Accumulating evidence has demonstrated that nucleic acid-based therapies are promising for atherosclerosis. However, nearly all nucleic acid delivery systems developed for atherosclerosis necessitate injection, which results in rapid elimination and poor patient compliance. Consequently, oral delivery strategies capable of targeting atherosclerotic plaques are imperative for nucleic acid therapeutics. Herein we report the development of yeast-derived capsules (YCs) packaging an antisense oligonucleotide (AM33) targeting microRNA-33 (miR-33) for the oral treatment of atherosclerosis. YCs provide stability for AM33, preventing its premature release in the gastrointestinal tract. AM33-containing YCs, defined as YAM33, showed high transfection in macrophages, thus promoting cholesterol efflux and inhibiting foam cell formation by regulating the target genes/proteins of miR-33. Orally delivered YAM33 effectively accumulated within atherosclerotic plaques in ApoE ^-/- mice, primarily by transepithelial absorption via M cells in Peyer's patches and subsequent translocation via macrophages through the lymphatic system. Inhibition of miR-33 by oral YAM33 significantly delayed the progression of atherosclerosis. Moreover, oral treatment with YCs co-delivering AM33 and atorvastatin afforded significantly enhanced anti-atherosclerotic effects. Our findings suggest that yeast-based microcapsules represent an effective carrier for oral delivery of nucleic acids, either alone or in combination with existing drugs, offering a promising approach for precision therapy of atherosclerotic diseases.

Objective:To investigate the effects of pulsed electric field (PEF) combined with gemcitabine (GEM) on the viability and stemness of HCCC-9810 cholangiocarcinoma stem cells.Methods:HCCC-9810 cholangiocarcinoma stem cells were established in serum-free, cytokine-rich medium and divided into four groups: the control group, GEM group, PEF group, and the pulsed electric field combined with gemcitabine (PEF+ GEM) group. Cell proliferation was detected using the Cell Counting Kit-8 (CCK8) assay. Cell viability and apoptosis rate were measured by flow cytometry. Cell invasion ability was assessed using the Transwell assay. The expression of stemness marker proteins CD133 and Octamer-binding transcription factor 4 (OCT4), as well as the expression of β-catenin, was detected by Western blotting.Results:Regarding cell viability, the GEM, PEF, and PEF+ GEM groups showed significantly lower cell viability and higher apoptosis rate than the control group at 24 h, 48 h, and 72 h (all P<0.05). At 48 h and 72 h, the PEF+ GEM group showed significantly lower cell viability (7.2%±0.3% and 5.9%±0.8%, respectively) than the GEM group (50.7%±0.6% and 31.0%±1.2%, respectively) and the PEF group (12.2%±0.2% and 12.8%±0.2%, respectively) (all P<0.05). Regarding stemness inhibition, the PEF+ GEM groups showed significantly lower expression levels of CD133 and OCT4 at 24 h, 48 h, and 72 h compared with the control group (all P<0.05). Notably, at 48 h, the PEF+ GEM group showed a significantly lower expression level of the OCT4 (0.61±0.02) than the GEM group (0.87±0.08) and the PEF group (1.00±0.10) ( P<0.01). Furthermore, at 24 h and 48 h, the GEM, PEF, and PEF+ GEM groups showed significantly lower expression levels of β-catenin compared with the control group (all P<0.05). Conclusion:Pulsed electric field combined with gemcitabine therapy demonstrated more effective anti-proliferation and cancer stemness inhibition effects on HCCC-9810 cholangiocarcinoma stem cells compared with either monotherapy.

