The intestinal microecology is closely related to the occurrence and development of hepatocellular carcinoma (HCC). The intestinal microbiota and its metabolites can regulate the tumor immune microenvironment through the "gut-liver axis", promoting cancer progression. Therefore, the intestinal microbiome is gradually demonstrating the potential as a biomarker for early diagnosis of HCC and prediction of the efficacy of immunotherapy. Targeted intervention on the intestinal microecology (such as probiotics, fecal microbiota transplantation, dietary regulation, etc.) may enhance the efficacy of immune checkpoint inhibitors (ICIs) and is becoming a promising combination therapy strategy. In the future, HCC treatment will rely on multi-omics integration, artificial intelligence-assisted diagnosis, and synthetic biology tools to promote the translation of precise gut flora intervention strategies from basic research to the clinic. This article summarized the latest research progress of intestinal microecology in HCC, explored its potential value and development direction for precision diagnosis and treatment of HCC, and provided a theoretical basis for the clinical application of related intervention strategies.

Objective:To explore the value of combining gut microbiota and clinical features for preoperative microvascular invasion (MVI) prediction in hepatocellular carcinoma (HCC).Methods:Clinical data and fecal samples were collected from 71 HCC patients who underwent curative resection at Ningbo Second Hospital between Jan 2023 and Aug 2024. Among them, 41 patients were assigned to the training set and 30 to the validation set. Gut microbiota composition was analyzed using 16S rRNA sequencing. Redundancy analysis (RDA) was used to evaluate the influence of clinical features on the microbiota. Differences in alpha and beta diversity between the MVI-negative and MVI-positive groups were assessed. Differential genera were identified using the Wilcoxon test and LEfSe analysis. A random forest model and Logistic regression were employed to screen key differential genera, followed by ROC analysis. Genera with high ROC values were further validated in the validation set.Results:RDA indicated that MVI was a key factor influencing gut microbiota composition. The random forest model (AUC=0.925), combined with Logistic regression analysis, identified four genera: Acidovorax ( OR=0.618), Tissierella ( OR=1.293), Chitinophaga ( OR=4.596), and Virgisporangium ( OR=0.960), as well as two clinical features: tumor diameter ( OR=0.668) and liver cirrhosis ( OR=14.011), as independent risk factors. ROC analysis showed that in the training set, the combination of Chitinophaga (AUC=0.71) and tumor diameter (AUC=0.75) had the best diagnostic performance (AUC=0.87). In the validation set, the combination of Virgisporangium (AUC=0.80) and tumor diameter (AUC=0.79) yielded the highest diagnostic performance (AUC=0.87). Conclusions:A genomics-based model combining gut microbiota and clinical features shows promising predictive value for noninvasive preoperative assessment of MVI status in HCC patients.

Objective:To explore the training method for personalized eyebrow shape design before eyebrow transplantation surgery and to evaluate its effectiveness.Methods:Thirty physicians with similar foundational knowledge in facial aesthetic design and equivalent educational backgrounds were prospectively selected from the Second Hospital of Lanzhou University and Beijing Biliansheng Medical Beauty Clinic from January to December 2023. All the physicians were right-handed. Based on prior experience in eyebrow transplantation surgery, they were divided into two groups: an observation group [ n=20; 10 males, 10 females; aged (27.8±3.9) years] without experience in personalized eyebrow transplantation surgery, and a control group [ n=10; 5 males, 5 females; aged (29.6±4.0) years] with at least 2 years of experience in personalized eyebrow transplantation surgery. All the physicians received 2 hours of theoretical instruction on personalized eyebrow shape design and 8 hours of simulated operation training. Differences in personalized eyebrow design scores between the two groups were compared before and after training. After the assessment, physicians in the observation group performed eyebrow shape design on 20 eyebrow transplantation patients with randomly assigned face shapes, and patient satisfaction was evaluated. Results:Before training, personalized eyebrow design scores were 1.16±0.29 for the observation group and 3.75±0.22 for the control group, showing a statistically significant difference ( P<0.001). After operation training, the scores were 4.51±0.11 for the observation group and 4.89±0.08 for the control group, with no statistically significant difference ( P>0.05). The difference between pre- and post-training scores within the observation group was statistically significant ( P<0.05). Physicians in the observation group designed eyebrow shapes for male and female patients with various face shapes. Immediately after surgery, 80% (16/20) of patients were very satisfied, and 20% (4/20) were satisfied. Conclusion:After training of combined theoretical lectures, simulated practice, and hands-on patient design, physicians in the observation group demonstrate improved ability in personalized eyebrow shape design and achieve high patient satisfaction.

