| BackgroundPsoriasis is a chronic inflammatory skin disorder characterized by the formation of red scaly plaques,predominantly affecting the scalp,elbows,knees,and other areas of the body.The precise pathogenesis of psoriasis remains unknown,and it is believed to be multifactorial,involving genetic,immune,infectious,and psychological factors.Psoriasis not only affects the physical appearance and quality of life of individuals but also contributes to psychological problems such as depression,anxiety,and social isolation.Conversely,psychological factors such as stress,anxiety,and depression may exacerbate psoriasis or contribute to its recurrence and worsening.However,conflicting results have been reported regarding the relationship between psychological factors and psoriasis,likely due to various confounding factors in research studies.The recent outbreak of the COVID-19 pandemic has significantly increased psychological stress,and its impact on psoriasis patients is a matter of concern.One approach that may help clarify the relationship between psychological factors and psoriasis is Mendelian Randomization(MR),which utilizes genetic instruments as instrumental variables and is based on genome-wide association analysis to explore the causal relationship between exposure and outcome factors.Using the MR method,we can further investigate the causal relationship between psychological disorders and psoriasis and enhance the reliability of the evidence.Oxidative stress is a crucial mechanism in the pathogenesis of psoriasis,characterized by an imbalance between free radicals and antioxidants in the body,leading to cellular damage and inflammatory responses.Kelch-like erythroid cell-derived protein with cap n collar homologyassociated protein 1(KEAP1)-nuclear factor erythroid-2-related factor 2(NRF2)is a sulfhydryl-based sensor-effector device that regulates antioxidant and anti-inflammatory effects and may contribute to skin homeostasis.Activation of NRF2 can impact various pathways involved in psoriasis,including proliferation and differentiation of keratinocytes.signal transduction pathways,cytokine release,and immune system stimulation.Therefore,targeting NRF2 may represent a promising therapeutic strategy for psoriasis treatment.Paeoniflorin,the active ingredient in Professor Bai Yanping’s formula for clearing heat and cooling blood,has demonstrated potent antioxidant properties by reducing the production and accumulation of reactive oxygen species and increasing antioxidant enzyme activity and content.In neurological diseases,paeoniflorin can activate the NRF2 pathway and exert antioxidant effects.Collagen peptides,small protein molecules composed of several amino acids,are one of the most critical structural proteins in the skin,promoting skin repair and acting as a moisturizer.Collagen peptides can also scavenge free radicals,protect skin cells from oxidative stress damage,and enhance the skin’s ability to absorb and retain moisture.The investigation of whether paeoniflorin and collagen peptides can activate NRF2 to treat psoriasis represents an exciting area of research,and whether the two compounds may have a synergistic effect in psoriasis treatment is a topic that warrants further investigation.Objectives1.Use Mendelian randomization to study the causal relationship between psoriasis and psychological disorders with low confounding factors.2.Compare NRF2 protein expression and MDA level in psoriasis patients’ skin lesions and healthy controls.3.Test the therapeutic effects of peonidin and collagen peptide on psoriasis in mice,and investigate their synergistic effects through NRF2 protein and oxidative stress factors.MethodsStudy One:In this study,a two-way,two-sample MR analysis was conducted to explore the potential causal relationship between psoriasis and psychological disorders,using summary statistics from genome-wide association studies(GWASs).Two GWAS datasets were selected for each disease to validate the findings,and instrumental variables in the form of singlenucleotide polymorphisms(SNP)were used to analyze the exposure and outcome factors.The data was weighted by inverse variance and various statistical methods,including Inverse variance weighted(IVW),weighted median(WM),and MR Egger’s statistics were employed to estimate the effect size and assess the horizontal pleiotropy of genes.Q-test and Leave-oneout analysis were also used to test the heterogeneity of the data and to ensure the reliability of the results.Study Two:This study enrolled 20 patients with blood fever psoriasis vulgaris and 15 healthy individuals as