Studies On The Effects Of PM_2.5 Exposure On The Severity Of Psoriasis And The KRT17 Related Regulation Mechanism

Part One Positive association between air pollutants and the severity of psoriasisObjective:To assess the relationship between six major air pollutants and the severity of psoriasis.Methods:A total of 254 patients with psoriasis in dermatological clinics were enrolled and scored psoriasis disease severity according to the Psoriasis area and severity index（PASI）scoring standard,including lesion area score and lesion severity score.The inverse distance weighting（IDW）method was used to assess the exposure level of individual air pollutants in the two periods before the onset of illness（lag0-30 and lag0-60 days）.The effect of air pollutant exposure on PASI score was observed using multilinear regression analysis in a generalized linear model.It was analyzed in a hierarchical manner by smoking,and different sets of covariate variables were introduced into the model for sensitivity analysis.Use principal component analysis to extract the most contributing contaminants from statistically significant contaminants for further study.Results:1.A total of 254 study subjects were included,with an age range of 8-90years among them,128 persons were under 40 years of age（50.39%）and126 aged 40 years and over（49.61%）.There were 90 males and 164 females.There were 70 cigarette smokers,accounting for 27.56%.A total of 190 cases occurred in winter and spring,accounting for 74.80%.2.Situation of air pollution:During lag0-30,the average daily exposure concentrations of PM_2.5,,PM₁₀,SO₂,NO₂,CO and O₃ were 57.17±35.62g/m³,106.60±62.54g/m³,17.50±17.98μg/m³,36.07±14.51μg/m³,0.90±0.49mg/m³and 106.99±44.52μg/m³,respectively.During lag0-60,average daily exposure concentrations of PM_2.5,PM₁₀,SO₂,NO₂,and CO and O₃ were57.56±33.71μg/m³,106.12±55.36μg/m³,17.98±18.16μg/m³,35.63±14.27μg/m³,0.91±0.47mg/m³ and 107.67±41.39 g/m³,respectively.3.The results of the single pollutant model in the two periods（lag0-30,lag0-60 days）showed a positive association between air pollutants PM_2.5,PM₁₀,CO,SO₂,NO₂ exposure concentration and PASI score（P<0.01）.Stratification by smoking showed that six air pollutants had a greater impact on psoriasis PASI score in smokers（P<0.01）.In the sensitivity analysis,the covariates in model 1 were six air pollutants,sex,age,and smoking factors;Based on these covariates in model 1 season of onset or recurrence and lesion site were added as covariates in model 2.Significant positive associations were fund between PM_2.5,PM₁₀,CO,NO₂,and SO₂ and PASI scores（P<0.01）.After season of onset or recurrence and site of lesion were added to the covariates,the results of model 2 were similar to those of model 1（P<0.01）,and the effects of various pollutants were slightly reduced.4.The principal component analysis of the six pollutants in the two periods,Lag0-30 and Lag0-60,found that the cumulative contribution of first principal component were 72.25%and 78.69%,which surpassed 70%with the eigenvalue>1.Thus to keep the first principal component,respectively.The factor loading matrix results showed that the original variables had close correlation with the first principal component.The factor score coefficient matrix showed that PM_2.5 had the highest score,during Lag0-30 and Lag0-60,0.217 and 0.205 respectively.It showed a positive association between the first principal component exposure and PASI score in the two periods,（β:1.18,95%CI:0.81,1.55）and（β:1.34,95%CI:0.95,1.73）,respectively.Summary:A positive association existed between the exposure levels of PM_2.5,PM₁₀,CO,SO₂,and NO₂ and the PASI scores.Of the six pollutants,PM_2.5may have the greatest effect on the severity of psoriasis.Part Two Effects of PM_2.5 long-term exposure on the development of psoriasis in miceObjective:To explore the the effects of PM_2.5exposure on the development of psoriasis disease by establishing the PM_2.5exposed psoriasis mouse model.Methods:Thirty C57BL/6 mice were randomly divided into three groups:healthy control mice+filtered air group（HC+FA）,psoriatic mice+filtered air group（PSO+FA）and psoriatic mice+PM_2.5 group(PSO+PM_2.5).They were exposed to filtered air or concentrated air with PM_2.5 for 8 weeks,6 hours a day,7 days a week,respectively,.The psoriasis mouse model was established by external application of imiquimod（IMQ）62.5mg/d on the dorsal skin of mice for 6 days at the 8th week and continuous exposure to PM_2.5.Morphometric and histological characteristics analysis were performed to investigate the changes of psoriasis lesion using PASI（Psoriasis area and severity index）score and H&E staining,respectively.The psoriasis pathological Baker Method score and measuring of epidermal thickness by K-Vi EWER were completed in a blinded fashion by a dermatopathologist.We graded the histopathological score on a scale from 0 to 10.Western blot was performed to assess