ObjectiveTo establish the mouse models of psoriasis-like lesions induced by continuous cold exposure or intermittent cold exposure combined with imiquimod （IMQ）， and to evaluate the interventional effects of Kaixuan Jiedu core prescription （KXJD） on the two models. MethodsMale C57BL/6J mice were selected and classified into two experimental batches. The first batch of 36 mice was randomized into a room temperature group， a continuous cold exposure （10 ℃/24 h） group， and an intermittent cold exposure （10 ℃/6 h） group. Each group was further divided into a normal subgroup and a model subgroup （topical application of IMQ to induce skin lesions）， with 6 mice in each subgroup， for modeling and evaluation. The second batch of 54 mice， with 6 in each group， were subjected to the same temperature grouping with an additional KXJD （30.42 g·kg^-1， continuous gavage for 5 days） group. Comprehensive evaluation of model characteristics and KXJD efficacy was conducted through Psoriasis Area and Severity Index （PASI） scoring， skin temperature measurement by infrared thermography， histopathological observation by hematoxylin-eosin （HE） staining， detection of vascular endothelial growth factor （VEGF） and platelet endothelial cell adhesion molecule 1 （CD31） by immunohistochemistry， detection of Claudin-1 and Occludin by immunofluorescence assay， determination of serum levels of tumor necrosis factor-α （TNF-α） and interleukin （IL）-10 by enzyme-linked immunosorbent assay （ELISA）， and quantification of mRNA levels of IL-17A， IL-23， IL-6， and chemokine ligand 20 （CCL20） in skin lesions by quantitative Real-time polymerase chain reaction （Real-time PCR）. ResultsModel mice in all temperature groups exhibited typical psoriasis-like skin lesions. Compared with the normal groups， the model groups showed increased PASI scores， decreased skin temperatures （P<0.05）， obvious epidermal thickening， parakeratosis， and dermal inflammatory cell infiltration， as well as elevated mRNA levels of IL-17A， IL-23， IL-6， and CCL20 （P<0.05）. Cold exposure further aggravated psoriasis. The total PASI score of the intermittent cold exposure model group was higher than that of the room temperature model group （P<0.05）. The serum IL-10 did not show a compensatory elevation， and the blood vessels presented a characteristic of elevated CD31 expression （P<0.05） without a synchronous increase in VEGF. The continuous cold exposure model group exhibited more significant dermal capillary tortuosity and dilation， with the highest mRNA levels of IL-17A， IL-23， IL-6， and CCL20 among all groups. Compared with the respective model groups， KXJD intervention alleviated skin lesions， reduced epidermal thickness and inflammatory cell infiltration， and increased skin temperature， with the temperature increase being particularly significant in the intermittent cold exposure+KXJD group （P<0.05）. Furthermore， KXJD down-regulated the expression of VEGF and CD31， restored the expression of Claudin-1 and Occludin， decreased the mRNA levels of IL-17A and IL-23 （P<0.05）， and reduced the serum TNF-α level. ConclusionThis study successfully established compound psoriasis-like mouse models induced by cold exposure combined with IMQ. It confirms that cold aggravates the severity of psoriasis by exacerbating the closure of Xuanfu （sweat pores）， microcirculation disorders， and immune imbalance. Moreover， different cold exposure patterns have distinct mechanism differences. Continuous cold exposure focuses on enhancing the inflammatory response via the IL-23/IL-17 axis and angiogenesis， simulating chronic aggravation under a long-term cold environment. Intermittent cold exposure tends to impair immune regulation and induce microvascular endothelial stress， corresponding to acute exacerbations caused by sudden temperature drops. KXJD can effectively alleviate psoriasis-like skin lesions under cold conditions by unblocking Xuanfu， regulating vasomotor function， and correcting abnormal immune-inflammatory responses.

