Abstract Psoriasis is a common chronic immune-mediated inflammatory skin disorder. Sophora flavescens Alt. (S. flavescens) has been widely acknowledged in the prevention and treatment of psoriasis. Kushenol F (KSCF) is a natural isopentenyl flavonoid extracted from the root of S. flavescens. We aimed to investigate the effect and mechanism of KSCF on imiquimod (IMQ)-induced psoriasis-like skin lesions in mice. A mouse model of psoriasis was induced with 5% IMQ for 5 days, and the mice were given KSCF dermally for 5 days. Changes in skin morphology, the psoriasis area, the severity index (PASI), and inflammatory factors of psoriasis-like skin lesions were evaluated. Metabolites in the psoriasis-like skin lesions were analyzed with ultra-high-performance liquid chromatography/mass spectrometry followed by a multivariate statistical analysis to identify the differential metabolites and metabolic pathway. The results of the present study confirmed that KSCF significantly reduced PASI scores, epidermal thickening, and epidermal cell proliferation and differentiation. KSCF also reduced the levels of interleukin (IL)-1β, IL-6, IL-8, IL-17A, IL-22, IL-23, and tumor necrosis factor (TNF)-α in the injured skin tissues while increasing IL-10 content. KSCF significantly regulated metabolites in the skin samples, and a total of 161 significant metabolites were identified. These differential metabolites involved sphingolipid and linoleic acid metabolism and steroid hormone biosynthesis. Collectively, KSCF inhibited the inflammatory response to prevent IMQ-induced psoriasis-like skin lesions in mice by call-backing the levels of 161 endogenous metabolites and affecting their related metabolic pathways. KSCF has the potential to be developed as a topical drug for treating psoriasis symptoms. Keywords: inflammation, Kushenol F, psoriasis, metabolomics, Sophora flavescens 1. Introduction Psoriasis is a chronic and systemic immune-mediated disease. The underlying pathomechanisms involve an interplay between the innate and adaptive immune systems [[38]1,[39]2]. Recently, psoriasis presents many challenges, including high prevalence, chronicity, and associated comorbidity [[40]3]. As a representative inflammatory skin disease occupied by large surface involvement, the occurrence and development of psoriasis is reported to be related to the disorder of many inflammation markers, such as IL-17A, IL-23, and TNF-α. Biologics targeting IL-17A, IL-23, and TNF-α have been developed and approved for the treatment of psoriasis [[41]4]. The progression of psoriasis has also been confirmed to be related to immunity [[42]5,[43]6,[44]7]. Common treatments for psoriasis include topical agents, systemic therapy, and ultraviolet light therapy. Topical agents mainly include calcipotriol (CVD), corticosteroids, and salicylic acid. Methotrexate, cyclosporine, and acitretin are commonly used as systemic therapy for psoriasis [[45]8,[46]9]. Although the therapeutic effects of these drugs have been widely recognized, adverse reactions still largely limit their clinical utilization [[47]10]. Hence, complementary and alternative medicine may offer options for some patients. Sophora flavescens Alt. (S. flavescens) is widely used in the clinic, can clear dampness-heat, kill insects, and dispel wind evil, which originates from the teachings of traditional Chinese medicine [[48]11]. S. flavescens has been clinically used in numerous countries to treat skin diseases [[49]12,[50]13]. Kushenol F (KSCF) is a natural isopentenyl flavonoid extracted from the root of S. flavescens. It has been reported to have anti-inflammatory and anti-bacterial properties [[51]14,[52]15,[53]16]. A previous study demonstrated that KSCF oral treatment could be an important therapeutic for treating atopic dermatitis. Although various studies have reported the biological effects of KSCF, the effect and mechanism of KSCF on psoriasis are unclear. Metabolomics can reveal the specific changes in physiological and biochemical states related to phenotypes in biological systems. The application of skin metabonomics is suitable for revealing the potential functional changes in a variety of metabolic pathways and signal networks in psoriasis-like organisms, which has become a significant approach to evaluate the efficacy and mechanism of drugs [[54]17]. The present study investigated the effects of KSCF on imiquimod (IMQ)-induced psoriasis-like skin lesions in mice using conventional pharmacology and metabolomics. The potential biomarkers and complex mechanisms of KSCF involved in psoriasis treatment are also discussed. The results demonstrated that KSCF inhibited the inflammatory response to prevent IMQ-induced psoriasis-like skin lesions in mice by call-backing the levels of 161 endogenous metabolites and affecting their related metabolic pathways. Thus, KSCF has the potential to be developed as a topical drug for treating psoriasis symptoms. 