ObjectiveTo observe the effects of Yiqi Huoxue Jiedu formula（YHJF） on immune cell exhaustion in the spleen of septic mice and to explore and validate its potential intervention targets. MethodsMice were randomly divided into the sham-operated， model， low-dose YHJF（4.1 g·kg^-1）， and high-dose YHJF（8.2 g·kg^-1） groups. Except for the sham-operated group， a cecal ligation and puncture（CLP） procedure was performed to establish a mouse sepsis model. The treatment groups received oral administration of the corresponding doses， while the sham-operated and model groups received an equal volume of physiological saline. After the intervention， the 7-day survival rate of each group was recorded， and spleen samples were collected 72 h post-intervention， and the spleen index was calculated. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate（dUTP） nick end labeling（TUNEL） staining was used to detect apoptosis in spleen cells. Enzyme-linked immunosorbent assay（ELISA） was performed to measure the levels of interleukin（IL）-4 and IL-10 in the serum. Transcriptomics and metabolomics were used to screen for differentially expressed genes（DEGs） and differential metabolites in the spleen， followed by bioinformatics analysis to identify key targets. Real-time quantitative polymerase chain reaction（Real-time PCR）， flow cytometry， and multiplex immunofluorescence were used to verify the expressions of key genes and proteins. ResultsThe high-dose YHJF group significantly improved the 7-day survival rate of septic mice（P0.05）. Compared with the sham-operated group， the model group showed a significant increase in apoptosis of spleen cells and a decrease in the spleen index at 72 h post-modeling， with markedly elevated peripheral serum IL-4 and IL-10 levels（P0.01）. Compared with the model group， the high-dose YHJF group showed a reduction in apoptosis of spleen cells， an increase in the spleen index， and a significant decrease in peripheral serum IL-4 and IL-10 levels（P0.05）. Spleen transcriptomics identified 255 DEGs between groups， potentially serving as intervention targets for YHJF. Gene Ontology（GO） enrichment analysis revealed that DEGs were mainly involved in biological processes such as natural killer（NK） cell-mediated positive immune regulation， cell killing， cytokine production， positive regulation of innate immune cells， and interferon production. Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis showed that DEGs were mainly involved in cytokine-cytokine receptor interactions， viral protein interactions with cytokines and cytokine receptors， chemokine signaling pathway， and nuclear transcription factor-κB（NF-κB） signaling pathway. Protein-protein interaction（PPI） network analysis identified CD160， granzyme B（GZMB）， and chemokine ligand 4（CCL4） as key targets for YHJF in treating sepsis. Metabolomics identified 46 differential metabolites that were significantly reversed by YHJF intervention， and combined transcriptomics and metabolomics analysis identified 17 differential metabolites closely related to CD160. Pathway enrichment revealed that these metabolites were mainly involved in glycerophospholipid metabolism， arachidonic acid metabolism， glycosylphosphatidylinositol（GPI） anchor biosynthesis， linoleic acid metabolism， and α-linolenic acid metabolism pathways. Verification results showed that， compared with the sham-operated group， the model group exhibited significantly elevated CD160 mRNA expression level in the spleen， along with markedly decreased CCL4 and GZMB mRNA expression， and had a significant increase in CD160 expression on the surface of natural killer T（NKT） cells in the spleen（P0.01）. Compared with the model group， the high-dose YHJF group had a significant decrease in CD160 mRNA expression in the spleen， a significant increase in CCL4 and GZMB mRNA expressions. Further flow cytometry and immunofluorescence revealed that compared with the sham-operated group， CD160 expression on the surface of splenic NKT cells in the model group was significantly increased（P0.01）， while high-dose YHJF intervention significantly reduced CD160 expression（P0.01）. ConclusionYHJF may alleviate NKT cell exhaustion in sepsis by downregulating the expression of the negative co-stimulatory molecule CD160， and this regulatory effect is closely related to fatty acid metabolism pathways. This study provides new insights and targets for further exploration of strengthening vital Qi and detoxifying strategy to improve immune cell exhaustion in acute deficiency syndrome of sepsis.

