ObjectiveTo improve the therapeutic effect of Buyang Huanwutang（BYHW） on diabetic kidney disease （DKD） and explore new methods for developing new Chinese medicine decoctions，we utilized 5-hydroxymethylcytosine （5hmC）-Seal sequencing technology and network pharmacology to modify BYHW. MethodsWe selected 14 diabetes mellitus （DM） patients and 15 DKD patients hospitalized in the Department of Endocrinology of Peking University Third Hospital in 2021. Circulating free DNA （cfDNA） in the patients’ plasma was sequenced. After data processing and screening， we performed temporal clustering analysis to select a DKD 5hmC gene set， which was then cross-validated with a DKD database gene set to obtain the DKD gene set. We retrieved target genes of the seven herbal components of BYHW from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and the Encyclopedia of Traditional Chinese Medicine （ETCM）， and performed cross-analysis with the DKD gene set to identify common genes shared by the disease and the Chinese medicines. A protein-protein interaction （PPI） network was constructed for the common genes to screen out the key genes. Chinese medicines targeting these key genes were searched against ETCM to identify removable Chinese medicines. Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis was performed on non-common DKD genes， and key genes in DKD-related pathways were selected based on machine learning. The GSE30529 dataset was used to verify the expression trends of 5hmC-modified genes and the feasibility of target genes as drug targets. TCMBank was used to search for target genes and obtain compounds targeting these genes and the corresponding Chinese medicines to construct a "key target-compound-Chinese medicine" network. Molecular docking was employed to verify the binding affinity of compounds with key targets. TCMSP and ETCM were used to search and count the candidate Chinese medicines targeting DKD-related genes， and a new decoction was formed by adding the selected Chinese medicines. A mouse model of DKD was established to examine the efficacy of the new decoction based on the mouse body mass， random blood glucose， urinary microalbumin （mALB）， serum creatinine （Scr）， and blood urea nitrogen （BUN） and by hematoxylin-eosin staining， periodic acid-Schiff staining， Masson staining， immunofluorescence assay， and Real-time PCR. ResultsThe cross-analysis results showed that the DKD gene set included 507 genes， of which 30 were target genes of BYHW. The PPI analysis indicated that the top 15% target genes regarding the degree were interleukin-6 （IL-6）， Toll-like receptor 4 （TLR4）， lactotransferrin （LTF）， lipoprotein lipase （LPL）， and sterol regulatory element-binding transcription factor 1 （SREBF1）. Persicae Semen and Pheretima in BYHW were unrelated to key genes and removed. Machine learning identified 10 potential target genes， among which TBC1 domain family member 5 （TBC1D5）， RAD51 paralog B （RAD51B）， and proteasome 20S subunit alpha 6 （PSMA6） had expression trends consistent with the GSE30529 dataset and could serve as drug targets. The "key target-compound-Chinese medicine" network and molecular docking results indicated that the compounds with good binding affinity to target proteins were arginine， glycine， myristicin， serine， and tyrosine， corresponding to 121 Chinese medicines. The top 10 Chinese medicines targeting DKD-related genes were Lycii Fructus， Ginseng Radix et Rhizoma， Dioscoreae Rhizoma， Rehmanniae Radix Praeparata， Isatidis Radix， Glehniae Radix， Ophiopogonis Radix， Allii Sativi Bulbus， Isatidis Folium， and Bolbostemmatis Rhizoma. Based on traditional Chinese medicine theory， the new decoction was obtained after removal of Persicae Semen and Pheretima and addition of Rehmanniae Radix Praeparata and Dioscoreae Rhizoma. Animal experiment results indicated that the modified BYHW improved the kidney function and inhibited renal fibrosis in DKD mice， with better effects than the original decoction. ConclusionThe BYHW modified based on 5hmC-Seal sequencing demonstrates better performance in inhibiting fibrosis and ameliorating DKD than the original decoction. This elucidates the biomedical theory behind the epigenetic modification of traditional Chinese medicine prescriptions， potentially offering new perspectives for the exploration of these prescriptions

ObjectiveTo improve the therapeutic effect of Buyang Huanwutang（BYHW） on diabetic kidney disease （DKD） and explore new methods for developing new Chinese medicine decoctions，we utilized 5-hydroxymethylcytosine （5hmC）-Seal sequencing technology and network pharmacology to modify BYHW. MethodsWe selected 14 diabetes mellitus （DM） patients and 15 DKD patients hospitalized in the Department of Endocrinology of Peking University Third Hospital in 2021. Circulating free DNA （cfDNA） in the patients’ plasma was sequenced. After data processing and screening， we performed temporal clustering analysis to select a DKD 5hmC gene set， which was then cross-validated with a DKD database gene set to obtain the DKD gene set. We retrieved target genes of the seven herbal components of BYHW from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and the Encyclopedia of Traditional Chinese Medicine （ETCM）， and performed cross-analysis with the DKD gene set to identify common genes shared by the disease and the Chinese medicines. A protein-protein interaction （PPI） network was constructed for the common genes to screen out the key genes. Chinese medicines targeting these key genes were searched against ETCM to identify removable Chinese medicines. Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis was performed on non-common DKD genes， and key genes in DKD-related pathways were selected based on machine learning. The GSE30529 dataset was used to verify the expression trends of 5hmC-modified genes and the feasibility of target genes as drug targets. TCMBank was used to search for target genes and obtain compounds targeting these genes and the corresponding Chinese medicines to construct a "key target-compound-Chinese medicine" network. Molecular docking was employed to verify the binding affinity of compounds with key targets. TCMSP and ETCM were used to search and count the candidate Chinese medicines targeting DKD-related genes， and a new decoction was formed by adding the selected Chinese medicines. A mouse model of DKD was established to examine the efficacy of the new decoction based on the mouse body mass， random blood glucose， urinary microalbumin （mALB）， serum creatinine （Scr）， and blood urea nitrogen （BUN） and by hematoxylin-eosin staining， periodic acid-Schiff staining， Masson staining， immunofluorescence assay， and Real-time PCR. ResultsThe cross-analysis results showed that the DKD gene set included 507 genes， of which 30 were target genes of BYHW. The PPI analysis indicated that the top 15% target genes regarding the degree were interleukin-6 （IL-6）， Toll-like receptor 4 （TLR4）， lactotransferrin （LTF）， lipoprotein lipase （LPL）， and sterol regulatory element-binding transcription factor 1 （SREBF1）. Persicae Semen and Pheretima in BYHW were unrelated to key genes and removed. Machine learning identified 10 potential target genes， among which TBC1 domain family member 5 （TBC1D5）， RAD51 paralog B （RAD51B）， and proteasome 20S subunit alpha 6 （PSMA6） had expression trends consistent with the GSE30529 dataset and could serve as drug targets. The "key target-compound-Chinese medicine" network and molecular docking results indicated that the compounds with good binding affinity to target proteins were arginine， glycine， myristicin， serine， and tyrosine， corresponding to 121 Chinese medicines. The top 10 Chinese medicines targeting DKD-related genes were Lycii Fructus， Ginseng Radix et Rhizoma， Dioscoreae Rhizoma， Rehmanniae Radix Praeparata， Isatidis Radix， Glehniae Radix， Ophiopogonis Radix， Allii Sativi Bulbus， Isatidis Folium， and Bolbostemmatis Rhizoma. Based on traditional Chinese medicine theory， the new decoction was obtained after removal of Persicae Semen and Pheretima and addition of Rehmanniae Radix Praeparata and Dioscoreae Rhizoma. Animal experiment results indicated that the modified BYHW improved the kidney function and inhibited renal fibrosis in DKD mice， with better effects than the original decoction. ConclusionThe BYHW modified based on 5hmC-Seal sequencing demonstrates better performance in inhibiting fibrosis and ameliorating DKD than the original decoction. This elucidates the biomedical theory behind the epigenetic modification of traditional Chinese medicine prescriptions， potentially offering new perspectives for the exploration of these prescriptions

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Mitochondria, functioning not only as the central hub of cellular energy metabolism but also as semi-autonomous organelles, orchestrate cellular fate decisions through their endogenous mitochondrial DNA (mtDNA), which encodes core components of the electron transport chain. Emerging research has identified microRNAs localized within mitochondria, termed mitochondria-located microRNAs (mitomiRs). Recent studies have revealed that mitomiRs are transcribed from nuclear DNA (nDNA), processed and matured in the cytoplasm, and subsequently transported into mitochondria. mitomiRs regulate mtDNA through diverse mechanisms, including modulation of mtDNA expression at the translational level and direct binding to mtDNA to influence transcription. Aberrant expression of mitomiRs leads to mitochondrial dysfunction and contributes to the pathogenesis of metabolic diseases. Restoring mitomiR expression to physiological levels using mitomiRs mimics or inhibitors has been shown to improve mitochondrial function and alleviate related diseases. Consequently, the regulatory mechanisms of mitomiRs have become a major focus in mitochondrial research. Given that mitomiRs are located in mitochondria, targeted delivery strategies designed for mtDNA can be adapted for the delivery of mitomiRs mimics or inhibitors. However, numerous intracellular and extracellular barriers remain, highlighting the need for more precise and efficient delivery systems in the future. The regulation of mtDNA expression mediated by mitomiRs not only expands our understanding of miRNA functions in post-transcriptional gene regulation but also provides promising molecular targets for the treatment of mitochondrial-related diseases. This review systematically summarizes recent research progress on mitomiRs in regulating mtDNA expression and discusses the underlying mechanisms of mitomiRs-mtDNA interactions. Additionally, it provides new perspectives on precision therapeutic strategies, with a particular emphasis on mitomiRs-based regulation of mitochondrial function in mitochondrial-related diseases.