2. Results and Discussion 2.1. KSCF Mitigated the Psoriatic Dermatitis Induced by IMQ Psoriasis is a skin disease with characteristics of well-demarcated, erythematous, raised lesions with silvery-white dry scales. As depicted in [55]Figure 1, after 5 days of IMQ treatment, erythema and silvery-white dry scales were observed on the dorsal skin, and the PASI was significantly increased (p < 0.001). However, after treatment with CVD and KSCF, the thickness and size of the scales on the dorsal skin were decreased. Erythema and PASI were also significantly decreased (p < 0.001). These results indicate that KSCF effectively relieved IMQ-induced psoriasis symptoms. Figure 1. [56]Figure 1 [57]Open in a new tab Effect of KSCF on psoriatic dermatitis induced by IMQ. KSCF mitigated skin lesions on the back (A) and decreased PASI values (B) in IMQ-induced mice. CVD, calcipotriol; H-KSCF, high-dose KSCF group; M-KSCF, medium-dose KSCF group; L-KSCF, low-dose KSCF group. ^# vs. control group; * vs. model group; ^### p < 0.001; ***p < 0.001. 2.2. Effect of KSCF on Inflammatory Reactions in Psoriasis-like Mice Psoriasis is a chronic inflammatory skin disease characterized by lymphocyte and neutrophil infiltration and the excessive keratinization and proliferation of keratinocytes [[58]18,[59]19]. The agents capable of attenuating keratinocyte hyperproliferation and excessive inflammatory responses are considered to be potentially useful for psoriasis treatment [[60]20,[61]21]. IMQ-induced psoriasis is usually accompanied by cellular inflammatory factor disorders in the dorsal skin. As shown in [62]Figure 2A, IMQ induced massive inflammatory cell infiltration in the dorsal skin, which was reduced by CVD and KSCF treatment. As shown in [63]Figure 2B, after 5 days of IMQ treatment, the levels of IL-1β, IL-6, IL-8, IL-17A, IL-22, IL-23, and TNF-α significantly increased in the dorsal skin (p < 0.05), whereas IL-10 levels significantly decreased (p < 0.0001). After treatment with CVD and KSCF, the levels of IL-1β, IL-6, IL-8, IL-17A, IL-22, IL-23, and TNF-α significantly decreased (p < 0.05) and IL-10 levels significantly increased (p < 0.0001). These results indicated that KSCF might stimulate the secretion of anti-inflammatory cytokines while inhibiting the release of pro-inflammatory cytokines, thereby alleviating IMQ-induced psoriasis. Figure 2. [64]Figure 2 [65]Open in a new tab Effect of KSCF on inflammatory reactions in psoriasis−like skin lesions in mice. KSCF reduced inflammatory cell infiltration in the dorsal skin (A) and regulated the secretion of inflammatory cytokines (B). CVD, calcipotriol group; H-KSCF, high-dose KSCF group; M-KSCF, medium-dose KSCF group; L-KSCF, low-dose KSCF group. ^# vs. control group; * vs. model group; ^#### p < 0.0001, ^### p < 0.001, ^## p < 0.01; **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. 2.3. Effect of KSCF on Metabolites in the Injured Skin of Psoriasis-like Mice The data of skin samples were collected with ultra-high-performance liquid chromatography/mass spectrometry (UHPLC/MS), and the metabolic profiles of each group were obtained. As depicted in [66]Supplementary Figure S1, the peak number and intensity significantly varied in the total ion current profiles of the control, model, CVD, and KSCF groups, indicating significant differences among the metabolic state of each group and endogenous metabolites. Thus, the endogenous metabolites of skin changed significantly after IMQ induction and administration. In this study, PCA and OPLS-DA were used to analyze metabolome differences in the dorsal skin between the control, model, and KSCF groups using an unsupervised statistical method. The metabolic state of the control and model groups was different ([67]Figure 3 and [68]Figure 4A). The KSCF group was far from the model group. The results demonstrated that KSCF effectively reversed the pathological changes triggered by IMQ treatment in psoriasis-like mice. Figure 3. [69]Figure 3 [70]Open in a new tab PCA score plots of psoriasis−like skin lesions in mice. I, positive ion mode; II, negative ion mode; KSCF, Kushenol F. Figure 4. [71]Figure 4 [72]Open in a new tab OPLS−DA score plots of psoriasis−like skin lesion samples. (A) OPLS−DA score plots. (B) Permutation test plot. I, positive ion mode; II, negative ion mode; KSCF, Kushenol F. Two hundred iteration permutation tests were performed on OPLS-DA in the positive and negative ion modes to further illustrate the reliability of the OPLS-DA model. The Q^2 values were less than 0.05, further confirming the accuracy of the multivariate statistical analysis ([73]Figure 4B). 