The protective effect of irisin against hemorrhagic injury is mediated by PI3K and p38 pathways in hemorrhage/resuscitation.

The objective of this study is to investigate whether PI3kinase (PI3K) and p38 mitogen-activated kinase contributes to the protection of irisin during hemorrhage/resuscitation. Experimental groups were divided by receiving the different treatments during resuscitation: I) Hemorrhage: Adult male CD-1 mice were subjected to hemorrhage at a mean arterial blood pressure of 35~45 mmHg for 60 min followed by 120 min of resuscitation (n=13); II) Hemorrhage + Irisin: receiving irisin (5µg/kg) (n=13); III) Hemorrhage + Irisin + PI3K inhibitor: receiving both Ly294002 (1mg/kg, i.v.) and irisin (n=6); IV) Hemorrhage + Irisin + p38 inhibitor: receiving SB202190 (1mg/kg, i.v.) and irisin (n=6). As compared to hemorrhage/resuscitation control, irisin improved the cardiac function and recovery of hemodynamics in association with the decreased systemic IL-1, IL-6, and TNF-α, which was completely abrogated by PI3K or p38 inhibitions. Furthermore, inhibition of PI3K or p38 abolished irisin-induced reduction of the infiltration of inflammatory cells and TUNEL-positive apoptosis in the cardiac and skeletal muscles. Irisin reduced TNF-α and IL6 expression in cardiac and skeletal muscle, which was abrogated by inhibition of PI3K or p38. Irisin-treated hemorrhage increases the phosphorylation of PI3K and p38 in both cardiac and skeletal muscle, which was mitigated by inhibition of PI3K or p38. Conclusion: PI3K and p38 play a critical role in modulating the protective effect of irisin during the hemorrhage/resuscitation. Significance Statement 1). This study has identified a critical pathway in regulation of trauma/hemorrhage by using a preclinical and reproducible model, in which Irisin, as a hormone factor, stimulates PI3K and p38 pathways to induce the protection against traumatic conditions. 2). The study holds promise to develop a new therapeutic strategy to target irisin and its pathway related to PI3K and p38 to treat trauma and its comorbidities to reduce mortality for clinical implication.

Inhibition of integrin alpha v/beta 5 mitigates the protective effect induced by irisin in hemorrhage.

INTRODUCTION: Irisin plays an important role in regulating tissue stress, cardiac function, and inflammation. Integrin αvß5 was recently identified as a receptor for irisin to elicit its physiologic function. It remains unknown whether integrin αvß5 is required for irisin's function in modulating the physiologic response to hemorrhage. The objective of this study is to examine if integrin αvß5 contributes to the effects of irisin during the hemorrhagic response. METHODS: Hemorrhage was induced in mice by achieving a mean arterial blood pressure of 35-45 mmHg for one hour, followed by two hours of resuscitation. Irisin (0.5  µg/kg) was administrated to assess its pharmacologic effects in hemorrhage. Cilengitide, a cyclic Arg-Gly-Asp peptide (cRGDyK) which is an inhibitor of integrin αvß5, or control RGDS (1 mg/kg) was administered with irisin. In another cohort of mice, the irisin-induced protective effect was examined after knocking down integrin ß5 with nanoparticle delivery of integrin ß5 sgRNA using CRSIPR/Cas-9 gene editing. Cardiac function and hemodynamics were measured using echocardiography and femoral artery catheterization, respectively. Systemic cytokine releases were measured using Enzyme-linked immunosorbent assay (ELISA). Histological analyses were used to determine tissue damage in myocardium, skeletal muscles, and lung tissues. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in tissues. RESULTS: Hemorrhage induced reduction of integrin αvß5 in skeletal muscles and repressed recovery of cardiac performance and hemodynamics. Irisin treatment led to significantly improved cardiac function, which was abrogated by treatment with Cilengitide or knockdown of integrin ß5. Furthermore, irisin resulted in a marked suppression of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), muscle edema, and inflammatory cells infiltration in myocardium and skeletal muscles, which was attenuated by Cilengitide or knockdown of integrin ß5. Irisin-induced reduction of apoptosis in the myocardium, skeletal muscles, and lung, which were attenuated by either the inhibition of integrin αvß5, or knockdown of integrin ß5. CONCLUSION: Integrin αvß5 plays an important role for irisin in modulating the protective effect during hemorrhage.

Proteoglycan 4 (Lubricin) and Regulation of Xanthine Oxidase in Synovial Macrophage as A Mechanism of Controlling Synovitis.

Background: Synovial macrophages (SMs) are important effectors of joint health and disease. A novel Cx3CR1 + TREM2 + SM population expressing the tight junction protein claudin-5, was recently discovered in synovial lining. Ablation of these SMs was associated with onset of arthritis. Proteoglycan 4 (PRG4) is a mucinous glycoprotein that fulfills lubricating and homeostatic roles in the joint. The aim of this work is to study the role of PRG4 in modulating synovitis in the context of SM homeostasis and assess the contribution of xanthine oxidase (XO)-hypoxia inducible factor alpha (HIF-1α) axis to this regulation. Methods: We used Prg4 FrlioxP/FrtloxP ;R26 FlPoER/+ , a novel transgenic mouse, where the Prg4 Frt allele normally expresses the PRG4 protein and was designed to flank the first two exons of Prg4 with a flippase recognition target and "LOXP" sites. Inducing flippase activity with tamoxifen (TAM) inactivates the Frt allele and thus creates a conditional knockout state. We studied anti-inflammatory SMs and XO by quantitative immunohistochemistry, isolated RNA and studied immune pathway activations by multiplexed assays and isolated SMs and studied PRG4 signaling dysfunction in relation to glycolytic switching due to pro-inflammatory activation. Prg4 inactivated mice were treated with oral febuxostat, a specific XO inhibitor, and quantification of Cx3CR1 + TREM2 + SMs, XO immunostaining and synovitis assessment were conducted. Results: Prg4 inactivation induced Cx3CR1 + TREM2 + SM loss (p < 0.001) and upregulated glycolysis and innate immune pathways in the synovium. In isolated SMs, Xdh (p < 0.01) and Hif1a (p < 0.05) were upregulated. Pro-inflammatory activation of SMs was evident by enhanced glycolytic flux and XO-generated reactive oxygen species (ROS). Febuxostat reduced glycolytic flux (p < 0.001) and HIF-1α levels (p < 0.0001) in SMs. Febuxostat also reduced systemic inflammation (p < 0.001), synovial hyperplasia (p < 0.001) and preserved Cx3CR1 + TREM2 + SMs (p < 0.0001) in synovia of Prg4 inactivated mice. Conclusions: PRG4 is a biologically significant modulator of synovial homeostasis via inhibition of XO expression and downstream HIF-1a activation. PRG4 signaling is anti-inflammatory and promotes synovial homeostasis in chronic synovitis, where direct XO inhibition is potentially therapeutic in chronic synovitis.