ObjectiveTo establish a C-C motif chemokine receptor 6 （CCR6） homozygous knockout mouse model in order to provide a crucial animal model foundation for subsequent in vivo functional studies. MethodsCcr6^-/- mice were generated using CRISPR-Cas9 technology. Genomic DNA was extracted from mouse tails， with genotyping performed by PCR and agarose gel electrophoresis. Pathological morphology of major organs （heart， liver， lung， kidney） was assessed through HE staining. Western blot was used to analyze CCR6 protein expression in blood， spleen， and bone marrow. To analyze the impact of CCR6 gene knockout on the proportion of major immune cell populations， the ratio of T cells and macrophages in the mouse spleen was detected using flow cytometry. ResultsThe results of agarose gel electrophoresis demonstrated that mice exhibiting a single specific band at the 307 bp position upon primer-based identification were confirmed as Ccr6^-/- mice. HE staining revealed no significant histopathological differences between Ccr6^+/+and Ccr6^-/- mice. Western blot demonstrated near-complete absence of CCR6 protein in target tissues. Flow cytometry results demonstrated that CCR6 gene deletion significantly increased the proportion of CD8⁺T cells， while the ratios of both CD4⁺T cells and macrophages remained unaltered. ConclusionA Ccr6^-/- mouse model is established using CRISPR-Cas9 technology， serving as an essential tool for elucidating CCR6′s regulatory role in tumor proliferation.

ObjectiveTo explore the clinical and phenomics characteristics of patients with phlegm-stasis binding syndrome and its accompanied patterns in stable angina pectoris （SAP） of coronary heart disease （CHD）. MethodsA multicenter cross-sectional study design was adopted. A total of 300 patients with SAP of CHD were enrolled and classified into 120 cases of phlegm-stasis binding syndrome， 125 cases of qi deficiency-accompanied syndrome， 38 cases of qi stagnation-accompanied syndrome， and 17 cases of toxin accumulation-accompanied syndrome according to traditional Chinese medicine （TCM） patterns. Data of patients with different TCM patterns were collected， including general condition， TCM symptoms， blood lipids， coagulation function， immune indicators， and serum metabolomics. Metabolic pathway enrichment analysis was used to compare differences in phenomics characteristics among groups. Metabolites with variable importance in projection （VIP） ≥1 and fold change （FC） ≥2 were considered as representative differential metabolites. ResultsPatients in each TCM pattern type were most commonly in the 60-75 years age group， with a relatively high proportion of males. Regarding TCM symptoms， patients with phlegm-stasis binding syndrome most commonly presented with wiry-choppy or wiry-slippery pulse， chest pain， and chest tightness； in patients with qi deficiency-accompanied syndrome， fatigue， chest pain， and weak pulse were most common； in patients with qi stagnation-accompanied sydnrome， wiry-choppy or wiry-slippery pulse， chest pain， and symptoms that increase or decrease with emotional changes， belching， or flatulence were most common； in patients with toxin accumulation-accompanied syndrome， bitter taste in the mouth， chest tightness， irritability， restlessness or manic delirium， and dry and hard stools or foul-smelling diarrhea were most common. Comparisons among the different TCM patterns showed statistically significant differences in coagulation parameters （P＜0.05）， whereas no statistically significant difference was found in blood lipid levels and immune indicators （P＞0.05）.Metabolomics analysis suggested that disorders of glycerophosphate metabolism and valine， leucine， and isoleucine metabolism are characteristic features of phlegm-stasis binding syndrome and its accompanied patterns. The representative differential metabolites between phlegm-stasis binding syndrome and qi deficiency-accompanied syndrome were 7,8-dihydrobiopterin （FC = 3.58） and oxypurinol （FC = 124.50）. Those between phlegm-stasis binding syndrome and qi stagnation-accompanied syndrome were cytidine 5′-diphosphocholine （FC = 2.62） and D-mannosamine （FC = 2.99）. Those between phlegm-stasis binding syndrome and toxin accumulation-accompanied syndrome were anserine （FC = 7.83） and canrenone （FC = 8.94）. ConclusionThere are certain differences in the clinical characteristics and phenomics characteristics among patients with SAP due to CHD exhibiting phlegm-stasis binding syndrome and its accompanied patterns. The phenomics characteristics mainly involve biological alterations such as lipid metabolism disorders， amino acid metabolism abnormalities， and multiple immune indicators activation， which may provide a reference for precise differentiation and treatment of SAP of CHD in TCM clinical practice.