Objective To explore the value of serum family with sequence similarity 19 member A5(FAM19A5)and vitronectin in evaluating the degree of coronary artery disease and major adverse cardiovas-cular events(MACE)in patients with coronary heart disease(CHD)complicated with heart failure(HF).Methods From March 2022 to March 2023,120 patients with CHD complicated with HF in this hospital were collected as the study group,and 76 healthy individuals who underwent physical examination in the hospital were as the control group.Patients in the study group were divided into mild group(41 cases),moderate group(59 cases)and severe group(20 cases)according to Gensini score and degree of coronary artery dis-ease.Meanwhile,they were divided into MACE group(36 cases)and non-MACE group(84 cases)according to whether MACE occurred 6 months after treatment.Serum FAM19A5 and vitronectin levels were detected by enzyme-linked immunosorbent assay(ELISA),and the correlation between serum FAM19A5 and vitronec-tin levels and Gensini score in CHD patients with HF was analyzed by Pearson method.Multivariate Logistic regression was used to analyze the influencing factors of MACE in CHD patients with HF,and receiver operat-ing characteristic(ROC)curve was used to evaluate the predictive value of FAM19A5 and vitronectin for MACE in CHD patients with HF.Results The serum FAM19A5 level in the study group was significantly decreased(P＜0.05),and the serum vitronectin level was significantly increased(P＜0.05).The serum FAM19A5 level in severe group was significantly lower than that in mild and moderate groups(P＜0.05),and the serum vitronectin level was significantly higher than that in mild and moderate groups(P＜0.05).The serum FAM19A5 level in MACE group was significantly lower than that in non-MACE group(P＜0.05),and the serum vitronectin level was significantly higher than that in no-MACE group(P＜0.05).Ser-um FAM19A5 level in CHD patients with HF was negatively correlated with Gensini scores(r=-0.755,P＜0.001),and vitronectin level was positively correlated with Gensini scores(r=0.697,P＜0.001).The risk factors for MACE in CHD patients with HF were cardiac function grade Ⅲ-Ⅳ,decreased FAM19A5 lev-el,increased LDL-C,vitronectin level and Gensini scores(P＜0.05).The area under the curve(AUC)of ser-um FAM19A5,vitronectin and the combination of FAM19A5 and Vitronectin were 0.882,0.877,and 0.962,respectively,which were superior to the AUC of FAM19A5 and Vitronectin alone(Z=2.612,2.188,P=0.009,0.029).Conclusion The serum levels of FAM19A5 and vitronectin are significantly correlated with the degree of coronary artery disease in CHD patients with HF,and the combination of Fam19A5 and Vitronectin has a high predictive value for the occurrence of MACE.

Objective To explore the evidence for Traditional Chinese Medicine(TCM)in the treatment of premature ovarian insufficiency(POI)based on evidence map and re-evaluation of systematic reviews.Methods CNKI,WanFang Data,VIP,SinoMed,PubMed,Embase,Cochrane Library and Web of Science database were electronically searched to collect systematic reviews(SR)/Meta-analysis on the treatment of POI with TCM from the inception to March 31,2025.The reporting quality,methodological quality,and evidence quality of the included studies were evaluated using the PRISMA 2020 Statement,AMSTAR 2 Checklist,and GRADE system,respectively.The interventions,number of studies,and evidence grades were comprehensively displayed using evidence map.Results A total of 15 SR/Meta-analysis were included,comprising 9 Chinese articles and 6 English articles.The PRISMA 2020 checklist evaluation revealed that 8 articles had certain deficiencies in reporting,while 7 articles demonstrated relatively complete reporting.Based on the AMSTAR 2 checklist,5 articles were rated as high-level and 10 as very low-level.A total of 10 primary outcome indicators were involved,reported 133 times.When classified using the GRADE system,there were 20 pieces of moderate-quality evidence,58 pieces of low-quality evidence,and 55 pieces of very low-quality evidence.The evidence map showed that TCM alone or in combination with hormone therapy could effectively treat POI,reduce follicle-stimulating hormone and luteinizing hormone levels,increase estradiol levels,and improve clinical manifestations and TCM syndrome manifestations.Conclusion TCM has certain advantages in the treatment of POI,enhancing the overall treatment effect,alleviating clinical symptoms of low estrogen,and regulating sex hormone levels to some extent.However,there are deficiencies in methodological quality and reporting quality,and the level of evidence is not high.Therefore,the findings should be used with caution in clinical practice.

