OBJECTIVES: Keratoconus (KC) is a progressive corneal ectasia disorder, arising from a myriad of causes including genetic predispositions, environmental factors, biomechanical influences, and inflammatory reactions. This study aims to identify potential pathogenetic gene mutations in patients with sporadic KC in the Han Chinese population. METHODS: Twenty-five patients with primary KC as well as 50 unrelated population-matched healthy controls, were included in this study to identify potential pathogenic gene mutations among sporadic KC patients in the Han Chinese population. Sanger sequencing and whole-exome sequencing (WES) were used to analyze mutations in the zinc finger protein 469 (ZNF469) gene. Bioinformatics analysis was conducted to explore the potential role of ZNF469 in KC pathogenesis. RESULTS: Five novel heterozygous missense variants were identified in KC patients. Among them, 2 compound heterozygous variants, c.8986G>C (p. E2996Q) with c.11765A>C (p. D3922A), and c.4423C>G (p. L1475V) with c.10633G>A (p. G3545R), were determined to be possible pathogenic factors for KC. CONCLUSIONS Mutations in the ZNF469 gene may contribute to the development of KC in the Han Chinese population. These mutation sites may provide valuable information for future genetic screening of KC patients and their families.
Adolescent ; Adult ; Female ; Humans ; Male ; Case-Control Studies ; China/ethnology* ; Exome Sequencing ; Genetic Predisposition to Disease ; Keratoconus/genetics* ; Mutation ; Mutation, Missense ; Transcription Factors/genetics* ; East Asian People/genetics*

ObjectiveBased on transcriptomics， to explore the mechanism of Hei Xiaoyaosan regulating the phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway to improve the learning and memory ability of insomnia rats with liver depression syndrome. MethodsSixty 8-week-old male SD rats were randomly divided into the blank group， model group， eszopiclone group （0.09 mg·kg^-1）， and low， medium， and high dose groups of Hei Xiaoyaosan （3.82， 7.65， 15.30 g·kg^-1）， with ten rats in each group. Except for the blank group， the other groups were induced insomnia rat model with liver depression by chronic restraint， tail clamping stimulation and intraperitoneal injection of p-chlorophenylalanine （PCPA）. Each treatment group received intragastric administration according to the specified dosage， once a day for 14 consecutive days. The pentobarbital sodium cooperative sleep test， open field test， and Morris water maze test were used to test the sleep quality， depressive-like behavior， and learning and memory abilities of rats. Additionally， enzyme-linked immunosorbent assay （ELISA） was used to detect the contents of 5-hydroxytryptamine （5-HT）， γ-aminobutyric acid （GABA）， brain-derived neurotrophic factor （BDNF）， glial cell line-derived neurotrophic factor （GDNF） and nitric oxide （NO） in hippocampus. Hematoxylin-eosin （HE） staining was performed to observe pathological changes of the hippocampal tissue， while terminal deoxynucleotidyl transferase deoxyuridine triphosphate （dUTP） nick end labeling （TUNEL） was used to evaluate apoptosis of hippocampal neurons. Transcriptomic sequencing technology was employed to identify differentially expressed genes in hippocampus between the model group and the blank group， as well as between the medium-dose group of Hei Xiaoyaosan and the model group. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis were performed on the intersecting genes. Subsequently， the enriched key genes and signaling pathways were analyzed and verified. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was utilized to assess the mRNA expression levels of phosphatase and tensin homolog （PTEN）， B-cell lymphoma-2 （Bcl-2）-like protein 11 （BCL2L11）， and mitogen-activated protein kinase 1 （MAPK1） in hippocampus， and Western blot was employed to evaluate the protein expressions of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， Bcl-2， Bcl-2-associated X protein （Bax）， and cleaved Caspase-3 in the same tissue. ResultsCompared with the blank group， the model group exhibited a reduction in body weight， an increase in sleep latency， and a decrease in sleep duration （P<0.01）. Additionally， rats showed obvious depression-like behavior， and their learning and memory abilities decreased. Furthermore， the contents of 5-HT， GABA， NO， BDNF and GDNF in hippocampus decreased （P<0.01）. Histological examination revealed a disorganized cell arrangement in the CA1 region of the hippocampus， characterized by irregular cell shapes， a reduced cell count， deeply stained and pyknotic nuclei， increased vacuolar degeneration， and