In recent years, the research on the microbial community within cancer has gradually attracted attention. The microbiota within the tumor affects the immune regulation of liver cancer and may promote the progression of liver cancer through inflammatory responses, metabolic regulation, etc. This article explores the sources and distribution characteristics of microorganisms in liver cancer, their potential mechanism of action in the occurrence and development of liver cancer, and evaluates the application prospects of intratumoral microorganisms in the early diagnosis, treatment response prediction and personalized treatment of liver cancer, especially their potential value in targeted therapy, providing new ideas for microbial intervention in future precision medicine.

Objective:To explore the training method for personalized eyebrow shape design before eyebrow transplantation surgery and to evaluate its effectiveness.Methods:Thirty physicians with similar foundational knowledge in facial aesthetic design and equivalent educational backgrounds were prospectively selected from the Second Hospital of Lanzhou University and Beijing Biliansheng Medical Beauty Clinic from January to December 2023. All the physicians were right-handed. Based on prior experience in eyebrow transplantation surgery, they were divided into two groups: an observation group [ n=20; 10 males, 10 females; aged (27.8±3.9) years] without experience in personalized eyebrow transplantation surgery, and a control group [ n=10; 5 males, 5 females; aged (29.6±4.0) years] with at least 2 years of experience in personalized eyebrow transplantation surgery. All the physicians received 2 hours of theoretical instruction on personalized eyebrow shape design and 8 hours of simulated operation training. Differences in personalized eyebrow design scores between the two groups were compared before and after training. After the assessment, physicians in the observation group performed eyebrow shape design on 20 eyebrow transplantation patients with randomly assigned face shapes, and patient satisfaction was evaluated. Results:Before training, personalized eyebrow design scores were 1.16±0.29 for the observation group and 3.75±0.22 for the control group, showing a statistically significant difference ( P<0.001). After operation training, the scores were 4.51±0.11 for the observation group and 4.89±0.08 for the control group, with no statistically significant difference ( P>0.05). The difference between pre- and post-training scores within the observation group was statistically significant ( P<0.05). Physicians in the observation group designed eyebrow shapes for male and female patients with various face shapes. Immediately after surgery, 80% (16/20) of patients were very satisfied, and 20% (4/20) were satisfied. Conclusion:After training of combined theoretical lectures, simulated practice, and hands-on patient design, physicians in the observation group demonstrate improved ability in personalized eyebrow shape design and achieve high patient satisfaction.

Objective:To explore the value of combining gut microbiota and clinical features for preoperative microvascular invasion (MVI) prediction in hepatocellular carcinoma (HCC).Methods:Clinical data and fecal samples were collected from 71 HCC patients who underwent curative resection at Ningbo Second Hospital between Jan 2023 and Aug 2024. Among them, 41 patients were assigned to the training set and 30 to the validation set. Gut microbiota composition was analyzed using 16S rRNA sequencing. Redundancy analysis (RDA) was used to evaluate the influence of clinical features on the microbiota. Differences in alpha and beta diversity between the MVI-negative and MVI-positive groups were assessed. Differential genera were identified using the Wilcoxon test and LEfSe analysis. A random forest model and Logistic regression were employed to screen key differential genera, followed by ROC analysis. Genera with high ROC values were further validated in the validation set.Results:RDA indicated that MVI was a key factor influencing gut microbiota composition. The random forest model (AUC=0.925), combined with Logistic regression analysis, identified four genera: Acidovorax ( OR=0.618), Tissierella ( OR=1.293), Chitinophaga ( OR=4.596), and Virgisporangium ( OR=0.960), as well as two clinical features: tumor diameter ( OR=0.668) and liver cirrhosis ( OR=14.011), as independent risk factors. ROC analysis showed that in the training set, the combination of Chitinophaga (AUC=0.71) and tumor diameter (AUC=0.75) had the best diagnostic performance (AUC=0.87). In the validation set, the combination of Virgisporangium (AUC=0.80) and tumor diameter (AUC=0.79) yielded the highest diagnostic performance (AUC=0.87). Conclusions:A genomics-based model combining gut microbiota and clinical features shows promising predictive value for noninvasive preoperative assessment of MVI status in HCC patients.

In recent years, the research on the microbial community within cancer has gradually attracted attention. The microbiota within the tumor affects the immune regulation of liver cancer and may promote the progression of liver cancer through inflammatory responses, metabolic regulation, etc. This article explores the sources and distribution characteristics of microorganisms in liver cancer, their potential mechanism of action in the occurrence and development of liver cancer, and evaluates the application prospects of intratumoral microorganisms in the early diagnosis, treatment response prediction and personalized treatment of liver cancer, especially their potential value in targeted therapy, providing new ideas for microbial intervention in future precision medicine.

