Role Of Adropin In The Regulation Of Hypertensive Cardiac Remodeling And The Underlying Mechanism

Background:Hypertensive myocardial remodeling is mainly characterized by left ventricular hypertrophy（LVH）and interstitial fibrosis,which is one of the main causes of heart failure.During the hypertensive myocardial remodeling,various stimulating factors including pressure load,neurohumoral and cytokines would cause the enhancement of oxidative stress and the activation of inflammatory responses in heart tissues,which further lead to cardiomyocyte hypertrophy,the activation of cardiac fibroblasts and the synthesis of extracellular matrix components.Therefore,inhibition of oxidative stress and inflammation is one of the important means to attenuate myocardial remodeling in hypertension.Adropin is a peptide hormone encoded by the energy homeostasis related gene（Enho）.Previous researches have reported that Adropin plays an important role in maintaining the balance of energy metabolism and improving insulin resistance.Recently,accumulating studies have found that Adropin could also participate in the occurrence and development of various diseases through the anti-oxidant stress and anti-inflammatory effects.However,its potential role and regulatory mechanism in hypertensive myocardial remodeling remain unclear.Our previous study found that Adropin might play a protective role in vascular endothelial function among obese adolescents.Based on the issue that Adropin plays important roles in various pathophysiological processes,including anti-oxidant stress,anti-inflammatory effects,maintaining the balance of energy metabolism,improving insulin resistance and protecting vascular function,our group speculated that Adropin might participate in the process of hypertensive myocardial remodeling.The present research explored the role of Adropin in hypertensive myocardial remodeling and its related regulatory mechanism at the clinical,animal and cellular levels.Part Ⅰ Changes of circulating Adropin level and its significance inhypert-ensive patients with left ventricular hypertrophyObjective:To investigate the changes of circulating Adropin level and its significance in hypertensive patients with left ventricular hypertrophy.Methods:A total of 86 hypertensive patients from the department of cardiovascular medicine of the Second Affiliated Hospital of Nanchang University and 12 healthy controls from the department of physical examination of the Second Affiliated Hospital of Nanchang University were enrolled finally in the present study.ELISA was used to detect the changes of serum Adropin levels between hypertensive patients and healthy controls.The changes of serum Adropin levels between hypertensive patients with left ventricular hypertrophy and hypertensive patients without left ventricular hypertrophy were further explored,and the relation of Adropin and left ventricular mass index,a marker of LVH,was evaluated using multiple linear regression,as well as the possible diagnostic value of Adropin in hypertensive LVH was detected by ROC curve analysis.Results:1.Compared with those healthy controls,the serum Adropin levels among hypertensive patients was significantly lower and the level of Adropin in the healthy controls was 9.06±2.80 pg/ml,while the level of Adropin in the hypertensive patients was 4.64±0.64 pg/ml.2.Compared with the control group of hypertensive patients without LVH,the age of the group of hypertensive patients with LVH was higher,while the expression level of Adropin was significantly lower.The level of Adropin in the control group of hypertensive patients without LVH was 4.79±0.65 pg/ml,and that in the group of hypertensive patients with LVH was 4.15±0.21 pg/ml.3.Multiple linear regression analysis showed that there was a negative correlation between Adropin and left ventricular mass index（LVMI）.After adjusting for the confounding factors of age,sex and body mass index（BMI）,LVMI decreased by 7.31 units for per one unit increment in serum Adropin level:β（95%CI）=-7.31（-13.95,-0.67）,P=0.034.Furthermore,we converted serum adropin from a continuous variable to a categorical（tertiles）.Compared to participants in T1（<4.36 pg/ml）of serum Adropin,there was a significantly lower LVMI level in T2（4.36 to<4.61 pg/ml）group and T3（≥4.61 pg/ml）group[T2:β（95%CI）=-18.82（-28.21,-9.42）,P<0.001;T3:β（95%CI）=-20.76（-30.36,-11.15）,P<0.001].After further adjusting for age,sex,BMI,systolic blood pressure,diastolic