psoriatic markers and to evaluate severity changes in psoriasis skin lesions.Results:1.It showed that PM_2.5 treatment induced more serious psoriasis characteristic,such as significant inflammatory infiltration,parakeratosis,dilated capillaries and thickened epidermis.2.Our results revealed that psoriatic mice with PM_2.5 treatment had higher PASI score and more severe psoriasis lesions.3.The pathological Baker score of skin lesions was more significantly increased in PSO+PM_2.5 group than PSO+FA group.Epidermal thickness was measured by K-Vi EWER with normal thickness observed in healthy control mice specimens approximately 16.86±1.88μm and the thickness degree of epidermal hyperplasia averaging 101.14±4.19μm and 154.85±7.52μm in PSO+FA group and PSO+PM_2.5 group,respectively.The epidermal thickness were significantly increased in the PM_2.5 exposure group compared with the psoriasis mice with filtered air group.4.Western blot showed that the expressions of S100a8 and S100a7a proteins in the skin lesion sections of psoriasis mice with PM_2.5 treatment were much higher,showing significant difference.Summary:PM_2.5 exposure can aggravate the disease severity of psoriasis model mice,suggesting that PM_2.5 exposure can exacerbate the disease progression of psoriasis.Part Three Ambient PM_2.5 aggravate the situation of psoriasis through KRT17 activation and AKT/m TOR/HIF-1αpathwayObjective:To investigate the molecular mechanism of the effect of PM_2.5exposure on psoriasis.Methods:Bioinformatics analysis was performed on the datasets of patients with psoriasis and normal populations,including differentially expressed gene analysis,functional enrichment analysis,and gene set enrichment analysis（GSEA）.The biogenesis analysis showed that KRT17was an upregulated gene in psoriasis,and the GSEA enrichment analysis pathway diagram was drawn to further explore the mechanism of the signaling pathway related to the KRT17 gene in the development of psoriasis disease.Thirty male C57BL/6 mice were randomly divided into three groups:healthy control mice+filtered air group（HC+FA）,psoriatic mice+filtered air group（PSO+FA）and psoriatic mice+PM_2.5 group(PSO+PM_2.5).Meanwhile,a KRT17 knockdown（KRT17 KD）mouse model was established by subcutaneous injection of KRT17 knockdown lentivirus.Another forty C57BL/6 mice were randomly divided into four groups:PSO+FA+KRT17-NC group,PSO+FA+KRT17-KD group,PSO+PM_2.5+KRT17-NC group and PSO+PM_2.5+KRT17-KD group.They exposed to concentrated air with PM_2.5or filtered air respectively for 8 weeks,6 hours a day,7 days a week.The psoriasis mouse model was established by external application of imiquimod（IMQ）62.5mg/d on the dorsal skin of mice for 6 days at the 8th week and continuous exposure to PM_2.5.Meanwhile,a KRT17 knockdown mouse model was established by subcutaneous injection of KRT17knockdown lentivirus.Immunofluorescence and Western blot method were used to detect KRT17expression.Ha Ca T cells were infected with KRT17 knockdown lentivirus to establish KRT17 knockdown cell model.Ha Ca T cells at 40%–60%confluence were stimulated with IL-17A（25 ng/ml）for 48 hours to establish psoriasis cell model.For the treatment of 740Y-P（AKT agonist）,culture medium containing 10μM 740Y-P was prepared,then the stably infected Ha Ca T cells were incubated with 740Y-P for 2 hr.KRT17 knockdown psoriasis cell model was established.Western blot was performed to identify the protein expression levels of AKT/m TOR/HIF-1αpathway.Results:1.It showed that KRT17 was an up-regulated m RNA in psoriasis samples and VEGF signaling pathways was associated with KRT17 in synthesis in psoriasis.2.It showed that KRT17 expression was increased by PM2.5 exposure,KRT17 positive area increased from（0.79±0.12）%to（1.73±0.24）%by immunofluorescence（P<0.01）.PM_2.5 exposure enhanced the expression of AKT/m TOR/HIF-1αpathway related proteins in psoriatic mouse skin lesions.3.The protein expression levels of AKT/m TOR/HIF-1αsignaling pathway in KRT17-KD group were diminished as compared with KRT17-NC group,It indicated that PM_2.5treatment induced AKT/m TOR/HIF-1αsignaling pathway activation and that was dependent on KRT17.4.Our results suggested that AKT signaling was involved in KRT17-induced m TOR/HIF-1α/VEGF signaling pathway activation in psoriasis.Summary:Epidermal overexpression of KRT17 in psoriasis lesion after ambient PM_2.5exposure resulted in increased disease severity and m TOR/HIF-1α/VEGF pathway activation via AKT signaling in mice.Conclusion:1.A positive association was found between the exposure levels of PM_2.5,PM₁₀,CO,SO₂,and NO₂ and the PASI scores.Of the six pollutants,PM_2.5may have the greatest effect on the severity of psoriasis.2.Disease severity increased in psoriasis mouse models after PM_2.5exposure.3.KRT17 activation and AKT/m TOR/HIF-1αsignaling pathway may play significant roles in the psoriasis deterioration induced by PM_2.5 exposure.