OBJECTIVE To investigate the effects of total flavonoids from Carthamus tinctorius L. （TFCTL） on hepatic stellate cell （HSC） activation based on the microRNA （miRNA）-204/NUAK family SNF1-like kinase 1 （NUAK1）/Hippo signaling axis， thereby elucidating the potential mechanism underlying their antifibrotic effects. METHODS The HSC-T6 cells were divided into control group， model group， TFCTL low-concentration group （20 μg/mL）， TFCTL medium-concentration group （40 μg/mL）， and TFCTL high-concentration group （60 μg/mL）. Except for control group， the remaining groups were treated with 5 ng/mL of transforming growth factor-β to induce the activation of hepatic stellate cells， followed by the addition of corresponding drug solutions/culture medium and incubation for 24 hours. Cell apoptosis was assessed， the expression levels of α-smooth muscle actin （α-SMA）， type Ⅰ collagen （Collagen Ⅰ） and proteins associated with the Hippo/Yes-associated protein （YAP） pathway [YAP， large tumor suppressor kinase 1 （LATS1）， and mammalian STE20-like kinase 1 （MST1）] were detected. Additionally， cell transfection was used to investigate the activity of the miRNA-204/NUAK1/Hippo signaling axis at both the genetic and protein levels. RESULTS After intervention with TFCTL， the apoptosis rate of HSC-T6 cells and the protein expressions of MST1 （except for the TFCTL high-concentration group） and LATS1 were significantly increased （P＜0.05）， while the protein expressions of α-SMA， CollagenⅠ， and YAP （except for the TFCTL medium-concentration group） were significantly decreased （P＜0.05）. Further results from cell transfection experiments revealed that after transfection with miRNA-204 mimics， the mRNA it’s protein expressions of α-SMA， CollagenⅠ， NUAK1， and YAP in HSC-T6 cells were significantly decreased （P＜0.05）， while the mRNA and protein expressions of LATS1 and the mRNA expression of MST1 were significantly increased （P＜0.05）. Conversely， the results were opposite following transfection with miRNA-204 inhibitors. CONCLUSIONS TFCTL can exert anti-hepatic fibrosis effects by up-regulating the expression of miRNA-204， thereby down- regulating the expressions of NUAK1， inactivating the Hippo/YAP pathway， which in turn suppresses the activation of HSC and promotes their apoptosis.

Objective: To explore the causal association between dietary components (carbohydrate, fat, protein, and sugar) and 119 genera of known gut microbiota using Mendelian randomization (MR) methods. Methods: Genome-wide association study (GWAS) data for dietary components were collected from the DietGen, while GWAS data for gut microbiota were collected from the MiBioGen. Single nucleotide polymorphism (SNP) loci associated with the four dietary components were used as instrumental variables, and 119 known gut microbiota genera were used as the outcomes. MR analysis was performed using inverse variance weighted (IVW) method. Heterogeneity was evaluated using Cochran's Q test, horizontal pleiotropy and exclude outliers were tested using MR-Egger regression and MR-PRESSO test. Common genetic pleiotropic genes between dietary components and gut microbiota were identified by MAGMA and PLACO analyses. Results: The MR analysis revealed causal associations between carbohydrates and 4 gut microbiota genera, fats and 14 genera, proteins and 14 genera, and sugars and 11 genera (all P<0.05). The MR-Egger regression analysis showed no horizontal pleiotropy among the selected SNPs, and the MR-PRESSO test did not identify any outliers (all P>0.05). The MAGMA and PLACO analyses revealed that 74.42% (32/43) of the causal associations had pleiotropic genes, with 1 to 10 pleiotropic genes identified. Multiple causal association groups shared the same pleiotropic genes. Conclusion There are potential genetic and causal associations between dietary components and gut microbiota.