blood pressure,total cholesterol,blood glucose,aspartate aminotransferase and alanine aminotransferase,LVMI decreased by 7.52 units for per one increment of serum Adropin level:β（95%CI）=-7.52（-14.32,-0.71）,P=0.034.Furthermore,we converted serum adropin from a continuous variable to a categorical（tertiles）.Compared to participants in T1 group（<4.36 pg/ml）of serum Adropin,there was a significantly lower LVMI level in T2（4.36 to<4.61 pg/ml）group and T3（≥4.61 pg/ml）group[T2:β（95%CI）=-17.59（-27.53,-7.65）,P=0.001;T3:β（95%CI）=-20.72（-30.41,-11.04）,P<0.001].4.ROC curve analysis suggested a significantly diagnostic value of serum Adropin level in hypertensive left ventricular hypertrophy:the area under the curve of serum Adropin was 0.936 and the cutoff value was 4.265 pg/ml,as well as the specificity was 89.5%,and the sensitivity was 91%.Conclusions:The level of serum Adropin in patients with hypertension was lower than that in healthy controls.The level of serum Adropin in hypertensive patients with left ventricular hypertrophy was lower than that in hypertensive patients without left ventricular hypertrophy.There was a negative relationship between serum Adropin level and LVMI,and it could be served as a marker for the diagnosis of hypertensive left ventricular hypertrophy.Part Ⅱ Effects of synthetic Adropin on cardiac remodeling inspontaneously hypertensive ratObjective:To investigate the changes of serum Adropin level in spontaneously hypertensive rats and to observe the effect of recombinant Adropin on myocardial remodeling in spontaneously hypertensive rats after 12 weeks of treatment.Methods:1.Six 16-week-old male spontaneously hypertensive rats（rat）were included as the experimental group（SHR）,and six male Wistar-Kyoto rats of the same age were included as the control group（WKY）.Blood pressure was measured by non-invasive sphygmomanometer,cardiac function indexes were detected by echocardiography,pathological changes of myocardial tissue were observed by HE staining,changes of myocardial cell size were observed by WGA staining,myocardial fibrosis level was observed by Masson staining,and mRNA levels of hypertrophy markers including ANP,BNP,β-MHC as well as the fibrosis markers including Collagen Ⅰ,Collagen Ⅲ and CTGF were detected by RT-PCR to verify the cardiac remodeling model of SHR.2.We constructed and synthesized the recombinant Adropin^34-76polypeptide having the biological activity of Adropin protein,as well as enrolled a total of 12male SHR rats with 4-week-old and 6 male WKY rats of the same age were selected as control group.SHR rats were further divided into two groups:SHR group（n=6）and SHR with Adropin treatment group（n=6）.Adropin（2.1μg/kg/d）was injected intraperitoneally in the Adropin treatment group,and the same dose of vehicle was injected in the SHR group.During the animal treatment,blood pressure was measured every two weeks.And after 12 weeks treatment,the indexes reflecting myocardial remodeling were detected.The levels of IL-6 and TNF-αin serum and myocardium were detected by methods of ELISA and Western Blot.DHE staining,MDA and SOD content detection were performed to further explore the oxidative stress level in rat myocardial tissues.Results:1.Compared to WKY with 16-week-old,the levels of SBP,DBP,heart rate,heart weight and heart index（HW/BW）in SHR with 16-week-old were significantly increased.Compared with WKY group,the levels of interventricular septal thickness at end diastole（IVS;d）and left ventricular posterior wall thickness（LVPW;d）at end diastole were significantly increased,and the left ventricular diameter end diastole（LVID;d）level was significantly lower in the SHR group,while the levels of left ventricular ejection fraction（EF）and fractional shortening（FS）between the two group were without significant difference.Histopathological staining was performed,compared with WKY group,HE staining suggested that the arrangement of myocardial tissue in SHR group was disordered or even broken,WGA staining suggested that the relative area of myocardial cells in SHR group was greater,Masson staining suggested that the level of cardiac fibrosis in SHR group was significantly increased,and RT-PCR suggested that the mRNA expression levels of ANP,BNP,β-MHC,Collagen Ⅰ,Collagen Ⅲ and CTGF in SHR group were significantly increased.Results from ELISA,immunohistochemistry,RT-PCR and WB detection showed that the expression levels of Adropin in serum and heart tissues