ObjectiveTo explore the clinical and phenomics characteristics of patients with phlegm-stasis binding syndrome and its accompanied patterns in stable angina pectoris （SAP） of coronary heart disease （CHD）. MethodsA multicenter cross-sectional study design was adopted. A total of 300 patients with SAP of CHD were enrolled and classified into 120 cases of phlegm-stasis binding syndrome， 125 cases of qi deficiency-accompanied syndrome， 38 cases of qi stagnation-accompanied syndrome， and 17 cases of toxin accumulation-accompanied syndrome according to traditional Chinese medicine （TCM） patterns. Data of patients with different TCM patterns were collected， including general condition， TCM symptoms， blood lipids， coagulation function， immune indicators， and serum metabolomics. Metabolic pathway enrichment analysis was used to compare differences in phenomics characteristics among groups. Metabolites with variable importance in projection （VIP） ≥1 and fold change （FC） ≥2 were considered as representative differential metabolites. ResultsPatients in each TCM pattern type were most commonly in the 60-75 years age group， with a relatively high proportion of males. Regarding TCM symptoms， patients with phlegm-stasis binding syndrome most commonly presented with wiry-choppy or wiry-slippery pulse， chest pain， and chest tightness； in patients with qi deficiency-accompanied syndrome， fatigue， chest pain， and weak pulse were most common； in patients with qi stagnation-accompanied sydnrome， wiry-choppy or wiry-slippery pulse， chest pain， and symptoms that increase or decrease with emotional changes， belching， or flatulence were most common； in patients with toxin accumulation-accompanied syndrome， bitter taste in the mouth， chest tightness， irritability， restlessness or manic delirium， and dry and hard stools or foul-smelling diarrhea were most common. Comparisons among the different TCM patterns showed statistically significant differences in coagulation parameters （P＜0.05）， whereas no statistically significant difference was found in blood lipid levels and immune indicators （P＞0.05）.Metabolomics analysis suggested that disorders of glycerophosphate metabolism and valine， leucine， and isoleucine metabolism are characteristic features of phlegm-stasis binding syndrome and its accompanied patterns. The representative differential metabolites between phlegm-stasis binding syndrome and qi deficiency-accompanied syndrome were 7,8-dihydrobiopterin （FC = 3.58） and oxypurinol （FC = 124.50）. Those between phlegm-stasis binding syndrome and qi stagnation-accompanied syndrome were cytidine 5′-diphosphocholine （FC = 2.62） and D-mannosamine （FC = 2.99）. Those between phlegm-stasis binding syndrome and toxin accumulation-accompanied syndrome were anserine （FC = 7.83） and canrenone （FC = 8.94）. ConclusionThere are certain differences in the clinical characteristics and phenomics characteristics among patients with SAP due to CHD exhibiting phlegm-stasis binding syndrome and its accompanied patterns. The phenomics characteristics mainly involve biological alterations such as lipid metabolism disorders， amino acid metabolism abnormalities， and multiple immune indicators activation， which may provide a reference for precise differentiation and treatment of SAP of CHD in TCM clinical practice.

OBJECTIVE To analyze the blood components of Suanzaoren Decoction after oral administration using UHPLC-Q Exactive Orbitrap-MS technology.METHODS Female Sprague-Dawley(SD)rats were used as experimental subjects,and Suanza-oren Decoction was administered orally.Serum samples were collected,and the aqueous extract of Suanzaoren Decoction and the serum were analyzed using UHPLC-Q Exactive Orbitrap-MS technology to identify the prototype components and metabolites absorbed into the blood by comparing and analyzing with the LuMet-TCM database.RESULTS It showed that a total of 458 components were iden-tified in the aqueous extract of Suanzaoren Decoction,and 26 chemical components were identified in the blood,including 23 prototype components and 3 metabolites.CONCLUSION The prototype components absorbed into the blood discovered in this study may be the active ingredients of Suanzaoren Decoction,providing a reference for the research on the pharmacodynamic material basis of Suanza-oren Decoction.