2.4. Identification of Differential Metabolites Changes in the metabolites can reflect the physiological and pathological states of the human body and might serve as an objective index to determine the efficacy and mechanism of drugs during disease intervention [[74]22]. The complete metabolic information was collected, and the mechanism of KSCF against IMQ-induced psoriasis, which involved the regulation of endogenous metabolites, was revealed using the UHPLC/MS technique. S-plot diagrams based on OPLS-DA analysis were plotted to obtain information on differential metabolites between the model group and the KSCF group. Substances with a VIP of >1 and p-value of <0.05 were selected as biomarkers ([75]Figure 5). A total of 161 potential biomarkers were detected in the dorsal skin, of which 102 metabolites were up-regulated, and 59 metabolites were down-regulated ([76]Table 1; their chemical structures are shown in [77]Supplementary Figure S2). These results suggested that KSCF might regulate these differential metabolites to relieve IMQ-induced psoriasis. Figure 5. [78]Figure 5 [79]Open in a new tab S−Plot of the model and KSCF administration group. I, positive ion mode; II: negative ion mode; red, VIP ≥ 1; green, VIP < 1. Table 1. Differential metabolites identified from psoriatic skin lesion samples. Retention Time (min) Molecular Weight (Da) VIP Potential Biomarker Formula Change Trend ESI− 4.843 308.124 2.604 Glutaminyltyrosine C[14]H[19]N[3]O[5] DOWN 6.468 453.202 2.054 2-Hydroxy-imipramine glucuronide C[25]H[32]N[2]O[7] UP 6.183 581.300 2.676 Serylarginine C[9]H[19]N[5]O[4] UP 2.626 263.076 1.478 3,4,5-trihydroxy-6-[(2-methylpropanoyl)oxy]oxane-2-carboxylic acid C[10]H[16]O[8] DOWN 2.821 374.095 2.080 7-Hydroxygliclazide C[15]H[21]N[3]O[4]S DOWN 2.829 299.070 1.075 Methionyl-Glutamate C[10]H[18]N[2]O[5]S DOWN 4.270 315.163 2.005 3-[8-hydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H-chromen-5-yl]propan oic acid C[19]H[24]O[4] DOWN 4.791 165.059 1.301 3-Mercapto-2-methyl-1-butanol C[5]H[12]OS DOWN 6.240 480.249 2.770 Dynorphin A (6–8) C[18]H[37]N[9]O[4] UP 6.278 457.197 2.666 Deoxypyridinoline C18H28N[4]O[7] UP 6.308 469.161 2.372 Melleolide B C[24]H[32]O[7] UP 6.308 667.171 1.090 Hydroxychlorpromazine C[17]H[19]ClN[2]OS UP 6.347 455.181 2.248 Armillane C[23]H[32]O[7] UP 6.378 437.170 2.123 Nevskin C[24]H[32]O[5] UP 6.431 177.023 1.656 3,3′-Thiobispropanoic acid C[6]H[10]O[4]S UP 6.442 295.031 2.518 Cis,trans-5′-Hydroxythalidomide C[13]H[10]N[2]O[5] UP 6.456 421.175 2.033 Euglobal IIb C[23]H[30]O[5] UP 6.462 465.202 1.909 Homofukinolide C[25]H[34]O[6] UP 6.493 486.187 1.533 4-Hydroxyvalsartan C[24]H[29]N[5]O[4] UP 5.583 451.185 1.071 7-[(2,6-dihydroxy-2,5,5,8a-tetramethyl-decahydronaphthalen-1-yl)methoxy ]-6-hydroxy-2H-chromen-2-one C[24]H[32]O[6] UP 6.601 467.217 2.471 Colupdox a C[25]H[36]O[6] UP 6.630 553.297 1.078 Phytolaccinic acid C[31]H[48]O[6] DOWN 6.969 492.117 2.575 2-amino-4-({1-[(carboxymethyl)-C-hydroxycarbonimidoyl]-2-[(2,3-dihydrox y-1-phenylpropyl)sulfanyl]ethyl}-C-hydroxycarbonimidoyl)butanoic acid C[19]H[27]N[3]O[8]S UP 7.023 609.361 1.208 Lactapiperanol C C[16]H[26]O[4] DOWN 7.434 810.583 1.023 PE(18:0/20:0) C[43]H[86]NO[8]P DOWN 7.480 606.500 2.048 1,1′-(1,4-Dihydro-4-nonyl-3,5-pyridinediyl)bis[1-dodecanone] C[38]H[69]NO[2] DOWN 8.936 453.202 1.579 Colupox a C[25]H[36]O[5] UP 8.915 701.421 1.880 PG(a-13:0/a-15:0) C[34]H[67]O[10]P UP 8.846 465.314 1.130 (2β,3α,9α,24R)-Ergosta-7,22-diene-2,3,9-triol C[28]H[46]O[3] DOWN 7.712 865.522 1.182 PG(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) C[46]H[77]O[10]P DOWN 7.365 820.589 1.091 PE(18:1(11Z)/22:1(13Z)) C[45]H[86]NO[8]P DOWN 7.157 463.293 2.768 Palmitoyl glucuronide C[22]H[42]O[7] DOWN 7.057 361.174 0.993 Perindoprilat C[17]H[28]N[2]O[5] DOWN 6.541 379.218 2.167 3′-Hydroxystanozolol C[21]H[32]N[2]O[2] DOWN 6.475 439.185 2.144 Lucidone A C[24]H[34]O[5] UP 6.456 423.154 2.463 5,7-dihydroxy-4-(1-hydroxypropyl)-6-(3-methylbut-2-en-1-yl)-8-(2-methyl