Objective This study aimed to investigate the tumorigenic properties of MMTV-PyMT breast cancer transgenic mice at different ages(in weeks)and the changes in the composition of immune cells in the tumor microenvironment.Methods Eight groups of 4,6,8,10,12,14,16 and 18 weeks of age MMTV-PyMT female mice(FVB mice as the background)and one group of 8 weeks of FVB female mice were prepared for routine blood testing,the pathological changes of the mammary gland and lung metastases were observed by histopathological sections,and the immune cells in blood,spleen,and tumor were analyzed by flow cytometry.Results MMTV-PyMT mice showed adenular ductal lesions at 4～6 weeks of age;the ductal portion expanded to the growth boundary at 8～9 weeks of age,and then gradually broke through the glandular boundary to form early breast cancer at 8～12 weeks of age,and advanced breast cancer at 10～14 weeks of age.At 12 weeks of age,metastases were visible in the lungs of some mice,and at 14 weeks of age,the number of metastases in the lungs increased significantly.As the age of the mice increased,the number of white blood cells,neutrophils,and platelets in their blood increased gradually,while the lymphocytes and erythrocytes showed a gradual downward trend.Flow cytometry showed that with the increase in age,the proportion of T cells in the spleen and tumor gradually decreased,the MDSCs in the blood,spleen,and tumor gradually increased,and the NK cells in the tumor also gradually increased.Conclusions This study analyzed routine blood tests,pathology,and immune cells in the tissues of MMTV-PyMT mouse models of different weeks of age,providing a novel perspective on the dynamic alterations of the tumor immune microenvironment during the malignant progression of breast cancer.

Objective:To establish the minimum number of negative lymph nodes (nLN) required for patients undergoing gastrectomy.Methods:This was a retrospective cohort study with inclusion criteria as follows: (1) radical gastrectomy; (2) histologically confirmed adenocarcinoma; (3) complete tumor staging information; and (4) known number of lymph nodes harvested. The exclusion criteria were: (1) other concurrent malignant tumors; (2) metastatic or recurrent gastric cancer; (3) initial surgery performed at another hospital; (4) preoperative neoadjuvant therapy; (5) distant metastasis; and (6) incomplete clinical data or follow-up information. Based on the above criteria, a total of 11 167 patients with gastric adenocarcinoma who underwent radical subtotal gastrectomy (RSG) or radical total gastrectomy (RTG) in the Department of Gastric Surgery, Fudan University Shanghai Cancer Center between January 1, 2000, and December 31, 2022, were included in the study. Among them, there were 7 596 cases in the RSG group and 3 571 cases in the RTG group. Restricted cubic spline (RCS) analysis was used to determine the ideal threshold for nLN for RSG and RTG patients. Survival analysis was conducted using Kaplan-Meier (KM) curves and log-rank tests, and propensity score matching (PSM) was utilized to balance parameters between two groups. Furthermore, subgroup analysis was conducted for RSG patients based on tumor location (upper, middle and lower) to determine the minimum number of nLN in each subgroup.Results:For patients who underwent RSG, the mean number of nLN was 21.9, with a median of 21. RCS analysis showed that more than 21 nLN was associated with better survival. Moreover, both pre- and post-PSM analysis confirmed that patients with nLN ≥21 had better survival benefits compared to those with nLN <21 (overall survival [OS]: P<0.001 before PSM, P=0.013 after PSM; disease-free survival [DFS]: P<0.001 before PSM, P=0.013 after PSM). For patients who underwent RTG, the mean number of nLN was 23.5, with a median of 22. Here RCS analysis indicated that more than 22 nLN was associated with better postoperative survival in RTG patients, and both pre- and post-PSM analysis confirmed that patients with nLN ≥22 had better survival benefits compared to those with nLN<22 (OS: P<0.001 both before and after PSM; DFS: P<0.001 both before and after PSM). Subgroup analysis showed that for RSG patients with tumor located in the upper part, having ≥17 nLN (OS: both P<0.001), and for RSG patients with tumor located in the middle and lower part, having ≥22 nLN (OS: both P<0.001), were associated with better prognoses. Conclusions:For patients who receive RSG, the minimal number of nLN is ideally ≥21 (upper ≥17, middle and lower ≥22). Similarly, for patients who receive RTG, the minimum number of nLN ideally is 22.

