| BackgroundAcne is the most common inflammatory skin disease occurring in the sebaceous glands.The prevalence of acne is 39.2%in China and 57.8%in Europe.It can reach more than 90%in adolescents and even 50%in adults.Acne usually occurs on the face and chest and back.Acne lesions are often accompanied by papules,pustules,nodules,cysts and other types of skin rashes,leading to physiological lesions and psychological pain.Propionibacterium acne(P.acnes)is considered to be the pathogen of acne and plays an important role in the occurrence and development of acne.It can induce local inflammation,and also involved in the process of hyperkeratosis of hair follicle epithelium.Inflammation is an important pathological process of acne,which is closely related to the activation of TNF pathway and complement system.migration of neutrophils.Controlling inflammation is the main method to treat acne.Clinically,Scutellaria baicalensis(C21H18O11)is a commonly used Chinese medicine for the treatment of acne and other inflammatory diseases.Baicalin(C21H18O11)is a flavonoid extracted from baicalin root,which is the main active component of Scutellaria baicalensis with strong anti-inflammatory effect.The molecular mechanism of baicalin in the treatment of acne remains to be studied further.ObjectiveKligman method was established in New Zealand rabbits to evaluate the therapeutic effect of baicalin on acne.The molecular mechanism of baicalin in the treatment of acne was systematically revealed by transcript sequencing analysis of rabbit ear samples before and after treatment and combining immunohistochemical experiment and Western blot.The purpose of the study is to provide experimental evidences for the efficacy of baicalin in the treatment of acne and give evidences for the clinical application of baicalin in the treatment of acne.Methods1.Theacne model of New Zealand rabbits was established by Kligman method to explore the therapeutic effect of Baicalin on acne.2.Theeffect of baicalin on the rabbit ear acne model was evaluated by using the method of H&E staining which can prove the success of acne model and the therapeutic effect of baicalin.3.Thecure rate of baicalin on acne were calculated by observing the number changes of acne model after the treatment of baicalin of high,moderate and low dose.4.Vernier caliper was used to measure the thickness and diameter changes of rabbit ear acne model and baicalin of high,moderate and low dose to evaluate the therapeutic effect of baicalin on acne.5.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of TNFA,IL1B,NFKB1 and IL8 and other inflammatory factors in serum of acne New Zealand rabbit model.6.The differential expressions of genes in acne samples of rabbit ears before and after baicalin treatment was detected by transcript sequencing analysis.7.Immunohistochemical method was used to detect the expressions of TNFA,IL1B,NFKB1 and IL8 in rabbit ear samples before and after treatment with baicalin.8.The expressions of TNFA,IL1B,NFKB1 and IL8 in rabbit ear samples before and after treatment with baicalin were detected by Western blot.Results1.Baicalin can treat acne in rabbit ear model.Compared with the normal control group,the swelling degree of rabbit ears was significantly enhanced in acne model.After treatment with baicalin,the thickness of rabbit ear in acne model,the diameter of acne lesion and the number of acne lesion decreased obviously.That is,the swelling degree and the appearance of acne in rabbit ear model were obviously improved.2.Histologic analysis of rabbit ear acne model.We found that compared with the control group,the rabbit ear acne model presented a typical successful appearance of acne:excessive keratinization in skin tissue,obvious hyperplasia in the granular layer of the epidermis and hair follicle epithelium,the accumulation of keratins at the orifice of the hair follicle and the dilation of the funnel part which like a kettle in the interior of the dermis and the phenomenon of more inflammatory cells infiltration in the interior of the dermis.After the treatment with baicalin of different concentrations,the expansion of hair follicles in rabbit ear acne model was obviously alleviated,and their internal keratins were loosened obviously.And the inflammatory cells also significantly reduced after the treatment of baicalin.3.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of TNFA,IL1B,IL6,IL8 in serum of acne model.The secretion of TNFA,IL1B,IL6,IL8 in serum of rabbit ear acne model was significantly increased.After the treatment with baicalin of high,moderate and low dose,the concentrations of TNFA,IL1B,IL6,IL8 decreased significantly and the difference was significant and statistically significant(P<0.05).4.Effects of Baicalin on gene expression in rabbit ear acne tissue.By transcriptomesequencing analysis,we found that there were a large number of differentially expressed genes between control group,acne model group and baicalin treatment group.Compared with the control group,there were 1325 up-regulated genes and 1187 down-regulated genes in acne model group.Compared with acne group,there were 350 up-regulatedgenes and 411 down-regulated genes in baicalin group.Among the genes up-regulated in acne group(compared with the control group),179 were down-regulated after baicalin treatment,accounting for 43.6%of the total number of down-regulated genes in baicalin treatment group.Compared with the control group,105 of the down-regulated genes in acne model group were up-regulated after baicalin treatment,accounting for 30%of the total down-regulated genes in baicalin treatment group.The effective mechanism of Baicalin in treating acne may be that the up-regulated genes in acne group are down-regulated and the down-regulated genes are up-regulated.5.Regulation effect of baicalin on TNF pathway in acne tissues of rabbit ears.KEGG enrichment pathway analysis of differentially expressed genes showed that TNF signaling pathway was the main enrichment pathway of differentially expressed genes in acne model group compared with control group.The expression of differentially expressed genes was analyzed.Transcript sequencing analysis showed that the expression levels of several key genes in TNF pathway of acne model such as TNFA,IL1B,IL8,TNFRSF1B.NFKB1,REL were up-regulated,while the expression levels of these genes were significantly decreased after the treatment of baicalin.The results of immunohistochemistry experiment and Western blot showed that baicalin could significantly inhibit the up-regulated TNFA?IL1B and NFKB1 proteins in TNF pathway.6.Inhibitory effect of Baicalin on infection pathway of Staphylococcus aureus in rabbit ear model of acne.The analysis of KEGG enrichment pathway of differentially expressed genes in control group,acne model group and baicalin medication group showed that the differentially expressed genes in baicalin group were mainly enriched in infection pathway of Staphylococcus aureus,phagocytic pathway,cell adhesion molecule,cytokine receptor interaction and complementation.Staphylococcus aureus infection pathway is the most prominent one in the body coagulation cascade pathway.Staphylococcus aureus infection pathways include neutrophil recruitment,antigen presentation,complement system activation and FC gamma receptor-mediated phagocytosis.According to the analysis of differentially expressed genes,four genes involved in neutrophil recruitment were up-regulated in acne group and down-regulated in baicalin group.Immunohistochemical results also showed that a large number of neutrophils were recruited to acne sites and inhibited by Daicalin.The complement system contains eight differentially expressed genes,including five classical pathway specific genes(C1QA,C1QB,C1QC,C1R and C1S),one bypass pathway specific gene(CFB),two classical pathway and lectin pathway shared genes(C2 and CBPA).Compared with the control group,thesegenes were all significantly up-regulated in acne group except for C2.While 7 genes were significantly down-regulated in baicalin group compared with the acne group,except for C4BPA..Conclusion1.Baicalincan inhibit acne inflammation in rabbit ear model.2.Baicalincan significantly inhibit the expressions of inflammatory cytokines in serum of acne model.3.Multipleinfections and inflammation-related pathways,such as TNF signaling pathway,pathway of Staphylococcus aureus infection were activated in the occurrence of acne.Baicalin treatment can significantly inhibit the activation of these pathways.Our study shows that baicalin can significantly inhibit multiple inflammatory pathways in the process of acne through its anti-inflammatory effects so that can improve acne symptoms.Our study further reveals the molecular mechanism of baicalin in the treatment of acne and provides experimental basis for the clinical application of baicalin. |
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