Protective Effect And Mechanisms Of Protocatechuic Acid On High-Fat Induced Endothelial Oxidative Damage

With the changes of modern life style,a large amount of high-sugar and high-fat diet can cause massive generation of reactive oxygen species(ROS)in the body,and the vascular endothelial oxidative damage induced by excessive ROS is the common pathological basis of man cardiovascular diseases(CVD).Antioxidant enzymes such as SOD,CAT,and GPx present in the body play a key role in scavenging excess ROS,slowing oxidative stress,and improving vascular endothelial dysfunction and CVD.A large number of clinical studies indicated that anthocyanins could effectively improve CVD.As the main metabolite of anthocyanins in the body,protocatechuic acid has strong antioxidant activity,which can effectively prevent and improve high-glucose and high-fat induced endothelial oxidative damages,but the specific mechanism is not completely clear.Therefore,palmitic aicd-induced HUVECs as a cell modle was employed to study the effect of protocatechuic acid on the oxidative damage of endothelial cells,and then~1H NMR、Western blot、transcriptome sequencing and q RT-PCR techniques were used to dig in-depth the genes and proteins related to protocatechuic acid-improved oxidative damage,and further researched the signaling pathways related to protocatechuic acid-activated antioxidant enzymes.All the above were hoped to uncover the molecular mechanisms of protocatechuic acid in improving endothelial oxidative damage,which laid a theoretical foundation for natural active compounds to exert antioxidant activity,prevent and improve CVD in vivo.The main research contents and results are as follows:1.The improvement of protocatechuic acid on high-fat induced oxidative damage of HUVECsIn order to further confirm the antioxidant activity of protocachuic acid,the effects of protocatechuic acid on endothelial oxidative damage were studied in the cell model of high-fat induced HUVECs.The results showed that 100μmol/L protocatechuic acid treatment could significantly reduce the content of ROS and oxidative damage products such as MDA,3-NT,and 8-OHd G in high-fat induced HUVECs,inhibit LDH leakage caused by cell membrane damage,restore mitochondrial membrane potential and improve mitochondrial function,promote NO synthesis,thus effectively improve the oxidative stress of endothelial cells.2.Protocatechuic acid activated the activities of antioxidant enzymes through AMPK/Nrf2 signaling pathway~1H NMR technique was employed to research the effect of protocatechuic acid on the metabolites of HUVECs.The results showed that protocatechuic acid could regulate the energy metabolism level of HUVECs and significantly increased the ratios of AMP/ATP and NAD~+/NADH,theregy activating AMPK through LKB1 pathway,promoting the expression of p-Nrf2 and transferring into nucleus,and increasing the expression and activities of antioxidant enzymes such as HO-1,SOD and GPx.Meanwhile,protocatechuic acid treatment could significantly increase the expression of PGC-1αin the nucleus and promote the synthesis of mitochondria,thereby effectively improving the high-fat induced endothelial oxidative damage.3.Screening and identification of genes related to protocatechuic acid improving oxidative damage in HUVECsThe effect of protocatechuic acid on RNA transcription in HUVECs was further studied by transcriptome sequencing,and the differentially expressed genes related to oxidative damage were screened and identified.The results showed that 1625 differentially expressed genes were screened among the blank group(CK group),the high-fat group(PA group)and the high-fat+protocatechuic acid group(Y100 group),and the number of genes with significant differences between the CK and PA groups,as well as CK and PA groups was the largest.KEGG analysis revealed that the genes affected by different treatment groups were mainly focused on metabolism,cancer,terpenoid synthesis,c GMP-PKG,type II diabetes mellitus,adhension factors and inflammation related pathways.Using q RT-PCR technology,20 genes with significantly differences were randomly selected for validation and the results showed that the expression differences were in good agreement with the sequencing results,which indicated that the transcriptome sequencing results in this study were reliable.4.The regulation of protocatechuic acid on the activities of AMPK and antioxidant enzymes through CD36-related pathwayFrom the results of transcriptome sequencing and q RT-PCR,we found that protocatechuic acid could significantly inhibit the expression of CD36 gene and protein in HUVECs under high-fat conditions,thereby inhibit the uptake of long-chain fatty acids by cells,thereby reducing the generations of acetyl-Co A and ATP,increasing the ratio of AMP/ATP,and there results further uncovered the upstream regulatory mechanism of protocatechuic acid to activate AMPK.Meanwhile,by increasing the expressions of deacetylase Sirt1 and Sirt3,as well as inhibiting the generation of acetyl-Co A,protocatechuic acid could significantly decrease the acetylation level of lysine in HUVECs,consequently increase the activities of antioxidant enzymes such as Mn SOD and reduce the product of ROS.These effects of protocatechuic acid obviously improved the high-fat induced endothelial oxidative damage.In addition,animal experiments have further validated the results of cell tests in vivo.In summary,protocatechuic acid could regulate CD36/AMPK signaling pathway to activate Nrf2 and PGC-1α,thereby promoting the expressions of antioxidant enzymes and mitochondrial synthesis,and on the other hand,reduce the generation of acetyl-Co A,promote the expressions of deacetylase Sirt1 and Sirt3 and decrease the acetylation level of lysine,thereby restoring the activities of antioxidant enzymes and protecting the function of mitochondia,improving high-fat induced oxidative damage of vascular endothelium.These results laid the theoretical foundation for further elucidating the antioxidant activity,preventing and improving CVD of natural products such as protocatechuic acid in vivo.