Objective: To investigate the epidemiological characteristics of hepatitis E in Shaoxing City, Zhejiang Province from 2006 to 2024, so as to provide the evidence for the prevention and control of hepatitis E. Methods: Data on hepatitis E incidence in Shaoxing City from 2006 to 2024 were collected through the Surveillance System of China Information System for Disease Control and Prevention. The epidemiological characteristics were analyzed using descriptive epidemiological methods. The trend in hepatitis E incidence was analyzed using the average annual percent change (AAPC) and annual percent change (APC). The spatial-temporal clustering characteristics of hepatitis E incidence were identified using spatial-temporal scanning analysis. Results: A total of 2 408 hepatitis E cases were reported in Shaoxing City from 2006 to 2024, with an average annual reported incidence of 2.55/100 000. The overall trend was not statistically significant (AAPC=3.181%, P>0.05). Specifically, it showed an upward trend from 2006 to 2011 (APC=17.371%, P<0.05), a downward trend from 2011 to 2019 (APC=-12.497%, P<0.05), and an upward trend from 2019 to 2024 (APC=18.076%, P<0.05). The epidemic season of hepatitis E was from January to May, with seasonal indices of 122.09%, 118.60%, 145.02%, 129.57%, and 106.15%, respectively. The top three average annual reported incidences were identified in Zhuji City, Xinchang County, and Shengzhou City, with rates of 4.18/100 000, 2.85/100 000, and 2.74/100 000, respectively. The average annual reported incidence of hepatitis E was higher in males than in females (3.52/100 000 vs. 1.56/100 000, P<0.05). A relatively large number of hepatitis E cases were reported among individuals aged 40-<70 years, with 1 639 cases (68.06%). Among them, the group aged 60-<70 years had the highest average annual reported incidence of hepatitis E, at 4.92/100 000. Farmers constituted the predominant occupational group, accounting for 1 515 cases (62.92%). Spatial-temporal scanning analysis identified two clusters in Shaoxing City from 2006 to 2024. The class Ⅰ cluster was located in Shengzhou City, with aggregation time from January 1, 2011 to May 1, 2014. The class Ⅱ cluster was located in Xinchang County, with aggregation time from December 1, 2012 to March 31, 2013. Conclusions The reported incidence of hepatitis E in Shaoxing City from 2006 to 2024 exhibited a pattern of an initial increase, followed by a decrease, and then a subsequent rise. The disease demonstrated higher prevalence during the winter and spring seasons. Key populations for targeted control and prevention include males, individuals aged 40-<70 years, and farmers. Shengzhou City and Xinchang County were identified as high-risk areas.

Objective: To investigate the association between childhood obesity and type 2 diabetes mellitus (T2DM) as well as coronary artery heart disease (CHD). Methods: Genome-wide association study (GWAS) data for childhood obesity were collected from the ECG consortium, encompassing information on children aged 2 to 18 years, including 18 613 cases and 12 696 controls. GWAS data for T2DM were collected from the DIAGRAM consortium, including 242 283 cases and 1 569 734 controls. GWAS data for CHD were collected from the CARDIoGRAMplusC4D consortium, including 10 801 cases and 137 371 controls. Pleiotropic genes associated with both T2DM and CHD were analyzed using the MAGMA, PLACO and conditional false discovery rate (cFDR) methods. Mendelian randomization (MR) analysis was performed using inverse variance weighted (IVW) method, exploring the causal relationships among childhood obesity, T2DM and CHD. Heterogeneity was evaluated using Cochran's Q test, horizontal pleiotropy and exclude outliers were tested using MR-Egger regression and MR-PRESSO test. The mediating variables among the three diseases were investigated by using a mediation analysis. Results: The results of MAGMA, PLACO and cFDR analyses identified 80 pleiotropic genes associated with both T2DM and CHD, primarily distributed on chromosomes 3, 17 and 19. The MR analysis revealed that childhood obesity increased the risk of T2DM (OR=1.151, 95%CI: 1.033-1.283) and CHD (OR=1.158, 95%CI: 1.068-1.255), T2DM increased the risk of CHD (OR=1.182, 95%CI: 1.139-1.227), and CHD increased the risk of T2DM (OR=1.124, 95%CI: 1.055-1.198). The MR-Egger regression analysis showed no horizontal pleiotropy, and the MR-PRESSO test did not identify any outliers (all P>0.05). Mediation analysis indicated that childhood obesity directly increased the risk of CHD (effect value=0.096, 95%CI: 0.012-0.180) and indirectly increased the risk of CHD through T2DM (effect value=0.023, 95%CI: 0.005-0.041), with the mediation effect accounting for 15.65% of the total effect. Conclusions There are potential causal associations between childhood obesity and T2DM as well as CHD, with a bidirectional causal relationship between T2DM and CHD. T2DM also plays a mediating role in the association between childhood obesity and CHD.

