ObjectiveTo investigate the effects and underlying mechanisms of Shenqi Wenfei prescription （SQWF） on chronic obstructive pulmonary disease （COPD）. MethodsA rat model of COPD with lung Qi deficiency was established using lipopolysaccharide （LPS） combined with cigarette smoke. Forty-eight SD rats were randomly divided into a blank group， a model group， low-， medium-， and high-dose SQWF groups （2.835， 5.67， 11.34 g·kg^-1）， and a Yupingfeng group （1.35 g·kg^-1）. Drug administration began on day 29 after modeling and continued for 2 weeks. The general condition of the rats was observed， and the lung function in each group was assessed. Hematoxylin-eosin （HE） staining was used to observe pathological changes in lung tissue. The proportion of inflammatory cells in bronchoalveolar lavage fluid （BALF） was measured. Apoptosis in lung tissue was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） staining. The release level of lactate dehydrogenase （LDH） in BALF was detected by a microplate assay. Reactive oxygen species （ROS） levels in lung tissue were detected using fluorescent probes. The levels of malondialdehyde （MDA）， total superoxide dismutase （SOD）， and reduced glutathione （GSH） in BALF were measured by biochemical methods. Ultrastructural changes in lung cells were observed via transmission electron microscopy. Double immunofluorescence staining was performed to detect the expression of thioredoxin-interacting protein （TXNIP） and nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） in lung tissue. Western blot analysis was used to detect the protein expression of TXNIP， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， cysteinyl aspartate-specific protease-1 （Caspase-1）， Caspase-1 p20， gasdermin D （GSDMD）， GSDMD N-terminal active fragment （GSDMD-N）， interleukin-1β （IL-1β）， and IL-18 in lung tissue. Serum IL-1β and IL-18 levels were measured by ELISA. ResultsCompared with the blank group， the model group showed lassitude， fatigue， tachypnea， and audible phlegm sounds， and lung function significantly declined （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were obvious. The level of inflammatory cells in BALF increased significantly （P0.05）. The number of TUNEL-positive cells increased （P0.01）. Levels of LDH， ROS， and MDA in BALF increased significantly （P0.01）， while GSH and SOD activities decreased significantly （P0.01）. Lung tissue cells showed irregular morphology， swollen mitochondria， disrupted cell membranes， and abundant vesicles， i.e.， pyroptotic bodies. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue were significantly elevated （P0.01）， and serum IL-1β and IL-18 levels also increased significantly （P0.01）. Compared with the model group， each medication group showed alleviation of qi deficiency symptoms and improved lung function （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were reduced. Inflammatory cell levels decreased （P0.05）. The number of TUNEL-positive cells decreased significantly （P0.01）. Levels of LDH， ROS， and MDA decreased significantly （P0.05）， while GSH and SOD activities significantly increased （P0.01）. Morphological and structural damage in lung tissue was improved to varying degrees. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue significantly decreased （P0.01）， and serum IL-1β and IL-18 levels also decreased significantly （P0.05）. ConclusionSQWF can improve lung function and alleviate inflammatory responses in COPD rats. Its mechanism may be related to regulating the ROS/TXNIP/NLRP3 pathway and inhibiting pyroptosis.

