1.Correlation between liver fibrosis degree and carotid plaque in patients with lean metabolic dysfunction-associated fatty liver disease
Shuai ZHANG ; Shoulu JIN ; Wanqing LI ; Xijing SHI ; Hao LIANG ; Hao DONG ; Dailong LU ; Ying ZHU ; Xiaoxing XIANG ; Jun LIU
Journal of Clinical Hepatology 2026;42(2):319-325
ObjectiveTo investigate the association between noninvasive liver fibrosis markers and carotid plaque (CP) in patients with lean metabolic dysfunction-associated fatty liver disease (MAFLD), and to provide a basis for screening high-risk populations. MethodsA total of 957 patients with lean MAFLD who underwent physical examination in Subei People’s Hospital from January 2021 to June 2023 was enrolled as the observation cohort, with the presence or absence of CP as the outcome, and fibrosis-4 (FIB-4) index and nonalcoholic fatty liver disease fibrosis score (NFS) were used to assess liver fibrosis degree. The independent-samples t test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. The multivariate logistic regression analysis, the restricted cubic spline analysis, the receiver operating characteristic curve, and the mediation effect analysis were used to investigate the association between liver fibrosis degree and CP. ResultsThe prevalence rate of CP was 36.6% in the lean MAFLD population. Compared with the non-CP group(n=607), the CP group (n=350) had a significantly higher proportion of male patients, a significantly higher proportion of patients with smoking/diabetes/hypertension, and significantly higher levels of age, creatinine, blood urea nitrogen, triglycerides, fasting blood glucose, aspartate aminotransferase, aspartate aminotransferase/alanine aminotransferase ratio, NFS, and FIB-4 index, as well as significantly lower levels of platelet count and albumin (all P<0.05). The multivariate logistic regression analysis showed that after adjustment for confounding factors, FIB-4 index (odds ratio[OR]=2.979, 95% confidence interval[CI]:2.141 — 4.219, P<0.001) and NFS (OR=1.747, 95%CI: 1.499 — 2.046, P<0.001) were positively correlated with CP. Both FIB-4 index and NFS had a good value in predicting CP. Hypertension had a significant indirect effect on the prevalence rate of CP through its impact on liver fibrosis markers, and its mediating effect accounted for 39.5% — 40.8% of the total effect (P<0.001). ConclusionIn patients with lean MAFLD, NFS and FIB-4 index are significantly positively correlated with the prevalence rate of CP, and they can be used as potential epidemiological predictive indicators. Liver fibrosis markers may play a mediating role in the association between hypertension and CP. Interventions targeting hypertension and liver fibrosis markers may help to prevent and delay the progression of CP.
2.Mechanism of Jianpi huatan formula improving polycystic ovary syndrome in rats by regulating the HMGB1/RAGE signaling pathway
China Pharmacy 2026;37(8):1009-1014
OBJECTIVE To explore the effects of the Jianpi huatan formula (JPHTF) on polycystic ovary syndrome (PCOS) in rats by regulating the high mobility group box 1 protein (HMGB1)/receptor for advanced glycation end products (RAGE) signaling pathway. METHODS The rats were randomly divided into the normal (Con) group, the PCOS group, the JPHTF-L group (5.54 g/kg), the JPHTF-H group (11.07 g/kg), the JPHTF-H+rHMGB1 group (11.07 g/kg of JPHTF+8 μg/kg of rHMGB1), and metformin group (0.27 g/kg), with 12 rats in each group. Except for the rats of Con group, which were given 1% sodium carboxymethyl cellulose intragastrically and fed with normal chow, the remaining rats were induced to develop PCOS models by using a high-fat diet combined with letrozole. Af ter successful modeling, rats in each drug group were administered the corresponding drugs by gavage or tail vein injection once a day for 4 consecutive weeks. 24 h after the intervention, body weight and ovarian coefficient were detected. The levels of fasting blood glucose (FBG), serum levels of fasting insulin (FINS), follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) and estradiol (E 2 ) were detected. