The Protective Effect And Mechanism Of Exogenous Phoenixin-14 On Rats With Heart Failure

Bcakground and ObjectiveChronic heart failure（Chronic heart failure,CHF）is the final stage and main cause of death of all kinds of cardiovascular diseases,and is one of the important challenges in the cardiovascular field in the 21st century.Its injury is closely related to pathophysiological processes such as oxidative stress and inflammation.Phoenixin（PNX）is a highly conserved secreted peptide first identified in 2013,widely distributed in the hypothalamus and heart.PNX14 is a highly expressed PNX subtype found in the heart,and it will increase greatly when myocardial ischemia.The administration of exogenous PNX14 can reduce the contraction of the heart and induce relaxation,reduce the infarct size,significantly improve the systolic function,and have a positive effect on myocardial ischemia and reperfusion injury.At the same time,the study also found that PNX14 can up-regulate Krüppel-like transcription factor 2（KLF2）.KLF2 is one of the extremely important transcription factors in the KLF family,which plays an important role in cardiovascular and endothelial functions.Based on the above research,we speculate that PNX14 may have a protective effect on heart failure.Therefore,this experiment is based on the heart failure model to explore the effects of PNX14 on the inflammatory response,oxidative stress and KLF2signaling pathway in rats with heart failure.MethodWe selected forty male Wistar rats of SPF grade 220g-260g in this experiment.After calculating,75 per centage of these rats were chosen as the model group to build the heart failure model,and the rest 25 per centage were selected as the normal group.The model group received isoproterenol（ISO）(60mg/kg/d^-1)injection through the abdominal cavity for 5 weeks.At the same time,the heart function of the rats was detected by color echocardiography.If the LVEF is less than 60%,it represents CHF.The model is successful.Rats in the normal group were given saline injections that lasted 5 weeks in the abdominal cavity,and the dose was equivalent to that of the model group.After we built the model successfully,the rats in the model group were stochastically split into the model group（CHF group）and the PNX14 injection model group（PNX14-CHFgroup）.The rats in the normal group were stochastically split into the normal control group（Ctrl group）and the PNX14 control group（PNX14-Ctrl group）.The rats in the normal group are randomly divided into the normal control group（Ctrl group）.And the PNX14 control group（PNX14-Ctrl group）.Among them,the PNX14-CHF and PNX14-Ctrl groups received the same dose of PNX14（80nmol/kg）by tail vein injection,and the CHF and Ctrl groups received the same dose of normal saline injection.Three days later,the heart tissue was taken for ultrastructural observation;the serum biomarkers of heart injury（BNP,CK-MB and LDH）were measured by ELISA,inflammatory factors TNF-α,oxidative stress indicators（SOD,MDA）and PNX14 were determined by ELISA method Content;the expression levels of KLF2 and Caspase3 protein in the heart of each group of rats were detected by immunoblotting method;The expressions of GPR173 and TUNEL were detected by immunohistochemistry;The mRNA expressions of PNX,GPR173,inflammatory factors（IL-6,NF-κB,VCAM-1）,antioxidant indexes C2,KLF2 and their downstream vascular protective factors（HO-1,eNOS）were detected by real-time fluorescence quantitative analysis（RT-q PCR）.Results1.Successfully prepared a rat model of heart failureEchocardiography:Compared with the Control group,CHF group had thinner myocardial thickness,enlarged cardiac cavity and decreased cardiac ejection capacity,which was statistically significant.2.Successfully prepared a rat model of heart failureUltrastructural changes under transmission electron microscope:in control group and PNX14-Ctrl group,mitochondria were rich and plump,membrane ridge structure was clear,stroma had no edema,myocardial nucleus was oval,normal structure,intercalated disc connection was close and clear,endothelial cell membrane was complete,nuclear shape was regular,nuclear membrane was complete,nucleolus was clear,chromatin was evenly distributed;In CHF group,the morphology of mitochondria was abnormal,the cristae were broken,fused or disappeared,and vacuolated.There was no obvious abnormality in the nucleus of myocardium.Intercalated disc connection was destroyed,the structure was loose and disordered,and some structures disappeared.The membrane of endothelial cells dissolved and fell off,and the morphology of nucleus was normal;Compared with CHF group,PNX14-CHF group had complete mitochondrial structure,relatively clear cristae,the whole intercalated disk connection could be seen,but some structures were fuzzy,and the morphology of endothelial cells was normal.3.Changes in the expression of PNX14 and its receptor GPR173（1）Expression of PNX14 in serum and mRNA expression of PNX14 in cardiac tissue:The CHF group was clearly higher than that of the Ctrl group（^*P<0.05）.