ObjectiveTo investigate the effects and underlying mechanisms of Shenqi Wenfei prescription （SQWF） on chronic obstructive pulmonary disease （COPD）. MethodsA rat model of COPD with lung Qi deficiency was established using lipopolysaccharide （LPS） combined with cigarette smoke. Forty-eight SD rats were randomly divided into a blank group， a model group， low-， medium-， and high-dose SQWF groups （2.835， 5.67， 11.34 g·kg^-1）， and a Yupingfeng group （1.35 g·kg^-1）. Drug administration began on day 29 after modeling and continued for 2 weeks. The general condition of the rats was observed， and the lung function in each group was assessed. Hematoxylin-eosin （HE） staining was used to observe pathological changes in lung tissue. The proportion of inflammatory cells in bronchoalveolar lavage fluid （BALF） was measured. Apoptosis in lung tissue was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） staining. The release level of lactate dehydrogenase （LDH） in BALF was detected by a microplate assay. Reactive oxygen species （ROS） levels in lung tissue were detected using fluorescent probes. The levels of malondialdehyde （MDA）， total superoxide dismutase （SOD）， and reduced glutathione （GSH） in BALF were measured by biochemical methods. Ultrastructural changes in lung cells were observed via transmission electron microscopy. Double immunofluorescence staining was performed to detect the expression of thioredoxin-interacting protein （TXNIP） and nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） in lung tissue. Western blot analysis was used to detect the protein expression of TXNIP， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， cysteinyl aspartate-specific protease-1 （Caspase-1）， Caspase-1 p20， gasdermin D （GSDMD）， GSDMD N-terminal active fragment （GSDMD-N）， interleukin-1β （IL-1β）， and IL-18 in lung tissue. Serum IL-1β and IL-18 levels were measured by ELISA. ResultsCompared with the blank group， the model group showed lassitude， fatigue， tachypnea， and audible phlegm sounds， and lung function significantly declined （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were obvious. The level of inflammatory cells in BALF increased significantly （P0.05）. The number of TUNEL-positive cells increased （P0.01）. Levels of LDH， ROS， and MDA in BALF increased significantly （P0.01）， while GSH and SOD activities decreased significantly （P0.01）. Lung tissue cells showed irregular morphology， swollen mitochondria， disrupted cell membranes， and abundant vesicles， i.e.， pyroptotic bodies. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue were significantly elevated （P0.01）， and serum IL-1β and IL-18 levels also increased significantly （P0.01）. Compared with the model group， each medication group showed alleviation of qi deficiency symptoms and improved lung function （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were reduced. Inflammatory cell levels decreased （P0.05）. The number of TUNEL-positive cells decreased significantly （P0.01）. Levels of LDH， ROS， and MDA decreased significantly （P0.05）， while GSH and SOD activities significantly increased （P0.01）. Morphological and structural damage in lung tissue was improved to varying degrees. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue significantly decreased （P0.01）， and serum IL-1β and IL-18 levels also decreased significantly （P0.05）. ConclusionSQWF can improve lung function and alleviate inflammatory responses in COPD rats. Its mechanism may be related to regulating the ROS/TXNIP/NLRP3 pathway and inhibiting pyroptosis.

OBJECTIVE To investigate the potential mechanism by which Juanxiao decoction improves bronchial asthma （hereinafter referred to as “asthma”） based on the nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 （NLRP3） inflammasome signaling pathway. METHODS Female SD rats were randomly assigned to normal group， model group and Juanxiao decoction low-， medium- and high-dose groups （0.36， 0.72 and 1.44 g/kg， calculated based on crude drug weight）， as well as positive control group （Dexamethasone acetate tablets， 0.2 mg/kg）， with 10 rats in each group. Except for the normal group， asthma models were established in the remaining groups via intraperitoneal injection of ovalbumin combined with aluminum hydroxide， followed by nebulized inhalation of ovalbumin. On day 14 of the experiment， rats in each group received intragastric administration of the corresponding solution or normal saline， once a day， for 7 consecutive days. Following the final administration， the following parameters were measured in each group： lung function indexes （forced vital capacity， forced expiratory volume in 0.3 second， peak expiratory flow）， serum levels of inflammatory markers （interleukin-1β， interleukin- 18）， and the percentages of inflammatory cells （lymphocytes， eosinophils， neutrophils） in bronchoalveolar lavage fluid. Histopathological changes in lung tissue were observed， and the protein and mRNA expressions of nuclear factor-kappa B （NF- κB）， NLRP3 and caspase-1 in lung tissue were detected. RESULTS Compared with the normal group， pathological changes such as alveolar wall thickening and inflammatory cell infiltration were observed in rats in the model group. All pulmonary function indicators were significantly reduced in rats in the model group and the administration groups. The levels of inflammatory markers， the percentages of inflammatory cells， and the protein and mRNA expressions of NF-κB， NLRP3 and caspase-1 were significantly elevated or up-regulated （P＜0.05）. Compared with the model group， pathological changes in rats in each dosage group of Juanxiao decoction were significantly alleviated， and all quantitative indicators showed dose-dependent improvements （P＜0.05）. CONCLUSIONS Juanxiao decoction can reduce airway inflammatory responses in asthmatic rats， alleviate lung function impairment， and improve pathological changes such as inflammatory cell infiltration. Those effects may be related to the inhibition of the NLRP3 inflammasome signaling pathway.