of SHR group was significantly decreased compared with WKY group.2.Rats of 4-week-old were divided into three groups:WKY group;SHR group;SHR+Adropin group.Compared with WKY group,the levels of SBP and DBP in SHR group increased significantly with age,and 12 weeks continuous Adropin treatment could significantly delay the incremental progression of SBP and DBP levels.Heart ultrasonography analysis found that there was no significant difference in IVS;d,LVPW;d,LVID;d,EF and FS levels among the three groups at baseline.After 12 weeks Adropin treatment,compared with WKY group,the levels of IVS;d and LVPW;d were significantly higher,and the LVID;d level was significantly lower in SHR group,while the levels of IVS;d and LVPW;d in SHR+Adropin group were significantly lower,and the LVID;d level was significantly higher than SHR group.There was no significant difference in EF and FS between the three groups.HE,WGA and Masson staining showed that the relative area of myocardial cells in SHR group was significantly increased,and the myocardial fibrosis level was significantly increased compared with WKY group,while the intervention of Adropin could significantly reduce the levels of cardiac hypertrophy and fibrosis in SHR group.RT-PCR was performed and showed that the mRNA expression levels of ANP,BNP,β-MHC,Collagen Ⅰ,Collagen Ⅲ and CTGF were higher in SHR group compared with WKY group,and Adropin intervention could significantly reduce them.Furthermore,ELISA and Western blot analysis showed that the expression levels of inflammatory cytokines including IL-6 and TNF-αin serum and myocardial tissues of SHR group were significantly increased compared with WKY group,while Adropin intervention could significantly reduce their expression levels.DHE staining,MDA and SOD content detection analysis found that,compared with WKY group,the levels of ROS and MDA in SHR group were significantly increased,as well as the SOD level was significantly decreased,while Adropin treatment could significantly reduce the levels of ROS and MDA,as well as increase the SOD level.Conclusions:Spontaneously hypertensive rats of 16-week-old were with cardiac remodeling and decreased expression level of Adropin.After 12 weeks of treatment,synthetic Adropin significantly could reduce cardiac remodeling,inflammation and oxidative stress in spontaneously hypertensive rats.Part Ⅲ Effects of Adropin on pathological cardiac remodeling andits underlying mechanism in miceObjective:To explore the changes of Adropin expression level during pathological cardiac remodeling induced by the common stimulants of hypertensive cardiac remodeling including pressure overload and Angiotensin Ⅱ stimulation in mice,and to observe the effects of synthetic Adropin injection intraperitoneally on pathological cardiac remodeling,as well as to explore the effects of Adropin knockout on pathological cardiac remodeling and its related mechanism.Methods:1.Twenty eight C57BL/6 mice of 8-week-old were included in this study.The model of pathological myocardial remodeling was established by transverse aortic constriction（TAC）and continuous infusion of angiotensin Ⅱ（Ang Ⅱ）with implanted capsule osmotic pump subcutaneously.Mice were divided into four groups:sham operation group（n=8）,TAC group（n=8）;normal saline（NS）infusion group（n=6）and angiotensin Ⅱ（Ang Ⅱ）infusion group（n=6）.Echocardiography was used to detect the indexes of cardiac function,HE staining was used to observe the pathological changes of myocardial tissue,WGA staining was used to observe the changes of relative myocardial cell size,and Masson staining was used to observe the myocardial fibrosis level.The Adropin expression level during pathological cardiac remodeling was detected by ELISA,RT-PCR and Western blot.2.We constructed and synthesized the synthetic Adropin^34-76polypeptide having biological activity of Adropin protein,as well as enrolled a total of 48 C57BL/6 mice of 8-week-old.Mice were further divided into 8 groups:sham operation and treated with vehicle group（Vehicle+Sham,n=6）,sham operation and treated with Adropin group（Vehicle+Adropin,n=6）,TAC operation and treated with vehicle group（Vehicle+TAC,n=6）,TAC operation and treated with Adropin（Adropin+TAC,n=6）;NS pump implantation and treated with vehicle（Vehicle+NS,n=6）,NS pump implantation and treated with Adropin（Adropin+NS,n=6）,Ang Ⅱ pump implantation and treated with vehicle（Vehicle+Ang Ⅱ,n=6）,Ang Ⅱ pump implantation and treated with