an elevated apoptosis rate （P<0.01）. Compared with the model group， the body weight of the high and medium dose groups of Hei Xiaoyaosan increased， the sleep latency shortened and the sleep time prolonged （P<0.05， P<0.01）. Additionally， depression-like behavior and learning and memory abilities of rats were significantly improved， the levels of 5-HT， GABA， NO， BDNF and GDNF in the hippocampus increased （P<0.05， P<0.01）. These interventions also ameliorated pathological damage in the hippocampal CA1 area and reduced the apoptosis of hippocampal neurons （P<0.01）. Transcriptomic sequencing results indicated that Hei Xiaoyaosan might exert a therapeutic effect by regulating PI3K/Akt pathway through key mRNAs such as PTEN， BCL2L11， and MAPK1. The roles of these key mRNAs and proteins within PI3K/Akt pathway were further validated. In comparison to the blank group， the expression levels of PTEN， BCL2L11 and MAPK1 mRNA in the hippocampus of rats in the model group were increased （P<0.01）， while the protein expression levels of p-PI3K， p-Akt and Bcl-2 were decreased （P<0.01）， and the protein expression levels of PTEN， Bax and cleaved Caspase-3 were increased （P<0.01）. Compared with the model group， the high-dose and medium-dose groups of Hei Xiaoyaosan could down-regulate the expressions of PTEN， BCL2L11 and MAPK1 mRNAs （P<0.01）， up-regulate the expressions of p-PI3K， p-Akt and Bcl-2 proteins （P<0.01）， and down-regulate the protein expressions of PTEN， Bax and cleaved Caspase-3 （P<0.05， P<0.01）. ConclusionHei Xiaoyaosan may regulate PI3K/Akt signaling pathway by down-regulating expressions of key genes such as PTEN， BCL2L11 and MAPK1， and thus improve the learning and memory abilities of insomnia rats with liver depression syndrome.

ObjectiveTo establish the quality standards for Sennae Folium（Cassia angustifolia） dispensing granules based on standard decoctions. MethodsHigh performance liquid chromatography（HPLC） specific chromatograms were established for 15 batches of Sennae Folium（C. angustifolia） standard decoctions and 10 of Sennae Folium（C. angustifolia） dispensing granules from different manufacturers， and the similarity evaluation， hierarchical cluster analysis（HCA） and principal component analysis（PCA） were performed. Linear calibration with two reference substances（LCTRS） and quantitative analysis of multi-components by single-marker（QAMS） were established for the common peaks in the specific chromatograms to determine the contents of main components in the decoction pieces， standard decoctions and dispensing granules， and to calculate their transfer rates from decoction pieces to standard decoctions and dispensing granules. ResultsThe similarities of specific chromatograms of 15 batches of Sennae Folium（C. angustifolia） standard decoctions and 10 batches of Sennae Folium（C. angustifolia） dispensing granules were all greater than 0.95， and a total of 8 characteristic peaks were calibrated， and five of them were identified， including kaempferol-3，7-O-diglucoside， apigenin-6，8-di-C-glucoside， quercetin-3-O-gentianoside， sennoside B and sennoside A. HCA and PCA results showed that there were certain differences in the composition of different batches of standard decoctions， but no clustering was observed in the production area. As the standard decoctions， the extract rate of 15 batches of samples was 26.54%-45.38%， the contents of kaempferol-3，7-O-diglucoside， apigenin-6，8-di-C-glucoside， quercetin-3-O-gentianoside， sennoside B and sennoside A were 12.16-19.26， 2.57-4.94， 3.27-5.11， 6.75-11.39， 4.69-7.79 mg·g^-1， and their transfer rates from decoction pieces to standard decoctions were 45.41%-79.02%， 29.12%-55.07%， 40.52%-67.90%， 24.72%-49.12%， 27.54%-49.34%， respectively. The extract rates of Sennae Folium（C. angustifolia） dispensing granules（C8-C10） were 38.10%-39.50%， the transfer rates of the above five components from decoction pieces to dispensing granules were 72.85%-73.58%， 53.43%-53.94%， 40.19%-40.74%， 24.62%-25.00%， 28.65%-29.11%， respectively， which were generally consistent with the transfer rates from decoction pieces to standard decoctions. ConclusionCompared with the relative retention time method， LCTRS has higher prediction accuracy and is more suitable for chromatographic columns. The established quality control standard of Sennae Folium（C. angustifolia） dispensing granules based on standard decoction is reasonable and reliable， and all indicators of samples from different manufacturers are within the range specified based on the standard decoction， which can provide reference for the quality control and process research of this dispensing granules.