AIM: To evaluate the efficacy and safety of 3% diquafosol sodium eye drops in children wearing orthokeratology lenses and with dry eye disease(DED)or at risk of DED.METHODS: Randomized controlled trials. Children with DED or at risk of DED were randomly assigned in a 1:1 ratio to receive either 3% diquafosol sodium eye drops 6 times daily or a blank control at Chongqing Aier Children's Eye Hospital from November 2023 to November 2024. The primary endpoint was the change in the Dry Eye Questionnaire-5(DEQ-5)score from baseline at 12 wk. Secondary assessments included non-invasive breakup time(NIBUT), tear meniscus height, Schirmer's test, corneal fluorescein staining score, and axial length.RESULTS: A total of 80 participants(80 eyes)were enrolled(40 in each group), the average age of the participants was 11.11±1.88 years, with 43 females(54%)and 37 males(46%), and all completed the trial. After 12 wk, the DEQ-5 scores for the diquafosol sodium group and the blank control group were 1.88±2.02 and 2.88±2.79, respectively(P=0.079). The diquafosol sodium group demonstrated a significant improvement in DEQ-5 dryness symptom scores(-0.33±0.66 vs. 0.05±0.81, P=0.023)and NIBUT(6.18±3.73 vs. -1.09±4.40 s, P<0.001)at 12 wk. Additionally, the diquafosol sodium group showed no axial length elongation, in contrast to the blank control group, which exhibited elongation(0.00±0.08 vs. 0.05±0.10 mm, P=0.013). No other significant differences were found in the secondary endpoints. No adverse events occurred during the trial.CONCLUSION: Although no statistically significant improvements were noted in the overall DEQ-5 scores, the 3% diquafosol sodium eye drops significantly improved dryness symptoms and NIBUT when compared to the blank control group.

ObjectiveThis paper aims to find the identified and validated clinical biomarker data building upon a clinical study of early-phase phase Ⅱ and investigate the correlation analysis of Huanglian Jiedu Wan on syndrome improvement and clinical biomarkers in the treatment of "excess heat-toxicity" based on a machine learning model. Additionally， the effective prediction of clinical biomarker values for the main symptoms of the "excess heat-toxicity" syndrome was assessed. MethodsA total of 229 patients meeting the inclusion criteria for "excess heat-toxicity" syndrome were randomly divided into the Huanglian Jiedu Wan group and the placebo group. Syndrome score transition matrices were constructed for the Huanglian Jiedu Wan group and the placebo group based on three main symptoms of "excess heat-toxicity" syndrome， such as oral ulcers， sore throat， and gum swelling and pain. Data from the patients with these three syndromes were also integrated for an overall analysis. The corresponding syndrome score transition matrices were further constructed to visualize symptom change trends of the patients in the two groups via heatmaps. Based on the identified and validated clinical biomarkers related to inflammation， oxidative stress， and energy metabolism in the early phase， Spearman correlation analysis was employed to analyze and evaluate the associations between clinical biomarkers and syndrome improvement. Key clinical biomarkers reflecting the effect of Huanglian Jiedu Wan were screened through the comparison of differences between groups. An extreme gradient boosting （XGBoost） algorithm was used to develop a prediction model for main symptom classification， with classification performance evaluated through 10-fold cross-validation. Feature importance analysis was applied to identify variables with the greatest contribution to the prediction result. ResultsThe syndrome transition matrix results indicated that the Huanglian Jiedu Wan group showed a superior effect to the placebo group in improving oral ulcers， sore throat， and overall symptoms， with significant effects observed especially in sore throat and overall symptom analyses （P<0.01）. Spearman correlation analysis revealed that several clinical biomarkers positively correlated with "excess heat-toxicity" syndrome and its main symptom improvement， were also called "heat-related biomarkers"， including succinic acid， α-ketoglutaric acid， glycine， lactic acid， adenosine monophosphate （AMP）， tumor necrosis factor-α （TNF-α）， interferon-γ （IFN-γ）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， interleukin-10 （IL-10）， and so on. Conversely， clinical biomarkers negatively correlated with symptom