Adropin（Adropin+Ang Ⅱ,n=6）.Adropin（450 nmol/kg/d）was injected intraperitoneally for two weeks,and the same dose of vechile was injected as the control.Ang Ⅱ group was subcutaneously infused with Ang Ⅱ（1500 ng/kg/d）for two weeks,and NS group was subcutaneously infused with the same dose of normal saline.After treatment,the indexes reflecting cardiac remodeling were detected.The level of oxidative stress in mice myocardial tissue was analyzed by DHE staining,MDA content and SOD protein expression detection.The role of Adropin treatment on Nrf2/HO-1 signaling pathway in pathological myocardial remodeling induced by Ang Ⅱ stimulation was further studied.3.Adropin knockout mice were constructed and the functional deletion research was further explored in Ang Ⅱ model.Ten Adropin knockout mice of 8-10 week old and 10 wild-type mice of 8-10 week old were included.Mice were divided into four groups:wild-type mice treated with NS pump implantation（WT+NS,n=5）,Adropin knockout mice treated with NS pump implantation(Adropin^-/-+NS,n=5),wild-type mice treated with Ang Ⅱ pump implantation（WT+Ang Ⅱ,n=5）,and Adropin knockout mice treated with Ang Ⅱ pump implantation(Adropin^-/-+Ang Ⅱ,n=5).After treatment,the indexes reflecting myocardial remodeling were detected.The levels of oxidative stress in myocardial tissues of mice in each group were analyzed by DHE staining,MDA content and SOD protein expression measurement,and the effects of Adropin knockout on Nrf2 expression in pathological myocardial remodeling induced by Ang Ⅱ stimulation was further studied.4.Adeno-associated virus AAV9-Nrf2-GFP was constructed and injected via tail vein for 4 weeks to specifically up-regulate the Nrf2 expression in myocardial tissues,and empty virus（vector）was used as the control in order to complete the functional restoration study in Adropin^-/-mice during Ang Ⅱ stimulation.Ten Adropin knockout mice of 8-10 week old were divided into two groups:vector injection and treated with Ang Ⅱ pump implantation（Vector+Ang Ⅱ,n=5）,AAV9-Nrf2-GFP injection and treated with Ang Ⅱ pump implantation（AAV9.Nrf2+Ang Ⅱ,n=5）.After treatment,the detection of various indicators reflecting myocardial remodeling was performed.DHE staining,MDA content and SOD protein expression analysis were performed to observe the level of oxidative stress in myocardial tissue of mice,and the protein expression of apoptosis related molecule cleaved Caspase3 was further investigated.Results:1.Cardiac ultrasonography analysis showed that,compared with Sham group,the levels of IVS;d and LVPW;d in TAC group were significantly increased,and the LVID;d level was decreased,while there was no significant difference for EF and FS between the two groups;compared with NS group,the levels of IVS;d,LVPW;d,EF and FS in Ang Ⅱ group were significantly increased,while the levels of LVID;d were decreased.Histopathological staining showed that,compared with sham group,HE staining showed there were disorder and rupture of myocardial tissue in TAC group,WGA staining showed that the relative area of myocardial cells in TAC group was greater,and Masson staining showed that the myocardial fibrosis level in TAC group was significantly increased.Ang Ⅱ group showed similar histopathological changes compared with NS group.ELISA,RT-PCR and Western Blot assay were performed and showed that the expression level of Adropin in serum and heart tissues of TAC mice was significantly lower than that of sham group,as well as the expression level of Adropin in serum and heart tissues of Ang Ⅱ mice was significantly lower than that of NS group.2.For TAC model,compared with Vehicle+Sham group,IVS;d and LVPW;d levels were significantly increased in Vehicle+TAC group,as well as LVID;d level was decreased,while the levels of IVS;d and LVPW;d in Adropin+TAC group were significantly decreased,and LVID;d level was increased than that in Vehicle+TAC group.HE,WGA and Masson staining showed that,compared to Vehicle+Sham group,the relative area of cardiomyocytes and the level of myocardial fibrosis in the Vehicle+TAC group were significantly increased,which were both attenuated by the intervention of Adropin.Western Blot analysis further found that,compared with the Vehicle+Sham group,the protein expression levels of ANP and Collagen Ⅰ in the Vehicle+TAC group were significantly increased,which were significantly decreased after Adropin treatment.DHE staining,MDA content and SOD protein expression analysis found