butanoyl)-2H-chromen-2-one C[22]H[28]O[6] UP 6.451 455.217 2.656 Cavipetin D C[25]H[38]O[5] UP 6.431 475.163 2.153 α-CEHC glucuronide C[22]H[30]O[10] UP 6.378 500.167 2.330 2-amino-4-({1-[(carboxymethyl)-C-hydroxycarbonimidoyl]-2-{[4-(2,6-dihyd roxyphenyl)-3-hydroxy-2-methylbutan-2-yl]sulfanyl}ethyl}-C-hydroxycarbo nimidoyl)butanoic acid C[21]H[31]N[3]O[9]S UP 6.371 453.165 2.610 Diosbulbin H C[23]H[30]O[7] UP 6.354 439.186 1.945 1α-O-Methylquassin C[23]H[32]O[6] UP 6.347 507.174 2.465 L-Nicotianine C[10]H[12]N[2]O[4] UP 6.284 520.193 2.082 Hydroxynefazodone C[25]H[32]C[l]N[5]O[3] UP 6.240 452.218 1.926 Chondroitin sulfate E (GalNAc4,6diS-GlcA), precursor 5a C[7]H[9]N[3]O UP 6.222 369.211 1.248 3-keto-Digoxigenin C[23]H[32]O[5] DOWN 6.218 476.279 1.123 LysoPE(0:0/18:2(9Z,12Z)) C[23]H[44]NO[7]P DOWN 6.134 457.160 1.105 Valechlorin C[22]H[31]C[l]O[8] UP 5.718 371.226 2.685 Digoxigenin C[23]H[34]O[5] DOWN 5.114 348.156 1.934 (+)-O-Methylarmepavine C[20]H[25]NO[3] DOWN 5.065 329.179 2.313 12,20-Dioxo-leukotriene B4 C[20]H[28]O[5] DOWN 4.974 357.211 1.896 17-Hydroxy-E4-neuroprostane C[22]H[32]O[5] DOWN 4.589 357.222 1.037 Kinetensin 1-3 C[15]H[30]N[6]O[4] UP 2.829 365.143 1.110 Valproic acid glucuronide C[14]H[24]O[8] DOWN 2.814 296.106 1.004 7-Aminonitrazepam C[15]H[13]N[3]O DOWN 1.685 295.040 1.398 N-Acetyldjenkolic acid C[9]H[16]N[2]O[5]S[2] UP 1.527 183.044 1.218 Mevalonic acid C[6]H[12]O[4] UP 6.371 383.161 1.501 21-Hydroxy-5b-pregnane-3,11,20-trione C[21]H[30]O[4]DOWN 6.278 581.263 1.973 S-(3-Methylbutanoyl)-dihydrolipoamide-E C[13]H[25]NO[2]S[2] UP 8.922 689.351 1.131 Zeranol C[18]H[26]O[5] UP ESI+ 6.444 453.200 3.257 Pro Ser Ser Tyr C[20]H[28]N[4]O[8] UP 4.727 828.534 0.976 3-O-Sulfogalactosylceramide (d18:1/18:1(9Z)) C[42]H[79]NO[11]S DOWN 3.285 404.222 1.210 Ser Ala Ala Arg C[15]H[29]N[7]O[6] UP 4.253 317.177 1.919 Capillartemisin A C[19]H[24]O[4] DOWN 5.069 331.193 2.827 16-Hydroxy-4-oxoretinoic acid C[20]H[26]O[4] DOWN 6.224 371.225 1.960 17-phenyl trinor-13,14-dihydro Prostaglandin A2 C[23]H[30]O[4] DOWN 6.254 395.252 1.328 (3R, 6′Z)-3,4-Dihydro-8-hydroxy-3-(6-pentadecenyl)-1H-2-benzopyran-1-one C[24]H[36]O[3] UP 6.304 565.350 1.002 PG(22:2(13Z,16Z)/0:0) C[28]H[53]O[9]P UP 6.324 313.077 2.457 Inosine-5′-carboxylate C[11]H[12]N[4]O[7] UP 7.031 626.533 1.139 Muricatenol C[37]H[68]O[6] UP 6.338 301.077 2.967 Temazepam C[16]H[13]C[l]N[2]O[2] UP 1.543 286.157 2.191 Tryptophyl-Valine C[16]H[21]N[3]O[3] UP 3.194 298.150 1.266 Histidinyl-Threonine C[10]H[16]N[4]O[4] DOWN 3.319 281.119 1.191 6-hydroxy-7E,9E-Octadecadiene-11,13,15,17-tetraynoic acid C[18]H[16]O[3]UP 4.064 534.323 1.347 Cys Lys Lys Arg C[21]H[43]N[9]O[5]S DOWN 4.235 255.129 1.186 Isoleucyl-Threonine C[10]H[20]N[2]O[4] DOWN 4.305 622.377 1.446 Tyr Arg Lys Arg C[27]H[47]N[11]O[6] DOWN 4.474 277.124 1.256 Isobutyrylcarnitine C[11]H[22]NO[4]^+ UP 4.604 386.246 1.473 Pseudoginsenoside RT3 C[41]H[70]O[13] DOWN 4.654 549.172 1.514 Myricatomentoside I C[26]H[32]O[10] DOWN 4.987 359.225 1.475 7,8-epoxy-17S-HDHA C[22]H[30]O[4] DOWN 5.118 696.452 1.502 PA(16:1(9Z)/15:0) C[34]H[65]O[8]P DOWN 5.170 828.533 1.338 PE(MonoMe(11,3)/MonoMe(11,3)) C[43]H[74]NO[10]P DOWN 5.994 242.134 0.972 Lysyl-Methionine C[11]H[23]N[3]O[3]S UP 6.016 548.287 1.511 11-Hydroxyprogesterone 11-glucuronide C[27]H[38]O[9]UP 6.082 465.259 1.132 (−)-Jolkinol B C[29]H[36]O[5] UP 6.136 502.163 2.001 3-(3-hydroxyphenyl)-2-(4-hydroxyphenyl)-5-[(E)-2-(3hydroxyphenyl)etheny l]-2,3-dihydro-1 benzofuran-6-ol C[28]H[22]O[5] UP 6.149 467.275 1.316 1,25-Dihydroxyvitamin D3-26,23-lactone C[27]H[40]O[5] UP 6.205 363.225 1.097 5α-Pregnan-20α-ol-3-one C[21]H[34]O[2] UP 6.224 453.221 1.813 Australigenin C[27]H[42]O[4] UP 6.283 534.221 1.608 Cyclocalopin D C[23]H[32]O[13] UP 6.297 365.241 1.267 Adipostatin A C[21]H[36]O[2] UP 6.324 574.253 1.565 Glaucarubolone 15-O-β-d-glucopyranoside C[26]H[36]O[13] UP 6.360 427.276 1.508 Asymmetric dimethylarginine C[8]H[18]N[4]O[2] UP 6.360 455.179 3.227 Gly Glu Ser Tyr C[19]H[26]N[4]O[9] UP 6.367 441.200 4.192 Gly Thr Tyr Thr C[19]H[28]N[4]O[8] UP 6.416 435.294 2.760 Alanyl-Lysine C[9]H[19]N[3]O[3] UP 6.437 527.345 2.800 Pro Val Arg Arg C[22]H[42]N[10]O[5] UP 6.451 355.125 2.024 Meta-O-Dealkylated flecainide C[15]H[19]F[3]N[2]O[3]UP 6.472 463.182 2.851 PA(8:0/8:0) C[19]H[37]O[8]P UP 6.479 441.200 3.151 (9S,10S)-10-hydroxy-9-(phosphonooxy)octadecanoate C[18]H[37]O[7]P UP 6.500 714.509 1.058 PA(18:1(9Z)/18:3(9Z,12Z,15Z)) C[39]H[69]O[8]P UP 6.521 523.411 2.648 Panaxydol linoleate C[35]H[54]O[3] DOWN 6.604 411.225 1.903 Pro