severity， were also called "heat-clearing related biomarkers" after administration of Huanglian Jiedu Wan， including malic acid， fumaric acid， cis-aconitic acid， adrenocorticotropic hormone （ACTH）， IL-1β， IL-4， IL-8， succinic acid， and citric acid. The XGBoost classification model using all 52 biomarkers as variables achieved an average test accuracy of 0.754 and an average F1 score of 0.777. Feature importance analysis identified the scores of glutamic acid in saliva and IL-6 were the highest in all the variables， with importance scores of 0.081 and 0.080， respectively. After screening out 14 key variables and optimizing the parameters， model performance improved to an average accuracy of 0.758 and an F1 score of 0.798. Feature importance analysis further determined that the glutamic acid in saliva and IL-6 showed obvious changes after screening the variables， confirming the good syndrome prediction ability of the model constructed by these key clinical biomarkers. ConclusionThis study systematically elucidates the correlation between syndrome improvement and clinical biomarkers of Huanglian Jiedu Wan in the treatment of "excess heat-toxicity" syndrome. An XGBoost classification model based on key clinical biomarkers is successfully established， achieving effective prediction of the symptoms related to the "excess heat-toxicity" syndrome such as oral ulcers and sore throat and providing a new insight for objective identification of traditional Chinese medicine syndromes.

ObjectiveThis paper aims to find the identified and validated clinical biomarker data building upon a clinical study of early-phase phase Ⅱ and investigate the correlation analysis of Huanglian Jiedu Wan on syndrome improvement and clinical biomarkers in the treatment of "excess heat-toxicity" based on a machine learning model. Additionally， the effective prediction of clinical biomarker values for the main symptoms of the "excess heat-toxicity" syndrome was assessed. MethodsA total of 229 patients meeting the inclusion criteria for "excess heat-toxicity" syndrome were randomly divided into the Huanglian Jiedu Wan group and the placebo group. Syndrome score transition matrices were constructed for the Huanglian Jiedu Wan group and the placebo group based on three main symptoms of "excess heat-toxicity" syndrome， such as oral ulcers， sore throat， and gum swelling and pain. Data from the patients with these three syndromes were also integrated for an overall analysis. The corresponding syndrome score transition matrices were further constructed to visualize symptom change trends of the patients in the two groups via heatmaps. Based on the identified and validated clinical biomarkers related to inflammation， oxidative stress， and energy metabolism in the early phase， Spearman correlation analysis was employed to analyze and evaluate the associations between clinical biomarkers and syndrome improvement. Key clinical biomarkers reflecting the effect of Huanglian Jiedu Wan were screened through the comparison of differences between groups. An extreme gradient boosting （XGBoost） algorithm was used to develop a prediction model for main symptom classification， with classification performance evaluated through 10-fold cross-validation. Feature importance analysis was applied to identify variables with the greatest contribution to the prediction result. ResultsThe syndrome transition matrix results indicated that the Huanglian Jiedu Wan group showed a superior effect to the placebo group in improving oral ulcers， sore throat， and overall symptoms， with significant effects observed especially in sore throat and overall symptom analyses （P<0.01）. Spearman correlation analysis revealed that several clinical biomarkers positively correlated with "excess heat-toxicity" syndrome and its main symptom improvement， were also called "heat-related biomarkers"， including succinic acid， α-ketoglutaric acid， glycine， lactic acid， adenosine monophosphate （AMP）， tumor necrosis factor-α （TNF-α）， interferon-γ （IFN-γ）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， interleukin-10 （IL-10）， and so on. Conversely， clinical biomarkers negatively correlated with symptom severity， were also called "heat-clearing related biomarkers" after administration of Huanglian Jiedu Wan， including malic acid， fumaric acid， cis-aconitic acid， adrenocorticotropic hormone （ACTH）， IL-1β， IL-4， IL-8， succinic acid， and citric acid. The XGBoost