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin-18 (IL-18) and IL-1β and the protein expressions of HMGB1, RAGE, phosphorylated nuclear factor-κB (p-NF-κB) and NF-κB in the ovarian tissues of rats were detected. The morphology of ovarian tissue was observed, and the numbers of cystic follicles and corpora lutea were counted. RESULTS Compared with PCOS group, polycystic changes of ovarian tissue in rats showed varying degrees of improvement in the JPHTF-L group, JPHTF-H group, and metformin group; body weight, ovarian coefficient, FBG, the number of cystic follicles, serum levels of FINS, HOMA-IR, T, LH, LH/FSH, the levels of TNF-α, IL-18 and IL-1β and protein expressions of HMGB1 and RAGE in ovarian tissue as well as phosphorylation level of NF-κB protein all significantly decreased; the number of corpora lutea and the serum levels of E 2 and FSH significantly increased ( P <0.05). Compared with JPHTF-H group, above indexes of rats were reversed significantly in JPHTF-H+rHMGB1 group ( P <0.05). CONCLUSIONS JPHTF can reduce the inflammatory response in PCOS rats, mitigate ovarian injury, regulate hormone balance, and improve insulin resistance and follicular development by inhibiting the HMGB1/RAGE signaling pathway.
3.Association of liver fibrosis markers and inflammation markers with the risk of gallstones in patients with metabolic dysfunction-associated fatty liver disease
Shuai ZHANG ; Shoulu JIN ; Wanqing LI ; Xijing SHI ; Hao LIANG ; Hao DONG ; Dailong LU ; Ying ZHU ; Xiaoxing XIANG ; Jun LIU
Journal of Clinical Hepatology 2026;42(3):579-585
ObjectiveTo investigate the association of liver fibrosis scores and inflammation markers with gallstones in patients with metabolic dysfunction-associated fatty liver disease (MAFLD), as well as the mediating role of liver fibrosis scores in the relationship between inflammation markers and gallstones. MethodsA total of 14 567 patients who received physical examination and were diagnosed with MAFLD in Subei People’s Hospital from January 2014 to June 2023 were enrolled in this study, and according to the results of abdominal color Doppler ultrasound, they were divided into gallstone group with 1 724 patients and non-gallstone group with 12 843 patients. Related clinical data were collected from all patients, including demographic data, medical history, family history, physical examination, Color Doppler ultrasound, and biochemical parameters. The biomarkers associated with metabolic disorders and insulin resistance included triglyceride-glucose index (TyG), TyG-body mass index (BMI) index, atherogenic index of plasma (AIP), and non-high-density lipoprotein cholesterol-to-high-density lipoprotein cholesterol ratio (NHHR); the biomarkers associated with inflammation and nutritional status included neutrophil-to-lymphocyte ratio (NLR), neutrophil percentage-to-albumin ratio (NPAR), and monocyte-to-lymphocyte ratio (MLR); the biomarkers for assessing liver fibrosis degree and liver function included albumin-bilirubin (ALBI) score, NAFLD fibrosis score (NFS), fibrosis-4 (FIB-4) index, and aspartate aminotransferase-to-platelet ratio index (APRI). The independent-samples t test was used for comparison of normally distributed continuous data between two groups, while the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. Multivariate Logistic regression analysis, restricted cubic spline analysis, and mediating effect analysis were used to assess the association of liver fibrosis markers and inflammation markers with the risk of gallstones. ResultsThe prevalence rate of gallstones was 11.8% among the MAFLD patients. There were significant differences between the gallstone group and the non-gallstone group in sex, age, smoking history, diabetes, hypertension, lymphocytes, platelets, glucose, albumin, serum uric acid, alanine aminotransferase, aspartate aminotransferase, red blood cell, NLR, NPAR, MLR, NFS, FIB-4 index, and ALBI score (all P<0.05). The multivariate Logistic regression analysis showed that NLR (odds ratio [OR]=1.091, 95% confidence interval [CI]: 1.028 — 1.160, P<0.05), NPAR (OR=1.073, 95%CI: 1.042 — 1.105, P<0.05), MLR (OR=1.142, 95%CI: 1.057 — 1.232, P<0.05), NFS (OR=1.239, 95%CI: 1.190 — 1.291, P<0.05), and FIB-4 index (OR=1.326, 95%CI: 1.241 — 1.417, P<0.05) were influencing factors for the prevalence rate of gallstones. The restricted cubic spline analysis showed a significant non-linear association between NFS/FIB-4 index and the risk of gallstone (non-linear P<0.05). The mediating effect analysis further showed that the association of NLR, MLR, and NPAR with gallstones was partially mediated by NFS or FIB-4 index, with a mediating effect accounting for 36.79%、28.09%、29.67% and 18.31%、17.70、11.57%, respectively. ConclusionNFS and FIB-4 index have a non-linear association with the prevalence rate of gallstones in MAFLD patients, and they also mediate the association of NLR, NPAR, and MLR with the risk of gallstone.