（2）mRNA expression of GPR173 in cardiac tissue:Expression of the PNX14-Ctrl and CHF groups increased compared to the Ctrl group（^*P<0.05 and^*P<0.05）.PNX14-CHF expression increased relative to that of the CHF group(^##P<0.01).（3）Expression of GPR173 immune organization in cardiac tissue:Expression of the PNX14-Ctrl and CHF groups with clear expression in blood vessels and cardiomyocytes in the Ctrl group increased compared to the Ctrl group(^***P<0.001 and^**P<0.01).PNX14-CHF expression increased relative to that of the CHF group (^###P<0.001).4.Protective action of CHF rats by PNX14（1）PNX14 reduces the expression of CHF rat Markers of heart injury（BNP,CK-MB and LDH）:Compared to the Ctrl group,the expression of the CHF group（BNP,CK-MB and LDH）all improved significantly(^****P<0.0001,^**P<0.01 and^**P<0.01).Compared to the CHF group,the expression of the PNX14-CHF group decreased relatively （^#P<0.05,^#P<0.05 and^#P<0.05）.（2）PNX14 reduces apoptosis of heart tissue in CHF rats1)Changes in cardiac tissue TUNEL cell apoptosis:Significant apoptosis appeared in the cardiac tissues of the CHF group compared to the Ctrl group(^****P<0.0001).Compared to the CHF group,the apoptosis expression of cardiac tissue in the PNX14-CHF group decreased relatively(^##P<0.01).2)Changes in expression of the rat heart Caspase3 protein level:Compared to the Ctrl group,CHF group expression was clearly increased(^****P<0.0001).The expression of the PNX14-CHF group was significantly lower than that of the CHF group(^####P<0.0001).5.PNX14 inhibits the expression of cardiac inflammatory response in CHF rats（1）mRNA expression changes of NF-κB and downstream inflammatory factor VCAM-1 in cardiac tissue:Rat NF-κB and vcam1 expression in the CHF group increased significantly compared to the Ctrl group(^***P<0.001,^****P<0.0001).Compared to the CHF group,expression of PNX14-CHF group NF-κB and VCAM-1 decreased （^#P<0.05 and^#P<0.05）.（2）Changes in serum inflammatory factor index TNF-α:CHF group expression was significantly higher than in ctrl group(^**P<0.01).Compared to the CHF group,the expression of the PNX14-CHF group decreased relatively（^#P<0.05）.（3）Changes in expression of mRNA of inflammatory factor index IL-6 in cardiactissue:CHF group expression significantly increased compared to Ctrl(^**P<0.01).The expression of the PNX14-CHF group was significantly lower than that of the CHF group（^#P<0.05）.6.PNX14 attenuates cardiac oxidative stress in CHF rats（1）Oxidative stress index SOD change in serum:CHF group expression was clearly reduced compared to Ctrl group(^****P<0.0001).Compared to the CHF group,the expression of the PNX14-CHF group increased relatively（^#P<0.05）.（2）Oxidative stress index MDA change in serum:CHF group expression was significantly improved compared to Ctrl group(^****P<0.0001).Compared to the CHF group,the expression of the PNX14-CHF group decreased relatively （^#P<0.05）.（3）mRNA expression changes of the antioxidant transcription factor Nrf-2 in cardiac tissue:Expression of the PNX14-Ctrl and CHF groups increased relative to that of the Ctrl group(^*P<0.05 and^***P<0.001).Expression of the PNX14-CHF group was significantly higher than that of the CHF group（^#P<0.05）.7.PNX14 activates KLF2 signaling pathway（1）Changes in the development of KLF2 in cardiac tissue:The expression of the PNX14-Ctrl and CHF groups increased relative to that of the Ctrl group(^*P<0.05 and^***P<0.001).Expression of the PNX14-CHF group was significantly higher than that of the CHF group（^#P<0.05）.（2）Changes in mRNA expression of downstream vascular protective factors HO-1 and eNOS of KLF2 in cardiac tissue:PNX14-Ctrl group HO-1 and eNOS expression increased relative to Ctrl group(^**P<0.01,^*P<0.05)and significantly increased in CHF group（^#P<0.05）.The expression levels of the PNX14-CHF group increased compared to the CHF group（^#P<0.05,^#P<0.05）ConclusionPNX14 protects rats with heart failure by reducing the inflammatory response and oxidative stress in rats with heart failure.We established isoproterenol（ISO）induced heart failure model to explore the effects of PNX14 on heart inflammation,oxidative stress and KLF2 signaling pathway in heart failure rats.