ObjectiveTo observe the effects of Yiqi Huoxue Jiedu formula（YHJF） on immune cell exhaustion in the spleen of septic mice and to explore and validate its potential intervention targets. MethodsMice were randomly divided into the sham-operated， model， low-dose YHJF（4.1 g·kg^-1）， and high-dose YHJF（8.2 g·kg^-1） groups. Except for the sham-operated group， a cecal ligation and puncture（CLP） procedure was performed to establish a mouse sepsis model. The treatment groups received oral administration of the corresponding doses， while the sham-operated and model groups received an equal volume of physiological saline. After the intervention， the 7-day survival rate of each group was recorded， and spleen samples were collected 72 h post-intervention， and the spleen index was calculated. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate（dUTP） nick end labeling（TUNEL） staining was used to detect apoptosis in spleen cells. Enzyme-linked immunosorbent assay（ELISA） was performed to measure the levels of interleukin（IL）-4 and IL-10 in the serum. Transcriptomics and metabolomics were used to screen for differentially expressed genes（DEGs） and differential metabolites in the spleen， followed by bioinformatics analysis to identify key targets. Real-time quantitative polymerase chain reaction（Real-time PCR）， flow cytometry， and multiplex immunofluorescence were used to verify the expressions of key genes and proteins. ResultsThe high-dose YHJF group significantly improved the 7-day survival rate of septic mice（P0.05）. Compared with the sham-operated group， the model group showed a significant increase in apoptosis of spleen cells and a decrease in the spleen index at 72 h post-modeling， with markedly elevated peripheral serum IL-4 and IL-10 levels（P0.01）. Compared with the model group， the high-dose YHJF group showed a reduction in apoptosis of spleen cells， an increase in the spleen index， and a significant decrease in peripheral serum IL-4 and IL-10 levels（P0.05）. Spleen transcriptomics identified 255 DEGs between groups， potentially serving as intervention targets for YHJF. Gene Ontology（GO） enrichment analysis revealed that DEGs were mainly involved in biological processes such as natural killer（NK） cell-mediated positive immune regulation， cell killing， cytokine production， positive regulation of innate immune cells， and interferon production. Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis showed that DEGs were mainly involved in cytokine-cytokine receptor interactions， viral protein interactions with cytokines and cytokine receptors， chemokine signaling pathway， and nuclear transcription factor-κB（NF-κB） signaling pathway. Protein-protein interaction（PPI） network analysis identified CD160， granzyme B（GZMB）， and chemokine ligand 4（CCL4） as key targets for YHJF in treating sepsis. Metabolomics identified 46 differential metabolites that were significantly reversed by YHJF intervention， and combined transcriptomics and metabolomics analysis identified 17 differential metabolites closely related to CD160. Pathway enrichment revealed that these metabolites were mainly involved in glycerophospholipid metabolism， arachidonic acid metabolism， glycosylphosphatidylinositol（GPI） anchor biosynthesis， linoleic acid metabolism， and α-linolenic acid metabolism pathways. Verification results showed that， compared with the sham-operated group， the model group exhibited significantly elevated CD160 mRNA expression level in the spleen， along with markedly decreased CCL4 and GZMB mRNA expression， and had a significant increase in CD160 expression on the surface of natural killer T（NKT） cells in the spleen（P0.01）. Compared with the model group， the high-dose YHJF group had a significant decrease in CD160 mRNA expression in the spleen， a significant increase in CCL4 and GZMB mRNA expressions. Further flow cytometry and immunofluorescence revealed that compared with the sham-operated group， CD160 expression on the surface of splenic NKT cells in the model group was significantly increased（P0.01）， while high-dose YHJF intervention significantly reduced CD160 expression（P0.01）. ConclusionYHJF may alleviate NKT cell exhaustion in sepsis by downregulating the expression of the negative co-stimulatory molecule CD160， and this regulatory effect is closely related to fatty acid metabolism pathways. This study provides new insights and targets for further exploration of strengthening vital Qi and detoxifying strategy to improve immune cell exhaustion in acute deficiency syndrome of sepsis.