OBJECTIVE To evaluate the clinical efficacy and safety of Jianwei xiaoshi oral liquid in the treatment of functional dyspepsia （FD） in children， and provide evidence-based basis for clinical use of the drug. METHODS Retrieved from CNKI， VIP， Wanfang， CBM， Cochrane Library and PubMed， randomized controlled trials （RCTs） about Jianwei xiaoshi oral liquid in the treatment of FD in children were collected from the inception to Apr. 2024. The control group was treated with conventional western drugs （including gastrointestinal motion-promoting， antacids or acid-suppressing drugs）， and the trial group was treated with Jianwei xiaoshi oral liquid alone or combined with conventional Western drugs （drug dosage and course of treatment were the same as the control group）. Meta-analysis was performed using RevMan 5.3 software after quality evaluation with the Cochrane risk bias assessment tool 6.1. RESULTS Totally 16 literature were employed which included 1 962 patients. The results of meta-analysis showed that the total clinical effective rate of Jianwei xiaoxi oral liquid in the treatment of FD in children of trial group was significantly higher than that of the control group [RR＝1.18， 95%CI （1.13， 1.22）， P＜0.000 01]. In this study， subgroup analysis was conducted on the usage and dosage， course of treatment， and combination or not in trial group， as well as the type of conventional Western drugs. The results showed that the total clinical effective rate of trial group was significantly higher than that of control group； the relief time of abdominal distension and abdominal pain in trial group [MD＝－2.54， 95%CI （－3.10， －1.98）]， loss of appetite relief time [MD＝－2.12， 95%CI （－2.63， －1.61）]， nausea and vomiting relief time [MD＝－1.70， 95%CI （－2.27， －1.14）]， and belching relief time [MD＝－1.61， 95%CI （－2.44， －0.78）] were shorter than that of the control group significantly （P＜0.05）. In addition， compared with control group， the levels of gastrin [SMD＝1.63， 95%CI （0.98， 2.29）] and motilin [SMD＝2.06， 95%CI （1.58， 2.54）] as well as gastric antral emptying rate [MD＝5.99， 95%CI （2.78， 9.21）] in trial group were increased significantly， while the level of somatostatin was decreased significantly [SMD＝－1.30， 95%CI （－1.57， －1.02）] （P≤0.000 3）. CONCLUSIONS Jianwei xiaoshi oral liquid， whether used alone or in combination with other medications， and regardless of the treatment course or dosage and usage， is effective in treating FD in children， with good safety.

