Adropin Reduces Myocardial Ischemia-reperfusion Injury Through RISK Pathway In Mice And Is Associated With Coronary Atherosclerosis In Clinical Research

Objective: Myocardial ischemia-reperfusion injury (MIRI) seriously weakens theeffect of reperfusion therapy, and ischemic postconditioning and pharmacologicalpostconditioning can reduce myocardial reperfusion injury through activating thereperfusion injury salvage kinase (RISK) pathway and the survivor activating factorenhancement (SAFE) pathway. Adropin, a newly identified endogenous bioactivesubstance, possesses non-metabolic properties including protection of endothelialfunction through RISK pathway. Nevertheless, it remains unclear whether Adropincan reduce reperfusion injury. In this study, we investigated the cardioprotective roleof Adropin during myocardial reperfusion injury and whether or not RISK or SAFEpathway is involved in the signaling pathways.Methods: MIRI model was established in20-week-old C57BL/6J wild type mice.Mice were divided into eight groups: all groups except the Sham group underwent45-min ischemia (I) and4-h reperfusion (R) induced by ligating and releasing the leftcoronary artery.1) Sham group: perfusion without ischemia;2) I/R group: I45-minplus R4-h;3) low-dose Adropin group (Adropin-L)： Adropin0.1mg/kg;4)moderate-dose Adropin group (Adropin-M)：Adropin0.2mg/kg;5) high-dose Adropingroup (Adropin-H)：Adropin0.4mg/kg;6) Ischemic postconditioning group (I-PostC):3intermittent cycles of10-s reperfusion and10-s ischemia immediately after the45-min ischemia;7) Adropin+LY294002group: besides Adropin0.2mg/kg, PI3Kinhibitor LY294002(40mg/kg) was given via intraperitoneal injection15min beforeischemia;8) Adropin+PD98059group: besides Adropin0.2mg/kg, and ERK1/2inhibitor PD98059(1mg/kg) was given via intraperitoneal injection15min beforeischemia. Adropin was given5min before reperfusion in each group. Akt1+/-mice andlittermate controls (Akt1+/+) were divided into three groups：I/R group (I45min/R4h inAkt1+/+mice), Akt1+/+group (Adropin0.2mg/kg was given in Akt1+/+mice)and Akt1+/-group (Adropin0.2mg/kg was given in Akt1+/-mice).We used TTC staining tomeasure infarct size, Western blot to determine levels of signaling protein expression(Akt, ERK1/2and GSK3β), Real-time PCR to detect the expression levels of Bcl-2and Bax genes and immunohistochemistry to detect Caspase-3activity.Results: Compared with the I/R group, Adropin-L group failed to reduce infarct size(45.8±5.1%vs.40.2±5.8%, P>0.05), while Adropin-M group (30.5±5.2%), I-Postcgroup (29.5±13%) and Adropin-H group (29.9±6.3%) significantly reduced infarctsize, and no significant difference was observed among the three groups. Comparedwith the I/R group, Adropin-M increased the phosphorylation of Akt, ERK1/2andGSK3β (p-Akt/t-Akt1.02±0.08vs.2.03±0.28, p-ERK1/2/t-ERK1/21.24±0.26vs.2.27±0.37, p-GSK3β/t-GSK3β0.51±0.11vs.1.40±0.21, all P<0.05) and Bcl-2/Baxratio (0.35±0.05vs.1.66±0.20, P<0.05). Compared with I/R group, only I-PostCgroup increased phosphorylation of STAT3(1.08±0.16vs.2.24±0.23, P<0.05), whileAdropin-M group didn’t increase p-STAT3level (P>0.05vs. I/R group).Theprotective effects of Adropin were partially inhibited by LY294002or PD98059(41.1±5.2%,42.3±4.2%vs. Adropin30.5±5.2%, P<0.001). Compared with I/R groupin littermate controls, Akt1+/+group significantly reduced infarct size (46.63±4.84%vs.31.23±4.65%, P<0.05) and increased p-Akt/t-Akt ratio, but Akt1+/-groupdecreased the phosphorylation of Akt and failed to reduce infarct size (44.83±3.92%),suggesting that Akt deficiency abolishes the cardioprotective role of Adropin.Conclusion: Adropin can reduce MIRI through RISK pathway but not SAFE pathwayand the cardioprotective role was dose-dependent. Objective: Reperfusion therapy is the main treatment for acute myocardial infarction,however, reperfusion injury seriously affect the effects of myocardial reperfusiontherapy. Pharmacological postconditioning can simulate ischemic postconditioning toreduce myocardial ischemia reperfusion injury (MIRI). Adropin, a newly identifiedsecreted polypeptide, plays an important role in protecting the vascular endotheliumby activating the reperfusion injury salvage kinase (RISK) pathway. It is unclear theeffects of Adropin on cardiomyocytes induced by ischemia/reperfusion injury. Thisstudy aimed to investigate the effects of simulated ischemia/reperfusion on H9c2cellsand the potential signaling transduction pathways.Methods: Hypoxia/reoxygenation model was established in H9c2cells and cells weredivided into eleven groups, all groups were undergone hypoxia for12h andreoxygenation for24h except the Control group:(1) Control