Objective:To investigate the infection rate of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in patients with severe aplastic anemia (SAA) after intensive immunosuppressive therapy in combination with a thrombopoietin receptor agonist (lST+TPO-RA) as well as assess the clinical impact of treatment.Methods:A retrospective, case series study was undertaken involving patients with SAA who were admitted to Soochow Hopes Hematonosis Hospital, The First Affiliated Hospital of Soochow University, and Zhengzhou Third People′s Hospital from June 2022 to February 2025. Thirty patients with complete CMV and EBV monitoring data after IST+TPO-RA treatment were enrolled. The first activation time of CMV and EBV, the maximum viral load, the first negative conversion time, and blood routine tests within 3 days before CMV and EBV positivity, during the positive period, and within 3 days after turning negative were recorded. The patients were followed up for 9 months after the completion of IST. One-way analysis of variance was used to compare the changes of blood routine before and after virus positivity and after turning negative. The χ2 test was used to compare the viral infection rate and the therapeutic effect of IST between the two groups. Results:The 30 SAA patients comprised 15 males and 15 females with an average age of (40.0±16.9) years. Of the 30 patients, 18 (60.0%) were infected with CMV and 6 (20.0%) with EBV. Among them, 17 cases received rabbit anti-human thymocyte immunoglobulin (r-ATG) treatment (r-ATG group), 13 cases received porcine anti-human lymphocyte immunoglobulin (p-ALG) treatment (p-ALG group). The CMV infection rate was significantly higher in the r-ATG group than in the p-ALG group (15/17 vs. 3/13, χ2=13.03, P<0.001); meanwhile, the rate of EBV infection was only slightly higher in the r-ATG group than in the p-ALG group, and the difference did not reach statistical significance (5/17 vs. 1/13, χ2=2.17, P=0.196). In patients infected with CMV, neutrophil, hemoglobin, and platelet counts were significantly decreased during the infection phase, followed by significant increases after CMV clearance ( F=14.48, 11.38, 4.73; all P<0.05). No significant differences in treatment efficacy were found between the r-ATG and p-ALG groups at 3, 6, and 9 months post-IST (all P>0.05). Conclusions:This preliminary study showed that the incidence of CMV and EBV infection in patients with SAA increased after IST, with CMV infections occurring significantly more frequently than EBV infections. The CMV infection rate was significantly higher in patients treated with r-ATG than in those receiving p-ALG. CMV infection was associated with notable alterations in hematological parameters, highlighting the need for close clinical monitoring.

OBJECTIVE To explore the relationship between white matter damage and sleepiness,decreased sleep quality,and cognitive decline in patients with OSA.METHODS There were 55 confirmed cases of OSA diagnosed by polysomnography(PSG)from January 2018 to May 2023 were selected,with 29 non-OSA controls also diagnosed by PSG.DKI scanning and Epworth sleepiness scale(ESS),Pittsburgh sleep quality index(PSQI),and Montreal cognitive assessment(MoCA)scores were performed for all subjects.Differences in kurtosis fractional anisotropy(KFA)of various brain regions were compared between the two groups to identify differential brain regions,and pairwise correlations were analyzed between KFA reduction and apnea-hypopnea index(AHI),lowest oxygen saturation(LSaO2)and various scale scores in OSA patients.RESULTS The KFA values in right external capsule,bilateral corona radiata,bilateral superior longitudinal fasciculus,corpus callosum,posterior cingulate gyrus of OSA group were lower than control group(P<0.05).For the OSA group:The correlation between ESS scores and KFA values of right posterior corona radiata,left superior longitudinal fasciculus,body of corpus callosum,splenium of corpus callosum are all negative(r=-0.287,-0.286,-0.276,-0.449,P<0.05).The correlation between PSQI scores and KFA values of right posterior corona radiata,left posterior corona radiata,splenium of corpus callosum are all negative(r=-0.390,-0.274,-0.348,P<0.05).The correlation between MoCA scores and KFA values of right posterior corona radiata,right superior longitudinal fasciculus,left anterior corona radiata,left posterior corona radiata,left superior corona radiata,left superior longitudinal fasciculus,genu of corpus callosum,body of corpus callosum,are all positive(r=0.290,0.389,0.298,0.278,0.340,0.473,0.344,0.344,P<0.05).The correlation between visuospatial and executive function scores and AHI,LSaO2,ESS scores and KFA values of right posterior corona radiata,left superior corona radiata,left superior longitudinal fasciculus and splenium of corpus callosum are all significant(r=-0.350,0.470,-0.343,0.401,0.284,0.387,0.274,P<0.05).CONCLUSION Patients with OSA exhibit damage to the white matter in certain brain regions.The damage to the corpus callosum,posterior corona radiata,and superior longitudinal fasciculus has the greatest impact on patients'sleepiness,reduced sleep quality,and cognitive impairment.In particular,the impairment in visuospatial and executive function is closely associated with white matter damage in the corona radiata and superior longitudinal fasciculus.