controls.The expression levels of NRF2 protein in blood fever psoriasis lesions and MDA levels in peripheral blood serum were measured using immunohistochemistry,and the data were compared to identify any differences between the two groups.Study Three:In this study,48 BALB/c male mice between 6 and 8 weeks of age were randomly divided into six groups,each containing eight mice.The blank control group was coated with petroleum jelly on the de-haired back,while the other groups were treated with different creams,including a cream containing 5%imiquimod,a positive control group treated with 0.025%Haxenaid solution,a paeoniflorin group treated with 1%paeoniflorin solution,a collagen peptide group treated with 3%collagen peptide solution,and a combination drug group treated with a mixture of solution containing 1%paeoniflorin and 3%collagen peptide.The mice were administered these drugs once daily for six days.On day 7,skin lesions and serum were collected from the back of the mice,and NRF2 and KEAP1 protein expression in the skin lesions were detected using immunohistochemistry.Nucleus and cytoplasm NRF2 protein expression levels in the skin lesions were measured by Western Blot,and the nucleoplasm ratio was calculated.Flow cytometry was used to measure the serum inflammatory factors in mice.ResultsStudy OneIn this study,we screened instrumental variables and obtained 40 and 46 instrumental variables for the dataset with psoriasis as an exposure factor and 7 and 9 instrumental variables for the dataset with psychological disorders as an exposure factor,respectively.Mendelian randomization analysis was performed using IVW as the primary statistical analysis method.and the results were expressed as Odds Ratios(OR)and 95%Confidence Interval(CI).The combined OR when psoriasis was used as an exposure factor was 1.34(CI 1.21-1.46,P<0.001)and 1.28(CI 1.17-1.39,P<0.001),respectively,and similar results were obtained using the WM and MR Egger’s statistics.The combined ORs were 1.23(CI 1.14-1.31,P<0.001)and 1.21(CI 1.11-1.31,P<0.001),respectively,when using mental illness as an exposure factor.The WM methods showed similar results,although some combinations did not show clear statistical significance with MR Egger(P>0.05),the direction of effect remained consistent with the other models.Multiple sensitivity analyses were performed,and the results of the MR analysis were supported,indicating the robustness and reliability of the results.Study TwoThe staining intensity of NRF2 protein did not differ significantly between the patient group and the healthy control group,but NRF2 in the skin tissue of healthy control patients showed more pronounced nuclear aggregation.MDA levels were significantly higher in the serum of patients with psoriasis(25.62 ± 0.39 μmol/L)compared with healthy controls(12.04± 0.72 μmol/L),with statistically significant differences in the correlation test(P<0.001)..Study Three1.In terms of lesion changes,mice in the model group showed typical psoriasis-like changes,such as erythema,hypertrophy,and scaling,etc.The erythema,hypertrophy,and scaling scores were reduced in each medication group compared with the model group.In terms of PASI scores,the erythema,hypertrophy,scaling scores and total scores of each drug group were significantly lower than those of the model group(P<0.05).Among them,the PASI score was most significantly improved in the Haxenaid solution group,followed by the combination drug group,the paeoniflorin group and the collagen group(P<0.05).2.Regarding pathological changes,mice in the model group showed typical psoriasis-like changes,including hyperkeratosis,echinoderm hypertrophy,and prolonged epidermal protrusions,etc.Epidermal hyperkeratosis and echinoderm hypertrophy in each medication group were less severe than those in the model group.Regarding the epidermal thickness of the mice,the model group was significantly higher than the blank control group(125.95±56.98μm vs.20.75 ± 1.60 μm,P<0.001),and the epidermal thickness of the paeoniflorin group,collagen peptide group,combined drug and positive control group were 76.69±9.86 μm,78.78± 9.90 μm,64.60 ± 6.07 μm,and 53.88 ± 6.13 μm,which were significantly lower than those of the model group(P<0.05).Compared with the combined drug group,the epidermal thickness of mice in the positive control group was significantly reduced(P<0.01),and that of mice in the paeoniflorin and collagen peptide groups was significantly increased(P<0.05).3.The immunohistochemical results showed that KEAP1 was more commonly expressed in the cytoplasm of the dermis in all groups of mice.While in the