Background::Clinical opportunistic screening is a cost-effective cancer screening modality. This study aimed to establish an easy-to-use diagnostic model serving as a risk stratification tool for identification of individuals with malignant gastric lesions for opportunistic screening.Methods::We developed a questionnaire-based diagnostic model using a joint dataset including two clinical cohorts from northern and southern China. The cohorts consisted of 17,360 outpatients who had undergone upper gastrointestinal endoscopic examination in endoscopic clinics. The final model was derived based on unconditional logistic regression, and predictors were selected according to the Akaike information criterion. External validation was carried out with 32,614 participants from a community-based randomized controlled trial.Results::This questionnaire-based diagnostic model for malignant gastric lesions had eight predictors, including advanced age, male gender, family history of gastric cancer, low body mass index, unexplained weight loss, consumption of leftover food, consumption of preserved food, and epigastric pain. This model showed high discriminative power in the development set with an area under the receiver operating characteristic curve (AUC) of 0.791 (95% confidence interval [CI]: 0.750–0.831). External validation of the model in the general population generated an AUC of 0.696 (95% CI: 0.570–0.822). This model showed an ideal ability for enriching prevalent malignant gastric lesions when applied to various scenarios.Conclusion::This easy-to-use questionnaire-based model for diagnosis of prevalent malignant gastric lesions may serve as an effective prescreening tool in clinical opportunistic screening for gastric cancer.

Aim To investigate the mechanism of py-roptosis mediated by the NLRP3/caspase-1/GSDMD signaling pathway and the intervention effect of Radix Angelica Sinensis and Radix Astragalus ultrafiltration extract(RAS-RA)in radiation-induced pulmonary fi-brosis.Methods Fifty Wistar rats were randomly di-vided into five groups,with ten rats in each group.Ex-cept for the blank control group,all other groups of rats were anesthetized and received a single dose of 40 Gy X-ray local chest radiation to establish a radiation-in-duced pulmonary fibrosis rat model.After radiation,the rats in the RAS-RA intervention groups were orally administered doses of 0.12,0.24 and 0.48 g·kg-1 once a day for 30 days.The average weight and lung index of the rats were observed after 30 days of contin-uous administration.Hydroxyproline(HYP)content in lung tissue was determined by hydrolysis method.The levels of IL-18 and IL-1 β in serum were detected by ELISA.Lung tissue pathological changes were ob-served by HE and Masson staining.Ultrastructural changes in lung tissue were observed by transmission e-lectron microscopy.The expression levels of NLRP3/caspase-1/GSDMD pyroptosis pathway-related proteins and fibrosis-related proteins in lung tissue were detec-ted by Western blot.Results Compared with the blank group,the HYP content in lung tissue and the levels of IL-18 and IL-1 β in serum significantly in-creased in the model group(P＜0.01).HE and Mas-son staining showed inflammatory cell infiltration and collagen fiber deposition.Transmission electron mi-croscopy revealed increased damaged mitochondria,disordered arrangement,irregular morphology,shallow matrix,outer membrane rupture,mostly fractured and shortened cristae,mild expansion,increased electron density of individual mitochondrial matrix,mild sparse structure of lamellar bodies,partial disorder,unclear organelles,and characteristic changes of pyroptosis.Western blot analysis showed increased expression of caspase-1,GSDMD,NLRP3,CoL-Ⅰ,α-SMA,and CoL-Ⅲ proteins(P＜0.01).Compared with the model group,the RAS-RA intervention group showed signifi-cant improvement in body mass index and lung index of rats,decreased levels of IL-18 and IL-1 β inflammatory factors(P＜0.01),improved mitochondrial structure,reduced degree of fibrosis,and decreased expression of caspase-1,GSDMD,NLRP3,COL-Ⅰ,COL-Ⅲ,and α-SMA proteins in lung tissue(P＜0.01).Conclusion RAS-RA has an inhibitory effect on radiation-in-duced pulmonary fibrosis,and its mechanism may be related to the inhibition of pyroptosis through the regu-lation of the NLRP3/caspase-1/GSDMD signaling pathway.