Intestinal flora imbalance is closely related to the pathogenesis of rheumatoid arthritis (RA). The existing studies have explored the monomer components such as tripterygium glycosides, total glycosides of Chaenomeles speciosa, and triterpenoid saponins of Clematis, Chinese materia medica such as Tripterygium wilfordii, Caulis Sinomenii, Radix Paeoniae Alba, Fructus Gardeniae, Fructus Chebulae, Radix Ginseng, Radix et Rhizoma Rhei, Rhizoma Atractylodis Macrocephalae, Pterostilbene, and Ginger, as well as the mechanisms of Danggui Sini Decoction, Danggui Niantong Decoction, Duhuo Jisheng Decoction, Yunpi Jiedu Tongluo Qushi Decoction, Qingre Huoxue Decoction, Compound Fengshining, Qingre Yangyin Chushi Decoction, Aconitum Decoction, Zhijing Powder, Jinwu Jiangu Capsule, and Fermented Chinese Medicine Qushi Chubi Decoction in intervening RA by regulating intestinal flora, suggesting that Chinese materia medica can restore intestinal homeostasis, reduce joint inflammation and play a role in the prevention and treatment of RA by regulating immune response, improving intestinal mucosal barrier and regulating intestinal metabolites.

Objective:To explore the impact of the Yiqi Huoxue formula combined with nucleos(t)ide analogs (NAs) antiviral therapy on the serum N-glycan abundance in patients with hepatitis B-related liver fibrosis so as to clarify the application value of the oligosaccharide chain test (GT) for dynamic monitoring of the liver fibrosis progression.Methods:Sixty-two cases diagnosed with chronic hepatitis B at the Department of Hepatology, the Third Hospital of Hebei Medical University, between January 2020 and December 2022 were enrolled and divided into a Yiqi Huoxue Formula (YQHX) combined with NAs group and an NAs monotherapy group ( n=31), with 31 cases in each group for a total of 96 weeks of follow-up. Patient's basic clinical characteristics and liver stiffness measurement (LSM) were collected. GT was used simultaneously to detect the serum N-glycan profile and abundance changes. The nonparametric Mann-Whitney U test was used to compare the differences between the two groups. Enumeration data were expressed as number of cases and percentages (%). The χ2 test was used to compare constituent ratios between two or more groups. Correlation analysis was performed using the Spearman method, with P<0.05 considered statistically significant. Results:The proportion of patients was significantly higher in the YQHX combined with NAs group than the NAs monotherapy group [61.29% (19/31) vs. 9.68% (3/31), P<0.05] with no progression in liver fibrosis staging following 96 weeks of follow-up. The abundance of the N-glycan marker peak 8 [triantennary N-glycan (NA3)] had resulted in significant change for liver fibrosis improvements ( P<0.05), which predicts liver fibrosis progression and reversal in populations sensitive to traditional Chinese medicine. Conclusion:The combined application of Yiqi Huoxue formula and NAs can significantly promote the improvement rate of hepatitis B-related liver fibrosis. Serum N-glycan peak 8 may serve as a potential biomarker for monitoring the reversal of hepatitis B-related liver fibrosis.