Background/Aims: Liver transplantation (LT) following total hepatectomy is a life-saving treatment for hepatocellular carcinoma (HCC). The HCC recurrence after LT hinders the effectiveness of the procedure. The objective of this study is to develop a pre-operative risk stratification model based on a liquid biopsy. Methods: We conducted a comprehensive multi-omics study of 260 HCC patients from three centers, including clinical data, low-coverage whole-genome sequencing of cell-free DNA (cfDNA) from plasma, as well as whole-exome, single-nucleus RNA, and spatial transcriptomics from matched tumor and non-tumor tissues. Results: We identified cfDNA-derived copy number alteration (CNA) signatures associated with post-transplant recurrence. By integrating cfDNA-derived CNA profiles with single-cell transcriptomic data, we traced recurrence-associated cfDNA to a distinct subpopulation of malignant cells within the primary tumor. These cells were embedded in a pro-metastatic microenvironment of specialized endothelial subtypes and cancer-associated fibroblasts. Notably, most recurrence-associated lesions were detectable in cfDNA prior to liver transplantation (LT). Building on these insights, we developed the ZJU Criteria based on CNA fragments and tumor markers, a pre-LT risk prediction tool that integrates conventional clinical factors with cfDNA-derived CNA signatures, and validated it using internal and independent external cohorts. Conclusion Our findings suggest that post-transplant recurrence commonly originates from advanced subclones that emerge late during tumor evolution. The ZJU Criteria provides an accurate, non-invasive strategy that significantly improves pre-LT risk stratification and clinical decision-making for patients with HCC.

ObjectiveHepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. The main risk factors associated with HCC include viral hepatitis (B and/or C), alcohol abuse, and metabolic dysfunction-associated steatotic liver disease (MASLD), which progressively advance to liver fibrosis, cirrhosis, and ultimately evolve into HCC. Surgical resection represents the most effective treatment for HCC, while recent advances in immunotherapy, including immune checkpoint inhibitors and adoptive cell therapies, have provided improved treatment prospects for patients with unresectable HCC. However, the complex metabolic heterogeneity of HCC limits the therapeutic efficacy. Metabolic intermediates acyl-CoA not only provide energy and substrates for numerous biochemical reactions but also serve as donors for protein lysine acylation, a major class of post-translational modification (PTM). Therefore, a deeper understanding of the molecular mechanisms underlying protein lysine acylation and hepatocarcinogenesis is urgently needed. MethodsThe levels of protein lysine acylation and silence information regulator 5 (SIRT5) expression levels in clinical HCC samples were analyzed by Western blot. Quantitative malonylome and succinylome of HCC samples were analyzed by antibody-based affinity enrichment coupled with tandem mass spectrometry. The proliferation of HCC cells was analyzed with Cell Counting Kit-8 (CCK-8) assays, the apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining coupled with flow cytometry, and the ability of cells to migrate was assayed by Transwell assays. The enzymatic activity of glutathione S-transferase Mu 1 (GSTM1) was quantified. Transgenic mice with hepatic overexpression of SIRT5 were constructed using CRISPR-Cas9, and primary hepatocarcinogenesis was induced by administration of diethylnitrosamine. ResultsWestern blot analysis indicated that the expression level of SIRT5 was elevated in clinical samples from HCC patients, and the levels of lysine malonylation, glutarylation, and succinylation were significantly reduced in HCC tissues. Knockout of SIRT5 in MHCC-97H and MHCC-97L hepatoma cells suppressed cell proliferation, and increased the percentage of apoptotic cells significantly. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially malonylome and succinylome of HCC samples revealed significant enrichment in two major classes of biological processes: core energy metabolism (e.g., glycolysis/gluconeogenesis, tricarboxylic acid metabolic process, fatty acid beta oxidation) and detoxification and oxidative stress response (e.g., response to toxic substance, chemical carcinogenesis, reactive oxygen species (ROS)). SIRT5 removes malonylation from lysine residues in GSTM1 and restores its detoxification activity, which is crucial for the survival of hepatocytes under stressed conditions. More importantly, in vivo experiment indicated that hepatic-specific overexpression of SIRT5 in mice accelerated diethylnitrosamine-induced liver fibrosis and hepatocarcinogenesis, indicating the critical role of SIRT5 in HCC progression. ConclusionThis study highlights the previously unrecognized SIRT5-GSTM1 axis as a key regulator in hepatocarcinogenesis, and suggests a potential target for the treatment of patients with HCC.

