| BACKGROUNDAcne vulgaris is a chronic disease that occurs in the sebaceous glands of hair follicles,and its mechanism is complex.It is of great advantage to use Traditional Chinese medicine with multi-component and multi-target characteristics to intervene in the pathophysiological process of acne.The licorice flavonoid is a component of the Traditional Chinese medicine licorice which has anti-inflammatory,antibacterial and anti-acne effects,and its anti-acne mechanism has not yet been elucidated."omics" is a research method for large-scale,systematic reflection of dynamic changes in living organisms.The multi-omics combination study of licorice flavonoids against acne can more systematically explain the mechanism of traditional Chinese medicine in the treatment of diseases.OBJECTIVEThe aim of this study was to investigate the chemical constituents of licorice flavonoids and their anti-acne mechanism.1.To study the intervention effect of licorice flavonoids on acne model rats.2.To explore the effects of licorice flavonoids on skin protein in acne rats by proteomics research methods,and to screen protein biomarkers related to acne pathology and drug intervention targets.3.Metabolomics analysis was used to study the changes of skin and serum metabolites in acne pathology and licorice flavonoids against acne,and to screen out endogenous small molecule biomarkers related to acne incidence and drug intervention.4.To investigate the effects of licorice flavonoids on the microbial composition and bacterial abundance of acne model rats,and to screen out the biomarkers related to acne disease and drug intervention.METHODS1.Construction of acne on the back of rats and intervention of licorice flavonoids.Rats were randomly divided into blank group,model group and licorice flavonoid group.Acne is induced on the back of the rat by oleic acid application and injection of P.acnes into the dermis.Give licorice flavonoid gel against acne.Skin was taken after the last modeling/administration,and pathological evaluation of tissue sections and electron microscopy sections was performed.The total protein of the skin was extracted,and the expression of acne-associated inflammatory factors was detected by immunoblotting.The components in the licorice flavonoids were identified by UPLCESI-Orbitrap-MS analysis system.2.Proteomics study of licorice flavone against acne.After the last modeling/administration,the total protein was extracted from the skin of the corresponding part of the back of the rat.The peptide is obtained by reductive alkylation and enzymatic treatment.The peptide molecules were detected by UPLCESI-Orbitrap-MS,and the database matching and protein species identification were performed by Proteome Discoverer software.Maxquant software quantifies proteins and R software performs statistical analysis of the data to screen for differential proteins between groups.Annotation of biological function of differential proteins between groups.Explain the regulation of protein biomarkers on acne.3.Metabolomics study of licorice flavonoids against acne.After the last modeling/administration,endogenous metabolic small molecules were extracted from rat serum and skin samples,and detected by UHPLC-ESI-TriodeF-MS.Statistical analysis was performed on the identified metabolites.Metabolites with statistically significant differences were screened as biomarkers and pathogen enrichment analysis was performed on biomarkers.4.Effect of licorice flavonoids on skin flora of acne rats.After the last modeling/administration,the rat’s back skin flora was scraped with a sterile cotton swab,and the total DNA of the skin flora sample was extracted,and the 16S rDNA V3-V4 region was subjected to PCR amplification.The amplified product was subjected to Illumina Hiseq sequencing analysis.Bioinformatics analysis was performed on the results of sequencing,including α\β diversity analysis,material composition difference analysis,and functional prediction.RESULTS1.The results of LC-MS analysis indicated that licorice flavonoids mainly consisted of flavonoids such as licorice chalcone A,licorice chalcone C,licorice flavonoid A,isoglycyrrhizin,Kanzonol C and glycyrrhizin etc.,and a little organic acids,Amino acids and alkaloids.Pathological tissue sections showed thickening of stratified squamous epithelium of acne skin,blockage of hair follicles,infiltration of inflammatory cells and enlargement of sebaceous glands.The ultrastructure of the skin was observed by transmission electron microscopy.It was found that the epithelium of the follicular funnel was thickened,a large number of lipid droplets were found in the stratum corneum,and cell edema was observed in the spinous cell layer.The lamellar bodies,keratohyalin granules and desmosome in the epidermis are increased.The pathological manifestations of acne after the intervention of licorice flavonoids were significantly improved.In addition,licorice flavonoids can reduce the expression levels of inflammatory factors IL-8 and TNF-α,which have been