4.A Nomogram for Predicting Metachronous Gastric Cancer After Endoscopic Submucosal Dissection of Early Gastric Cancer Following Successful Helicobacter pylori Eradication
Shangtao MAO ; Miao LIU ; Tao ZHAO ; Qiong YAN ; Ying XIANG ; Hai WU ; Wenjun LI ; Hongji TAO ; Duanming ZHUANG ; Lei WANG ; Guifang XU
Journal of Gastric Cancer 2026;26(2):279-294
Purpose:
Due to the preservation of the entire stomach after endoscopic resection, the occurrence of metachronous gastric cancer (MGC) remains a possibility. In this study, we investigated the incidence and risk factors for MGC in patients with early gastric cancer who underwent endoscopic submucosal dissection (ESD) and successfully eradicatedHelicobacter pylori.
Materials and Methods:
A retrospective analysis was conducted of 1,191 patients who underwent ESD and successfully eradicated H. pylori at the Affiliated Drum Tower Hospital of Nanjing University. Endoscopic surveillance was performed at 3, 6, and 12 months post-resection, and annually thereafter. MGC was defined as the development of a new cancer at a site other than the primary gastric cancer site, at least 1 year after the initial endoscopic resection.
Results:
A total of 77 patients were diagnosed with MGC during a median follow-up of 41.5 months. Kaplan-Meier analysis showed a 5-year cumulative incidence of MGC of 9.4% after successful H. pylori eradication. Multivariate analysis of the training set using Cox proportional hazards models identified male sex, severe atrophic gastritis, multiple gastric cancers before H. pylori eradication, and smoking history as independent risk factors for MGC.The nomogram exhibited favorable discrimination, with area under the curves of 0.767 and 0.822 in the training set and 0.724 and 0.745 in the testing set at 3 and 5 years, respectively.
Conclusions
Patients with gastric cancer who undergo endoscopic resection, even after successful H. pylori eradication, should undergo annual and continuous endoscopic surveillance for MGC.