BACKGROUND:Multiple studies have confirmed that mitogen-activated protein kinase(MAPK)signaling pathway is involved in cell proliferation,and microRNA(miR)is involved in the occurrence and development of hypertrophic scars.Therefore,the role of miR-27a-3p and MAPK signaling pathways in pathological scar formation has been further explored. OBJECTIVE:To explore the effect of miR-27a-3p on the proliferation of human hypertrophic scar fibroblasts through the MAPK signaling pathway. METHODS:The primary fibroblasts were isolated and collected from the skin samples.The primary fibroblasts were observed by inverted microscope and verified by immunofluorescence.The relative expression level of miR-27a-3p in tissues was detected by qRT-PCR.The target genes of hsa-miR-27a-3p were predicted using the database,and then the predicted target genes were enriched by gene ontology function analysis and biological pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes.There were seven groups:blank control,negative control,miR-27a-3p mimic,miR-27a-3p inhibitor,miR-27a-3p mimic+p38 MAPK inhibitor,miR-27a-3p mimic+extracellular regulated protein kinase inhibitor,miR-27a-3p mimic+c-Jun N-terminal kinase inhibitor.Western blot was used to detect the levels of extracellular regulated protein kinase,c-Jun N-terminal kinase inhibitor.and p38 kinase and their phosphorylation levels.Cell counting kit-8 and EdU were used to detect cell proliferation. RESULTS AND CONCLUSION:Compared with normal skin fibroblasts,hypertrophic scar fibroblasts had stronger proliferative activity(P<0.05)and faster proliferation level(P<0.001).Compared with normal skin,miR-27a-3p was highly expressed in hypertrophic scars(P<0.001).Compared with the negative control group,overexpression of miR-27a-3p could promote cell proliferation activity(P<0.001)and proliferation levels(P<0.001).Compared with the negative control group,knockdown of miR-27a-3p could inhibit the proliferation activity(P<0.05)and proliferation levels(P<0.001).Compared with the negative control group,overexpression of miR-27a-3p promoted the phosphorylated levels of extracellular regulated protein kinase,c-Jun N-terminal kinase,and p38 mitogen-activated protein kinase(P<0.05).Compared with the negative control group,knockdown of miR-27a-3p inhibited the phosphorylated levels of extracellular regulated protein kinase,c-Jun N-terminal kinase,and p38 MAPK(P<0.05).Compared with the miR-27a-3p mimic group,specific inhibitors of extracellular regulated protein kinase,c-Jun N-terminal kinase,and p38 MAPK reversed the effects of miR-27a-3p on the proliferative activity(P<0.01)and proliferation level(P<0.001)of fibroblasts.To conclude,these results suggest that miR-27a-3p promotes the proliferation of human hypertrophic scar fibroblasts by activating the MAPK signaling pathway.

Laparoscopic subtotal cholecystectomy (LSC) has been a safe and viable alternative to conversion to laparotomy in cases of severe cholecystitis. The objective of this study is to determine the utility of intraoperative choledochoscopy in LSC for the exploration of the gallbladder, cyst duct, and subsequent stone clearance of the cystic duct in cases of severe cholecystitis. A total of 72 patients diagnosed with severe cholecystitis received choledochoscopy-assisted laparoscopic subtotal cholecystectomy (CALSC). A choledochoscopy was performed to explore the gallbladder cavity and/or cystic duct, and to extract stones using a range of techniques. The clinical records, including the operative records and outcomes, were subjected to analysis. No LSC was converted to open surgery, and no bile duct or vascular injuries were sustained. All stones within the cystic duct were removed by a combination of techniques, including high-frequency needle knife electrotomy, basket, and electrohydraulic lithotripsy. A follow-up examination revealed the absence of residual bile duct stones, with the exception of one common bile duct stone, which was extracted via endoscopic retrograde cholangiopancreatography. In certain special cases, CALSC may prove to be an efficacious treatment for the management of severe cholecystitis. This technique allows for optimal comprehension of the situation within the gallbladder cavity and cystic duct, facilitating the removal of stones from the cystic duct and reducing the residue of the non-functional gallbladder remnant.

