The Role And Mechanism Of Klotho In Delaying Cardiac Aging By Regulating Macrophage Polarization

Objective: The effect of Klotho delaying cardiac aging by regulating macrophage polarization.Methods: The aging model of mice and MCM cells were induced by Dgalactose(D-gal).(1)Animal experiment:(1)Adult group;(2)Adult+Klotho group:intraperitoneal injection of the mouse recombinant Klotho(100 μg/kg/d,14 d)from week9;(3)Aged group: subcutaneous injection of D-gal(40 mg/kg/d,10 W)on the back of the neck;(4)Aged+Klotho group: subcutaneous injection of D-gal on the back of the neck and intraperitoneal injection of Klotho.After 10 weeks,echocardiography was used to detect cardiac function;The activity of β-galactosidase was assessed;The content of superoxide dismutase(SOD)and malondialdehyde(MDA)were detected;P53 and P21 were marked by the immunofluorescence technique;F4/80+IL-10 was marked by double the immunefluorescence technique;The m RNA expression of P53,P21,CD206,IL-10 and TGF-β were detected by RT-q PCR;The expression of P53,P21,Toll-like receptor 4(TLR4),myeloid differentiation factor 88(Myd88),P65 and P50 were detected by Western blot;The levels of CD206,IL-10 and TGF-β in cardiac tissue and TNF-α,IL-1β and IL-6 in serum were detected by ELISA.(2)Cell experiment: RAW264.7 cells were treated with Klotho(2000pmol/L)for 24 h.The levels of M2a/M2 c markers CD206,IL-10 and TGF-β were detected by RT-q PCR and ELISA,then the M2a/M2 c macrophage culture supernatant was collected and used to interfere with D-gal(10 mg/m L)-induced aging MCM cells.The control groups were established at the same time.After 24 hours,cells were collected and the activity of β-galactosidase was detected,the m RNA expression of P53 and P21 were detected by RTq PCR.Results:(1)At animal levels: Compared with Adult group,in Aged group: The immune-fluorescence technique,Western blot and RT-q PCR results showed that the aging markers P53 and P21 were increased(P < 0.001),the activity of β-galactosidase was upregulated(P < 0.001)in cardiac tissue;Compared with Aged group,Aged+Klotho group:the aging markers P53,P21 and the activity of β-galactosidase were decreased(P < 0.001)in cardiac tissue.Compared with Adult group,in Aged group: ejection fraction(EF),fractional shortening(FS),interventricular septum(IVS)and left ventricular end-systolic posterior wall(LVPW-s)were decreased(P < 0.001),while left ventricular end-diastole dimension(LVEDD)and left ventricular end-systolic dimension(LVESD)were increased(P < 0.001);Compared with Aged group,Aged+Klotho group: EF,FS,IVS and LVPW-s were increased(P < 0.001),while LVEDD and LVESD were decreased(P < 0.001),the heart function improved.Compared with Adult group,in Aged group: The oxidative stress indicator SOD activity was decreased(P < 0.001),while the content of MDA was increased(P < 0.001)in serum,the inflammatory markers of TNF-α,IL-1β and IL-6 in serum and TLR4、Myd88、P65 and P50 in cardiac tissue were up-regulated(P < 0.001);Compared with Aged group,Aged+Klotho group: antioxidative enzymes SOD activity was increased(P < 0.001),while the content of membrane lipid peroxidation product MDA was decreased(P < 0.001)in serum,the levels of inflammatory markers TNF-α,IL-1β,IL-6,TLR4,Myd88,P65 and P50 were down-regulated(P < 0.001).Compared with Adult group,in Aged group:The immunofluorescence technique results showed that the macrophage marker F4/80 was increased(P < 0.001),while the M2a/M2 c macrophage marker F4/80+IL-10 was decreased(P < 0.001).RT-q PCR and ELISA results showed that M2a/M2 c macrophage markers CD206,IL-10 and TGF-β were decreased(P < 0.001)in cardiac tissue;Compared with Aged group,Aged+Klotho group: M2a/M2 c macrophage markers F4/80+IL-10,CD206,IL-10 and TGF-β were increased(P < 0.001)in cardiac tissues.(2)At the cellular level:Compared with control group,the Klotho treated RAW264.7 cells group: RT-q PCR and ELISA results showed that the expression of CD206 was up-regulated(P < 0.01)and the expression of IL-10 and TGF-β were increased significantly(P < 0.001);Compared with control group,in D-gal treated group: the level of P53 m RNA,P21 m RNA and the activity of β-galactosidase were increased(P < 0.001);Compared with D-gal treated group,the group co-processed by D-gal and Klotho: the level of P53 m RNA,P21 m RNA and the activity of β-galactosidase were decreased(P < 0.001).Conclusion: Klotho supplementation in peripheral circulation improves oxidative stress and inflammation in aging heart,protects cardiac function,and delays cardiac aging.The mechanism may be that Klotho promotes M2a/M2 c macrophage polarization in aging heart.