Study On The Inhibitory Effect Of Lentinus Edodes Mycelium Polysaccharide On Cell Damage Caused By Advanced Glycation End Products Based On Whole Transcriptome Sequencing

Diabetes mellitus(DM)is a chronic disease with high morbidity,disability and mortality.It can cause many complications,among which diabetes angiopathy(DA)is one of the main causes of death in DM patients.Long-term hyperglycemia can promote the formation of advanced glycation end products(AGEs),which is closely related to the process of DA.Therefore,it is of great significance to find natural products that can effectively inhibit the formation of AGEs and its cytotoxicity for the prevention and treatment of DA.Edible and medicinal fungi are an important part of natural drug resources and a kind of microorganisms that have been used by humans earlier.Polysaccharide is one of the main active ingredients in edible and medicinal fungi.It has many biological activities such as antioxidant,hypoglycemic and immune enhancement.Because of its good effect,low toxicity and wide source,it has become a hot spot of common concern in the pharmaceutical and food industries.Lentinus edodes mycelium polysaccharide(LMP)prepared by our group in the early stage can effectively improve the oxidative stress and inflammatory damage induced by glucose toxicity and inhibit the generation of AGEs,but the inhibitory mechanism of LMP on AGEs-induced cell damage remains to be further explored.In this study,AGEs-induced human umbilical vein endothelial cells(HUVECs)injury and LMP protection were used to explore the inhibitory mechanism of LMP on AGEs-induced HUVEC cell injury.The effects of LMP and AGEs on HUVEC cell injury were investigated by detecting cell viability,lactate dehydrogenase release and intracellular ROS levels.The cells in the Control group,AGEs group and LMP group were detected by RNA sequencing(RNA-Seq),and the differentially expressed genes(DEGs)at the levels of Lnc RNA,Circ RNA,mi RNA and m RNA were analyzed.GO functional annotation and KEGG metabolic pathway analysis were performed on DEGs.Based on the results of RNA-Seq,the Lnc RNA/Circ RNA-mi RNA-m RNA regulatory network was constructed,and the expression of corresponding RNA was verified by q PCR at the cellular level.Finally,q PCR and Western Blot were used to analyze the expression of Lnc RNA/Circ RNA-mi RNA-m RNA related genes and key proteins in their downstream signaling pathways,and to explore the inhibitory effect of LMP on AGEs-induced HUVEC cell injury.The results of cell level showed that LMP effectively improved the decrease of HUVEC cell viability,the increase of lactate dehydrogenase release and the increase of ROS level induced by AGEs,indicating that LMP has a good inhibitory effect on AGEs-induced cell damage.The results of RNA-Seq showed that there were 1342 and790 differentially expressed Lnc RNAs,102 and 99 differentially expressed Circ RNAs,214 and 122 differentially expressed mi RNAs,2828 and 3052 differentially expressed m RNAs in Control group vs AGEs group and AGEs group vs LMP group,respectively.The results of q PCR verification were consistent with those of RNA-seq.The signaling pathways significantly enriched by differentially expressed RNA between groups include NF-κB inflammatory pathway,apoptosis,Ras signaling pathway and PI3K-Akt signaling pathway,which are closely related to cell inflammation and apoptosis.Based on RNA-seq results,Lnc RNA,Circ RNA,mi RNA and m RNA were screened for common differential expression in Control group vs AGEs group and AGEs group vs LMP group.The relationship pairs of target genes(m RNA,Lnc RNA,Circ RNA)corresponding to common differentially expressed mi RNAs in RNA-seq results were predicted,a ce RNA network was constructed,and the ZNF609/MSTRG.335979-mi R-483-3p-RAPGEF1 regulatory axis was screened.The results of cell level showed that compared with Control group,the expression levels of ZNF609,MSTRG.335979 and RAPGEF1 in AGEs group were significantly decreased,and the expression of mi R-483-3p was increased.Compared with AGEs group,LMP group effectively inhibited the above gene expression.At the same time,Western Blot results showed that AGEs group activated p38 protein phosphorylation,induced the activation of p38 MAPK signaling pathway,promoted the nuclear translocation of downstream NF-κB p65,and increased the protein expression of inflammatory factors TNF-a,IL-6,COX-2 and i NOS.After incubation with LMP,the phosphorylation of p38,the nuclear translocation of NF-κB p65 and the expression of related inflammatory factors were decreased,indicating that LMP effectively inhibited AGEs-induced oxidative stress and inflammatory response.In addition,the expression of Bax,Cyt-c,Cleaved Caspase-3 and Cleaved Caspase-9 in AGEs injury group was significantly increased,and the expression ratio of Bax/Bcl-2 protein was significantly increased.After LMP added,the above trends were significantly inhibited,indicating that LMP could effectively improve AGEs-induced apoptosis.In summary,this study revealed that LMP inhibited differentially expressed Lnc RNA,Circ RNA,mi RNA and m RNA in HUVEC cells damaged by AGEs through RNA-seq,and constructed a ce RNA regulatory network to clarified the connection of related molecules and the biological processes affected.At the same time,it was proved that LMP effectively inhibited AGEs-induced HUVEC cell damage,which may be related to the ZNF609/MSTRG.335979-mi R-483-3p-RAPGEF1 regulatory axis and its downstream p38 MAPK-related cell inflammation and apoptosis pathways.In this study,RNA-seq was used to obtain the full transcriptome sequence of the protective effect of LMP on HUVEC cells treated with AGEs for the first time,and combined with cell biology experiments to reveal the specific mechanism of LMP inhibiting AGEsinduced HUVEC damage.It provides new ideas for exploring the mechanism of DA induced by AGEs and the development of related prevention and treatment drugs,and also provides experimental basis for the development and utilization of Lentinula edodes resources.