5.Effects of Portulaca oleracea cream on skin pruritus and barrier function in allergic contact dermatitis mice
Xiaoxue WANG ; Xia CHEN ; Xiang PU ; Guanwei FAN ; Xiangyan KONG ; Ying TANG ; Nana WU ; Jiangli LUO
China Pharmacy 2025;36(11):1352-1357
OBJECTIVE To study the effects and mechanism of Portulaca oleracea cream on skin pruritus and barrier function in allergic contact dermatitis (ACD) mice. METHODS Low-concentration and high-concentration P. oleracea creams were prepared, with the P. oleracea extract solution (1 g/mL, calculated by crude drug) concentrations of 10% and 20%. Sixty BALB/c mice were randomly allocated into blank group, model group, Mometasone furoate cream group (positive control), blank matrix cream group, P. oleracea low-concentration and high-concentration cream groups. Except for blank group, ACD model was induced in each group using 2,4-dinitrochlorobenzene solution. The blank group and model groups received normal saline, while the remaining groups were treated with their respective creams, once a day, at a dose of approximately 0.5 g per application, continuously for 14 days. At 24 h post-final administration, skin lesions of mice were observed and scored; pathological changes of skin tissue were observed; serum levels of immunoglobulin E(IgE) and tumor necrosis factor-α (TNF-α) were quantified. mRNA expression of MAS-related G protein-coupled receptors (including MrgprA3, MrgprC11, and MrgprD) in dorsal root ganglion (DRG) was assessed; while protein expressions of skin barrier function-related proteins Claudin-1 and Occludin in skin tissues were determined. RESULTS Compared with blank group, mice in the model group exhibited severe skin damage, characterized by loss of epidermal architecture, hyperkeratosis of the skin tissue, and the infiltration of a large number of inflammatory cells. The skin injury scores, as well as the serum levels of IgE and TNF-α, and the mRNA expression levels of MrgprA3, MrgprC11, and MrgprD in DRG, were all significantly elevated compared to the blank group (P<0.05 or P<0.01); in contrast, the protein expression levels of Claudin-1 and Occludin in the skin tissue were markedly reduced (P<0.05). Compared with model group, mice in P. oleracea low-concentration and high- concentration cream groups demonstrated significant alleviation of skin damage, as evidenced by reduced epidermal hyperplasia, mitigated spongiosis in the dermis, and decreased infiltration of inflammatory cells; these quantitative indicators were almost significantly reversed (P<0.05 or P<0.01). No significant differences were observed in the aforementioned skin injuries, pathological alterations, or quantitative indicators between the blank matrix cream group and the model group. CONCLUSIONS P. oleracea may ameliorate skin lesions and restore skin barrier function of ACD mice, the mechanism of which may be associated with downregulating mRNA expressions of MrgprA3, MrgprC11 and MrgprD in DRG, and up-regulating the protein expressions of Claudin-1 and Occludin in skin tissue.
6.Association between insulin resistance and idiopathic central precocious puberty in girls and the diagnostic value of insulin resistance
Jin-Bo LI ; Ya XIAO ; Shu-Qin JIANG ; Xiang-Yang LUO ; Hong-Ru ZHANG ; Jun SUN ; Wen-Hui SHI ; Ying YANG ; Wei WANG
Chinese Journal of Contemporary Pediatrics 2025;27(12):1487-1492
Objective To explore the relationship between insulin resistance and idiopathic central precocious puberty(ICPP)in girls and the diagnostic value of insulin resistance.Methods Clinical data of 245 girls aged 4 to 7.5 years with low luteinizing hormone(LH)levels(0.2-0.83 IU/L),normal body weight(body mass index standard deviation score between-2 and+2),and early breast development who visited the Department of Pediatric Endocrinology,Henan Provincial Maternal and Child Health Hospital from January 2022 to March 2025 were retrospectively analyzed.According to the Expert Consensus on the Diagnosis and Treatment of Central Precocious Puberty(2022),patients were assigned to an ICPP group(n=123)or a control group(n=122).Correlations between the homeostasis model assessment of insulin resistance(HOMA-IR)and selected indices were assessed.Multivariable logistic regression was used to evaluate the association between HOMA-IR and ICPP,and the diagnostic performance of various indices for ICPP was evaluated.Results HOMA-IR was higher in the ICPP group than in the control group(P<0.001)and was positively correlated with LH peak(rs=0.467,P<0.05)and the LH peak/FSH peak ratio(rs=0.444,P<0.05).The multivariable logistic regression model including age,BMI,and basal LH showed that HOMA-IR was closely associated with ICPP(OR=2.756,95%CI:1.940-3.913).Receiver operating characteristic curve analysis showed that the areas under the curve for basal LH,HOMA-IR,and their combination in diagnosing ICPP were 0.735,0.735,and 0.805,respectively(P<0.05),and the combined model had a greater area under the curve than either basal LH or HOMA-IR alone(both P<0.05).Conclusions HOMA-IR is closely associated with ICPP in girls with low LH and normal body weight,and combining HOMA-IR with basal LH improves early identification and diagnostic efficiency in this population.