classification model using all 52 biomarkers as variables achieved an average test accuracy of 0.754 and an average F1 score of 0.777. Feature importance analysis identified the scores of glutamic acid in saliva and IL-6 were the highest in all the variables， with importance scores of 0.081 and 0.080， respectively. After screening out 14 key variables and optimizing the parameters， model performance improved to an average accuracy of 0.758 and an F1 score of 0.798. Feature importance analysis further determined that the glutamic acid in saliva and IL-6 showed obvious changes after screening the variables， confirming the good syndrome prediction ability of the model constructed by these key clinical biomarkers. ConclusionThis study systematically elucidates the correlation between syndrome improvement and clinical biomarkers of Huanglian Jiedu Wan in the treatment of "excess heat-toxicity" syndrome. An XGBoost classification model based on key clinical biomarkers is successfully established， achieving effective prediction of the symptoms related to the "excess heat-toxicity" syndrome such as oral ulcers and sore throat and providing a new insight for objective identification of traditional Chinese medicine syndromes.

Objective To investigate the causal relationship between metabolic syndrome and breast cancer by using a bidirectional two-sample Mendelian randomization (MR) approach. Methods Genome-wide association study (GWAS) summary statistics for metabolic syndrome and breast cancer were acquired from the Integrative Epidemiology Unit GWAS database and the GWAS Catalog, with populations encompassing the United States and East Asia. A bidirectional causal design was employed: a forward analysis with metabolic syndrome as the exposure and breast cancer as the outcome, followed by a reverse analysis wherein their roles were interchanged. The inverse-variance weighting (IVW) method was primarily used for effect estimation, supplemented by MR-Egger regression, the weighted median method, the simple mode method, and the weighted mode method. Instrument variable strength was screened using the F-statistic (F>10). Robustness of the results was assessed through heterogeneity tests, horizontal pleiotropy tests, forest plots, and leave-one-out sensitivity analyses. Results The IVW analysis indicated no significant causal relationship between metabolic syndrome and breast cancer (OR=1.00, 95%CI: 0.97-1.03), P>0.05). Sensitivity analyses yielded consistent results, suggesting the good robustness of the study findings. Conclusion This study found no evidence to support a causal relationship, either positive or negative, between metabolic syndrome and breast cancer.

Traditional Chinese medicine and its bioactive components have garnered increasing attention as potential therapeutic options for allergic asthma. By targeting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, these natural compounds exhibit unique advantages in multilevel immunomodulation and inflammation suppression compared with single-target synthetic drugs. Accumulating pharmacological evidence supports their capacity to restore pathway homeostasis, positioning them as promising candidates for complementary strategies in asthma management. Allergic asthma, a heterogeneous respiratory disorder affecting approximately 150 million individuals worldwide, arises from a complex interplay of genetic predisposition, environmental exposures, and lifestyle factors. Its pathological progression is marked by aberrant activation of the PI3K/AKT signaling cascade, with the mechanistic target of rapamycin serving as a key downstream regulatory node. This evolutionarily conserved pathway orchestrates fundamental cellular processes that contribute to three hallmark pathological features of allergic asthma: chronic airway inflammation, structural remodeling of the bronchial architecture, and airway hyperresponsiveness. This review has 3 primary objectives: (1) to evaluate the role of the PI3K/AKT pathway in allergic asthma pathogenesis, (2) to analyze the molecular mechanisms of representative traditional Chinese medicine preparations and their active ingredients, and (3) to identify novel bioactive inhibitors derived from natural products. Collectively, these investigations provide a conceptual framework for the development of next-generation targeted therapies and for optimizing clinical management strategies for allergic asthma.