ObjectiveTo establish the specific chromatogram and quantitative analysis of multi-components by single-marker（QAMS） based on linear calibration using two reference substances（LCTRS）， explore the consistency between Hedyotis Herba dispensing granules and standard decoction， and evaluate the quality of the dispensing granules. MethodsHigh performance liquid chromatography（HPLC） specific chromatogram was established based on 15 batches of Hedyotis Herba standard decoction and 10 batches of the dispensing granules， and LCTRS was used to locate chromatographic peaks. The actual retention times of 7 characteristic peaks in the specific chromatogram was measured on 24 different types of C₁₈ columns， taking deacetyl asperulosidic acid and asperulosidic acid as the dual standard compounds， the retention times of the other 5 characteristic peaks were predicted and validated. Based on this， QAMS was developed to determine the contents of four components（deacetyl asperulosidic acid， deacetyl asperulosidic acid methyl ester， asperulosidic acid， and p-coumaric acid）. Then， the relative correction factors of deacetyl asperulosidic acid， deacetyl asperulosidic acid methyl ester and p-coumaric acid were calculated using the reference peak of asperulosidic acid in the dual standard compounds， and each component was quantified accordingly. Finally， the consistency between the dispensing granules and standard decoction was assessed by taking extract rate of the standard decoction， consistency of the specific chromatograms， contents and transfer rates of the indicator components as indexes， and the quality of the dispensing granules was evaluated. ResultsThere were 7 common peaks in the characteristic chromatogram of samples of Hedyotis Herba standard decoction and the dispensing granules， and four of them were identified by reference standards， namely deacetyl asperulosidic acid（peak 1）， deacetyl asperulosidic acid methyl ester（peak 3）， asperulosidic acid（peak 6） and p-coumaric acid（peak 7）. The similarity between the dispensing granules and the standard decoction was >0.9. The absolute deviation in the predicted retention time for each component by LCTRS was lower than that of the relative retention time method. The extract rate of the 15 batches of Hedyotis Herba standard decoction ranged from 7.89% to 14.60%， the contents of deacetyl asperulosidic acid， deacetyl asperulosidic acid methyl ester， asperulosidic acid and p-coumaric acid were 6.62-19.70， 3.83-17.99， 1.57-6.69， 1.62-4.52 mg·g^-1， and the transfer rates of these components from decoction pieces to the standard decoction were 22.89%-39.60%， 34.03%-62.24%， 24.25%-43.70%， and 40.58%-73.71%， respectively. The extract rate， index component contents and transfer rates from decoction pieces to the three batches of Hedyotis Herba dispensing granules（P1-P3）， produced by manufacturer A， were similar to those of the standard decoction prepared from the same batch of decoction pieces， and all fell within the specified range. The contents of the 4 indicator components in 7 batches of the dispensing granules（P4-P10） from manufacturers B-E were all within the range of the content converted from the standard decoction based on the quantity of the dispensing granules. ConclusionThe established specific chromatogram and QAMS based on LCTRS are reasonable and reliable. Based on the evaluation indicators of standard decoction yield， consistency of specific chromatograms， contents and transfer rates of the four index components， the 10 batches of Hedyotis Herba dispensing granules from various manufacturers have exhibited good consistency with the standard decoction， indicating that the current production process is relatively reasonable.

ObjectiveTo establish the specific chromatogram of Chuanxiong Rhizoma dispensing granules（CRdg）， and to evaluate its quality by chemometrics and two reference substances for determination of multiple components（TRSDMC）. MethodsHigh performance liquid chromatography（HPLC） specific chromatograms were established using 13 batches of CRdg from 7 manufacturers， and preliminary quality evaluation was performed by similarity evaluation and chemometrics analysis. Eight characteristic peaks in the specific chromatogram of CRdg were measured on 22 different types of C₁₈ columns， and the actual retention times were recorded. Taking chlorogenic acid（peak 1） and senkyunolide A（peak 8） as double standard compounds， the retention times of the eight characteristic peaks were predicted by linear calibration using two reference substances（LCTRS）， and the method was validated on three other columns of different brands. Taking chlorogenic acid as reference peak， the relative correction factor method（RCFM） was used to quantify cryptochlorogenic acid， caffeic acid， ferulic acid， senkyunolide I and senkyunolide A， and the results were compared with the external standard method（ESM）. ResultsThe similarities of specific chromatograms of 13 batches of CRdg were all >0.90， and a total of 8 characteristic peaks were calibrated， and six of them were identified， including chlorogenic acid（peak 1）， cryptochlorogenic acid（peak 2）， caffeic acid（peak 3）， ferulic acid（peak 5）， senkyunolide I（peak 6） and senkyunolide A（peak 8）. Through chemometric analysis， it was found that ferulic acid， chlorogenic acid， senkyunolide I and cryptochlorogenic acid were the main components causing quality difference in CRdg， and the accuracy of LCTRS in predicting the retention time of 8 characteristic peaks was superior to that of the relative retention time method（RRT）. Further comparison of the results obtained from RCFM and ESM showed that there was no statistically significant difference between the two methods. ConclusionA quality evaluation method for CRdg based on HPLC specific chromatogram and TRSDMC is established， its qualitative accuracy is better than that of RRT， the quantitative accuracy is similar to that of ESM， and 4 quality-differentiated components among different manufacturers are found. This method is stable and reliable， and has reference value for the quality evaluation of other dispensing granules.

