| BackgroundCondyloma acuminate(CA)is a common sexually transmitted disease(STD)caused by human papillomavirus(HPV)infection,which often occurs in the skin and mucosa of the external genital areas.Clinical manifestations are cauliflower-like or papilloma-like neoplasms.Condyloma acuminata ranks third among sexually transmitted diseases in China,the incidence of condylomata acuminata have been increasing remarkably,while the incidence rate is higher in European and American countries.Now,more than 200 human papillomaviruses have been identified and sequenced,and about one-third of them can infect genital squamous epithelial cells.CA mainly occurs in the vulva,penis,and perianal parts,persistent HPV infection in these areas may lead to further infection of the vagina,urethra,cervix and anal canal.Long-term infection with HPV is believed to have a direct correlation with the development of malignant proliferation,intraepithelial neoplasia and cancer.In additon,due to the presences of HPV latent infection and subclinical infection,it is difficult to diagnose early in clinical,which makes the treatments of CA are often unsatisfactory and relapses easily,affecting the daily life of patients and causing significant frustration for them.Therefore,in recent years,the treatment of CA has received more and more attention.5-aminolevulinic Acid Photodynamic Therapy(ALA-PDT),as a rapidly developing and non-invasive new technology,has been widely applied to the treatment of CA.It has good tissue selectivity and few side effects,at the same time it can reduce the recurrence rate significantly.ALAPDT can also be used to treat other diseases related to HPV infection,such as vaginal/vulvar precancerous lesions,cervical cancer,etc.,but the specific mechanism of ALA-PDT for these diseases is still unclear.Purpose1.Analysis of HPV subtypes distribution characteristics in different anatomical sites of patients with CA.2.The characteristics of HPV viral loads during the treatment of extraluminal(skin type)sites and intraluminal(mucosal type)sites by ALA-PDT.3.To detect the expression of STA1/p-STAT1 in condyloma acuminata and HPV-infected cells.4.To explore the effect of ALA-PDT on JAK/STAT1 signaling pathway in HPV-infected cells.5.To study the role of STAT1 in ALA-PDT regulation of proliferation and apop-tosis of HPV-infected cells.Method:1.Collect clinical basic informations and datas of patients with CA1.1 Collect basic informations of patients diagnosed with CA who attended the XXX Hospital between January 2017 and May 20181.2 Before treatment,photographing,locating,and taking materials,PCR was used to detect HPV type and load in cells scraped from the lesion surface at different sites.1.3 HPV test was performed after each ALA-PDT treatment to record the change of HPV viral loads.1.4 Changes of viral loads assisted in making treatment plans and ALA-PDT rounds.1.5 SPSS.20 and Graphpad Prism were used to analysis datas.2.Expression of STA1/p-STAT1 in CA and HPV-infected cells2.1 Collected warts of patients with CA included in the study,foreskins from healthy people as a control.The expression of STA1/p-STAT1 in the warts and foreskin tissue were detected by immunohistochemistry.2.2 Use human immortalized keratinocytes(HacaT cells),cervical cancer cell line Siha(HPV 16),Hela(HPV18),incubated in 5%CO2 incubator at 37 ℃ and cultured with 10%fetal bovine serum and 1%streptomycin mixture added into DMEM(high sugar)medium.2.3 Experiment cells grouping:three kinds of cells were randomly divided into two groups:control group and IFN-y group.2.4 Western-blot was used to detect the expression of JAK1/p-JAK1,STAT1/pSTAT1 and its downstream molecules IRF-1 in experimental groups.3.The effect of ALA-PDT on JAK/STAT1 signaling pathway in HPVinfected cells3.1 Detected the expression of key proteins in JAK-STAT1 signaling pathway in Siha/Hela cells after ALA-PDT treatment.Siha/Hela cells were grouped into four groups:blank control group;ALA group;Laser group;ALA+PDT group.ALA concentration was 0.1mmol/L,wavelength of red light was 635nm and irradiation energy was 0.6J/cm2.Western-blot assay was used to detect the expression of JAK1/p-JAK1,STAT1/p-STAT1 and its downstream molecules.IRF-1.3.2 Effects of different ALA concentrations,different light energy and different time interval on the expression of pathway proteins:Western-blotting detected expression of JAK1/p-JAK1,STAT1/p-STAT1 and its downstream molecule IRF1 in each concentration gradient group,light energy gradient group and time gradient group.4.The role of STAT1 in ALA-PDT regulations of proliferation and apoptosis of HPV-infected cells4.1 Changes in cell proliferation activity after transfection of siRNA-STAT1:experimental cells were grouped into:① control group;② si-con group;③ siSTAT1 group;④ALA-PDT+si-STAT1 group;⑤ALA-PDT+si-con group.CCK8 method was used to detected cell proliferation activity of each group.4.2 Detection of apoptosis after transfection of siRNA-STAT1:The experimental groups were the same as 4.1,and the apoptosis of each group was detected by flow cytidine Annexin V/PI double staining.Result:1.Multiple sites’ HPV infection predominates in patients with CA,and perianal is the major predilection site for multi-sites,multi-types HPV infection.1.1 Among the 106 cases of CA patients,the incidence of perianal was the highest,41.51%(44/106),followed by penis(33.02%,35/106),urethra(21.70%),anal(18.88%),cervix(18.88%),vulva(17.92%),glans(14.15%),vagina(12.26%),scrotum(5.57%)had the lowest incidence.1.2 Multi-sites infections predominate in patients.Single site infection accounted for 39.6%(42/106),penis was the most common(19.8%,21/106);multi-sites infection accounted