ObjectiveTo investigate the target proteins directly bound by neochlorogenic acid （NA） and the molecular mechanisms that ameliorate the proliferation and inflammatory response of psoriatic keratinocytes. MethodsM5-induced HaCaT cells were used as a psoriatic keratinocyte proliferation and inflammatory cell model. The synthesized NA probe （NA-P） and NA prodrug were first evaluated for cell viability using a cell proliferation/cell counting kit-8（CCK-8）. The potency of NA and NA-P was evaluated in the safe concentration range， and the effects of 0-100 μmol·L^-1 NA and probe on M5-induced proliferation of HaCaT cells were detected using CCK-8. The effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the expression of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-23 （IL-23）， and interleukin-17A （IL-17A） inflammatory factors were detected by enzyme-linked immunosorbent assay （ELISA）， and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the effects of NA on the mRNA expression of keratin 16 （K16） in HaCaT cells， S100 calcium-binding protein A9 （S100A9）， S100 calcium-binding protein A7 （S100A7）， IL-6， IL-17A， and chemokine 1 （CXCL1）. In vitro fluorescence labeling and competition experiments using NA-P were performed， and target protein angling and analysis using pull-down experiments combined with liquid chromatography-mass spectrometry （Pull-down/LC-MS/MS） were conducted. Target validation was performed using pull-down experiments combined with protein immunoblotting （Pull down-WB）， cellular heat transfer analysis combined with protein immunoblot （CETSA-WB） experiments， and molecular docking. Finally， Real-time PCR was utilized to detect the effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the mRNA expression of IL-1β， nucleotide-binding oligomeric structural domain-like receptor protein 3 （NLRP3）， apoptosis-associated speckled-like protein （ASC）， and cysteine protease-1 （Caspase-1） in HaCaT cells. Protein immunoblot （Western blot） was used to detect the effects of phosphorylated p65 （p-p65）， p65， phosphorylated human nuclear factor-κB inhibitory protein α （p-IκBα）， human nuclear factor κB inhibitory protein α （IκBα）， and heat shock protein 90 （HSP90） expression. ResultsIn the 200 μmol·L^-1 safe concentration range， HaCaT cell proliferation， increased expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors， and increased mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 were observed in the M5 group compared with the blank group. Cell proliferation in 5-100 μmol·L^-1 NA and NA-P groups was inhibited， and the expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors was decreased in the NA-L， NA-M， NA-H， and NA-P-H groups. The mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 was decreased （P<0.05）. High-confidence targets were screened for HSP90 protein by Pull-down/LC-MS/MS using 200 μmol·L^-1 NA competing with 100 μmol·L^-1 NA-P. Compared with that in the blank group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1， as well as the expression of p-p65/p65， p-IκBα/IκBα， and HSP90 protein， were increased in HaCaT cells in the M5 group （P<0.05）. Compared with that in the M5 group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1 of cells in the NA-L group， the NA-M group， the NA-H group， and the NA-P-H group was decreased （P<0.05）. p-p65/p65 and p-IκBα/IκBα were decreased in the NA-M and NA-H groups （P<0.05）， and there was no change in HSP90 protein. Pull down-WB showed that NA could directly target HSP90 protein， and NA binding to HSP90 protein enhanced its thermal stability. Molecular docking of NA with HSP90 family proteins HSP90AA1， HSP90B1， and HSP90AB1 all resulted in highly stable binding. ConclusionNA can inhibit the proliferation and inflammatory response of psoriatic keratinocytes by a mechanism that may be achieved by targeting HSP90 to modulate the NF-κB/NLRP3 signaling pathway.