Objective To explore the evidence for Traditional Chinese Medicine(TCM)in the treatment of premature ovarian insufficiency(POI)based on evidence map and re-evaluation of systematic reviews.Methods CNKI,WanFang Data,VIP,SinoMed,PubMed,Embase,Cochrane Library and Web of Science database were electronically searched to collect systematic reviews(SR)/Meta-analysis on the treatment of POI with TCM from the inception to March 31,2025.The reporting quality,methodological quality,and evidence quality of the included studies were evaluated using the PRISMA 2020 Statement,AMSTAR 2 Checklist,and GRADE system,respectively.The interventions,number of studies,and evidence grades were comprehensively displayed using evidence map.Results A total of 15 SR/Meta-analysis were included,comprising 9 Chinese articles and 6 English articles.The PRISMA 2020 checklist evaluation revealed that 8 articles had certain deficiencies in reporting,while 7 articles demonstrated relatively complete reporting.Based on the AMSTAR 2 checklist,5 articles were rated as high-level and 10 as very low-level.A total of 10 primary outcome indicators were involved,reported 133 times.When classified using the GRADE system,there were 20 pieces of moderate-quality evidence,58 pieces of low-quality evidence,and 55 pieces of very low-quality evidence.The evidence map showed that TCM alone or in combination with hormone therapy could effectively treat POI,reduce follicle-stimulating hormone and luteinizing hormone levels,increase estradiol levels,and improve clinical manifestations and TCM syndrome manifestations.Conclusion TCM has certain advantages in the treatment of POI,enhancing the overall treatment effect,alleviating clinical symptoms of low estrogen,and regulating sex hormone levels to some extent.However,there are deficiencies in methodological quality and reporting quality,and the level of evidence is not high.Therefore,the findings should be used with caution in clinical practice.

Objective This study aimed to investigate the tumorigenic properties of MMTV-PyMT breast cancer transgenic mice at different ages(in weeks)and the changes in the composition of immune cells in the tumor microenvironment.Methods Eight groups of 4,6,8,10,12,14,16 and 18 weeks of age MMTV-PyMT female mice(FVB mice as the background)and one group of 8 weeks of FVB female mice were prepared for routine blood testing,the pathological changes of the mammary gland and lung metastases were observed by histopathological sections,and the immune cells in blood,spleen,and tumor were analyzed by flow cytometry.Results MMTV-PyMT mice showed adenular ductal lesions at 4～6 weeks of age;the ductal portion expanded to the growth boundary at 8～9 weeks of age,and then gradually broke through the glandular boundary to form early breast cancer at 8～12 weeks of age,and advanced breast cancer at 10～14 weeks of age.At 12 weeks of age,metastases were visible in the lungs of some mice,and at 14 weeks of age,the number of metastases in the lungs increased significantly.As the age of the mice increased,the number of white blood cells,neutrophils,and platelets in their blood increased gradually,while the lymphocytes and erythrocytes showed a gradual downward trend.Flow cytometry showed that with the increase in age,the proportion of T cells in the spleen and tumor gradually decreased,the MDSCs in the blood,spleen,and tumor gradually increased,and the NK cells in the tumor also gradually increased.Conclusions This study analyzed routine blood tests,pathology,and immune cells in the tissues of MMTV-PyMT mouse models of different weeks of age,providing a novel perspective on the dynamic alterations of the tumor immune microenvironment during the malignant progression of breast cancer.