epidermis layer,the expression of KEAP1 was little in the mice of the blank control group,while the expression of epidermal KEAP1 protein was significantly increased in the model group,and the expression of epidermal KEAP1 was decreased in all the dosing groups.Compared with the blank control group,epidermal KEAP1 was significantly reduced in the model group(P<0.001),and epidermal KEAP1 was significantly reduced in each drug administration group compared with the model group,and the difference was statistically significant(P<0.001).Epidermal KEAP1 was significantly reduced in the combined drug group compared with the paeoniflorin group and collagen group(P<0.001)and significantly increased compared with the positive control group(P<0.05).The expression of NRF2 in the epidermis was detected in the skin tissues of all groups of mice,among which,the nuclear expression of NRF2 protein was reduced in the model group mice compared with the blank control group,while the nuclear expression of NRF2 was increased in each drug group mice,which may be related to the migration and activation of NRF2 protein after drug administration.4.Western Blot results showed that the expression of NRF2 in Nucleus(NRF2N)was significantly reduced in the model group compared with the blank control group,and the ratio of NRF2 in Cytoplasm(NRF2C)was significantly lower compared with the blank group(P<0.001),while the nuclear expression and nucleoplasmic ratio of NRF2 protein were significantly lower in all other drug groups.The nuclear expression and nucleoplasmic ratio of NRF2 protein in all other drug groups were significantly higher than those in the model group(P<0.001).The nuclear expression and nucleoplasmic ratio of NRF2 protein in the paeoniflorin and collagen groups were significantly lower compared with the combined drug group(P<0.05),and the nuclear expression of NRF2 protein in the positive control group was significantly higher than that in the combined drug group(P<0.01),but the nucleoplasmic ratio in the positive control group was higher than that in the combined drug group,but the two groups did not show statistical differences(P>0.05).5.Oxidative stress-related marker assay showed that the MDA level of mice in the model group was significantly higher than that in the blank group(P<0.001),and the MDA level in the paeoniflorin group,collagen group,combined drug group and positive control group was significantly lower than that in the model group(P<0.001).MDA was significantly lower in the combination group than in the paeoniflorin and collagen groups(P<0.001),but did not show statistical differences compared with the positive control group(P>0.05).As for the antioxidant-related factors,the levels of SOD,CAT and GSH in the model group were significantly lower than those in the blank group(P<0.001),and the expression levels of SOD,CAT and GSH in the paeoniflorin group,collagen group,combined drug group and positive control group were significantly higher than those in the model group(P<0.05).The SOD,CAT,and GSH levels of mice in the combined drug group were significantly lower than those in the positive control group(P<0.05),but significantly higher than those in the paeoniflorin group as well as the collagen group(P<0.05).Neither paeoniflorin nor collagen peptide showed statistical differences in the regulation of the expression of MDA,SOD,GSH and CAT(P>0.05).6.As for serum inflammatory factors,the levels of TNF-α,IL-6,IL-17a and IL-23 were significantly higher in the model group than in the blank control group(P<0.001),while the levels of TNF-α,IL-6,IL-17a and IL-23 were significantly lower in each drug group and the positive control group(P<0.001).The levels of TNF-α,IL-6,IL-17a,and IL-23 were significantly lower in the mice of the combined dosing group compared with those of the paeoniflorin and collagen groups(P<0.01),and the serum inflammatory factor levels in the mice of the combined dosing group did not show statistical differences compared with those of the positive control group(P>0.05).Conclusions1.There is a bidirectional causal association between psoriasis and psychological disorders,and the psychological stress caused by the new crown epidemic may adversely affect the condition of psoriasis patients.2.Paeoniflorin and collagen peptide can play a therapeutic role in psoriasis through antioxidation and anti-inflammation,and their mechanism may be related to the regulation of KEAP1/NRF2 signaling pathway.Both showed synergistic effects in antioxidant,antiinflammatory as well as anti-psoriasis. |
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