7.Ultrasound radiomics combined with machine learning for early diagnosis of seronegative hashimoto’s thyroiditis
Wenjun WU ; Chang LIU ; Shengsheng YAO ; Daming LIU ; Yuan LUO ; Yihan SUN ; Ting RUAN ; Mengyou LIU ; Li SHI ; Mingming XIAO ; Qi ZHANG ; Zhengshuai LIU ; Xingai JU ; Jiahao WANG ; Xiang FEI ; Li LU ; Yang GAO ; Ying ZHANG ; Liying GONG ; Xuanyu CHEN ; Wanli ZHENG ; Xiali NIU ; Xiao YANG ; Huimei CAO ; Shijie CHANG ; Zuoxin MA ; Jianchun CUI
Chinese Journal of Endocrine Surgery 2025;19(3):313-319
Objective:To evaluate the value of ultrasound radiomics combined with machine learning for early diagnosis of seronegative Hashimoto’s thyroiditis (SN-HT) .Methods:This retrospective study included 164 patients from Liaoning Provincial People’s Hospital , Lixin County People’s Hospital, Linghai Dalinghe Hospital, Fengcheng Phoenix Hospital, who underwent thyroidectomy for solitary nodules with normal thyroid function between Nov. 2016 and Jan. 2024. Postoperative pathology confirmed Hashimoto’s thyroiditis (HT) in some cases, who were further categorized into antibody-positive and antibody-negative groups based on serum antibody status. Patients without Hashimoto’s thyroiditis served as the control group. A total of 298 ultrasound images were analyzed. Radiomics features were extracted from hypoechoic non-nodular areas within 0.5 cm surrounding the tumor. Two senior pathologists and two senior ultrasound physicians independently assessed lymphocytic infiltration, eosinophilic changes of follicular epithelium, and the proportion of hypoechoic areas in pathology and ultrasound images, respectively. A machine learning model, CCH-NET, was developed using linear regression and t-distributed stochastic neighbor embedding (t-SNE) techniques. The dataset was divided into a training set (80%) and a validation set (20%) to compare the diagnostic accuracy of CCH-NET with that of senior ultrasound physicians. Results:In internal validation, CCH-NET achieved a diagnostic accuracy of 88.89% for both antibody-positive and antibody-negative groups, significantly higher than the 66.67% accuracy of senior ultrasound physicians ( P<0.01). In external validation, CCH-NET achieved 75.00% and 66.67% accuracy for the two groups, compared to 50.00% by senior ultrasound physicians. For the control group, both methods achieved 93.33% accuracy. The AUC of CCH-NET was 0.848, outperforming senior ultrasound physicians (0.681) ,demonstrating superior diagnostic performance. Conclusion:The radiomics-based CCH-NET model, using non-nodular hypoechoic areas as a specific indicator, can accurately identify early SN-HT in euthyroid patients. It significantly outperforms senior ultrasound physicians, improving diagnostic accuracy and reducing missed diagnoses.