ObjectiveTo establish the specific chromatogram of Chuanxiong Rhizoma dispensing granules（CRdg）， and to evaluate its quality by chemometrics and two reference substances for determination of multiple components（TRSDMC）. MethodsHigh performance liquid chromatography（HPLC） specific chromatograms were established using 13 batches of CRdg from 7 manufacturers， and preliminary quality evaluation was performed by similarity evaluation and chemometrics analysis. Eight characteristic peaks in the specific chromatogram of CRdg were measured on 22 different types of C₁₈ columns， and the actual retention times were recorded. Taking chlorogenic acid（peak 1） and senkyunolide A（peak 8） as double standard compounds， the retention times of the eight characteristic peaks were predicted by linear calibration using two reference substances（LCTRS）， and the method was validated on three other columns of different brands. Taking chlorogenic acid as reference peak， the relative correction factor method（RCFM） was used to quantify cryptochlorogenic acid， caffeic acid， ferulic acid， senkyunolide I and senkyunolide A， and the results were compared with the external standard method（ESM）. ResultsThe similarities of specific chromatograms of 13 batches of CRdg were all >0.90， and a total of 8 characteristic peaks were calibrated， and six of them were identified， including chlorogenic acid（peak 1）， cryptochlorogenic acid（peak 2）， caffeic acid（peak 3）， ferulic acid（peak 5）， senkyunolide I（peak 6） and senkyunolide A（peak 8）. Through chemometric analysis， it was found that ferulic acid， chlorogenic acid， senkyunolide I and cryptochlorogenic acid were the main components causing quality difference in CRdg， and the accuracy of LCTRS in predicting the retention time of 8 characteristic peaks was superior to that of the relative retention time method（RRT）. Further comparison of the results obtained from RCFM and ESM showed that there was no statistically significant difference between the two methods. ConclusionA quality evaluation method for CRdg based on HPLC specific chromatogram and TRSDMC is established， its qualitative accuracy is better than that of RRT， the quantitative accuracy is similar to that of ESM， and 4 quality-differentiated components among different manufacturers are found. This method is stable and reliable， and has reference value for the quality evaluation of other dispensing granules.

Objective To construct a conceptual framework and methodology for developing disability eligibility/standard using the bio-psycho-social model of disability,and classification theory and methods of the International Classification of Functioning,Disability and Health(ICF).Methods Based on the ICF's bio-psycho-social model,and aliged with the United Nations Convention on the Rights of Persons with Disabilities and the World Health Organization technical documents in the fields of disability statis-tics and services,this study explored the systematic implementation of ICF to construct a conceptual framework and methodology for disability eligibility/standards,including core concepts and terminology of functioning and disability,methods and system of classification and grading,coding system,standardization of disability survey questionnaires and protocols,and assessment/evaluation tools,as well as standard system of disability data.Results The establishment of disability standards should be based on specific application contexts and functions.Stan-dards for surveys and statistics emphasized data collection and analysis,applied to the design of survey question-naires and methodologies.Standards for service eligibility focused on determining service qualification through structured assessments/evaluation of functioning and disability.Comprehensive standards served both purposes.A well-rounded disability standard integrated a clear theoretical framework,scientific classification,grading and coding systems,and standardized disability reporting and evaluation/assessment methods/tools.The conceptual framework and methodological system for disability standards based on ICF comprised five core aspects:a core conceptual and terminological system for disability;disability classification and grading methods and systems;a scientifically structured classification,grading,and coding system;standardized disability reporting and assess-ment/evaluation methods and procedures;and standardized disability data systems.Applying ICF in practice in-volved systematically integrating the theoretical framework into the development of disability classification and grading systems,constructing coding systems using ICF,implementing standardized disability reporting and as-sessment/evaluation tools derived from the ICF,designing and standardizing disability survey instruments,ques-tionnaires/protocols,and assessment/evaluation tools and protocols,and establishing ICF-based disability data standards.Conclusion ICF provides a robust conceptual framework and methodology for the development of disability eligibility/standards.The bio-psycho-social model,conceptual framework and methodological approaches of classification,grading and coding of functioning and disability,and ICF-based standardized survey and assessment/evaluation tools,enable the establishment of core disability concepts and terminology,disability classification and grading systems,disability coding systems,disability survey questionnaires and protocols and assessment/evaluation tools and programs,and disability data standards.The systematic application of the ICF framework and method-ology enhances the scientific rigor,inclusivity and applicability of disability eligibility/standards.