ObjectiveTo investigate the ameliorative effects of Kaixuan Jiedu core prescription （KXJD） on skin lesions in psoriasis-like mouse models under cold environment exposure， and to analyze its influences on transient receptor potential （TRP） channels and related neuroimmune regulatory factors. MethodsThirty-six C57BL/6J mice were randomized into 6 groups， with 6 mice in each group. Two feeding conditions were set： Normal temperature and cold ［simulating a cold environment at （10±0.5） ℃， for 6 h daily］. Mice were induced to develop psoriasis-like lesions by applying imiquimod externally. The model mice were allocated into model groups and KXJD （30.42 g·kg^-1， continuous gavage for 5 days） groups. Normal mice were used as the control group. Specifically， mice were allocated into normal temperature， normal temperature model， normal temperature+KXJD， cold exposure control， cold exposure model， and cold exposure+KXJD groups. The pathological changes in skin lesions were observed by hematoxylin-eosin （HE） staining. The expression of cluster of differentiation （CD） 3⁺ T lymphocytes， CD11c⁺ dendritic cells （DCs）， phosphorylated extracellular signal-regulated kinase （p-ERK）， and substance P （SP） were detected by immunofluorescence assay. The protein level of transient receptor potential cation channel subfamily M member 8 （TRPM8） in the skin tissue was determined by Western blot. The expression of TRPM8， transient receptor potential cation channel subfamily V member 1 （TRPV1）， transient receptor potential cation channel subfamily A member 1 （TRPA1）， and transient receptor potential cation channel subfamily V member 2 （TRPV2） at the protein and mRNA levels was determined by immunohistochemistry and Real-time PCR， respectively. The levels of calcitonin gene-related peptide （CGRP） and neuropeptide Y （NPY） in the serum were analyzed by enzyme-linked immunosorbent assay （ELISA）. The enrichment analysis of differentially expressed genes （DEGs） and TRP pathway network construction were conducted based on the GEO database. The co-expression of TRPM8 and CGRP in the skin lesions was verified by immunofluorescence double labeling. ResultsBoth the normal temperature and cold exposure model groups showed typical psoriasis-like skin lesions. Compared with the normal temperature and cold exposure control groups， the model groups had excessive epidermal keratinization， thickened spinous layer， and inflammatory infiltration in the dermis， with increased pathological scores （P<0.05）， increased infiltration of CD3⁺ and CD11c⁺ cells and expression of p-ERK and SP， upregulated mRNA levels of TRPM8， TRPA1， and TRPV2， downregulated mRNA level of TRPV1 （P<0.05）， and reduced content of CGRP and increased content of NPY in the serum. Compared with the normal temperature and cold exposure model groups， KXJD reduced the pathological manifestations and pathological scores of psoriasis-like skin lesions （P<0.05）， and inhibited the infiltration of CD3⁺ and CD11c⁺ cells and the expression of p-ERK and SP. Gene enrichment analysis suggested that the DEGs of psoriasis were significantly enriched in the interleukin （IL）-17 signaling pathway and TRP channel inflammatory regulation. Compared with the normal temperature and cold exposure model groups， KXJD reversed the abnormal mRNA levels of genes related to the TRP channel subfamilies （P<0.05）， increased the CGRP level， and decreased the NPY level. Immunofluorescence double labeling further confirmed that compared with the model groups， KXJD down-regulated the co-expression of TRPM8 and CGRP in the skin lesions. ConclusionKXJD may ameliorate psoriasis-like skin lesions by downregulating the overexpressed cold-sensitive receptor TRPM8 in skin lesions and correcting the disorder of neuropeptide （such as SP and CGRP） release mediated by it， thereby inhibiting the IL-23/helper T cell 17 （Th17） core inflammatory pathway， suppressing the infiltration of inflammatory cells and the activation of the ERK signaling pathway， and regulating the Xuanfu （sweat pore）-TRPM8-neuroimmune response axis.