8.Effect of Pricking-Cupping Therapy on Endotoxin-Induced Fever in Rabbits Based on Mitochondrial Energy Metabolism
Wenyu LIU ; Xin WANG ; Ying WANG ; Jin WU ; Bowen XIANG ; Ying PEI
Journal of Nanjing University of Traditional Chinese Medicine 2025;41(1):102-108
OBJECTIVE To observe the fever-reducing effect of pricking-cupping method on endotoxin-induced fever in rab-bits,and to further explore the fever-reducing mechanism of pricking-cupping method from the perspective of skeletal muscle mito-chondrial energy metabolism.METHODS A total of 72 rabbits were randomly divided into blank group,model group,Western medi-cine group,pricking group,cupping group and pricking-cupping group,with 12 rabbits in each group.The rabbit model of endotoxin-in-duced fever was reproduced and the corresponding therapeutic intervention was carried out.The body temperature of rabbits was recor-ded continuously for 6 h.The mitochondrial structure was observed by transmission electron microscopy.Oxygraph-2k system was used to detect mitochondrial function.ATP content in thigh muscle tissue of rabbits in each group was detected.The expression of uncoupling protein 2(UCP2)and mitochondrial fusion protein 2(Mfn2)in thigh muscle of rabbits was detected by Wes automated western blot quantitative analysis system.RESULTS The model was successfully replicated,and the body temperature of rabbits in the four treatment groups was lower than that in the model group.The skeletal muscle mitochondrial morphology of rabbits in the blank group was intact,and the mitochondrial morphology was closest to normal after the treatment of pricking-cupping.Moreover,the oxygen con-sumption of mitochondrial CII OXPHOS stage in the pricking-cupping group was significantly higher than that in the model group.Compared with the model group,the ATP content in the muscle tissue of rabbits in the pricking-cupping group was significantly increased,Mfn2 protein expression was significantly increased,and UCP2 protein expression was significantly decreased.CONCLU-SION The pricking-cupping method has a good fever-reducing effect on endotoxin-induced fever in rabbits,and its mechanism may be related to inhibiting UCP2 protein expression in skeletal muscle,promoting Mfn2 protein expression,promoting the oxidative phos-phorylation process in skeletal muscle mitochondria,and increasing ATP synthesis in mitochondria.
9.A Rubric System for Evaluating the TCA-based Ideological and Political Teaching Model:Its Construction and Application
Ying WANG ; Ling-Hui LV ; Ren-Ji WEI ; Xiang ZHANG ; Yi RU ; Bo YAN ; Lan SHEN ; Mao SUN ; Liang LIANG ; Jing ZHAO
Chinese Journal of Biochemistry and Molecular Biology 2025;41(1):53-67
The success of"New Medical Sciences"in higher education requires effective tools in evalua-ting students'performance in courses.Previously,we reported a teamwork(T),critique(C)and ap-preciation(A)(TCA)ideological and political model,a teaching model widely applied in Basic Medical courses.TCA is an abbreviation derived from Tricarboxylic Acid Cycle in Biochemistry as an analogy for nurturing the abilities of thinking and teamwork(T),critique(C)and appreciation(A),which hope-fully could provide students with moral norms for cognition,science and life.This paper further explores the tools to assess the educational outcomes of the TCA model,by which teachers can collect feedback and reflect on teaching quality and effectiveness.Addressing the challenges of individual differences in large classes,fragmented learning feedback,and the difficulty of measuring meta-cognition in educational evaluation,this study employs strategies of value-added assessment,matrix assessment and norm-transfer-able assessment to evaluate the TCA abilities from the aspects of thinking quality,thinking creativity,co-operation ability,iterative thinking,dialectical thinking and job responsibility.By modifying/using 18 e-valuation tools in Education and Psychology,we have established a rubric system composed of 30 primary indicators(with 11 newly designed,10 partly modified and 9 directly adopted),along with 49 secondary indicators and 98 tertiary indicators to enhance the feasibility of the evaluation process.This rubric sys-tem was applied to Biochemistry teaching among the five-year-program undergraduates at Air Force Medi-cal University.Specifically,thinking and teamwork are evaluated by creative works from"the magic bio-chemical-circle",while critiques are assessed in large classes under the guidance of basic and clinical teachers,coupled with appreciation measured by job responsibility in a task-driven virtual reality(VR)project.The results indicate that Biochemistry teaching not only accumulates knowledge in students,but also achieves the goals in nurturing values and cognition.The inclusion of creative performance evalua-tion,cooperative learning and clinical case studies,can enhance students'interpersonal skills,coopera-tion,quality of thinking,creative thinking,iterative thinking and dialectical thinking to varying degrees.TCA-based Biochemistry teaching has a long-lasting impact on character education,and is capable of in-ducing positive long-term changes in students'cognition and lifestyle.Taken together,with the help of this rubric system,teachers can promptly acknowledge the effectiveness of their teaching,thereby facilita-ting their teaching strategies.