Objective:To explore the effect of thymol on improving the core symptoms of autism through regulating scinderin expres-sion in autism spectrum disorder(ASD)rats by constructing valproic acid-induced ASD rat model and intervening with thymol.Methods:The ASD-like behaviors of rats were observed using three box social experiment,open field experiment,and youth social ex-periment.RNA-seq was used to detect the expression of RNA in the prefrontal cortex of rats.The protein level of scinderin in the pre-frontal cortex of rats was measured by Western blot.Results:Compared with the rats in the control group,the rats in the model group showed ASD-like behaviors,including decreased social ability,impaired social preference,decreased exploration ability,and repeated stereotyped behavior.Compared with the control group,the model group showed reduced standing times(P<0.001)and increased self combing time(P=0.003).Moreover,the expression level of scinderin in the prefrontal cortex increased in model rats.After thymol treat-ment,the ASD-like behaviors in the model group were significantly improved.Compared with the model group,the number of standing times in the intervention group increased(P<0.001)and the self grooming time decreased(P=0.004).The expression of scinderin basi-cally returned to the normal level.Conclusion:The abnormal expression of scinderin in the prefrontal cortex of ASD model rats can lead to ASD-like behaviors.Thymol intervention can reduce the expression of scinderin and improve the core symptoms of ASD.

AIM:To elucidate the pharmacody-namic and network pharmacological mechanisms of total flavonoids of Carthamus tinctorius L.,to ex-plore their key targets and related pathways,and to clarify their mechanism of action against hepatic fibrosis.METHODS:The total flavonoids of Cartha-mus tinctorius L.were determined by LC-MS/MS and analysed for their compositions;the active in-gredients were screened by TCMSP database,SWISS ADME database and literature search;the targets related to total flavonoids of Carthamus tinctorius L.were screened by Swiss Target Predic-tion database;and the targets related to hepatic fi-brosis were screened by GeneCards database;the anti-hepatic fibrosis targets of total flavonoids of Carthamus tinctorius L.were obtained by taking the intersection of Venny.2.1.0;the protein interac-tions were analysed by STRING database;the visu-alization analysis was carried out by Cytoscape soft-ware;the GO function and KEGG pathway analysis was carried out by Metascape platform;and molec-ular docking was verified by using AutoDock soft-ware for the core targets and active ingredients.The mechanism of anti-hepatic fibrosis of total fla-vonoids of Carthamus tinctorius L.was verified by animal model and in vitro cell experiments.RE-SULTS:A total of 41 flavonoid components were identified in Carthamus tinctorius L.Through the network pharmacological analysis,149 anti-hepatic fibrosis targets of total flavonoids of Carthamus tinctorius L.were obtained,including 23 core tar-gets.The GO enrichment analyses involved a total of three aspects,namely,biological process(BP),cellular component(CC),and molecular function(MF).KEGG enrichment results showed that PI3K/Akt and MAPK are pathways involved in the devel-opment of hepatic fibrosis.Molecular docking veri-fied that the active ingredients Quercetin,Acacetin and Glabridin were tightly bound to Akt1 and HI-FIA,respectively.In animal model experiments,it was observed by HE and Masson staining that fibro-plasia was reduced,collagen deposition was re-duced,inflammatory cell infiltration was reduced,and fibrotic liver tissues were improved in total fla-vonoids of Carthamus tinctorius L.administration group.In isolated cell experiments:Western blot-ting results suggested that total flavonoids of Car-thamus tinctorius L.could decrease the hepatic fi-brosis marker factor α-SMA,Collagen1(P＜0.01)and PI3K,Akt protein expression(P＜0.01).CONCLU-SION:Total flavonoids of Carthamus tinctorius L.ex-erted anti-hepatic fibrosis effects through multi-components,multi-targets and multi-pathways,and their mechanism of action may be achieved by regulating the PI3K/Akt signalling pathway.