Objective:To investigate the effects of pulsed electric field (PEF) combined with gemcitabine (GEM) on the viability and stemness of HCCC-9810 cholangiocarcinoma stem cells.Methods:HCCC-9810 cholangiocarcinoma stem cells were established in serum-free, cytokine-rich medium and divided into four groups: the control group, GEM group, PEF group, and the pulsed electric field combined with gemcitabine (PEF+ GEM) group. Cell proliferation was detected using the Cell Counting Kit-8 (CCK8) assay. Cell viability and apoptosis rate were measured by flow cytometry. Cell invasion ability was assessed using the Transwell assay. The expression of stemness marker proteins CD133 and Octamer-binding transcription factor 4 (OCT4), as well as the expression of β-catenin, was detected by Western blotting.Results:Regarding cell viability, the GEM, PEF, and PEF+ GEM groups showed significantly lower cell viability and higher apoptosis rate than the control group at 24 h, 48 h, and 72 h (all P<0.05). At 48 h and 72 h, the PEF+ GEM group showed significantly lower cell viability (7.2%±0.3% and 5.9%±0.8%, respectively) than the GEM group (50.7%±0.6% and 31.0%±1.2%, respectively) and the PEF group (12.2%±0.2% and 12.8%±0.2%, respectively) (all P<0.05). Regarding stemness inhibition, the PEF+ GEM groups showed significantly lower expression levels of CD133 and OCT4 at 24 h, 48 h, and 72 h compared with the control group (all P<0.05). Notably, at 48 h, the PEF+ GEM group showed a significantly lower expression level of the OCT4 (0.61±0.02) than the GEM group (0.87±0.08) and the PEF group (1.00±0.10) ( P<0.01). Furthermore, at 24 h and 48 h, the GEM, PEF, and PEF+ GEM groups showed significantly lower expression levels of β-catenin compared with the control group (all P<0.05). Conclusion:Pulsed electric field combined with gemcitabine therapy demonstrated more effective anti-proliferation and cancer stemness inhibition effects on HCCC-9810 cholangiocarcinoma stem cells compared with either monotherapy.

Objective:To establish the minimum number of negative lymph nodes (nLN) required for patients undergoing gastrectomy.Methods:This was a retrospective cohort study with inclusion criteria as follows: (1) radical gastrectomy; (2) histologically confirmed adenocarcinoma; (3) complete tumor staging information; and (4) known number of lymph nodes harvested. The exclusion criteria were: (1) other concurrent malignant tumors; (2) metastatic or recurrent gastric cancer; (3) initial surgery performed at another hospital; (4) preoperative neoadjuvant therapy; (5) distant metastasis; and (6) incomplete clinical data or follow-up information. Based on the above criteria, a total of 11 167 patients with gastric adenocarcinoma who underwent radical subtotal gastrectomy (RSG) or radical total gastrectomy (RTG) in the Department of Gastric Surgery, Fudan University Shanghai Cancer Center between January 1, 2000, and December 31, 2022, were included in the study. Among them, there were 7 596 cases in the RSG group and 3 571 cases in the RTG group. Restricted cubic spline (RCS) analysis was used to determine the ideal threshold for nLN for RSG and RTG patients. Survival analysis was conducted using Kaplan-Meier (KM) curves and log-rank tests, and propensity score matching (PSM) was utilized to balance parameters between two groups. Furthermore, subgroup analysis was conducted for RSG patients based on tumor location (upper, middle and lower) to determine the minimum number of nLN in each subgroup.Results:For patients who underwent RSG, the mean number of nLN was 21.9, with a median of 21. RCS analysis showed that more than 21 nLN was associated with better survival. Moreover, both pre- and post-PSM analysis confirmed that patients with nLN ≥21 had better survival benefits compared to those with nLN <21 (overall survival [OS]: P<0.001 before PSM, P=0.013 after PSM; disease-free survival [DFS]: P<0.001 before PSM, P=0.013 after PSM). For patients who underwent RTG, the mean number of nLN was 23.5, with a median of 22. Here RCS analysis indicated that more than 22 nLN was associated with better postoperative survival in RTG patients, and both pre- and post-PSM analysis confirmed that patients with nLN ≥22 had better survival benefits compared to those with nLN<22 (OS: P<0.001 both before and after PSM; DFS: P<0.001 both before and after PSM). Subgroup analysis showed that for RSG patients with tumor located in the upper part, having ≥17 nLN (OS: both P<0.001), and for RSG patients with tumor located in the middle and lower part, having ≥22 nLN (OS: both P<0.001), were associated with better prognoses. Conclusions:For patients who receive RSG, the minimal number of nLN is ideally ≥21 (upper ≥17, middle and lower ≥22). Similarly, for patients who receive RTG, the minimum number of nLN ideally is 22.