for 60.4%(64/106),and perianal area combined with other anatomical sites accounted for(37.7)%,40/106).1.3 Up to 4 types of HPV infected the same one site simultaneously:single type infection accounted for 57.4%,double infection 29.8%,triple infection 8.2%,and quadruple infection 4.6%.HPV6 and HPV 11 are the most common infection subtypes,whether in single infection or multiple infections.1.4 The number of HPV subtype detected in the perianal area was the most,which were 15 species,penis had 14 subtypes,cervix,vulva and vagina had 13 subtypes,anal and glans had 12 subtypes,urethra had 10 subtypes.In addition to the vagina and cervix,the detection rate of low-risk HPV in other parts was higher than that of high-risk HPV.2.The HPV viral loads of the extraluminal(skin type)site were significantly higher than the intraluminal(mucosal type)site,and ALA-PDT can effectively reduce the HPV viral loads whether in the extraluminal sites or intraluminal sites.2.1 Comparing the external sites of the cavity:perianal,vulva,penis,glans,scrotum and internal sites of cavity:intra-anal,cervix,vagina,urethra,the initial viral loads of intraluminal infection sites were significantly higher than that of the extraluminal infection sites(P<0.05).2.2 After the first cycle treatment of ALA-PDT,the HPV loads of the extraluminal and intraluminal infection sites were significantly decreased(P<0.05).There was no significant difference in the viral loads between the extraluminal and intraluminal sites after treatment(P>0.05).The HPV negative rate in the intraluminal site(58.35%)was higher than the extraluminal site(51.03%)after the first cycle ALA-PDT,but there was no statistical difference.2.3 The cycles of ALA-PDT for intracavitary sites(6.1±2.12cycles)was higher than that of extraluminal sites(4.7±1.19cycles),with significantl statistical difference(P<0.0001).3.HPV infection inhibited the expression of STAT1/p-STAT1.3.1 The expression of STAT1/p-STA1 was significantly decreased in CA.The results of immunohistochemistry showed that the expression of STAT1/p-STAT1 in CA was significantly lower than that in healthy controls(P<0.001).3.2 The expression of JAK1 and STAT1 in HPV-infected cells(Hela/Siha)was lower than that in normal keratinocytes.3.3 HPV can inhibit the up-regulation and phosphorylation of JAK1,STAT1 by IFN-y.Western-blot’ results suggested that IFN-γ significantly up-regulated the expression of JAK1/p-JAK1,STAT1/p-STAT1 and its downstream IRF-1 in HacaT cells,but in Siha/Hela cells,there was no significant change in the proteins after IFNγ treatment.4.ALA-PDT can up-regulate the expression of JAK/STAT1 signaling pathway in HPV-infected cells.4.1 Western-blot’ results suggested that ALA-PDT can significantly up-regulate the expression of JAK1/p-JAK1,STAT1/p-STAT1 total protein and phosphorylated protein in Siha/Hela cells,while the IRF-1 protein was also significantly up-regulated with statistical differences.4.2 Effect of energy on the JAK/STAT1 pathway.In Siha/Hela cells,JAK/pJAK1 decreased with the increasing energy of red light at 635 nm,while expressions of STAT1/p-STAT1 and IRF-1 gradually increased with the increasing energy.4.3 Effect of different ALA concentrations on JAK/STAT1 pathway.In Hela cells,JAK1 expressed significantly higher at 0.25 mmol,and there was no difference in other concentrations.In Siha cells,JAK1 expressed significantly higher in 0.25 mmol and 2 mmol/L,and there was no difference in other concentrations.The of STAT1/p-STAT1 and IRF-1 did not change with the different concentration of ALA in Siha/Hela cells4.4 Effect of time on the JAK/STAT1 pathway.In Siha cells,JAK/p-JAK1,STAT1/p-STAT1 and IRF-1 increased gradually with the time of ALA-PDT treatment,and peaked at 24h after ALA-PDT.In Hela cells,JAK1 began to peak at 12h,and there was no statistical difference after 12h.STAT1/p-STAT1 peaked at 24h,while IRF-1 peaked at 48h.4.5 ALA-PDT up-regulates the expression of MHC-I and MHC-related molecule MICB.After ALA-PDT treatment,expression of mRNA of MHC-I and MICB were significantly up-regulated in Hela/Siha cells(P<0.0001),while MHC-related molecules MICA expression was not significantly changed.5.STAT1 played a key role in ALA-PDT regulation of proliferation and apoptosis of HPV-infected cells.5.1 In Hela/Siha cells,ALA-PDT significantly inhibited the proliferation of two cells.After transfection with siRNA-STAT1,the antiproliferative effect of ALA-PDT was nhibited significantly(P<0.05).5.2 In Hela/Siha cells,ALA-PDT can significantly promote the apoptosis of two cells.After transfection with siRNA-STAT1,inhibiting the expression of STAT1,the proapoptotic effect of ALA-PDT was inhibited.Conclusion:This study confirmed that multi-sites and multi-types HPV infections were dominant in patients with condyloma acuminata,and perianal’s multiple infections are the most common.Simultaneously,the number of HPV subtypes found in the perianal was the highest,reaching 15 types.ALA-PDT can effectively reduce the HPV loads in the extraluminal and intraluminal(mucosal)sites,but the cycles of ALA-PDT required for intraluminal sites were slightly more than the extraluminal sites.HPV infection can inhibit the expression and activation of JAK/STAT1.ALA-PDT was able to activate the pathway and promote the expression of downstream antiviral related molecule IRF-1,which was related to energy-time-concentration.At the same time,ALA-PDT up-regulated expressions of MHC-1 and MICB to improve cell antigen presentation ability and CTL effect.In addition,ST AT1 played an important role in the regulation of proliferation and apoptosis of HPV-infected cells by ALAPDT. |
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