Pro Thr Pro C[19]H[30]N[4]O[6] UP 6.646 722.519 1.386 PE(18:4(6Z,9Z,12Z,15Z)/P-18:1(11Z)) C[41]H[72]NO[7]P UP 6.660 678.492 1.312 PE(18:4(6Z,9Z,12Z,15Z)/P-16:0) C[39]H[70]NO[7]P UP 6.674 634.465 1.471 Tsugarioside B C[37]H[60]O[7] UP 6.856 670.523 1.605 PE(15:0/18:0) C[38]H[76]NO[8]P UP 7.010 904.712 1.184 PC(o-20:0/22:0) C[50]H[102]NO[7]P UP 7.066 407.153 1.119 3b,16a-Dihydroxyandrostenone sulfate C[19]H[28]O[6]S UP 8.944 425.205 4.129 O-Desmethylcarvedilol C[23]H[24]N[2]O[4] UP 9.287 423.189 2.131 Cys Val Thr Thr C[16]H[30]N[4]O[7]S UP 8.685 895.754 1.939 TG(18:1(11Z)/22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)) C[61]H[102]O[6] DOWN 6.940 539.360 1.721 3-methyl-4-(methylamino)-1,2-diphenylbutan-2-ol C[18]H[23]NO UP 6.870 626.496 1.675 PE(15:0/P-16:0) C[36]H[72]NO[7]P UP 6.870 614.353 1.399 LysoPC(22:2(13Z,16Z)) C[30]H[58]NO[7]P UP 6.849 714.550 1.546 PC(14:1(9Z)/18:0) C[40]H[78]NO[8]P UP 6.828 802.604 1.578 PC(18:0/22:4(7Z,10Z,13Z,16Z)) C[48]H[88]NO[8]P UP 6.737 696.496 1.312 CL(i-12:0/18:2(9Z,11Z)/i-18:0/i-19:0) C[76]H[144]O[17]P[2] UP 6.730 740.523 1.185 CL(18:0/18:0/16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) C[83]H[148]O[17]P[2] UP 6.646 840.578 1.939 PG(18:0/22:4(7Z,10Z,13Z,16Z)) C[46]H[83]O[10]P DOWN 6.604 163.076 1.060 4-Hydroxy-2,6,6-trimethyl-3-oxo-1,4-cyclohexadiene-1-carboxaldehyde C[10]H[12]O[3] UP 6.590 295.066 1.816 2-hydroxy-3-[3-(3-methylbut-2-en-1-yl)-4-(sulfooxy)phenyl]propanoic acid C[14]H[18]O[7]S UP 6.583 459.187 2.136 Ser Glu Ser His C[17]H[26]N[6]O[9] UP 6.542 355.194 1.101 Nigakihemiacetal B C[22]H[30]O[6] DOWN 6.493 764.516 1.715 CL(16:0/18:0/18:0/22:5(7Z,10Z,13Z,16Z,19Z)) C[83]H[152]O[17]P[2] DOWN 6.472 700.493 1.413 PA(15:0/18:2(9Z,12Z)) C[36]H[67]O[8]P UP 6.472 385.136 3.206 2′,7-Dihydroxy-4′-methoxy-8-prenylflavan C[21]H[24]O[4] UP 6.465 351.130 1.754 N-Ribosylhistidine C[11]H[17]N[3]O[6] UP 6.451 521.396 1.727 Ginsenoyne A linoleate C[35]H[52]O[3] DOWN 6.388 285.081 1.654 (2S)-2-hydrazinyl-3-(4-hydroxy-3-methoxyphenyl)-2-methylpropanoic acid C[11]H[16]N[2]O[4] DOWN 6.381 439.184 3.069 Gln Glu Tyr C[19]H[26]N[4]O[8] UP 6.345 375.115 1.271 Cyclic N-Acetylserotonin glucuronide C[18]H[20]N[2]O[8] DOWN 6.338 361.135 2.023 7-Hydroxy-6-methyl-8-ribityl lumazine C[12]H[16]N[4]O[7] UP 6.324 538.255 1.523 Rubraflavone B C[30]H[34]O[5] UP 6.311 618.283 2.136 3′-Deaminofusarochromanone C[15]H[19]NO[4] UP 6.297 424.221 2.812 Sphingosine 1-phosphate C[18]H[38]NO[5]P UP 6.290 508.280 2.746 LysoPE(0:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) C[27]H[44]NO[7]P UP 6.283 510.296 4.312 LysoPE(0:0/22:5(7Z,10Z,13Z,16Z,19Z)) C[27]H[46]NO[7]P UP 6.276 583.277 3.375 (4E)-6-hydroxy-1,7-diphenylhept-4-en-3-one C[19]H[20]O[2] UP 6.241 451.279 0.944 PA(19:1(9Z)/0:0) C[22]H[43]O[7]P UP 6.231 498.259 3.159 PE(18:3/0:0) C[23]H[42]NO[7]P UP 6.199 437.264 1.065 1-Oleoyl Lysophosphatidic Acid (sodium salt) C[21]H[41]O[7]P UP 6.186 175.123 2.811 α-Methyltryptamine (AMT) C[11]H[14]N[2] UP 6.174 569.298 3.370 11-α-O-β-d-Glucopyranosyl-16α-O-methylneoquassin C[29]H[44]O[11] UP 6.075 487.359 1.085 Lys Ile Val Lys C[23]H[46]N[6]O[5] UP 6.005 618.340 1.903 Pentasine C[30]H[49]N[6]O[10]^+ UP 6.000 349.209 1.127 Oryzalexin E C[20]H[32]O[2] UP 5.554 183.082 1.526 2-Methyl-4-propyl-1,3-oxathiane C[8]H[16]OS UP 5.112 740.479 1.500 PS(14:0/20:3(8Z,11Z,14Z)) C[40]H[72]NO[10]P DOWN 5.006 240.142 0.993 Isopropyl β-d-glucoside C[9]H[18]O[6] UP 4.945 336.130 0.976 Gynocardin C[12]H[17]NO[8] DOWN 4.897 352.161 1.890 (+/−)-Rollipyrrole C[16]H[20]N[2]O[3] DOWN 4.615 338.145 1.145 N-Acetyl-3-hydroxyprocainamide C[15]H[23]N[3]O[3] DOWN 4.391 233.097 1.094 L-Targinine C[7]H[16]N[4]O[2] UP 3.611 402.242 1.022 N-Didesmethylmifepristone (RU 42848) C[27]H[31]NO[2]DOWN 3.396 261.150 1.327 Methyl dihydrophaseate C[16]H[24]O[5] UP 3.222 229.123 1.660 Blennin B C[15]H[20]O[4] UP 2.566 267.140 1.527 (S)-17-Hydroxy-9,11,13,15-octadecatetraynoic acid C[18]H[20]O[3] UP 1.360 217.134 1.518 Girgensonine C[13]H[16]N[2]O DOWN 0.013 300.208 1.040 2-hydroxydesipramine C[18]H[22]N[2]O UP 6.800 524.283 1.270 11-β-Hydroxyandrosterone-3-glucuronide C[25]H[38]O[9] UP [80]Open in a new tab 2.5. Metabolic Pathway Analysis The differential metabolites were subjected to a pathway enrichment analysis to explore the potential metabolic pathways of KSCF exerting medicinal effects. [81]Figure 6 shows the path influence diagrams of the metabolic pathway analysis. Pathways with a p-value of <0.05 were considered KSCF-involved pathways. The difference between the model and KSCF