ObjectiveTo investigate the ameliorative effects and mechanisms of Kaixuan Jiedu core prescription （KXJD） on neuroimmunological inflammation in imiquimod （IMQ）-induced psoriasis-like mice. MethodsA total of 24 C57BL/6J mice were randomly divided into four groups （n=6）： Normal， model， KXJD， and tropomyosin receptor kinase A （TrkA） inhibitor GW441756 groups. The mice in the model， KXJD， and GW441756 groups were topically treated with 5% IMQ cream （62.5 mg·d^-1） on the back to induce psoriasis-like inflammation. The KXJD group received KXJD by gavage （30.42 g·kg^-1）， the GW441756 group received intraperitoneal injection of GW441756 （10 mg·kg^-1）， and the normal and model groups received an equal volume of normal saline by gavage， with continuous intervention for 5 days. The severity of skin lesions was evaluated using the psoriasis area and severity index （PASI）. Hematoxylin-eosin （HE） staining was used to measure epidermal thickness and observe pathological changes in the lesioned skin. Immunohistochemistry was employed to detect the expression of proliferating cell nuclear antigen （Ki67） and interleukin-17A （IL-17A） in the lesioned skin. Enzyme-linked immunosorbent assay （ELISA） was used to quantify the levels of interleukin-23 （IL-23） and calcitonin gene-related peptide （CGRP） in the lesioned tissues. Western blot was used to detect the expression of TrkA and phosphorylated TrkA （p-TrkA）. Immunofluorescence assay was performed to detect the expression of TrkA receptor， protein gene product 9.5 （PGP9.5）， cluster of differentiation 11c （CD11c）， and CGRP in the lesions. Flow cytometry was used to detect the activation of splenic dendritic cells （DCs）. ResultsCompared with the normal group， the model group exhibited typical psoriasis-like inflammation， characterized by erythema， infiltration and scaling， with histopathological findings of epidermal hyperkeratosis and acanthosis. The model group showed significantly increased expression of Ki67， IL-17A， IL-23 and p-TrkA （P<0.05， P<0.01）， increased fluorescence intensity of CD11c， and significantly decreased CGRP expression （P<0.05）. The splenic DC activation was significantly enhanced， as indicated by the increased mean fluorescence intensity （MFI） of CD86 （P<0.05）. Compared with the model group， both the KXJD and GW441756 groups showed amelioration of the psoriasis-like skin inflammation， with significantly down-regulated expression of IL-17A， IL-23 and p-TrkA （P<0.05， P<0.01）， significantly up-regulated expression of CGRP （P<0.01）， reduced CD11c⁺ DC infiltration， and restored splenic DC activation balance （down-regulated CD86 MFI and up-regulated CD80 and CD40 MFI）. Furthermore， the inhibitory effect of KXJD on Ki67 was significantly superior to that of the GW441756 group （P<0.01）. ConclusionKXJD may alleviate IMQ-induced psoriasis-like inflammation in mice by targeting and inhibiting TrkA receptor phosphorylation， regulating CGRP expression in the lesions， and ameliorating aberrant activation of dendritic cells， while also significantly inhibiting keratinocyte proliferation.

ObjectiveTo investigate the effects and molecular mechanisms of Kaixuan Jiedu core prescription （KXJD） on neuroinflammation and immuno-inflammation in the mouse model of imiquimod （IMQ）-induced psoriasis-like skin lesions by regulating the nerve growth factor （NGF）-tropomyosin receptor kinase A （TrkA）/transient receptor potential vanilloid 1 （TRPV1）-protease-activated receptor-2 （PAR-2） signaling pathway. MethodsA psoriasis-like skin lesion model was established in male C57BL/6J mice with IMQ. A total of 24 mice were randomized into four groups （n=6 per group）： Blank control， model， methotrexate （MTX， 1 mg·kg^-1）， and KXJD （30.42 g·kg^-1）. Immunohistochemistry was employed to detect the expression of cluster of differentiation 3 （CD3）， EGF-like module-containing mucin-like hormone receptor-like 1 （F4/80）， lymphocyte antigen 6 complex locus G （Ly-6G）， substance P （SP）， calcitonin gene-related peptide （CGRP）， NGF， phosphorylated TrkA （p-TrkA）， phosphorylated TRPV1 （p-TRPV1）， and PAR-2 in skin lesions. Serum levels of interleukin-17A （IL-17A） and interleukin-23 （IL-23） were measured by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence assay and Western blot were employed to determine the expression of phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK） and phosphorylated nuclear factor-κB p65 （p-NF-κB p65）. Reverse transcription quantitative polymerase chain reaction （Real-time PCR） was used to measure the mRNA levels of interleukin-1β （IL-1β）， tumor necrosis factor （TNF）， interleukin-6 （IL-6）， and C-X-C motif chemokine ligand 1 （CXCL1） in skin lesions. Correlation analysis was performed. ResultsCompared with the blank control group， the model group showed increased positive areas of CD3⁺， F4/80⁺， and Ly-6G⁺ in skin lesions （P<0.05）， elevated serum levels of IL-17A and IL-23 （P<0.05）， increased positive areas of SP and CGRP in skin lesions （P<0.05）， increased positive areas of NGF， p-TrkA， p-TRPV1， and PAR-2 （P<0.05）， elevated ratios of p-p38 MAPK/p38 MAPK and p-NF-κB p65/NF-κB p65 （P<0.05）， and upregulated mRNA levels of IL-1β， TNF， IL-6， and CXCL1 （P<0.05）. Compared with those in the model group， all the above indicators were reversed in the KXJD group （P<0.05）. MTX only downregulated the expression of p-TRPV1 and PAR-2 （P<0.05）， with no significant effects on the expression of NGF， p-TrkA， or CGRP. ConclusionKXJD inhibits the NGF-TrkA/TRPV1-PAR-2 signaling pathway to reduce neuropeptide release and simultaneously downregulates the p38 MAPK/NF-κB signaling pathway and the expression of downstream pro-inflammatory factors to ameliorate psoriasis-like neuroinflammation and immuno-inflammation. The therapeutic effect of KXJD on psoriasis-like inflammation is closely associated with the inhibition of the NGF-TrkA/TRPV1-PAR-2 signaling pathway. This study provides experimental evidence for the modern interpretation of the Xuanfu theory.

Objective To investigate the gene expression profile in rat pancreatic ductal stem cells(PDSCs)when induced to differentiate into insulin-secreting cells(IPCs),with the goal of identifying key genes involved in this differentiation process.Methods The expanded PDSCs were categorized into a normal control(NC)group and an induced(Tre)group.PDSCs continued expansion culture in NC group,and cultured in induction medium for 28 days to facilitate the differentiation of PDSCs into IPCs in Tre group.Dithizone staining was employed to morphologically assess whether the cells exhibited a reddish-brown coloration,indicating a positive result.The immunofluorescence staining method was used to detect the expression of insulin(Ins)and PDX1 in the cells following induction.Additionally,ELISA was conducted to measure the Ins release from IPCs,thereby verifying the responsiveness of the induced cells to glucose-stimulated Ins secretion.Concurrently,cells were collected on induction days 0 and 28 for RNA sequencing(RNA-seq),and differentially expressed genes(DEGs)were analyzed and functionally annotated.The analysis revealed that regulatory factor X3(RFX3)was overexpressed in PDSCs,and the impact of RFX3 upregulation on differentiation induction was subsequently verified.Results Compared with NC group,DTZ staining was positive,PDX1 and Ins proteins were expressed,and an increased release of Ins in response to sugar stimulation was demonstrated in the Tre group.RNA-seq analysis identified 4270 DEGs,and functional enrichment analysis utilizing the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed associations with Ins response,positive regulation of Ins secretion,pancreatic endocrine cell development,and overall pancreatic development.Additionally,functionally related genes such as ALDHA2,CREB5,EIF6,FOXO1,RFX3,WNT5a,OGT,GPR39,SMAD6,and TRPM2 were identified,indicating involvement in the cell cycle,TGF-β1 signaling pathway,FOXO signaling pathway,and Wnt signaling pathway in the regulation of the differentiation of pancreatic ductal stem cells(PDSCs)into insulin-producing cells(IPCs).Furthermore,the upregulation of RFX3 can inhibit the expression of TGF-β1 within 72 hours,thereby promoted the formation and release of Ins from insulin-positive cells.Conclusions Multiple genes and signaling pathways associated with pancreatic β-cell function collectively regulate the differentiation of rat PDSCs into IPCs.Notably,the upregulation of RFX3 enhances this differentiation process.