up-regulated in the skin of model rats,and inhibit acne inflammation.2.In the proteomics study,a total of 314 differential proteins（FC>1.5）were screened between the blank group and the model group,of which 27 had significant differences（P<0.05）.The differential proteins between the groups involved 41 KEGG pathways such as Metabolic pathways,PI3K-Akt signaling pathway,Estrogen signaling pathway,Tight junction,Protein processing in endoplasmic reticulum,Leukocyte transendothelial migration and Necroptosis.This revealed abnormalities in protein biosynthesis,lipid metabolism,inflammatory cell infiltration,and skin barrier function in acne rats.A total of 359 differential proteins were screened between the licorice flavonoid group and the model group,and 15 of them had significant differences.Intergroup differential proteins are involved in the skin’s Metabolic pathways,Tight junction,Focal adhesion,PI3K-Akt signaling pathway,Apoptosis,Leukocyte transendothelial migration,Phagosome,Purine metabolism,Drug metabolism-other enzymes and other 50 KEGG pathway processes.Licorice flavonoids improve acne symptoms by affecting the aforementioned pathways,inhibit inflammation of acne skin,inhibit lipid synthesis and promote recovery of skin barrier function.3.In the skin metabolome study,124 differential metabolites were screened between the blank group and the model group,among which PE 16,Aspartate,Leucine,Captopril,Aminobenzoyl-glutamate,and M-Tyrosine were significantly different（VIP>0.8,P<0.05）.A total of 92 differential metabolites were screened between the licorice flavonoid group and the model group,among which PE 16,Leucine,Captopril,5Fluro-2-pyrimidone,M-Tyrosine and Uridine were significantly different Enrichment analysis of differential metabolites of the skin showed abnormalities in Alanine,aspartate and glutamate metabolism,Arginine and proline metabolism,Aminoacyl-tRNA biosynthesis,Histidine metabolism,beta-Alanine metabolism,and phospholipid metabolism in acne rat skin.After treatment with licorice flavonoids,it could regulate the metabolic disorder of acne rat skin and make it tend to be normal.4.In the serum metabolome study,a total of 116 metabolites were screened for differences between the blank and model groups,12 of which were significantly different,including Coprostanone,DL-beta-Hydroxybutyric acid,gammaGlutamylleucine,LPE 18:1,LPC 16,Stearic acid,LPC 18:2,Citric acid,LPE 16:0,LPE 18:2,Palmic acid,Phosphatidylserine 18.A total of 102 differential metabolites were screened between the licorice flavonoid group and the model group,11 of which were significant including Stearic acid,Cholic acid,Trans-Vaccenic acid,Phosphatidylcholine lyso 16,Oleic Acid,LPE 18:1,Coprostanone,Thymidine,LPhenylalanine,L-Proline and L-Arginine.Pathway enrichment analysis of differential metabolites showed abnormalities in Phenylalanine,tyrosine and tryptophan biosynthesis,Phenylalanine metabolism,Biosynthesis of unsaturated fatty acids,Fatty acid biosynthesis,Valley,leucine and isoleucine biosynthesis in acne rats.Licorice flavonoids can regulate the disorder of serum metabolism in acne rats,and make amino acid metabolism,fatty acid metabolism and phospholipid metabolism tend to be normal.CONCLUSION1.Licorice flavonoids can effectively improve the pathological condition of acne and inhibit the inflammation of acne skin.2.The PI3K-AKT signaling pathway is activated in the skin of acne rats.Increased mitochondrial activity in skin metabolism,increased energy supply,facilitates protein processing in the endoplasmic reticulum and facilitates lipid synthesis.Upregulation of Msn mediates transendothelial migration of leukocytes,resulting in infiltration of skin tissue cells.Licorice flavonoids inhibit the expression of 14-3-3 protein and promote the activation of FoxO.On the other hand,it inhibits mitochondrial activity and anabolic pathways of proteins and lipids and promotes ester hydrolysis.Licorice flavonoids regulate the transendothelial migration of leukocytes by down-regulating the expression of Msn,inhibiting the inflammatory infiltration of skin,and finally improving the pathological state of acne.3.In the acne disease of rats,the metabolism of amino acids,fatty acids,unsaturated fatty acids,and phospholipids in the skin and serum is abnormal,resulting in inflammation of the whole body and skin,and abnormal metabolism of proteins and lipids.The administration of licorice flavonoids can regulate the metabolic disorder of acne.4.The structure and the diversity of the skin flora in acne rats decreased.Biomarkers,as Staphylococcus,Corynebacterium,Propionibacterium,Enterococcus and other flora,affect the process of keratinization,sebaceous gland lipid metabolism and inflammatory reaction in acne skin.Licorice flavonoids can restore the disordered flora structure of acne skin and promote the homeostasis of skin flora,reflecting the importance of skin flora for maintaining skin immunity and skin barrier function. |
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