groups was clearly reflected in three pathways ([82]Table 2), which are mainly related to lipid metabolism and steroidogenic activity. Figure 6. [83]Figure 6 [84]Open in a new tab Pathway analysis of KSCF treatment. Table 2. Metabolic pathway of psoriatic skin lesion samples. No. Pathway p-Value Details 1 Sphingolipid metabolism 0.001 KEGG 2 Linoleic acid metabolism 0.036 KEGG 3 Steroid hormone biosynthesis 0.021 KEGG [85]Open in a new tab Sphingolipids have structural and biological functions in the human epidermis, are importantly involved in the maintenance of the skin barrier, and regulate cellular processes, such as the proliferation, differentiation, and apoptosis of keratinocytes [[86]23,[87]24]. In this study, 3-O-sulfogalactosylceramide (d18:1/18:1(9Z)) and sphingosine 1-phosphate were identified as metabolites with significant differences after modeling and drug administration, suggesting that these key metabolites could be targets for KSCF against IMQ-induced psoriasis. Therefore, it was inferred that KSCF improved IMQ-induced psoriasis by regulating sphingolipid metabolism pathways. Abnormal linoleic acid metabolism has been shown to be a key pathway in psoriasis. The combination of a moisturizer containing a linoleic acid–ceramide complex and glucocorticoids was shown to significantly improve the therapeutic efficacy of psoriasis and delay recurrence [[88]25]. Our results confirmed that the levels of PC (14:1(9Z)/18:0) and PC (18:0/22:4(7Z,10Z,13Z,16Z)) were close to the normal group after KSCF administration. These results suggested that KSCF improved IMQ-induced psoriasis by regulating the linoleic acid metabolism pathways. The skin has an endogenous steroidogenic capacity, and modulating local steroidogenic activity may be a new approach to treating inflammatory skin diseases [[89]26,[90]27]. In this study, 5α-Pregnan-20α-ol-3-one and 21-Hydroxy-5b-pregnane-3,11,20-trione were identified as metabolites with significant differences after modeling and drug administration. After KSCF administration, steroid hormone biosynthesis was similar to the normal group, suggesting that KSCF treatment affected steroid hormone biosynthesis to relieve IMQ-induced psoriasis. Our results indicated that KSCF inhibited the inflammatory response to prevent IMQ-induced psoriasis-like skin lesions in mice by call-backing the levels of endogenous metabolites and affecting their related metabolic pathways ([91]Figure 7). It is very likely that the flavonoid extract from S. flavescens also shows the therapeutic effects on psoriasis because it contains many active compounds, such as KSCF, which is worthy of further study. The deep molecular mechanism of KSCF regulating endogenous metabolites in three key pathways is also worthy to be further explored by detecting the key proteins in the pathways. It is reported that another flavonoid (kurarinone) and alkaloids (matrine and oxymarine) isolated from S. flavescens regulated the inflammatory response to intervene in psoriasis [[92]7,[93]28,[94]29]. However, the differences in the efficacy and mechanism of these compounds in the psoriasis treatment and the relationship between the chemical structures of them and their anti-psoriasis activity are still unclear, which is worthy of further study. Figure 7. [95]Figure 7 [96]Open in a new tab KSCF regulated endogenous metabolites in three key pathways against IMQ-induced psoriasis. 1, steroid hormone biosynthesis; 2, sphingolipid metabolism; 3, linoleic acid metabolism. Red, up. 3. Materials and Methods 3.1. Chemicals, Reagents, and Materials KSCF was provided from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). CVD was purchased from A&M Pharmaceuticals (Hong Kong, China). Hematoxylin stain was obtained from Wuhan Google Biotechnology Co., Ltd. (Wuhan, China). Vaseline for medical use was purchased from Shandong Lircon Medical Technology Co., Ltd. (Dezhou, China). IMQ was acquired from Sichuan MED-SHINE Pharmaceutical Co., Ltd. (Chengdu, China). Four percent paraformaldehyde solution was provided from Beijing lanjieke Technology Co., Ltd. (Beijing, China). IL-6, IL-8, IL-1β, IL-10, IL-22, IL-23, IL-17A, and TNF-α ELISA Kits were purchased from Jiangsu Meimian Industrial Co., Ltd. (Yancheng, China). The reference substance of KSCF (purity > 98%) was purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). High purity deionized water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). HPLC-grade formic acid, methyl alcohol, and acetonitrile were acquired from Fischer Control Equipment International Co., Ltd. (Hong Kong, China). H&E Staining kits were purchased from Beijing Solarbio Science&Technology Co., Ltd. (Beijing, China). Seventy-five percent ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents used were of analytical reagent grade or higher. 