Evaluation Of Photodynamic Therapy For The Treatment Of Condyloma Acuminate And The Mechanism Of Its Combined Therapy Promoting Cell Death In HPV Infected Cells

Background:Sustained human papillomavirus(HPV)infection and condyloma acuminate(CA)caused by it are difficult problems in clinical treatment.The efficacy of 5-aminolevulinic acid photodynamic therapy(ALA-PDT)in the treatment of CA is significant with high virus clearance rate and low recurrence rate,but there is no fully accepted standard to guide the treatment and evaluate the efficacy,and the treatment cost is high.Podophyllotoxin(POD),as the first-line drug in the clinical treatment of CA,has a significant effect.However,long-term use of large doses might induce adverse reactions.We still need a more efficient,safe and effective strategy to treat HPV infectious diseases such as CA.Objective:To find the best endpoint of ALA-PDT in the treatment of CA,we used the HPV DNA detection,including genotyping and viral load measurement to guide the treatment and evaluate the efficacy of ALA-PDT in the treatment of CA under different HPV infection.And to investigate whether ALA-PDT combined with POD could promote the HPV-infected cell death more effectively than single treatment at the same intensity or concentration,and explore the further mechanism.Methods:Collected the information of CA patients treated by HPV DNA detection-guided ALA-PDT.Grouped patients according to the HPV infection status before the first ALA-PDT,set the endpoint as getting two consecutive negative results of HPV DNA detection and evaluated the difference of the efficacy of ALA-PDT under different infection status.After reaching the endpoint,the patients were followed up for at least six months to observe the recurrence rate of this strategy.CCK-8 was used to explore the appropriate combination of ALA-PDT and POD.To prove that ALA-PDT combined with POD had a stronger ability to kill HPV infected cells than single treatment at the same strength and concentration.The morphological changes of cells and nuclei were observed by microscope.Annexin V-FITC&PI flow cytometry was used to detect apoptosis rate,Edu was used to detect cell proliferation change,LDH release test was used to detect cell necrosis.To investigate whether the combined therapy could damage mitochondria,activate endogenous and exogenous apoptosis pathway,used flow cytometry to detect ROS production level in cells,JC-1 test to detect mitochondrial membrane depolarization,Western blot to detect apoptosis-related proteins.By using flow cytometry to detect intracellular calcium level,Western blot to detect endoplasmic reticulum stress-related proteins,we found that ALA-PDT combined with POD could strongly induce endoplasmic reticulum stress.Immunofluorescence staining and Western blot showed that ALA-PDT combined with POD could significantly induce autophagy.Results:CA patients with multisite and multitype HPV infection need more sessions of ALA-PDT to achieve virus clearance,particularly those suffering from internal CA or HR-HPV infection.The recurrence rate was effectively reduced by ending ALA-PDT after obtaining twice negative HPV DNA detection results in a row.ALA-PDT combined with POD inhibited the proliferation of HPV infected cells,promoted apoptosis and necrosis.Compared with any single treatment,the activations of the caspase-dependent apoptosis pathway,endoplasmic reticulum stress,and autophagy were more powerful in ALA-PDT combined with POD.Conclusion:HPV DNA detection-guided ALA-PDT is an effective strategy for clinical treatment of CA,which conforms to the concept of precision medicine and is conducive to the promotion of individualized treatment.ALA-PDT combined with POD can effectively improve the ability of promoting cell death of HPV infected cells through a variety of pathways,which is a promising combined therapy.