Objective To explore the change of nucleoplasm distribution of glucocorticoid receptor alpha(GRα)in peripheral blood of children with primary nephrotic syndrome(PNS)during the course of the disease,aiming at evaluating the correlation between nuclear transport abnormality and different GC responses.Methods A total of 45 children with PNS were enrolled as subjects in this prospective study,and divided into steroid-sensitive nephrotic syndrome(SSNS,n=36)and steroid-resistant nephrotic syndrome(SRNS,n=9)groups,according to their response to GC.The SSNS group was further subclassified into non-frequently relapsing nephrotic syndrome(NFRNS,n=21)and frequently relapsing nephrotic syndrome(FRNS,n=15)based on relapse frequency during 12-month follow-up.Peripheral blood samples were collected before GC treatment,6-week and 6-month after GC treatment.GRα nuclear localization was detected by immunofluorescence assay,and their correlations with clinical-laboratory indicators were analyzed.Results Before the GC treatment,the average fluorescence intensity showed no significantly difference among different groups(P＞0.05),the GRαin the three groups were localized mainly in cytoplasm,and the nucleocytoplasmic ratio showed no significantly difference among the three groups(P＞0.05).6-week after the GC treatment,the average fluorescence intensity showed no significantly difference among the three groups(P＞0.05),the GRα in SSNS group were localized mainly in nucleus,while those in SRNS group were localized mainly in cytoplasm.Furthermore,nucleocytoplasmic ratio in NFRNS group and SRNS group demonstrated significant differences,while those in NFRNS group and FRNS group showed no significant difference(P＞0.05).6-month after the GC treatment,the average fluorescence intensity in NFRNS group and FRNS group showed no significant difference(P＞0.05),GRα in the two groups were localized mainly in nucleus,and their nucleocytoplasmic ratio had significantly differences(P＜0.05).The GRα nucleocytoplasmic ratio in children with PNS was negatively correlated with 24-hour urine protein(24 h-UTP),TNF-α,while positively correlated with serum albumin(Alb).Conclusion There are differences in nuclear transport ability among PNS children of SRNS,NFRNS and FRNS groups,and these differences are correlated with the differency of GC responses.

Objective To explore the change of nucleoplasm distribution of glucocorticoid receptor alpha(GRα)in peripheral blood of children with primary nephrotic syndrome(PNS)during the course of the disease,aiming at evaluating the correlation between nuclear transport abnormality and different GC responses.Methods A total of 45 children with PNS were enrolled as subjects in this prospective study,and divided into steroid-sensitive nephrotic syndrome(SSNS,n=36)and steroid-resistant nephrotic syndrome(SRNS,n=9)groups,according to their response to GC.The SSNS group was further subclassified into non-frequently relapsing nephrotic syndrome(NFRNS,n=21)and frequently relapsing nephrotic syndrome(FRNS,n=15)based on relapse frequency during 12-month follow-up.Peripheral blood samples were collected before GC treatment,6-week and 6-month after GC treatment.GRα nuclear localization was detected by immunofluorescence assay,and their correlations with clinical-laboratory indicators were analyzed.Results Before the GC treatment,the average fluorescence intensity showed no significantly difference among different groups(P＞0.05),the GRαin the three groups were localized mainly in cytoplasm,and the nucleocytoplasmic ratio showed no significantly difference among the three groups(P＞0.05).6-week after the GC treatment,the average fluorescence intensity showed no significantly difference among the three groups(P＞0.05),the GRα in SSNS group were localized mainly in nucleus,while those in SRNS group were localized mainly in cytoplasm.Furthermore,nucleocytoplasmic ratio in NFRNS group and SRNS group demonstrated significant differences,while those in NFRNS group and FRNS group showed no significant difference(P＞0.05).6-month after the GC treatment,the average fluorescence intensity in NFRNS group and FRNS group showed no significant difference(P＞0.05),GRα in the two groups were localized mainly in nucleus,and their nucleocytoplasmic ratio had significantly differences(P＜0.05).The GRα nucleocytoplasmic ratio in children with PNS was negatively correlated with 24-hour urine protein(24 h-UTP),TNF-α,while positively correlated with serum albumin(Alb).Conclusion There are differences in nuclear transport ability among PNS children of SRNS,NFRNS and FRNS groups,and these differences are correlated with the differency of GC responses.