Objective:To explore the mechanisms of action of Huanglian Jiedu decoction combined with Xijiao Dihuang decoction (HLJDT-XJDH) in regulating fibroblasts in the treatment of psoriasis. Methods:A mouse model of psoriasis was established by topical application of imiquimod 5% cream on the shaved back; HLJDT-XJDH at different doses of 7.7 and 30.6 g/kg was administered via gavage for intervention, and methotrexate (2 mg/kg) served as a positive control; after 7 days, the severity of skin lesions was assessed using the psoriasis area and severity index (PASI), while histopathological changes of skin tissues were evaluated using hematoxylin-eosin (HE) staining and Baker scoring. For in vitro experiments, fibroblasts were divided into a control group, a model group, a low-dose (5% drug-containing serum) intervention group, and a high-dose (20% drug-containing serum) intervention group; cells in the control group were cultured with 20% normal rat serum for 24 hours; in the model group, cells cultured with 20% normal rat serum were stimulated with 5 ng/ml tumor necrosis factor (TNF) -α and 50 ng/ml interleukin (IL) -17A for 24 hours to mimic fibroblasts during the occurrence of psoriasis; cells in the low- and high-dose intervention groups received the same stimulation as the model group, and were cultured for 24 hours with 5% and 20% HLJDT-XJDH-containing serum, respectively, but not with the 20% normal rat serum. After the above treatment, these cells were co-cultured with keratinocytes (HaCaT cells) using a Transwell system. In addition, on the basis of the control group, fibroblasts were divided into the model group, 20% drug-containing serum intervention group, and 20% drug-containing serum intervention + OE-SFRP2 group; TNF-α and IL-17A were used to stimulate the cells to simulate the psoriatic state; the treatment in the 20% drug-containing serum intervention group was carried out as previously described; in the 20% drug-containing serum intervention + OE-SFRP2 group, cells were transfected with the vector for 48 hours to establish an overexpression model, followed by culture with 20% drug-containing serum for 24 hours, without co-culture with HaCaT cells.. Cell counting kit-8 (CCK-8) assay was performed to assess cell viability, flow cytometry to measure apoptosis rates, enzyme-linked immunosorbent assay (ELISA) to detect levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) as well as chemokine ligand (CXCL) 1 and CXCL12 in mouse serum or cell culture supernatant, qPCR to determine the mRNA expression of inflammatory cytokines, chemokines, cell cycle- and proliferation-related factors, as well as SFRP2 in mouse skin tissues or cells, and Western blot analysis to determine the protein expression of SFRP2, Wnt3a, and β-catenin in fibroblasts. One-way analysis of variance was employed for intergroup comparisons, and post-hoc analysis was conducted using Tukey's test. Results:In vivo mouse experiments showed that compared with the normal control group, the model group exhibited typical psoriatic characteristics in skin morphology, including significant inflammatory infiltration in skin tissues and marked epidermal thickening; compared with the normal control group, the serum levels of TNF-α (531.16 ± 28.27 pg/ml vs. 239.58 ± 10.39 pg/ml), IL-1β (111.40 ± 5.16 pg/ml vs. 80.35 ± 3.87 pg/ml), and IL-6 (109.17 ± 4.84 pg/ml vs. 71.73 ± 2.04 pg/ml) significantly increased in the model group, along with their mRNA expression levels in mouse skin tissues (all P < 0.001) ; compared with the model group, the treatment group showed alleviated psoriatic manifestations, and significant reductions in the levels of inflammatory factors TNF-α (low-dose, high-dose, and positive control groups: 