group: H9c2cells werecultured under normal conditions;(2) SI/R group: hypoxia for12h and reoxygenationfor24h;(3) low-dose Adropin group (Adropin-L): Adropin10ng/ml;(4)moderate-dose Adropin group (Adropin-M): Adropin25ng/ml;(5) high-dose Adropingroup (Adropin-H): Adropin50ng/ml;(6) LY294002group: PI3K specific inhibitorLY294002was added in the medium (40μmmol/L) before hypoxia;(7)Adropin+LY294002group: LY294002was added in the medium before hypoxia (40μmmol/L) and Adropin (25ng/ml) was added before reperfusion;(8) PD98059group:ERK1/2specific inhibitor PD98059was added in the medium (25μmmol/L) beforehypoxia;(9) Adropin+PD98059group: PD98059was added in the medium (25μmmol/L) before hypoxia and Adropin (25ng/ml) was added before reperfusion;(10)AG490group: JAK2inhibitor AG490was added in the medium (100μmol/L) beforehypoxia;(11) Adropin+AG490group: AG490was added in the medium (100μmol/L)before hypoxia and Adropin (25ng/ml) was added before reperfusion. Cell viability was measured by MTT assay, cell early apoptosis by flow cytometry, and Caspase-3activity by colorimetric detection. ELISA was used to detect CK-MB, TNFα, IL-10,MDA and SOD levels in H9c2cells and Western blot was applied to detect theexpression levels of Akt, ERK1/2, GSK3β, Bcl-2and Bax.Results: Compared with SI/R group (55.91±4.74%), Adropin-L group failed toimprove cell viability (57.04±4.15%), while Adropin-M group significantly increasedcell viability (71.10±3.45%), and Adropin-H group further increase cell viability(74.38±3.94%), but there was no difference between Adropin-M and Adropin-Hgroups. Compared with SI/R group, Adropin-M group reduced early apoptosis ofH9c2cells (35.96±2.52%vs.21.88±2.43%, P <0.001), Caspase-3activity (150.26±4.45%vs.118.25±2.66%, P <0.001) and promoted the expression of antiapoptoticprotein of Bcl-2, inhibited the expression of proapoptotic protein of Bax and increasedthe ratio of Bcl-2/Bax (0.71±0.20vs.2.34±0.19, P <0.001). Compared with the SI/Rgroup, Adropin group significantly increased p-Akt/t-Akt (1.03±0.12vs.2.12±0.22,P<0.05), p-ERK1/2/t-ERK1/2(0.71±0.16vs.1.23±0.19, P<0.05) and p-GSK3β/t-GSK3β (1.03±0.16vs.2.23±0.19, P<0.05). LY294002or PD98059inhibited thephosphorylation of these above protein and abolished the cardioprotective role ofAdropin in improving cell viability, reducing early apoptosis and Caspase-3activity.Compared with the Control group, AG490significantly inhibited the phosphorylationof STAT3(1.00±0.13vs.0.43±0.18, P<0.05), however, no significant difference ofp-STAT3/t-STAT3was observed between the Adropin group and the Control group. Inaddition, AG490did not significantly inhibit the protective role of Adropin inimproving cell viability and reducing early apoptotic cells.Conclusion: Adropin reduced the hypoxia/reoxygenation-induced injury in H9c2cells by RISK signaling pathway instead of SAFE pathway, suggesting that Adropinmay be a potential protective agent against MIRI. Objective: Diabetes increases the risk and severity of atherosclerosis. Adropin, ametabolic homeostasis-related protein, has been implicated in the maintenance ofmetabolic homeostasis. We examined the relationship between serum adropin leveland angiographic severity of coronary atherosclerosis in diabetic and non-diabeticpatients.Methods: A total of392patients with suspected coronary artery disease, whounderwent coronary angiography, were assigned into the type2diabetic andnon-diabetic groups and also classified into four groups according to the quartiles ofadropin level. Venous serum samples were collected for Adropin measurement byenzyme-linked immunosorbent assay and for biochemistry assay. The angiographicseverity of coronary atherosclerosis was assessed by Gensini, Friesinger, andSYNTAX scores.Results: Compared with non-diabetic patients, diabetic patients had lower serumAdropin level and higher Gensini, Friesinger and SYNTAX scores (all P<0.001).Serum Adropin level was inversely correlated with the Gensini, Friesinger andSYNTAX scores (rs=-0.389,-0.390and-0.386, respectively, all P<0.001) among allpatients. Low Adropin level was an independent predictor of clinically relevantcoronary atherosclerosis (SYNTAX score>11), both in diabetic patients (odds ratio0.66,95%confidence interval0.53–0.83; P<0.001) and in nondiabetic patients (oddsratio0.51,95%confidence interval0.35–0.74; P<0.001).Conclusion: Serum Adropin level was significantly lower in type2diabetic patientsthan in nondiabetic patients and was inversely and independently associated withangiographic severity of coronary atherosclerosis, suggesting that serum Adropinserves as a novel predictor of coronary atherosclerosis.