Objective To analyze the diagnostic value of ultrasound blood quantitative parameters combined with murine sarcoma filtering toxin oncogenic homolog B(BRAF)gene mutation in Hashimoto's thyroiditis(HT)with thyroid carcinoma.Methods A total of 86 patients with HT and thyroid nodules in Sir Run Run Hospital,Nanjing Medical University from June 2020 to June 2022 were selected as the research objects.The patients with thyroid cancer were included into the malignant group(n=26),and the patients with benign thyroid nodules were included into the benign group(n=60)according to the pathological examination results.The patients in the malignant group was divided into the metastasis group(n=10)and the non-metastasis group(n=16)according to whether lymph node metastasis occurred.All subjects underwent color Doppler ultrasonography and BRAF gene mutation detection.The pulsatility index(PI),resistance index(RI),peak systolic velocity(S),end diastolic velocity(D),peak systolic velocity to end diastolic velocity ratio(S/D)and BRAF gene mutation between the malignant group and the benign group were compared.The detection rate of blood flow signals by CDFI,the detection rate of blood flow signals of grade 2 to 3 and the mutation of BRAF gene between the metastasis group and the non-metastasis group were compared.The value of the above blood parameters and BRAF gene mutations in the diagnosis of HT complicated with thyroid cancer was evaluated by receiver operating characteristic(ROC)curve,and the value of the above blood parameters and BRAF gene mutation in the diagnosis of lymph node metastasis of thyroid cancer was evaluated.Results The PI,RI,S,D,S/D and positive rate of BRAF gene mutation in the malignant group were significantly higher than those in the benign group(P<0.05).The detection rate of blood flow signals by CDFI,the detection rate of blood flow signals of grade 2 to 3,and the positive rate of BRAF gene mutation in the metastasis group were significantly higher than those in the non-metastasis group(P<0.05).ROC curve analysis showed that the area under the curve(AUC)of RI,PI,S,D,S/D and BRAF gene mutation in the combined assessment of HT complicated with thyroid cancer was 0.951,with the sensitivity of 88.46%and the specificity of 93.33%,and the efficiency of combined assessment was better than that of the independent assessment of each index.Meanwhile,the AUC of the detection rate of blood flow signals by CDFI,the detection rate of blood flow signals of grade 2 to 3,and BRAF gene mutation in the combined assessment of lymph node metastasis of thyroid cancer was 0.938,and the efficiency of combined assessment was better than that of the independent assessment of each index.Conclusion The ultrasound blood quantitative parameters combined with BRAF gene mutation has certain value in the evaluation of HT with thyroid cancer and lymph node metastasis,and the combined evaluation of each index is more effective.

Background and Aims:Sonodynamic therapy(SDT)is an emerging tumor treatment method that primarily utilizes low-intensity ultrasound(US)to activate sonosensitizers,leading to the production of cytotoxic reactive oxygen species(ROS)that kill cells.This study was conducted with an attempt to develop a novel nanoparticle(NPs)formulation containing a sonosensitizer and the small interfering RNA(siRRS1)of tumor-related gene RRS1 to observe its anticancer effects on colon cancer,to provide new strategies for colon cancer treatment. Methods:RRS1 RNA-Seq data were downloaded from the TCGA and GEO databases,and clinical pathological data were collected from 80 patients with colon cancer at Hunan Provincial People's Hospital.The expression of RRS1 in colon cancer and its relationship with prognosis were analyzed.The proliferation and apoptosis of colon cancer cells transfected with siRRS1 were observed.The sonosensitizer tetra(4-carboxyphenyl)porphyrin(TCPP),gene carrier DSPE-PEI,and siRRS1 were assembled into TCPP@DSPE-PEI-siRRS1 NPs(T@D-siRRS1 NPs).After structural characterization and examination of cellular uptake and ROS generation capabilities in vitro,the anticancer effects of T@D-siRRS1 NPs on colon cancer were assessed in cell experiments and tumor-bearing mouse models. Results:Both database analysis and clinical specimen testing indicated that RRS1 expression in colon cancer tissues was higher than that in adjacent normal tissues,and high expression was associated with poor prognosis(HR=1.88,95％CI=1.27-2.79,P=0.002).Univariate and multivariate analyses showed that RRS 1 mRNA expression was an independent risk factor for overall survival in colon cancer patients(HR=1.354,95％CI=1.447-2.215,P=0.031).Knockdown of RRS1 significantly reduced the proliferation capacity of colon cancer cells and increased apoptosis(both P＜0.05).Characterization results showed that the particles were uniform and stable;T@D-siRRS1 NPs entered cells through endocytosis,where TCPP could generate singlet oxygen(1O2)under ultrasound irradiation.Subsequent in vitro and in vivo experimental results demonstrated that transfection with NPs carrying siRRS1 or TCPP(T@D-control siRNA,T@D-siRRS1)combined with US resulted in varying degrees of growth inhibition in colon cancer cells,with the T@D-siRRS1 NPs+US treatment showing the strongest effect(all P＜0.05).Moreover,no significant damage was observed in the vital organs of mice treated with T@D-siRRS1 NPs+US. Conclusion:T@D-siRRS1 NPs plus US integrate SDT and gene therapy,producing an effective synergistic cytotoxic effect on colon cancer cells.Additionally,T@D-siRRS1 NPs demonstrate good safety and biocompatibility,suggesting potential for clinical application.