420.80 ± 29.30 pg/ml, 322.33 ± 9.40 pg/ml, 322.97 ± 12.16 pg/ml, respectively), IL-1β (98.69 ± 4.49 pg/ml, 89.02 ± 1.56 pg/ml, 88.88 ± 2.08 pg/ml, respectively), and IL-6 (94.07 ± 3.76 pg/ml, 80.54 ± 3.30 pg/ml, 83.21 ± 3.18 pg/ml, respectively), as well as in their mRNA expression levels (all P < 0.001). In in vitro fibroblast experiments, compared with the control group, the model group exhibited a significant elevation in the supernatant levels of IL-1β (126.42 ± 3.56 pg/ml vs. 34.81 ± 0.44 pg/ml), IL-6 (459.44 ± 9.35 pg/ml vs. 115.51 ± 7.26 pg/ml), CXCL1 (2 434.88 ± 127.63 pg/ml vs. 762.85 ± 30.60 pg/ml) and CXCL12 (3 542.14 ± 35.86 pg/ml vs. 2 095.86 ± 45.12 pg/ml), the expression levels of their mRNAs (all P < 0.001), as well as the protein expression levels of SFRP2, Wnt3a, and β-catenin; after intervention with HLJDT-XJDH-containing serum, all the above indices significantly decreased (all P < 0.001). However, when 20% drug-containing serum intervention was administered simultaneously, the expression of inflammatory factors and chemokines in fibroblasts was significantly higher in the SFRP2 overexpression group than in the non-overexpression group (all P < 0.01). When fibroblasts were co-cultured with HaCaT cells, the model group showed significantly increased cell viability but a decreased apoptosis rate of HaCaT cells compared with the control group, while the low- and high-dose intervention groups showed significantly decreased cell viability but increased apoptosis rates of HaCaT cells compared with the model group (all P < 0.05) . Conclusion:HLJDT-XJDH may exert therapeutic effects in psoriasis by downregulating the SFRP2/Wnt/β-catenin signaling pathway, thereby inhibiting fibroblast activation and inflammatory process, which subsequently suppresses the proliferation of keratinocytes and the activation of inflammatory cells.

Objective:To explore the mechanisms of action of Huanglian Jiedu decoction combined with Xijiao Dihuang decoction (HLJDT-XJDH) in regulating fibroblasts in the treatment of psoriasis. Methods:A mouse model of psoriasis was established by topical application of imiquimod 5% cream on the shaved back; HLJDT-XJDH at different doses of 7.7 and 30.6 g/kg was administered via gavage for intervention, and methotrexate (2 mg/kg) served as a positive control; after 7 days, the severity of skin lesions was assessed using the psoriasis area and severity index (PASI), while histopathological changes of skin tissues were evaluated using hematoxylin-eosin (HE) staining and Baker scoring. For in vitro experiments, fibroblasts were divided into a control group, a model group, a low-dose (5% drug-containing serum) intervention group, and a high-dose (20% drug-containing serum) intervention group; cells in the control group were cultured with 20% normal rat serum for 24 hours; in the model group, cells cultured with 20% normal rat serum were stimulated with 5 ng/ml tumor necrosis factor (TNF) -α and 50 ng/ml interleukin (IL) -17A for 24 hours to mimic fibroblasts during the occurrence of psoriasis; cells in the low- and high-dose intervention groups received the same stimulation as the model group, and were cultured for 24 hours with 5% and 20% HLJDT-XJDH-containing serum, respectively, but not with the 20% normal rat serum. After the above treatment, these cells were co-cultured with keratinocytes (HaCaT cells) using a Transwell system. In addition, on the basis of the control group, fibroblasts were divided into the model group, 20% drug-containing serum intervention group, and 20% drug-containing serum intervention + OE-SFRP2 group; TNF-α and IL-17A were used to stimulate the cells to simulate the psoriatic state; the treatment in the 20% drug-containing