Network Pharmacological Analysis Of Salvia Miltiorrhiza Bge In The Treatment Of Oral Leucoplakia And Cell Experimental Study Of Salvia Miltiorrhiza Bge Injection

Objective:Oral leukoplakia(OLK)is one of the most common potentially malignant diseases of the oral cavity.The main symptom is the appearance of white patches on the oral mucosa and there is no effective treatment to stop its further development.Salvia miltiorrhiza Bge(SMB),a traditional Chinese herbal medicine,has been used to promote blood circulation and resolve blood stasis as well as antioxidant,anti-fibrotic,anti-inflammatory,anti-tumour and other activities.Clinical studies have shown that Salvia miltiorrhiza can improve the symptoms of OLK and has the potential to treat OLK,but the specific mechanism of action on OLK has not been clarified.In this study,a network pharmacology approach was used to explore the mechanism of SMB in the treatment of OLK,and the effect of Salvia miltiorrhiza Bge Injection(SMBI)on oral leukoplakia cells(DOK)was investigated using in vitro cellular assays,which validated the network pharmacology analysis and provided an important theoretical basis for future clinical trials.Methods:Part 1: Network pharmacology study of Danshen on oral leukoplakia:Using TCMSP and Swiss Target Prediction database to obtain the active ingredients of SMB and their corresponding targets;searching Gene Cards and OMIM database to collect the disease targets of OLK,using Venn to intersect the disease targets of OLK with the component targets of SMB to obtain the potential therapeutic targets of SMB acting on OLK;The STRING platform was used to analyze the intersecting targets to build a PPI network and screen the core targets;Cytoscape software was used to build a "SMB-component-intersecting target-OLK" network and screen the main components of SMB;DAVID database was then used to perform GO and KEGG analysis;finally,molecular Finally,molecular docking technique was used to detect the binding force.Part 2: In vitro cell experiment study of Danshen on oral leukoplakia:Oral leukoplakia(DOK)cells and human normal oral mucosa keratin-forming cells(HOK)were used as cell models and treated with different concentrations of Salvia miltiorrhiza Bge Injection(SMBI)to verify the results of network pharmacology analysis.The CCK-8 and clonogenic assays were used to observe the proliferation ability of cells;the migration ability of cells was observed by scratch and Transwell assays;the cell cycle and apoptosis were observed by flow cytometry;the expression changes of core target m RNAs were detected by RT-q PCR,and finally the protein expression levels of key pathway target molecules were detected by Western blot.The expression levels of key pathway target molecules were detected by Western blot to verify the accuracy of the network pharmacology enrichment analysis.Results:Part 1: Analysis results of network pharmacology:The network pharmacology analysis identified 65 SMB ingredients that had an effect on OLK,with cryptotanshinone,salvianolic acid A,tanshinone I,and salvianolic acid B being the main components.There were 77 intersected targets,and the core targets were AKT1,CCND1,ESR1,HSP90AA1,and STAT3.GO function analysis revealed a total of 505 biological processes,mainly involving apoptosis,protein transcription,and energy.KEGG analysis identified 115 signaling pathways,with the PI3K-Akt signaling pathway being the most critical.Molecular docking results showed a strong binding affinity between the main active components of SMB and the core targets.Part 2: Results of in vitro cell experiments:After CCK8 and clone formation experiments,it was found that SMBI inhibited the proliferation of both DOK and HOK cells,and the difference in the effect on HOK cell growth was not statistically significant when the concentration was ≤60 mg/ml(ns);the effect on DOK showed a dose-and time-dependent effect with a significant difference(P<0.05),and the inhibitory effect on DOK cells was stronger at the same concentration effect(P<0.05);as verified by scratch and Transwell assays,SMBI dose-dependently inhibited DOK cell migration(P<0.05)and showed inhibitory effect on HOK cells only after 48 hours of action at high concentration(60 mg/ml)(P<0.05);as detected by flow cytometry,SMBI concentration-dependently blocked DOK cell cycle in S phase and SMBI concentration-dependently blocked DOK cell cycle in S phase and induced apoptosis in DOK cells by flow cytometry(P<0.05),but only at high concentrations affected HOK cell cycle and apoptosis differently(P<0.05);RT-q PCR results showed that the expression levels of five core target genes m RNA decreased with increasing SMBI concentration and were higher in HOK cells than in DOK(P<0.05);Western blot The results showed that SMBI dose-dependently decreased the phosphorylation levels of PI3 K,AKT and m TOR in DOK cells to inhibit the activation of the key signaling pathway PI3K-Akt,and showed the inhibitory effect on HOK cells only at high concentrations.Conclusion:Based on network pharmacology screening,the main constituents of SMB that display anti-OLK effects were identified as cryptotanshinone,salvianolic acid A,tanshinone I,and salvianolic acid B.The core targets were found to be AKT1,CCND1,ESR1,HSP90AA1,and STAT3,while the most critical signaling pathway was the PI3K-Akt pathway.Additionally,molecular docking analysis revealed a robust binding affinity between the main components and the core targets.The results of cytological experiments showed that SMB inhibited the proliferation and migration of DOK cells,induced apoptosis,blocked cells in S phase,affected the expression of core target m RNA,and inhibited the activation of key pathway PI3K-Akt,but had a weak effect on HOK cells,which laid a theoretical foundation and scientific basis for the application of SMB in clinical research of OLK.