ObjectiveTo investigate the target proteins directly bound by neochlorogenic acid （NA） and the molecular mechanisms that ameliorate the proliferation and inflammatory response of psoriatic keratinocytes. MethodsM5-induced HaCaT cells were used as a psoriatic keratinocyte proliferation and inflammatory cell model. The synthesized NA probe （NA-P） and NA prodrug were first evaluated for cell viability using a cell proliferation/cell counting kit-8（CCK-8）. The potency of NA and NA-P was evaluated in the safe concentration range， and the effects of 0-100 μmol·L^-1 NA and probe on M5-induced proliferation of HaCaT cells were detected using CCK-8. The effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the expression of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-23 （IL-23）， and interleukin-17A （IL-17A） inflammatory factors were detected by enzyme-linked immunosorbent assay （ELISA）， and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the effects of NA on the mRNA expression of keratin 16 （K16） in HaCaT cells， S100 calcium-binding protein A9 （S100A9）， S100 calcium-binding protein A7 （S100A7）， IL-6， IL-17A， and chemokine 1 （CXCL1）. In vitro fluorescence labeling and competition experiments using NA-P were performed， and target protein angling and analysis using pull-down experiments combined with liquid chromatography-mass spectrometry （Pull-down/LC-MS/MS） were conducted. Target validation was performed using pull-down experiments combined with protein immunoblotting （Pull down-WB）， cellular heat transfer analysis combined with protein immunoblot （CETSA-WB） experiments， and molecular docking. Finally， Real-time PCR was utilized to detect the effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the mRNA expression of IL-1β， nucleotide-binding oligomeric structural domain-like receptor protein 3 （NLRP3）， apoptosis-associated speckled-like protein （ASC）， and cysteine protease-1 （Caspase-1） in HaCaT cells. Protein immunoblot （Western blot） was used to detect the effects of phosphorylated p65 （p-p65）， p65， phosphorylated human nuclear factor-κB inhibitory protein α （p-IκBα）， human nuclear factor κB inhibitory protein α （IκBα）， and heat shock protein 90 （HSP90） expression. ResultsIn the 200 μmol·L^-1 safe concentration range， HaCaT cell proliferation， increased expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors， and increased mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 were observed in the M5 group compared with the blank group. Cell proliferation in 5-100 μmol·L^-1 NA and NA-P groups was inhibited， and the expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors was decreased in the NA-L， NA-M， NA-H， and NA-P-H groups. The mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 was decreased （P<0.05）. High-confidence targets were screened for HSP90 protein by Pull-down/LC-MS/MS using 200 μmol·L^-1 NA competing with 100 μmol·L^-1 NA-P. Compared with that in the blank group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1， as well as the expression of p-p65/p65， p-IκBα/IκBα， and HSP90 protein， were increased in HaCaT cells in the M5 group （P<0.05）. Compared with that in the M5 group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1 of cells in the NA-L group， the NA-M group， the NA-H group， and the NA-P-H group was decreased （P<0.05）. p-p65/p65 and p-IκBα/IκBα were decreased in the NA-M and NA-H groups （P<0.05）， and there was no change in HSP90 protein. Pull down-WB showed that NA could directly target HSP90 protein， and NA binding to HSP90 protein enhanced its thermal stability. Molecular docking of NA with HSP90 family proteins HSP90AA1， HSP90B1， and HSP90AB1 all resulted in highly stable binding. ConclusionNA can inhibit the proliferation and inflammatory response of psoriatic keratinocytes by a mechanism that may be achieved by targeting HSP90 to modulate the NF-κB/NLRP3 signaling pathway.