serum intervention group was carried out as previously described; in the 20% drug-containing serum intervention + OE-SFRP2 group, cells were transfected with the vector for 48 hours to establish an overexpression model, followed by culture with 20% drug-containing serum for 24 hours, without co-culture with HaCaT cells.. Cell counting kit-8 (CCK-8) assay was performed to assess cell viability, flow cytometry to measure apoptosis rates, enzyme-linked immunosorbent assay (ELISA) to detect levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) as well as chemokine ligand (CXCL) 1 and CXCL12 in mouse serum or cell culture supernatant, qPCR to determine the mRNA expression of inflammatory cytokines, chemokines, cell cycle- and proliferation-related factors, as well as SFRP2 in mouse skin tissues or cells, and Western blot analysis to determine the protein expression of SFRP2, Wnt3a, and β-catenin in fibroblasts. One-way analysis of variance was employed for intergroup comparisons, and post-hoc analysis was conducted using Tukey's test. Results:In vivo mouse experiments showed that compared with the normal control group, the model group exhibited typical psoriatic characteristics in skin morphology, including significant inflammatory infiltration in skin tissues and marked epidermal thickening; compared with the normal control group, the serum levels of TNF-α (531.16 ± 28.27 pg/ml vs. 239.58 ± 10.39 pg/ml), IL-1β (111.40 ± 5.16 pg/ml vs. 80.35 ± 3.87 pg/ml), and IL-6 (109.17 ± 4.84 pg/ml vs. 71.73 ± 2.04 pg/ml) significantly increased in the model group, along with their mRNA expression levels in mouse skin tissues (all P < 0.001) ; compared with the model group, the treatment group showed alleviated psoriatic manifestations, and significant reductions in the levels of inflammatory factors TNF-α (low-dose, high-dose, and positive control groups: 420.80 ± 29.30 pg/ml, 322.33 ± 9.40 pg/ml, 322.97 ± 12.16 pg/ml, respectively), IL-1β (98.69 ± 4.49 pg/ml, 89.02 ± 1.56 pg/ml, 88.88 ± 2.08 pg/ml, respectively), and IL-6 (94.07 ± 3.76 pg/ml, 80.54 ± 3.30 pg/ml, 83.21 ± 3.18 pg/ml, respectively), as well as in their mRNA expression levels (all P < 0.001). In in vitro fibroblast experiments, compared with the control group, the model group exhibited a significant elevation in the supernatant levels of IL-1β (126.42 ± 3.56 pg/ml vs. 34.81 ± 0.44 pg/ml), IL-6 (459.44 ± 9.35 pg/ml vs. 115.51 ± 7.26 pg/ml), CXCL1 (2 434.88 ± 127.63 pg/ml vs. 762.85 ± 30.60 pg/ml) and CXCL12 (3 542.14 ± 35.86 pg/ml vs. 2 095.86 ± 45.12 pg/ml), the expression levels of their mRNAs (all P < 0.001), as well as the protein expression levels of SFRP2, Wnt3a, and β-catenin; after intervention with HLJDT-XJDH-containing serum, all the above indices significantly decreased (all P < 0.001). However, when 20% drug-containing serum intervention was administered simultaneously, the expression of inflammatory factors and chemokines in fibroblasts was significantly higher in the SFRP2 overexpression group than in the non-overexpression group (all P < 0.01). When fibroblasts were co-cultured with HaCaT cells, the model group showed significantly increased cell viability but a decreased apoptosis rate of HaCaT cells compared with the control group, while the low- and high-dose intervention groups showed significantly decreased cell viability but increased apoptosis rates of HaCaT cells compared with the model group (all P < 0.05) . Conclusion:HLJDT-XJDH may exert therapeutic effects in psoriasis by downregulating the SFRP2/Wnt/β-catenin signaling pathway, thereby inhibiting fibroblast activation and inflammatory process, which subsequently suppresses the proliferation of keratinocytes and the activation of inflammatory cells.