Objective:To investigate the mechanism of Fusobacterium nucleatum ( Fn) in regulating the formation of neutrophil extracellular trap (NET) to aggravate colitis. Methods:With a completely randomized design, 15 C57BL/6 mice were randomly divided into negative control group, dextran sulfate sodium (DSS) A group (DSS-induced colitis), and DSS+ Fn A group (DSS-induced colitis with Fn infection); another 15 C57BL/6 mice were randomly divided into DSS B group, DSS+ Fn B group, and GSK484 group (DSS-induced colitis with Fn infection and followed by intraperitoneal injection of peptidylarginine deiminase 4 (PAD4)inhibitor GSK484), with 5 mice in each group. The Fn-infected neutrophils (HL-60 cell) model and PAD4-inhibited cell model were established and divided into neutrophil-like control cell (induced with 1.25% dimethylsulfoxide), Fn+ neutrophil-like cell (neutrophil-like cells co-cultured with Fn), and GSK484 cell ( Fn+ neutrophil-like cell co-cultured with GSK484) with a completely randomized design. The neutrophil-like control and Fn+ neutrophil-like cells were divided into 2 batches to conduct experiments before and after PAD4 inhibition separately.The expression of zonula occludens-1 (ZO-1), neutrophil elastase (NE), reactive oxygen species, PAD4, and citrullinated histone H3(cit-H3) were analyzed by immunohistochemistry staining, Western blotting, and flow cytometry assay. Two-tailed t test was used for statistical analysis. Results:The results of immunohistochemical staining showed that compared with those of the DSS A group, the expression levels of NE, PAD4 and cit-H3 of the DSS+ Fn A group were upregulated, and the expression level of ZO-1 was downregulated; compared with those of the DSS+ Fn B group, the expression level of ZO-1 of the GSK484 group was upregulated, and the expression levels of cit-H3 and PAD4 were downregulated. The results of Western blotting demonstrated that, before the PAD4 inhibition, the expression levels of NE, PAD4 and cit-H3 of the Fn+ neutrophil-like cell were all higher than those of the neutrophil-like control cell (1.52±0.11 vs. 1.00±0.19, 1.21±0.06 vs. 1.00±0.07, 1.59±0.16 vs. 1.00±0.16), and the differences were statistically significant ( t=-4.13, -3.86, and -4.47; P=0.014, 0.014, and 0.018); after the PAD4 inhibition, the expression levels of PAD4, cit-H3, and NE of the GSK484 cell were all lower than those of the Fn+ neutrophil-like cell (0.95±0.09 vs. 1.27±0.04, 1.15±0.34 vs. 2.29±0.50, 1.22±0.14 vs. 1.68±0.12), and the differences were statistically significant ( t=5.61, 3.24, and 4.49; P=0.005, 0.032, and 0.011). The results of flow cytometry assay indicated that the positive rate of reactive oxygen species of the DSS+ Fn A group was higher than that of the DSS A group ((21.15±2.93)% vs. (11.14±1.42)%), and the positive rate of reactive oxygen species of the Fn+ neutrophil-like cell was also higher than that of the neutrophil-like control cell before the PAD4 inhibition ((51.69±6.94)% vs.(31.95±3.31)%), and the differences were statistically significant( t=5.33 and 4.45, P=0.006 and 0.011). Conclusion:Fn can promote neutrophil to release NET by upregulating reactive oxygen/PAD4/cit-H3 signaling pathway, which disrupt the intestinal barrier and aggravate colitis.

Objective:To investigate whether F.nucleatum affects the chemotherapy resistance of colorectal cancer by regulating ABC subfamily G subtype 2 (ABCG2), in view of the fact that the correlation between the two has not been reported. Methods:Oxaliplatin was used to interfere with colorectal cancer cells and co-cultured with F.nucleatum to establish a chemotherapy-induced model of microbial infection. Calcein AM/PI cell staining, trypan blue staining, and cell counting kit 8 (CCK-8) method were used to detect cell activity. Real-time fluorescence quantitative polymerase chain reaction and western blot were used to detect ABCG2 mRNA and protein expression levels in colorectal cancer cells. The target gene was knocked down by constructing shRNA plasmids. The HCT-116 cell F.nucleatum infection model was constructed and transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analyses were performed to determine the differential gene expression enrichment pathways. Genistein (GST) was used as an E-cadherin blocker, and triclabendazole sulfoxide (TCBZSO) was used as an ABCG2 blocker. Immunofluorescence was used to detect E-cadherin and β-catenin protein expressions and intracellular localization levels. The subcutaneous xenograft model of nude mice was constructed in vivo, and the expression level of ABCG2 protein in tissues was detected by immunohistochemical staining. Results:CCK-8 results showed that F. nucleatum+oxaliplatin group [HT-29, (92.26±1.66)%; HCT-116, (82.13±1.84)%] cell relative survival rate was higher than that of oxaliplatin group [HT-29, (79.64±3.72)%; HCT-116, (67.56±2.96)%; P＜0.001]. The relative survival rate of oxaliplatin and F. nucleatum co-culture group with ABCG2-knockdown HCT-116 cells [(61.44±1.48)%] was lower than that of F. nucleatum and oxaliplatin co-cultured with HCT-116 cells [(69.29±4.45)%, P=0.015]. GO enrichment analysis showed that HCT-116 cells co-cultured with F.nucleatum were significantly enriched in "β-catenin binding", "cadherin binding" and "regulation of Wnt signaling pathway". RT-qPCR results showed that the relative expression of ABCG2 mRNA in F.nucleatum + genistein group was significantly lower than that in F.nucleatum group ( P＜0.001). The results in vivo showed that the tumor weight in the F.n+L-OHP+TCBZSO group [(0.12±0.02)g] was lower than that in the F.n+L-OHP group [(0.33±0.05)g, P＜0.001]. Immunohistochemistry suggested that the promotion of ABCG2 protein expression by F. nucleatum was blocked after TCBZSO intervention. Conclusion:F. nucleatum up-regulates ABCG2 expression through E-cadherin/β-catenin signaling pathway to promote colorectal cancer resistance to oxaliplatin.

