediated transcriptional activation of PPAR-γ and Nrf2 in RAW264.7 and BMDMs,and inhibited the accumulation of anti-inflammatory cytokines downstream of PPAR-γ.Knockdown of STAT6 using sh RNA similarly reduced the m RNA expression of PPAR-γ,Nrf2 and their downstream genes.The luciferase activity of the mutant PPAR-and Nrf2 groups was inhibited,and 200 ng/m L Irisin promoted STAT6 binding to the PPAR-γ/Nrf2 promoter.4.Irisin promotes the activation of STAT6 and its downstream signaling through JAK2 phosphorylation.Irisin significantly increased the expression of JAK2 m RNA and promoted the rapid phosphorylation of JAK2.JAK2 inhibitor FLLL32 attenuated Irisininduced phosphorylation of JAK2 and STAT6,inhibited the increase of PPAR-γ,Arg-1and IL-10 m RNA,and decreased the m RNA expression of Nrf2 and downstream antioxidant genes mediated by Irisin in RAW264.7 cells.Irisin receptor integrin αVβ5inhibitor Cilengitide attenuated Irisin-induced increase of JAK2 and STAT6 phosphorylation and m RNA expression of PPAR-γ,Nrf2,Arg-1 and IL-10 in RAW264.7cells.Similarly,knockdown of integrin αVβ5 inhibited the increased m RNA expression of PPAR-γ,Nrf2,Arg-1 and IL-10 in RAW264.7 cells treated with Irisin.200 ng/m L Irisin promotes the interaction of JAK2 and integrin αVβ5 in macrophages.Conclusion:1.Irisin alleviates LPS-induced acute lung injury in mice by regulating macrophage anti-inflammatory differentiation and increases the expression of M2 macrophage markers,suggesting that Irisin may be a novel anti-inflammatory factor.2.Irisin up-regulates the expression of PPAR-γ and Nrf2 and promotes the differentiation of macrophages into anti-inflammatory cells.3.Irisin regulates PPAR-γ and Nr F2-dependent downstream molecule expression via activating STAT6.4.Irisin may regulate macrophage anti-inflammatory differentiation by promoting the interaction between JAK2 and integrin αVβ5.