Cancer-related cognitive impairment(CRCI)refers to cognitive dysfunction that occurs during or after chemotherapy in patients with cancer.However,the pathogenesis of CRCI remains unclear,and effective treatments are lacking in clinical practice.Based on the"toxin damaging brain collaterals"theory,this study systematically explores the traditional Chinese medicine(TCM)etiology,pathogenesis,and treatment strategies of CRCI.In TCM,CRCI is attributed to a"deficiency of brain collaterals"in patients with cancer.Chemotherapy drugs,as exogenous pathogens,invade the brain when the body is weakened and interact with endogenous phlegm,blood stasis,and turbid toxins.This creates a vicious cycle of"toxin,blood stasis,phlegm,deficiency"ultimately leading to the malnourishment of the sea of marrow and the dysfunction of the spiritual mechanism.Modern biological research aligns with this TCM perspective,as neurotoxicity,oxidative stress,and inflammatory responses associated with CRCI correspond to the TCM concepts of"toxin damaging brain collaterals."Pathological changes such as increased microvascular permeability and neuronal network disruption are similar to the TCM pathogenesis characteristics of"toxin and blood stasis blocking the collaterals"and"emptiness of the sea of marrow."Given the progressive nature of CRCI pathogenesis,TCM therapeutic principles focus on strengthening healthy qi,enhancing cognitive function,eliminating toxins,and unblocking collaterals.Acupuncture,moxibustion,and Daoyin serve as supplementary external treatments,forming a comprehensive treatment approach of"treating the viscera through the collaterals and regulating the body to nourish the spirit."This framework provides novel insights for TCM diagnosis and CRCI treatment.

Objective To investigate the effects of kaempferol on the oxidative damage and apoptosis of human lens epithelial cells induced by hydrogen peroxide(H2O2),as well as its regulatory role in the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway.Methods Human lens epithelial cells HLE-B3 were cultured in vitro and divided into the blank group(no intervention),the model group(400 pmol?L-1 H2O2),the kaempferol group(400 μmol?L-1 H2O2+40 pmol?L-1 kaempferol),the AG490 group(400 pmol?L-1 H2O2+50 μmol?L-1 JAK2/STAT3 signaling pathway inhibitor AG490),the inhibitor group(400 pmol?L-1 H2O2+40 pmol?L-1 kaempferol+50 μmol?L-1 AG490),and the activator group(400 pmol?L-1 H2O2+40 pmol?L-1 kaempferol+0.5 μmol?L-1 JAK2/STAT3 signaling pathway activator C-A1).After 24 h of intervention,Hoechst 33258 staining was used to determine the ap-optosis rate of these cells.Malondialdehyde(MDA)and superoxide dismutase(SOD)kits were used to detect the levels of MDA and SOD.The protein expression levels of Cysteine aspartic protease 3(Caspase-3),B-cell lymphoma-2(Bcl-2),JAK2,STAT3,p-JAK2,and p-STAT3 were detected by Western blot.Results Compared with the blank group,the apop-tosis rate,the MDA level,and the protein expression levels of Caspase-3,p-JAK2,and p-STAT3 increased(all P＜0.05)in the model group,while the protein expression levels of SOD and Bcl-2 decreased(all P＜0.05).Compared with the model group,the apoptosis rate,the MDA level,and the protein expression levels of Caspase-3,p-JAK2,and p-STAT3 decreased in the kaempferol and AG490 groups(all P＜0.05),while the protein expression levels of SOD and Bcl-2 increased(all P＜0.05).Compared with the kaempferol group,the apoptosis rate,the MDA level,and the protein expression levels of Caspase-3,p-JAK2,and p-STAT3 decreased in the inhibitor group,while the protein expression levels of SOD and Bcl-2 in-creased(all P＜0.05);the changes in the above indicators in the activator group were opposite to those in the inhibitor group(all P＜0.05).Conclusion Kaempferol can protect HLE-B3 cells from H2O2-induced oxidative damage and inhibit their apoptosis by inhibiting JAK2/STAT3 signaling pathway.