3.2. Animals and Experimental Protocol All experimental procedures in this study complied with the National Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Yunnan University of Chinese Medicine, with ethics number R062021158. Healthy male BALB/c mice (20 ± 2 g), aged 6–8 weeks, were obtained from Sipeifu Biotechnology Co., Ltd. (Beijing, China). The mice were kept in the Laboratory of Yunnan University of Traditional Chinese Medicine (Kunming, China), following standard housing conditions. All mice were adaptively fed for one week. The mice were randomized into six groups (n = 6 mice per group), including control group (75% ethanol), model group (75% ethanol), positive group (CVD, 62.5 mg/kg), low-dose KSCF group (L-KSCF, 200 mg/kg), medium-dose KSCF group (M-KSCF, 400 mg/kg), and high-dose KSCF group (H-KSCF, 600 mg/kg). Except for the control group, mice were topically treated with 5% IMQ cream at a daily dose of 62.5 mg for a duration of 5 days on their dorsal region. KSCF and CVD were dissolved in 75% ethanol. Starting from the day of modeling, the corresponding drugs were sprayed continuously for 5 days (0.3 mL/day/mouse) in the administration group. Seventy-five percent ethanol (0.3 mL/day/mouse) was sprayed for the control group and the model group. 3.3. Psoriasis Area and Severity Index (PASI) Assessment According to clinical PASI score standard, the severity of the skin inflammation was evaluated once daily, including the measurements for skin erythema, scaling, and thickening. Erythema, scaling, and thickening were scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The cumulative score (erythema plus scaling plus thickening) served as a measure of the severity of inflammation (scale 0–12) [[97]30]. 3.4. Image Acquisition and Skin Sample Collection After euthanizing the mice through carbon dioxide asphyxiation, images were captured on the dorsal side of each mouse group. Then, the dorsal skin of the mice was carefully excised using surgical scissors and divided into two sections. One section was immediately immersed in liquid nitrogen for cryopreservation, while the other section was fixed in 4% paraformaldehyde universal tissue fixative. 3.5. Histopathology Analyses The dorsal skin of mice was fixed in 4% paraformaldehyde universal tissue fixative for 24 h, washed with PBS, dehydrated with gradient ethanol and transparentized with xylene, and embedded in paraffin. After hematoxylin and eosin staining, histological parameters were observed under a light microscope, and images were taken at 200× magnification. 3.6. Measurement of Skin Inflammatory Factors Approximately 50 mg of injured skin from the dorsal region of each mouse were weighed and placed on ice. The surface blood stains on the skin samples were rinsed with pre-cooled normal saline solution. After air-drying the filter paper, the skin samples were promptly sectioned into pieces and transferred into a covered 2 mL centrifuge tube. The tube was then subjected to centrifugation (Centrifuge 5430R, Eppendorf, Germany) at 1500× g for 15 min, and subsequently, the supernatant of the homogenized tissue was collected for further utilization. The levels of cytokines IL-1β, IL-6, IL-8, IL-10, IL-17A, IL-22, IL-23, and TNF-α in the dorsal skin lesions of all groups of mice were detected according to the kit manufacturer’s instructions. The values were determined by measuring the absorbance value (OD) at 450 nm using a microplate reader (Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China). 3.7. Metabolomic Analysis 3.7.1. Sample Pre-Treatment Skin samples: 50 mg skin sample and a 6 mm diameter grinding bead was added to a 2 mL centrifuge tube. An amount of 400 μL of extraction solution (methanol: water = 4:1 (v:v)) was used for metabolite extraction. Samples were ground with Wonbio-96c frozen tissue grinder (Shanghai Wanbo Biotechnology Co., Ltd., Shanghai, China) for 6 min (−10 °C, 50 Hz) followed by low-temperature ultrasonic extraction for 30 min (5 °C, 40 kHz). Then, the sample was allowed to stand for 30 min at −20 °C and centrifuged for 15 min (4 °C, 3500× g). The supernatant was transferred to a clean tube and dried gently with nitrogen. The residues were redissolved in 200 µL of methanol for UHPLC/MS analysis. Quality control sample: A random injection sequence was employed to detect signal fluctuation. An amount of 2 μL of skin samples were taken and thoroughly mixed. Then, the mixture was centrifuged at 3500× g for 15 min at 4 °C. The supernatant was collected to perform UHPLC/MS analysis. 