Objective:To investigate the mechanism of Fusobacterium nucleatum ( Fn) in regulating the formation of neutrophil extracellular trap (NET) to aggravate colitis. Methods:With a completely randomized design, 15 C57BL/6 mice were randomly divided into negative control group, dextran sulfate sodium (DSS) A group (DSS-induced colitis), and DSS+ Fn A group (DSS-induced colitis with Fn infection); another 15 C57BL/6 mice were randomly divided into DSS B group, DSS+ Fn B group, and GSK484 group (DSS-induced colitis with Fn infection and followed by intraperitoneal injection of peptidylarginine deiminase 4 (PAD4)inhibitor GSK484), with 5 mice in each group. The Fn-infected neutrophils (HL-60 cell) model and PAD4-inhibited cell model were established and divided into neutrophil-like control cell (induced with 1.25% dimethylsulfoxide), Fn+ neutrophil-like cell (neutrophil-like cells co-cultured with Fn), and GSK484 cell ( Fn+ neutrophil-like cell co-cultured with GSK484) with a completely randomized design. The neutrophil-like control and Fn+ neutrophil-like cells were divided into 2 batches to conduct experiments before and after PAD4 inhibition separately.The expression of zonula occludens-1 (ZO-1), neutrophil elastase (NE), reactive oxygen species, PAD4, and citrullinated histone H3(cit-H3) were analyzed by immunohistochemistry staining, Western blotting, and flow cytometry assay. Two-tailed t test was used for statistical analysis. Results:The results of immunohistochemical staining showed that compared with those of the DSS A group, the expression levels of NE, PAD4 and cit-H3 of the DSS+ Fn A group were upregulated, and the expression level of ZO-1 was downregulated; compared with those of the DSS+ Fn B group, the expression level of ZO-1 of the GSK484 group was upregulated, and the expression levels of cit-H3 and PAD4 were downregulated. The results of Western blotting demonstrated that, before the PAD4 inhibition, the expression levels of NE, PAD4 and cit-H3 of the Fn+ neutrophil-like cell were all higher than those of the neutrophil-like control cell (1.52±0.11 vs. 1.00±0.19, 1.21±0.06 vs. 1.00±0.07, 1.59±0.16 vs. 1.00±0.16), and the differences were statistically significant ( t=-4.13, -3.86, and -4.47; P=0.014, 0.014, and 0.018); after the PAD4 inhibition, the expression levels of PAD4, cit-H3, and NE of the GSK484 cell were all lower than those of the Fn+ neutrophil-like cell (0.95±0.09 vs. 1.27±0.04, 1.15±0.34 vs. 2.29±0.50, 1.22±0.14 vs. 1.68±0.12), and the differences were statistically significant ( t=5.61, 3.24, and 4.49; P=0.005, 0.032, and 0.011). The results of flow cytometry assay indicated that the positive rate of reactive oxygen species of the DSS+ Fn A group was higher than that of the DSS A group ((21.15±2.93)% vs. (11.14±1.42)%), and the positive rate of reactive oxygen species of the Fn+ neutrophil-like cell was also higher than that of the neutrophil-like control cell before the PAD4 inhibition ((51.69±6.94)% vs.(31.95±3.31)%), and the differences were statistically significant( t=5.33 and 4.45, P=0.006 and 0.011). Conclusion:Fn can promote neutrophil to release NET by upregulating reactive oxygen/PAD4/cit-H3 signaling pathway, which disrupt the intestinal barrier and aggravate colitis.