that,compared with Vehicle+Sham group,the level of reactive oxygen species and MDA content in heart tissues of Vehicle+TAC group were significantly increased,and the SOD protein expression level was significantly decreased.Adropin treatment could significantly reduce the reactive oxygen species and MDA levels in heart tissues,and increase the SOD expression.For Ang Ⅱ model,there were similar effects of Adropin treatment on improving pathological myocardial remodeling and oxidative stress.Western Blot analysis was used to analyze the effects of Adropin on the Nrf2/HO-1 signaling pathway in Ang Ⅱ-induced cardiac remodeling.Compared to Vehicle+NS group,the protein expression levels of Nrf2 and HO-1 in nucleus were significantly increased,and the total NQO-1 protein expression level was decreased in Vehicle+Ang Ⅱ group,while Adropin treatment could further increase the levels of Nrf2 and HO-1 in nucleus,as well as increase the total NQO-1 level in heart tissues.Adropin treatment could also reduce the protein expression level of apoptosis related molecule cleaved Caspase3 induced by Ang Ⅱ.3.Adropin knockout mice were constructed to verify the effects of Adropin deletion on cardiac remodeling induced by Ang Ⅱ stimulation.Western Blot analysis showed that there was no expression of Adropin protein in heart tissues of Adropin knockout mice compared with wild-type（WT）mice,which proved the successful construction of Adropin knockout mouse model.Cardiac ultrasonography analysis showed that,compared with WT+NS group,the levels of IVS;d and LVPW;d were significantly increased,and the LVID;d level was decreased in WT+Ang Ⅱ group,while the levels of IVS;d and LVPW;d were significantly further increased,as well as the LVID;d level was further decreased in Adropin^-/-+Ang Ⅱ group than that in WT+Ang Ⅱ group.HE,WGA and Masson staining analysis showed that the relative area of myocardial cells and the myocardial fibrosis level in WT+Ang Ⅱ group were significantly increased than that in WT+NS group,while the levels of cardiac hypertrophy and fibrosis were further increased in Adropin^-/-+Ang Ⅱ group.Western Blot analysis further found that the protein expression levels of ANP and Collagen Ⅰ in WT+Ang Ⅱ group were significantly increased than that in WT+NS group,both of which were further increased due to Adropin knockout.DHE staining,MDA content and SOD protein expression analysis showed that,compared with WT+NS group,the levels of reactive oxygen species and MDA content were significantly increased,and the level of SOD protein expression was significantly decreased in WT+Ang Ⅱ group,while Adropin knockout further increased the levels of reactive oxygen species and MDA content,as well as further decreased SOD expression.RT-PCR and Western Blot analysis found that,compared with WT+Ang Ⅱ group,the mRNA and protein expression of Nrf2 in myocardium of Adropin^-/-+Ang Ⅱ group were significantly decreased,indicting that Adropin might affect pathological cardiac remodeling induced by Ang Ⅱ through Nrf2 signaling pathway.4.AAV9-Nrf2-GFP was used to verify the functional restoration effects of Nrf2overexpression on cardiac remodeling induced by Ang Ⅱ in Adropin knockout mice.Frozen section fluorescence imaging,RT-PCR and Western Blot analysis showed that there was fluorescent protein expression in the heart tissue in AAV9-Nrf2-GFP and Vector group compared with NS group,as well as the mRNA and protein levels of Nrf2 were significantly higher in the AAV9-Nrf2-GFP group than that in Vector group,proving the ability of AAV9-Nrf2-GFP to up-regulate Nrf2 expression in heart tissues in the Adropin^-/-mice.Cardiac ultrasonography analysis showed that,compared with Vector+Ang Ⅱ group,the levels of IVS;d and LVPW;d in AAV9.Nrf2+Ang Ⅱ group were significantly decreased,while the LVID;d levels were increased,which were with statistical significance.HE,WGA and Masson staining analysis showed that,compared to Vector+Ang Ⅱ group,the relative area of cardiomyocytes and the level of cardiac fibrosis were significantly decreased in AAV9.Nrf2+Ang Ⅱ group.RT-PCR analysis showed that the mRNA levels of ANP,BNP,β-MHC,Collagen Ⅰ,Collagen Ⅲ and CTGF in AAV9.Nrf2+Ang Ⅱ group were significantly lower than that in Vector+Ang Ⅱ group.Compared to Vector+Ang Ⅱ group,the levels of ROS and MDA were significantly lower,and the SOD level was higher in AAV9.Nrf2+Ang Ⅱ group.Furthermore,Nrf2 overexpression intervention could reduce the protein