Preliminary Study On The Mechanism Of Deoxyshikonin Inducing Apoptosis In CaSki Cells Through PI3K-Akt Pathway Based On Network Pharmacology

Background:Human Papillomavirus(HPV)infection is the second most common cause of cancer in the world after breast cancer,which poses a serious threat to human health and a heavy burden on many countries,especially in underdeveloped countries.At present,there is still a lack of uniform and effective prevention and treatment of HPV infection.In the clinical treatment of HPV infection,the Traditional Chinese Medicine Lithospermum Erythrorhizon has a good effect,with the value of clinical promotion.Because of the complexity of the components and the unknown mechanism of action,traditional Chinese medicine is difficult to be accepted by the Western medical community,which limits the promotion and application of Traditional Chinese Medicine in the international scope.The purpose of this study was to identify the specific components of Traditional Chinese medicine in the treatment of HPV-related diseases and in-depth study of relevant mechanisms.Objective:In this study,network pharmacology,molecular docking and cell experiments were used to explore the mechanism of deoxyshikonin,a major component of Lithospermum erythrorhizon(L.erythrorhizon),inducing the apoptosis of CaSki cells infected with HPV 16 through PI3K-Akt pathway,to provide scientific basis for the application of L.erythrorhizon in the treatment of HPV infection-related diseases.Methods:(1)Network pharmacology was used to predict the potential target of Lithospermum Erythrorhizon on HPV,and KEGG and GO analysis were carried out to find out the main signal pathway.(2)The main active components and target proteins of L.Erythrorhizon were identified by molecular docking technique.(3)The best active component,the best concentration interval and the best inhibitory cell line were obtained by MTT test.(4)Apoptosis detection experiment(Hoechst fluorescence staining and Flow cytometry)was used to test whether the best active component of L.erythrorhizon could induce apoptosis of the best inhibitory cell.(5)Western blotting was used to detect the effects of the best active component of L.erythrorhizon at different concentrations on the expression of PI3K-Akt signaling pathway-related proteins.Results:(1)A total of 41 compounds and 148 corresponding targets were searched,of which 59 were related to diseases.GO functional enrichment analysis yielded 20 entries of GO_BP,10 entries of GO_CC and 19 entries of GO_MF and most of them were mainly related to apoptosis.KEGG pathway analysis yielded 20 signal pathways,among which PI3K-Akt signal pathway was the main enrichment pathway(2)Molecular docking validation manifested that ursolicacid,deoxyshikonin and order derivatives had good affinities with candidate targets(STAT3,TP53,JUN,MAPK8,TNF).(3)MTT test showed that the active components of acetylshikonin,deoxyshikonin and ursolic acid had good inhibitory activity on CaSki cells,especially deoxyshikonin.(4)With the increase of deoxyshikonin concentration,the proportion of late apoptotic cells increased.(5)Western blotting showed that deoxyshikonin could down-regulate p-PI3K(Tyr458)and p-Akt(Tyr308).The reaction of them were positively dose-related.Conclusion:Network pharmacology shows that L.erythrorhizon may treat HPV infection-related diseases through multi-target,multi-biological process and multi-signal pathways.Experimental studies suggest that L.erythrorhizon may promoting the apoptosis of CaSki cells via PI3K-Akt signaling pathway.