Objective To explore the objective facial appearance characteristics of patients with coronary heart disease(CHD).Methods From April 7,2019 to December 1,2022,313 patients with CHD were recruited from Dongzhimen Hospital,Beijing University of Chinese Medicine,Dongfang Hospital,Beijing University of Chinese Medicine,and the First Affiliated Hospital of Hunan University of Chinese Medicine,together with 293 healthy controls.Standardized facial images were obtained using the tongue-face diagnostic instrument.The face was divided into six regions:the forehead,left cheek,right cheek,nose,lips,and chin.Nine color parameters were extracted from each region,including red(R),green(G),blue(B),hue(H),saturation(S),value(V),lightness(L),red-green axis(a),and yellow-blue axis(b).Comparisons between groups were performed.Results Compared with the healthy group,in the forehead region,values of R,S,V,a,and b were higher in the coronary heart disease group,whereas B was lower(P<0.05);in the left cheek,nose,and chin regions,R,G,B,V,and L decreased,whereas S,a,and b increased(P<0.05);in the right cheek region,R,G,B,H,V,and L decreased,while S,a,and b increased(P<0.05);in the lips region,R,G,B,H,V,L,and a decreased,whereas S and b increased(P<0.05).Conclusion Compared with healthy individuals,patients with CHD present with a darker,more saturated facial complexion with reduced brightness,overall manifesting as"dark red complexion"and"dense but not bright color,"suggesting the pathogenesis of qi and blood circulation stagnation and internal blood stasis retention.The objective expression of facial features may have greater application value in syndrome differentiation and auxiliary diagnosis in traditional Chinese medicine.

To establish an indirect enzyme linked immunosorbent assay(ELISA)method for the detection of Zaire Ebola virus(ZEBOV)specific antibodies,the full-length of ZEBOV GP1 gene was amplified by PCR and cloned into pET-30a(+)vector to generate the pET-30a(+)-GP1 plasmid.After expressed in the E.coli expression system,the purified GP1 protein was used as coating antigen to establish the indirect ELISA method for detection of ZEBOV antibody.The con-ditions including concentration of coating antigen and serum dilution were determined by chess-board titration.Specificity,sensitivity,and reproducibility of the established ELISA detection meth-od were evaluated.GP1 protein was successfully prepared by prokaryotic expression,and was used as the coatingantigen for indirect ELISA.By optimizing the reaction conditions,the optimal concen-tration of the coating antigen was determined to be 0.5 g/L;the optimal dilution of serum was cal-culated to be 1∶3 200;the optimal dilution of enzyme-labeled secondary antibody was measured to be 1∶20 000.The established method exhibited excellent specificity,sensitivity,and reproducibili-ty.In the present study,the GP1 protein was successfully expressed in the E.coli expression sys-tem and the high purity GP1 protein was used as the coating protein to establish an indirect ELISA assay for ZEBOV antibody.This method is highly specific,sensitive,and reproducible,which provides technical support for the fur-ther study of the biological function of GP1 and the detection of ZEBOV antibody in serum.

To establish an indirect enzyme linked immunosorbent assay(ELISA)method for the detection of Zaire Ebola virus(ZEBOV)specific antibodies,the full-length of ZEBOV GP1 gene was amplified by PCR and cloned into pET-30a(+)vector to generate the pET-30a(+)-GP1 plasmid.After expressed in the E.coli expression system,the purified GP1 protein was used as coating antigen to establish the indirect ELISA method for detection of ZEBOV antibody.The con-ditions including concentration of coating antigen and serum dilution were determined by chess-board titration.Specificity,sensitivity,and reproducibility of the established ELISA detection meth-od were evaluated.GP1 protein was successfully prepared by prokaryotic expression,and was used as the coatingantigen for indirect ELISA.By optimizing the reaction conditions,the optimal concen-tration of the coating antigen was determined to be 0.5 g/L;the optimal dilution of serum was cal-culated to be 1∶3 200;the optimal dilution of enzyme-labeled secondary antibody was measured to be 1∶20 000.The established method exhibited excellent specificity,sensitivity,and reproducibili-ty.In the present study,the GP1 protein was successfully expressed in the E.coli expression sys-tem and the high purity GP1 protein was used as the coating protein to establish an indirect ELISA assay for ZEBOV antibody.This method is highly specific,sensitive,and reproducible,which provides technical support for the fur-ther study of the biological function of GP1 and the detection of ZEBOV antibody in serum.