Objective:To investigate the protective effect of achyranthes bidentata(AB)on sperm quality in mice with sper-matogenic disorder through the glycolytic metabolic pathway and its action mechanism.Methods:We equally randomized 40 Kun-ming mice into a normal control,a model control,a low-dose AB(3.5 g/kg)and a high-dose AB group(7.0 g/kg),and established the model of spermatogenic disorder in the latter three groups of mice by intraperitoneal injection of doxorubicin(30 mg/kg).Two days after modeling,we collected the testis and kidney tissues and blood samples from the mice for observation of the pathological changes in the testis tissue by HE staining,detection of perm motility with the sperm quality analyzer,examination of the apoptosis of testis cells by flow cytometry,measurement of the levels of testosterone(T),malondialdehyde(MDA),superoxide dismutase(SOD)and cata-lase(CAT)in the serum and testis tissue by ELISA,and determination of expressions of the key enzymes of glycolysis hexokinase Ⅱ(HK2),pyruvate kinase M2(PKM2),platelet phosphofructokinase(PFKP),lactate dehydrogenase A(LDHA)and the meiosis pro-teins REC8 and SCP3 by Western blot,and the mRNA expressions of glycolytic phosphofructokinase 1(PFK1),phosphoglycerate ki-nase 1(PGK1),tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1β)by fluorescence quantitative PCR(FQ-PCR).Results:Compared with the model controls,the mice in the AB groups showed significant increases in the testis coefficient,kidney in-dex,sperm concentration,sperm motility,spermatogonia,primary spermatocytes,spermatids,sperm count and the serum T level(P＜0.05 orP＜0.01),but dramatic decreases in the apoptosis of testis cells and percentage of morphologically abnormal sperm(P＜0.01).Achyranthes bidentata also significantly elevated the levels of SOD and CAT,and down-regulated the mRNA expressions of MDA,TNF-α and IL-1β(P＜0.05 or P＜0.01),and up-regulated the protein expressions of HK2,PKM2,PFKP,LDHA,REC8 and SCP3,and expressions of the glycolysis key genes Pfk1 and Pgk1(P＜0.05 orP＜0.01).Conclusion:Achyranthes bidentata ameliorates doxorubicin-induced spermatogenic disorder in mice by regulating the glycolytic pathway and reducing oxidative stress and the expressions of inflammatory factors.

Objective To investigate the clinical prognosis of untreated residual coronary artery dissection treated with drug coated balloon(DCB).Methods A retrospective analysis was conducted on the clinical and imaging data of patients with primary coronary artery lesions(2.5-4.0 mm)treated with DCB under angiography guidance at Xuzhou Cancer Hospital,Xuzhou New Health Geriatric Hospital,and Peixian Guotai Hospital from September 2017 to April 2023.According to the observation of coronary artery dissection through angiography,the patients were divided into a dissection group and a non dissection group.The main endpoint of this study was the major adverse cardiovascular event(MACE)during a 12-month follow-up.Results A total of 381 patients were enrolled in the three research centers,with 30 cases(30 lesions)in the dissection group and 351 cases(367 lesions)in the non dissection group.There was no significant difference between the two groups in terms of age,gender,hypertension,hyperlipidemia,diabetes,smoking,previous myocardial infarction,previous percutaneous coronary intervention,coronary artery bypass grafting and other baseline clinical characteristics(all P＞0.05).Except for the reference vessel diameter(P=0.049)and DCB pressure(P=0.032),there was no statistically significant difference in the characteristics of coronary angiography lesions between the two groups of patients(both P＞0.05).During a 12-month follow-up,there was no statistically significant difference(P＞0.05)in the incidence of MACE between the dissection group and the non dissection group after DCB treatment for primary coronary artery lesions in situ.Conclusions Untreated residual dissection after DCB treatment of de novo coronary lesions does not lead to an increase in clinical MACE.

Aortic regurgitation and mitral regurgitation are more common in elderly heart valve disease,and both may be present in some patients.Severe aortic regurgitation complicated with severe mitral regurgitation often requires surgical valve replacement,but in patients at high risk of surgery,the risk of perioperative mortality is significantly increased.Therefore,for such patients,minimally invasive interventions can significantly improve long-term patient outcomes while reducing surgical risk.This article report a case of transcatheter aortic valve replacement combined with transcatheter edge-to-edge repair in the treatment of severe aortic regurgitation combined with mitral valve prolapse,in order to explore new treatment ideas for similar cases.