Objective To prepare tubeimoside Ⅲ nanoemulsion(TBMⅢ-NE)and evaluate its adjuvant effect in vaccines.Methods TBMⅢ-NE was prepared using low-energy emulsification.Dynamic light scattering was used to characterize the particle size and polydispersity index of the obtained TBMⅢ-NE,and transmission electron microscopy(TEM)was employed to observe the morphology.CCK-8 assay was utilized to determine the cytotoxicity of TBMⅢ-NE on bone marrow-derived dendritic cells(BMDCs).The in vitro safety of TBMⅢ-NE was evaluated using a hemolysis assay.The ability of TBMⅢ-NE to promote the phagocytosis of antigens by DC2.4 cells was observed using confocal laser microscopy.After co-incubation of TBMⅢ-NE with BMDCs,the expression levels of CD40,CD86,MHC-Ⅰ,and CCR7 on the surface of BMDCs were detected using flow cytometry,and the levels of cytokines in the supernatant of BMDCs were measured using enzyme-linked immunosorbent assay(ELISA).After female BALB/c mice were immunized with the SARS-CoV-2 antigen RBD in combination with TBMⅢ-NE,ELISA was conducted to determine the serum levels of specific IgG,IgG2a,and IgG1 antibodies.The number of specific IFN-γ-secreting cells in mouse splenocytes was detected using enzyme-linked immunospot(ELISpot)assay.Results The prepared blank nanoemulsion(BNE)and TBMⅢ-NE were in a particle size of 25.46 and 25.89 nm,and a polydispersity index of 0.214 and 0.125,respectively.TEM displayed that TBMⅢ-NE was in uniform sphere and well dispersed.When the TBMⅢ-NE adjuvant was diluted by 400-fold,the survival rate of BMDCs was approximately 86%.Compared with free TBMⅢ,the hemolytic toxicity of TBMⅢ-NE was significantly reduced(P<0.01).TBMⅢ-NE promoted the phagocytosis of antigens by DC2.4 cells and significantly increased the expression of CCR7 on the surface of BMDCs(P<0.05),indicating its potential to promote more dendritic cells to effectively migrate to lymph nodes.TBMⅢ-NE also promoted the expression of IL-6 and IL-1β in the supernatant of BMDCs(P<0.05).When combined with RBD,TBMⅢ-NE significantly increased the levels of specific IgG,IgG2a,and IgG1 antibodies in mouse serum(P<0.01)and promoted the secretion of specific IFN-γ in splenocytes(P<0.01),indicating that TBM Ⅲ-NE could enhance specific cellular immune responses.Conclusion A stable and highly effective TBMⅢ-NE that can induce humoral and cellular immune responses is successfully prepared.

Objective To investigate the interaction between a novel antimicrobial compound,HL-J6,and penicillin-binding protein 1(PBP1)of Staphylococcus aureus.Methods With MRSA252 genomic DNA as the template and PBP1F and PBP1R as primers,the expression plasmid pET30a-pbp1-39-608 was constructed by amplifying the target gene fragment followed by cloning into the Nde I/Xho I restriction sites of the pET30a vector.Then the obtained plasmids were transformed into Escherichia coli for the expression of PBP1-39-608 protein,and the product was purified by affinity chromatography.The inhibitory effect of HL-J6 on the transpeptidase activity of PBP1-39-608 was measured using peptidoglycan side chain backbone peptide,with thiol ester analog S2d as the substrate.The affinity between HL-J6 and PBP1-39-608 was detected using microscale thermophoresis(MST),and the binding interaction was confirmed by cellular thermal shift assay(CETSA).Molecular docking and dynamics simulation were performed using AutoDock Vina and Desmond software,respectively,to elucidate the binding mode of HL-J6 with the PBP1-39-608 protein and the key amino acid residues involved.Results The recombinant plasmid pET30a-pbp1-39-608 was successfully constructed,and PBP1-39-608 protein was produced after induction and purified,yielding a protein with an approximate molecular mass of 65×103.HL-J6 inhibited the transpeptidase activity of PBP1-39-608 in a time-dependent manner(P<0.001).The dissociation constant Kd of the binding between HL-J6 and PBP1-39-608 was 64.92 μmol/L.Molecular docking results showed that HL-J6 bound to the active pocket of PBP1-39-608 by interacting with key residues such as ILE-348,ASN-370,THR-516 and PHE-423,with a binding score of-8.38 kcal/mol(<-5.00 kcal/mol).Dynamics simulation results indicated that the complex became stable after 50 ns.Conclusion HL-J6 effectively inhibits the transpeptidase activity of Staphylococcus aureus PBP1,and shows stable interaction with the protein.