Objective To explore the objective facial appearance characteristics of patients with coronary heart disease(CHD).Methods From April 7,2019 to December 1,2022,313 patients with CHD were recruited from Dongzhimen Hospital,Beijing University of Chinese Medicine,Dongfang Hospital,Beijing University of Chinese Medicine,and the First Affiliated Hospital of Hunan University of Chinese Medicine,together with 293 healthy controls.Standardized facial images were obtained using the tongue-face diagnostic instrument.The face was divided into six regions:the forehead,left cheek,right cheek,nose,lips,and chin.Nine color parameters were extracted from each region,including red(R),green(G),blue(B),hue(H),saturation(S),value(V),lightness(L),red-green axis(a),and yellow-blue axis(b).Comparisons between groups were performed.Results Compared with the healthy group,in the forehead region,values of R,S,V,a,and b were higher in the coronary heart disease group,whereas B was lower(P<0.05);in the left cheek,nose,and chin regions,R,G,B,V,and L decreased,whereas S,a,and b increased(P<0.05);in the right cheek region,R,G,B,H,V,and L decreased,while S,a,and b increased(P<0.05);in the lips region,R,G,B,H,V,L,and a decreased,whereas S and b increased(P<0.05).Conclusion Compared with healthy individuals,patients with CHD present with a darker,more saturated facial complexion with reduced brightness,overall manifesting as"dark red complexion"and"dense but not bright color,"suggesting the pathogenesis of qi and blood circulation stagnation and internal blood stasis retention.The objective expression of facial features may have greater application value in syndrome differentiation and auxiliary diagnosis in traditional Chinese medicine.

Objective To investigate the gene expression profile in rat pancreatic ductal stem cells(PDSCs)when induced to differentiate into insulin-secreting cells(IPCs),with the goal of identifying key genes involved in this differentiation process.Methods The expanded PDSCs were categorized into a normal control(NC)group and an induced(Tre)group.PDSCs continued expansion culture in NC group,and cultured in induction medium for 28 days to facilitate the differentiation of PDSCs into IPCs in Tre group.Dithizone staining was employed to morphologically assess whether the cells exhibited a reddish-brown coloration,indicating a positive result.The immunofluorescence staining method was used to detect the expression of insulin(Ins)and PDX1 in the cells following induction.Additionally,ELISA was conducted to measure the Ins release from IPCs,thereby verifying the responsiveness of the induced cells to glucose-stimulated Ins secretion.Concurrently,cells were collected on induction days 0 and 28 for RNA sequencing(RNA-seq),and differentially expressed genes(DEGs)were analyzed and functionally annotated.The analysis revealed that regulatory factor X3(RFX3)was overexpressed in PDSCs,and the impact of RFX3 upregulation on differentiation induction was subsequently verified.Results Compared with NC group,DTZ staining was positive,PDX1 and Ins proteins were expressed,and an increased release of Ins in response to sugar stimulation was demonstrated in the Tre group.RNA-seq analysis identified 4270 DEGs,and functional enrichment analysis utilizing the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed associations with Ins response,positive regulation of Ins secretion,pancreatic endocrine cell development,and overall pancreatic development.Additionally,functionally related genes such as ALDHA2,CREB5,EIF6,FOXO1,RFX3,WNT5a,OGT,GPR39,SMAD6,and TRPM2 were identified,indicating involvement in the cell cycle,TGF-β1 signaling pathway,FOXO signaling pathway,and Wnt signaling pathway in the regulation of the differentiation of pancreatic ductal stem cells(PDSCs)into insulin-producing cells(IPCs).Furthermore,the upregulation of RFX3 can inhibit the expression of TGF-β1 within 72 hours,thereby promoted the formation and release of Ins from insulin-positive cells.Conclusions Multiple genes and signaling pathways associated with pancreatic β-cell function collectively regulate the differentiation of rat PDSCs into IPCs.Notably,the upregulation of RFX3 enhances this differentiation process.