expression level of apoptosis related molecule cleaved Caspase3 induced by Ang Ⅱ stimulation in Adropin knockout mice.Conclusions:Adropin treatment could attenuate the cardiac pathological remodeling induced by pressure overload and Ang Ⅱ in mice,and Adropin knockout could aggravate the cardiac pathological remodeling induced by Ang Ⅱ in mice,as well as that Nrf2/HO-1signaling pathway might play a role during the progress above.Part Ⅳ Roles of Adropin in the proliferation and collagen synthesis of cardiac fibroblasts induced by Ang ⅡObjective:To investigate whether Adropin is involved in the proliferation and collagen synthesis of cardiac fibroblasts（CFs）induced by Ang Ⅱ and at the cellular level.Methods:1.To explore the effects of Ang Ⅱ treatment on the expression of Adropin in CFs,cells were divided into four groups:Control group,Ang Ⅱ 0.1μM treatment group,Ang Ⅱ 1μM treatment group and Ang Ⅱ 2μM treatment group.The treatment time was 24 hours.RT-PCR and WB were used to detect the mRNA and protein expression levels of Adropin.The effect of recombinant Adropin on protein expression level of Adropin in CFs stimulated by Ang Ⅱ was observed by WB analysis.2.Functional experiments were carried out in Ang Ⅱ induced proliferation and collagen synthesis of cardiac fibroblasts using the method of adding recombinant Adropin（0.5μg/mL）into the cultured cells.The experiment was divided into four groups:Vehicle treatment group（Vehicle）,Adropin treatment group（Adropin）,vehicle treatment and with Ang Ⅱ stimulation group（Vehicle+Ang Ⅱ）,as well as Adropin treatment and with Ang Ⅱ stimulation group（Adropin+Ang Ⅱ）.After the treatment,the proliferation of cardiac fibroblasts was detected by MTT method,and the expression of molecular markers of cardiac fibrosis Collagen Ⅰ,Collagen Ⅲ and CTGF were detected by Western blot.3.To investigate the effects of Adropin treatment on the markers of oxidative stress including ROS,MDA and SOD during the progressof collagen synthesis of CFs induced by Ang Ⅱ.WB was performed to explore the effects of recombinant Adropin on Nrf2/HO-1 signaling pathway.Nrf2 DNA binding activity analysis was performed to investigate the effects of Adropin on the DNA binding activity of Nrf2 during the process of cardiac fibroblasts injury stimulated by Ang Ⅱ.Results:1.RT-PCR and WB analyses showed that the mRNA and protein expression levels of Adropin decreased with the increase of Ang Ⅱ concentration,while the decreased protein expression level of Adropin in the CFs stimulated by Ang Ⅱ could be reversed via adding recombinant Adropin.2.MTT test analysis was conducted and suggested that,compared with the Vehicle group,the proliferation level of CFs was significantly increased after the stimulation of Ang Ⅱ,which was significantly reduced by Adropin treatment.Western Blot analysis suggested that,compared with Vehicle group,the protein expression levels of Collagenan I,Collagena Ⅲ and CTGF in CFs were increased after the stimulation of Ang Ⅱ,which could be significantly reduced via the Adropin treatment.3.The level of oxidative stress in the cultured cells was detected.Compared with vehicle group,the levels of intracellular ROS and MDA in Ang Ⅱ group were significantly higher,which were significantly attenuated through Adropin treatment.Compared to vehicle group,the level of intracellular SOD level was significantly lower in Ang Ⅱ group,which were significantly reversed through Adropin treatment.The effects of Adropin on Nrf2/HO-1 signaling pathway of cardiac fibroblasts during the stimulation with Ang Ⅱ were further analyzed.Compared with vehicle group,the expression level of Nrf2 and HO-1 protein in the nucleus were significantly higher in Ang Ⅱ group,both of which were further significantly increased by Adropin treatment.Compared to vehicle group,the expression level of total Nrf2 protein was increased,and the expression level of NQO-1 was decreased in Ang Ⅱ group,both of which were increased after Adropin treatment.Furthermore,DNA binding analysis of Nrf2 was performed and found that,compared to vehicle group,the transcription activity of Nrf2 was significantly increased after Ang Ⅱ stimulation in CFs,which was further elevated by Adropin treatment.Conclusions:Adropin treatment might improve the antioxidant capacity of CFs and reduce the level of reactive oxygen stress via enhancing Nrf2/HO-1 signaling pathway,thus,inhibiting the proliferation and collagen synthesis of CFs stimulated by Ang Ⅱ.