Hyperthermia Induces HPV Positive Cell Death In Human Papillomavirus Infectious Diseases Via Apoptosis

Objective: Human papillomavirus(HPV)is a common sexually transmitted infection virus.HPV is strongly associated with the development of diseases and cancers,including anogenital warts,recurrent respiratory papillomatomatosis,and anogenital and oropharyngeal cancers,resulting in a significant burden of disease.The skin manifestations of HPV infection are predominantly skin warts and genital warts,which may affect daily activities,such as physical pain and impaired patient’s psychosocial state.Newly treatments such as thermotherapy(e.g.,thermotherapy)play an important role in immune diseases.Hyperthermia has been successfully utilized to treat viral warts and cervical intraepithelial neoplasia caused by high-risk HPV,and thermotherapy possesses the advantages of low pain rate,wide application population and low recurrence rate compared with traditional laser,cryotherapy or other methods.This study focuses on the effect of thermotherapy on the death of HPV positive cells,so as to deeply explore the clearance effect of local hyperthermia on HPV infection,and provide a certain theoretical basis for the treatment of viral warts by heat treatment.Methods: HPV positive cell lines(SiHa and CaSki cells)and condyloma acuminatum(CA)tissues were studied in our study.CA tissues were acquired from patients undergoing surgical operation.Cells or CA tissues were treated with water bath at 44(±0.1)℃ for30 minutes.1.To research whether thermotherapy contributes to immunogenic death of HPV positive cells,we detected immunogenic death related markers in untreated and heattreated SiHa cells and CaSki cells,including heat shock protein A6(HSPA6),high mobility group protein B1(HMGB1),calreticulin(CRT),extracellular ATP and mitochondrial DNA.2.Explore the effect of thermotherapy on cell apoptosis and cell cycle.Investigate the effects of thermotherapy in combination with TLR4 inhibitor TAK-242 on cell cycle and apoptotic protein p21.3.The above-mentioned proteins before and after hyperthermia were verified at the tissue level by single-cell data sequencing of the patient’s CA tissues.Results: 1.Heat treatment at 44℃ led to immunogenic death of HPV positive cells.The expression of total HSPA6 in SiHa cells and CaSki cells increased at 1h post-thermotherapy,and remained high level for 24 h.The total HMBG1 of SiHa and CaSki cells remained unchanged before or after heat treatment.HMGB1 protein in CaSki cells was released outside the cell 24 h after hyperthermia,while the level of extracellular HMGB1 in SiHa cells did not change at 1 h or 24 h after hyperthermia.Heat made no change to total CRT expression in SiHa and CaSki cells,while flow cytometry analyses exhibited an increase of membrane surface expression in SiHa and CaSki cells 24 h post-thermotherapy.There was no increase in supernatant ATP release in SiHa and CaSki cells 1 h after heat treatment in comparison with untreated cells,but an increase was discovered in cell supernatants 24 h after hyperthermia.Compared to untreated cells,heat-treated SiHa cells possessed increased mitochondrial DNA levels after 1 h,while CaSki cells subjected to thermotherapy saw increased mitochondrial DNA expression after 24 h.2.Hyperthermia contributed to cell apoptosis,SiHa and CaSki cells treated at 44℃ for 30 min showed elevated early apoptotic cells 24 h after heat treatment.SiHa and CaSki cells experienced cell cycle arrest 24 h post-hyperthermia,which was manifested as a decrease in the proportion of cells at G1 phase and an increase in the proportion of cells at G2/M phase.At 1 h and 6 h after thermotherapy,p21 protein expressed more in both SiHa and CaSki cells compared with untreated cells.SiHa and CaSki cells treated with hyperthermia combined with TLR4 inhibitor had reduced HSPA6 expression,increased p53 expression,increased p21 expression,and an increased proportion of early apoptotic cells compared to single heat treatment.HSPA6 knocked down by si RNA increased the level of p21 and p53 reduction by si RNA decreased p21 level.Conclusions: 1.Hyperthermia induces immunogenic death of HPV positive cells,which is manifested by increased HSPA6 levels,extracellular release of HMGB1 and ATP,increased surface expression of CRT and increased mitochondrial DNA level after thermotherapy.2.Hyperthermia combined with TLR4 inhibitor aggravates p21-dependent apoptosis by reducing the expression of HSPA6 protein and increasing the expression of p53 protein.