Effects And Mechanisms Of Phloretin On Oxidative Damage In HUVECs And C2C12 Cells

In vivo reactive oxygen species(ROS)have important functions,such as immune and signal transduction.However,excessive oxidative damage caused by reactive oxygen species in the body could cause endothelial dysfunction and muscle damage.As a dihydrochalcone structural flavonoid compound(open loop structure after hydrogenation),phloretin can effectively remove excessive free radicals in vivo or in vitro,thereby improving oxidative damage and inhibiting free radical-induced cells apoptosis.However,its specific mechanism of action is unclear.Therefore,in this study,the effect of phloretin on the oxidative damage of HUVECs was firstly studied at the cellular level,using palmitic acid(PA)as a model,and then the techniques of transcriptome sequencing,q RT-PCR and Western blot were used to screen and verify the oxidative damage-related genes and proteins and study their enriched signaling pathways on HUVECs and C2C12 cells,in order to reveal the molecular mechanism of its antioxidant effects of phloretin,which would lay a theoretical foundation for the study of oxidative damage of animal organisms by using exogenous active substances1.Effect of phloretin treatment on related indexes of oxidative damage in HUVECs cellsHUVEC is one of the most commonly used cell lines for studying endothelial cell functions.In this study,firstly,MTT assay was used to detect the effect of different concentrations of phloretin(1,10,50,and 100 μmol/L)on HUVEC cell viability.The results showed that 1-50 μmol/L concentration of phloretin had no significant effect on the activity of HUVEC cells(p>0.05),but 100 μmol/L concentration of phloretin treatment significantly decreased the cell viability(p<0.05).Furthermore,based on previous reports,this experiment used 100 μmol/L palmitic acid to stimulate cells for 12 h to induce oxidative stress in the cells(namely as PA group),and 1 and 10 and 50 μmol/L concentrations of phloretin for the cells to preprotection was also performed to observe the effect of phloretin on oxidative stress in cells.The results showed that,compared with the blank group(namely as CK group),the cell morphology of the PA group changed significantly,and after adding different concentrations of phloretin treatment,the cell morphology gradually returned to normal,especially the high-concentration treatment group(50 μmol/L),which was no significant difference between the cell morphology and the blank group.At the same time,compared with the blank group,the transcription level of PCNA in the PA group was significantly reduced,while the treatment of phloretin significantly increased the expression of PCNA,but the transcription level of apoptosis marker genes was not significantly difference,indicating that phloretin can be restored cell proliferation inhibited by PA.Furthermore,compared with the PA group,phloretin significantly reduced intracellular ROS and MDA(malondialdehyde)levels,while up-regulating mitochondrial membrane potential and antioxidant enzyme activity of SOD and Gpx-1(p<0.05).In summary,phloretin can improve the oxidative damage produced by PA stimulated HUVECs cells in a concentration-dependent manner.2.Revealing key genes involved in phloretin alleviating HUVECs cellular oxidative damage based on transcriptome sequencing resultsThe HUVECs cells from different treatments(CK group,PA group,50 μmol/L phloretin recovery group,each group include three replications)were collected and subjected to transcriptome sequencing.According to the results of bioinformatics analysis,there were1425 differentially expressed genes between the CK group and the PA group,and 754 genes were significantly upregulated while 671 genes were significantly downregulated.Meanwhile,CK group compared with the G50 group,there were 1640 differentially expressed genes,which consist of 707 significantly up-regulated genes and 933 down-regulated genes.Additionaly,182 differentially expressed genes were existed in the PA group and the G50 group,and 46 genes were significantly up-regulated while 136 genes were significantly down-regulated.Furthermore,GO and KEGG classification and functional enrichment analysis were performed on the differentially expressed genes.GO results showed that the differentially expressed genes were mainly enriched in function and biological processes,cell metabolism,etc.The KEGG results showed that the differentially expressed genes were mainly enriched in c AMP,apoptosis and cytoskeletal regulation and other signaling pathways.Based on the above analysis,20 differentially expressed genes(such as CALCRL,SCD,ATP2B4,etc.)were selected,which belong to the c AMP pathway,the AMPK pathway and genes related to endothelial function,and the q RT-PCR was used to verify and the resuls of13 genes were consistent with those of sequencing in the HUVECs cell line,indicating that the sequencing results were better.3.Revealing the molecular mechanism of phloretin alleviating oxidative damage in HUVECs cellsAccording to the results of sequencing analysis,most of the differential genes were significantly enriched in the c AMP signaling pathway,whereas the AMPK-related pathway genes were not significantly difference.It is known that the c AMP signaling pathway can regulate phosphorylation of AMP phosphokinase(AMPK)through activation of serine/threonine kinase B1(LKB1)and activate downstream antioxidant pathways.Therefore,in order to further determine the anti-oxidative effects of phloretin,Western blotting and q RT-PCR were used to detect the expression of key proteins in HUVECs cell lines.The results showed that phloretin significantly activates LKB1 protein,which in turn increases the phosphorylation of AMPK and increases nuclear factor E2(Nrf2)phosphorylation compared to PA,and further up-regulated the expression of the downstream anti-oxidative stress heme oxygenase-1(HO-1)gene(p<0.05).At the same time,the activity of AMPK downstream protein SIRT1 was also significantly up-regulated.Further,STO-609 was used to inhibit AMPK’s other upstream kinase CAMKK,and found that phloretin can still upregulate AMPK protein activity,indicating that phloretin activates LKB1 and up-regulates AMPK protein activity.At the same time,using the AMPK inhibitor Compound C,the results showed that phloretin could not activate the downstream Nrf2 protein,suggesting that phloretin activates Nrf2 protein through AMPK.In summary,phloretin can activate the LKB1/AMPK/Nrf2/HO-1 signaling pathway and alleviate oxidative damage in HUVECs.4.Revealing the molecular mechanism of phloretin alleviating oxidative damage in C2C12 cellsIn order to further determine the anti-oxidative damage and regulatory mechanism of phloretin,based on the previous reports,this study used 500 μmol/L hydrogen peroxide to stimulate cells to investigate the effect of phloretin on oxidative stress in C2C12 cells.In this experiment,CCK8 kit was used to detect the effect of phloretin on C2C12 cell viability.The results showed that 0.1-10 μmol/L phloretin had no significant effect on the viability of C2C12 cells(p>0.05).Further,compared with the hydrogen peroxide group,phloretin significantly reduced intracellular ROS levels.Moreover,the expression of LKB1/AMPK/Nrf2 pathway protein was detected by Western blotting.The results showed that treatment with 1 μmol/L and 10 μmol/L phloretin significantly increased the expression of LKB1 protein,increased the phosphorylation of AMPK,and increased the phosphorylation of Nrf2(p<0.05).At the same time,CCK8 kit and q RT-PCR were used to test the effect of phloretin on the expression of proliferation and apoptosis marker genes in C2C12 cells.The results showed that phloretin can significantly promote the expression of Cyclin D1 and Cyclin E,but there was no significant effect on the expression of apoptotic marker gene.In summary,phloretin can activate the LKB1/AMPK/Nrf2 pathway,improve the oxidative damage of C2C12 cells,and also participate in regulating the proliferation of C2C12 cells.In summary,phloretin can alleviate the oxidative damage of cells by regulating the LKB1/AMPK/Nrf2 pathway,and protect mitochondrial function,and then play its anti-oxidation function.These results provide a theoretical basis for phloretin as a commonly animal feed additive,and lay a foundation for the study of active substances improving animal health.