Alginate Oligosaccharides Regulate The MicroRNA-153-3p To Delay D-gal-induced H9c2 Cardiomyocytes Senescence

Objective This study aimed to investigate the anti-aging effect of Alginate oligosaccharide(AOS)on H9c2 cardiomyocytes by simulating mitochondrial damage induced by D-galactose(D-gal)in vivo,and to explore the possible molecular mechanism of its anti-aging effect by targeting microRNA-153-3p(miR-153-3p).Methods 1.D-gal was administered to establish the aging model of H9c2 cells.H9c2 cardiomyocytes were cultured with a medium containing different concentrations of D-gal for 48 hours.After the experiment,the appropriate concentration of D-gal was selected according to the results of SA-β-gal,flow cytometry,and CCK-8 methods to establish H9c2 cardiomyocytes aging.2.Protective effect of AOS on D-gal-induced H9c2 cardiomyocytes.After being randomly divided into Control group,D-gal group,and D-gal+AOS group.D-gal group and the D-gal+AOS group were deal with D-gal(40 g/L)to establish the aging model.Then the D-gal+AOS group was deal with AOS(100 μg/mL)for 48 hours.At the end of the experiment,the cell senescence was evaluated by SA-β-gal staining;ROS levels were measured using a reactive oxygen species(ROS)detection kit;the changes of mitochondrial membrane potential were tested by mitochondrial membrane potential detection kit(JC-1).Western blot was used to examine the protein expression of p53,p21,and PGC-1α;RT-qPCR was used for detecting the gene expression of miR-153-3p and PGC-1α before and after AOS treatment.3.miR-153-3p participates in AOS delaying D-gal-induced cardiomyocyte senescence.The target genes that may interact with miR-153-3p were predicted by the Targetscan database.H9c2 cardiomyocytes with good growth were transfected with miR-153-3p mimic and miR-NC,respectively.After transfection,the interaction between miR-153-3p and PGC-1α was verified by RT-qPCR and Western blot,and then the cardiomyocytes were randomly divided into the following groups: Control group,AOS group,D-gal group,D-gal+AOS group,D-gal+AOS+miR-NC(miR-NC)group,D-gal+AOS+miR-153-3p mimic(miR-153-3pmimic)group.The D-gal,D-gal+AOS,miR-NC,and miR-153-3p mimic groups were treated with D-gal to construct cardiomyocyte senescence model;then the miR-NC and miR-153-3p mimic groups were transfected into miR-NC and miR-153-3p mimic.After 48 hours later,the cells in the AOS,D-gal+AOS,miR-NC,and miR-153-3p mimic groups were deal with AOS for 48 hours,while the Control group was only dealt with the synchronous fluid exchange.After overexpression of miR-153-3p mimic,the level of miR-153-3p and PGC-1α in D-gal-induced H9c2 cardiomyocytes before and after AOS treatment was detected by RT-qPCR;the number of senescent cells,the ROS levels,and mitochondrial membrane potential changes in each group were detected;the proteins expression of p53,p21,PGC-1α,Tfam,and Nrf1 were evaluated by Western blot.Results 1.Protective effects of AOS on D-gal-induced H9c2 cardiomyocytes.After continuous treatment with 40 g/L D-gal for 48 hours,compared to the Control group,H9c2 cardiomyocytes’ viability was significantly decreased(p<0.01).SA-β-gal staining results showed that the number of aging cardiomyocytes was obviously increased(p<0.01).Flow cytometry results show that there is a significant increase in apoptosis rate when the D-gal concentration is 60 g/L(p<0.01).Based on these experimental results,the cell aging model was determined to be established by using D-gal with a concentration of 40 g/L.Compared with the D-gal group,the morphological changes in the D-gal+AOS group were significantly improved.The results showed that the senescent cardiomyocytes were reduced,ROS levels were declined and mitochondrial membrane potential was recovered.Western blot results exhibited that compared with the D-gal group,AOS down-regulated the expression of aging associated proteins(p<0.01),while up-regulated the expression of PGC-1α(p<0.01).RT-qPCR results showed that compared with the D-gal group,AOS reduced the expression of miR-153-3p(p<0.01)and increased the expression of PGC-1α(p<0.01).2.AOS delays D-gal-induced H9c2 cardiomyocyte senescence by downregulating the expression of miR-153-3p.SA-β-gal staining displayed that the quantity of senescent cardiomyocytes in the D-gal group increased when compared with the Control group(p<0.01);AOS significantly relieved the situation when compared to the D-gal group(p<0.01).Compared with the miR-NC group,overexpression of miR-153-3p increased the proportion of senescent cells in the miR-153-3p mimic group(p<0.01).The mitochondrial membrane potential test showed that,the mitochondrial membrane potential in the D-gal group was obviously lower than that of in the Control group;it was increased after AOS treatment;compared with the miR-NC group,overexpression of miR-153-3p alleviated the significant decrease in the mitochondrial membrane potential of cardiomyocytes in the miR-153-3p mimic group.ROS detection results demonstrated that the ROS content of the D-gal group was higher than that of Control group(p<0.01);and it was cut down by AOS treatment(p<0.01);compared to the miR-NC group,the ROS level of miR-153-3p mimic group increased(p<0.01).RT-qPCR results revealed that the expression of miR-153-3p in D-gal group was noteworthily increased(p<0.01),and decreased after AOS treatment(p<0.01)when compared with D-gal group,and that in miR-NC group was up-regulated(p<0.01).The Targetscan database predicts that miR-153-3p can regulate the expression of PGC-1α.RT-qPCR results showed that miR-153-3p expression in myocardial cells of miR-153-3p mimic group was increased(p<0.01),while PGC-1α gene and protein expression were decreased(p<0.01).The aging-related protein expression in cardiomyocytes results were displayed by Western blot.When compared with the Control group,the expressions of cardiomyocyte senescence-related proteins increased significantly after D-gal treatment(p<0.01),in contrast,there has an obvious decline in the expressions of factors related to mitochondrial biosynthesis(p<0.01);AOS down-regulated the expression of senescence-related proteins(p<0.01),and up-regulated the expression of factors related to mitochondrial biosynthesis(p<0.01)when compared to the D-gal group.Overexpression of miR-153-3p significantly increased the expression of senescence-related proteins(p<0.01),and down-regulated the expression of factors related to mitochondrial biosynthesis(p<0.05,p<0.01)when compared with the miR-NC group.Conclusion AOS can delay D-gal-induced H9c2 cardiomyocytes senescence,the mechanism of which may be related to AOS inhibiting the expression of miR-153-3p in aging cardiomyocytes,and then improving cardiomyocyte mitochondrial biosynthesis function.