10.Comparison and Analysis of Tumorigenicity of Tumor Cells in Bile between PTBD and ERBD of Hilar Cholangiocarcinoma
Kai-Hua ZHU ; De-Xiang ZHANG ; Ying TAO ; Shu-Long ZHANG ; Kun FAN ; Hou-Bao LIU
Chinese Journal of Biochemistry and Molecular Biology 2025;41(1):112-124
Hilar cholangiocarcinoma is insidious in onset and often causes obstructive jaundice due to bile stasis,leading to impaired liver function.For tumors with malignant obstructive jaundice,biliary drainage is often performed before surgery in clinical practice.Currently,the commonly used drainage methods are percutaneous transhepatic biliary drainage(PTBD)and endoscopic retrograde biliary drain-age(ERBD),but there are controversies over the advantages and disadvantages of the two drainage methods.PTBD drainage can often lead to tumor implantation metastasis,but the underlying mechanism remains unclear.We detected tumor cells in PTBD and ERBD bile samples from hilar cholangiocarcino-ma patients,subsequently explored their tumorigenicity and mechanisms through tumorsphere assay in vitro and xenograft tumor models in vivo.The experiments included benign gallstones group(30 cases)as a negative control,PTBD group(14 cases)and ERBD group(13 cases).Tumorsphere formation was i-dentified in 3 cases(23%)among the 13 cases of ERBD group,in 6 cases(42%)among the 14 cases of PTBD group,but there were no tumor cells or formed tumorspheres in the 30 cases of benign gallstone group.The tumor sphere formation ability of cells in the PTBD group was significantly higher than that in ERBD group.Subcutaneous xenograft tumor assays showed that tumor growth in the PTBD group was sig-nificantly higher than that in the ERBD group.Tumor cells in PTBD bile possessed stronger tumorigenici-ty compared with the ERBD group.Mechanically,stem cell transcription factor Nanog mRNA levels were significantly higher in the PTBD group compared to the ERBD group.Both tumorsphere formation and xenograft tumor growth were reduced by Nanog knockdown in three cases of the PTBD group,indicating the important roles of Nanog in tumorigenicity of PTBD group tumor cells.The half-life of Nanog mRNA was longer in PTBD group cells than in ERBD group cells,suggesting potential post-transcriptional regu-lation on Nanog mRNA.The Nanog m6A level was higher in PTBD group tumor cells compared to the ERBD group.Analysis of methyltransferases and demethylases,ALKBH5(α-ketoglutarate dependent dioxygenase alkb family homolog 5)mRNA levels were lower in the PTBD group than in the ERBD group and significantly correlated with the m6A level of Nanog.ALKBH5 knockdown led to an increase in the m6A level of Nanog,while ALKBH5 overexpression decreased the m6A level of Nanog.Dual-luciferase activity assays demonstrated that ALKBH5 knockdown significantly enhanced luciferase activity,whereas ALKBH5 overexpression reduced it.Further studies confirmed that ALKBH5 knockdown upregulated both the mRNA and protein levels of Nanog,whereas overexpressing ALKBH5 downregulated them.ALKBH5 mediated the demethylation modification of Nanog mRNA,and the lower levels of ALKBH5 expression in the PTBD group promoted Nanog's m6A modification.Overexpressing ALKBH5 decreased tumorsphere growth,while ALKBH5 knockdown increased it,which was subsequently reduced by the simultaneous Nanog knockdown again.In sum,tumor cells in PTBD and ERBD drainage bile from hilar cholangiocar-cinoma patients exhibited tumorigenicity.Compared to the ERBD group,tumor cells in PTBD bile with lower ALKBH5 expression levels enhanced Nanog's m6A modification to upregulate Nanog expression levels,resulting in stronger tumorigenicity.These findings are significant for elucidating propensity to tumor implantation metastasis from PTBD drainage.

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