3.7.2. UHPLC/MS Analysis UHPLC analyses were performed using Ultimate 3000 Ultra High Performance Liquid Chromatography (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a Thermo Scientific Hypersil GOLD column (50 mm × 2.1 mm, 1.9 μm). The column temperature was maintained at 40 °C to obtain better sample separation. The mobile phases consisted of water containing 0.1% formic acid (A) and acetonitrile (B) at a 0.4 mL/min of flow rate. The gradient program was set as follows: 0–3.5 min, 0% B → 24.5% B; 3.5–5 min, 24.5% B → 65% B; 5–7.4 min, 65% B → 100% B; 7.4–7.6 min, 100% B → 51.5% B; 7.6–10 min, 51.5% B → 0% B. The MS data were collected using a Thermo Scientific Q-Exactive TM hybrid quadrupole-orbitrap mass spectrometer with a heated electrospray ionization probe (Thermo Fisher Scientific, San Jose, CA, USA). MS conditions were as follows: CUR, 15 psi; Gas1 and Gas2, 50 psi; IS, 5500 V; gas temperature, 500 °C. 3.8. Statistical Analysis All UHPLC/MS raw files were exported in comma-separated value (CSV) format using the Progenesis QI (Waters Corporation, Milford, CT, USA) software. All data were uploaded to XCMS online for peak alignment, normalization, and retrieval. The data were analyzed with principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) using the SIMCA-P 14.1 software (Sweden, Umeå, Umetrics). The potential biomarkers were selected according to the parameters of variable importance in the projection (VIP > 1 and p < 0.05) from OPLS-DA. The structural information of the metabolites was identified using the HMDB databases. Finally, the metabolic pathways were searched using Kyoto Encyclopedia of Genes and Genomes (KEGG) through MetaboAnalyst 5.0 online, and the pathways with an impact value greater than 0.1 were considered KSCF-involved pathways. All experimental data were presented as means ± S.D. Each experiment was performed in triplicate. The overall significance of the results was examined with one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Kolmogorov–Smirnov test was used for the pre-test of the analysis of ANOVA to ensure the rationality of the statistical analysis. Student’s t-test delivered the p-values shown. The differences between the compared groups were considered statistically significant at p < 0.05, p < 0.01, p < 0.001, p < 0.0001. 4. Conclusions KSCF inhibited the inflammatory response to prevent IMQ-induced psoriasis-like skin lesions in mice by call-backing the levels of 161 endogenous metabolites and affecting the three related metabolic pathways, including sphingolipid and linoleic acid metabolism and steroid hormone biosynthesis. Thus, KSCF has the potential to be developed as a topical drug for treating psoriasis. Supplementary Materials The following supporting information can be downloaded at: [98]https://www.mdpi.com/article/10.3390/molecules29112410/s1, Figure S1: Representative total ion current profiles of the skin samples in the negative and positive ion mode; Figure S2: The chemical structures of 161 potential biomarkers. [99]molecules-29-02410-s001.zip^ (1.3MB, zip) Author Contributions Z.C. and J.C. wrote the manuscript. J.C., X.Y. (Xunqing Yin) and T.A. performed the experiments. Z.C., Y.Y. and X.H. provided technical support and helpful discussions. Y.Y. and X.Y. (Xingxin Yang) wrote and modified the manuscript. X.Y. (Xingxin Yang) and Y.L. designed the study. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All experimental procedures in this study complied with the National Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Yunnan University of Chinese Medicine, with ethics number R062021158. Informed Consent Statement Not applicable. Data Availability Statement Data are available upon request. Conflicts of Interest The authors declare that they have no competing interests. Funding Statement This study was supported by grants from the National Natural Science Foundation of China (Grant numbers 82174037, 82060707, 82104381, and 81960710) and the Application and Basis Research Project of Yunnan China (Grant numbers 202001AV070007, 202001AZ070001-006, 202201AW070016, 2019IB009 and 202101AY070001-077). Footnotes Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References