ObjectiveTo investigate whether intestinal deficiency of lanosterol synthase （LSS）， a key enzyme in cholesterol synthesis， influences the progression of metabolic dysfunction-associated steatotic liver disease （MASLD） by regulating intestinal cholesterol absorption and immune response. MethodsLSS heterozygous knockout （LSS^+/-） mice and wild-type （WT） controls were generated using CRISPR/Cas9 technology and fed either a high-fat diet （HFD） or regular chow （CHOW）. The model was validated by genotyping. Hepatic steatosis was assessed by HE and oil red O staining. Immunohistochemistry was used to detect the localization and expression of NPC1L1 and CD36 proteins in the intestine. Western blot analysis was performed to measure JNK phosphorylation and TLR4 protein levels in intestinal tissues. Real-time quantitative polymerase chain reaction （qPCR） was employed to examine the mRNA expression of TLR4 and IL-6. ResultsLSS^+/- mice were successfully validated by genotyping and reduced intestinal LSS protein expression. HE and oil red O staining of liver sections showed that， compared with WT mice fed a CHOW diet， WT mice fed a HFD exhibited a marked increase in hepatic lipid vacuoles. In contrast， compared with HFD-fed WT mice， HFD-fed LSS^+/- mice displayed significantly attenuated hepatic lipid deposition and reduced serum ALT levels （P<0.05）. Immunohistochemical analysis revealed that， compared with WT mice， the expression of the cholesterol absorption protein NPC1L1 in the intestinal villi of LSS^+/- mice was downregulated under both CHOW and HFD conditions （P_HFD<0.001）. Conversely， the expression of the fatty acid transporter CD36 was upregulated in the intestines of LSS^+/- mice （P_CHOW<0.05， P_HFD<0.01）. Western blot analysis demonstrated that， compared with WT mice， TLR4 protein expression in the intestines of LSS^+/- mice significantly increased under both CHOW and HFD conditions （both P<0.05）. JNK phosphorylation level was significantly elevated in LSS^+/- mice under CHOW condition （both P<0.05）. Under HFD condition， total JNK protein expression increased， but its phosphorylation level showed no significant change. qPCR analysis showed that， compared with WT mice， the mRNA levels of TLR4 （P_CHOW<0.01， P_HFD<0.000 1） and IL-6 （P_CHOW<0.001， P_HFD<0.01） were significantly upregulated in the intestines of LSS^+/-mice. ConclusionLSS deficiency counteracts hepatic lipid deposition by orchestrating a synergistic reprogramming involving restricted intestinal cholesterol absorption， enhanced fatty acid utilization， and activation of immune pathways， suggesting intestinal LSS as a potential therapeutic target of MASLD.

Objective:To study the role of cadherin 1 (CDH1) gene DNA methylation in autoimmune thyroiditis (AIT) in population from different water-iodine regions.Methods:From May to June 2019, the information of AIT cases and healthy individuals in Shandong Province were collected in three types of water-iodine regions: iodine-fortification (IF) region, iodine-adequate (IA) region and iodine-excess (IE) region. A case-control study design was applied to match 176 AIT cases (case group) with age, gender, body mass index, and place of residence in a 1 ∶ 1 ratio to 176 healthy individuals (control group). Fasting urine and whole blood samples were collected to test the contents of urinary iodine, thyroid function indicators [serum free triiodothyronine (FT 3), free thyroxine (FT 4), thyroid stimulating hormone (TSH)], and serum iodine. The DNA methylation levels of the target region of the CDH1 gene and its four CpG sites in whole blood were determined using methylation sequencing technology for target regions (MethylTarget TM). Results:The DNA methylation level of the target region of CDH1 gene in the case group was 0.832 ± 0.044, and that in the control group was 0.828 ± 0.049, there was no statistically significant difference between the two groups ( t = 0.76, P = 0.448). There was no statistically significant difference in DNA methylation levels of the four CpG sites in the target region of CDH1 gene between the case group and the control group ( P > 0.05). There was no statistically significant difference in the DNA methylation level of the CDH1 gene target region between the case group and the control group in IF, IA and IE regions ( P > 0.05). The detection results of DNA methylation levels at CpG sites in the target region of CDH1 gene in different water iodine regions showed that the DNA methylation level at site 83 in case group in IF region was higher than that in the control group ( t = 2.30, P = 0.023). However, there was no statistically significant difference in the DNA methylation levels of the four CpG sites between the case group and the control group in IA and IE regions ( P > 0.05). The DNA methylation level of CDH1 gene target region in AIT patients was not significantly correlated with urinary iodine, serum iodine, and serum FT 3, FT 4, and TSH contents ( P > 0.05), but was significantly negatively correlated with age ( r =-0.19, P = 0.014). Conclusions:The DNA methylation level at CpG site 83 of CDH1 gene in AIT patients in IF region is significantly higher than that in control population, indicating that DNA methylation at this locus may be involved in the occurrence and development of AIT after iodine fortification. The DNA methylation level of CDH1 gene is negatively correlated with age.

Objective To investigate the effect of lifestyle on cognitive decline in rural people aged 40 years and older in Xi'an.Methods This was a prospective cohort study.People aged 40 years and older in two villages in Huyi District were selected as the study population.They completed the baseline survey from October 2014 to March 2015 as well as two follow-up visits in 2016 and 2018,respectively.A comprehensive score of lifestyle was calculated based on factors including smoking,drinking,exercise,and diet collected at the baseline.The Mini-Mental State Examination(MMSE)was used to evaluate global cognitive function at both baseline and follow-up;a≥4-point decrease in MMSE score from the baseline was defined as marked cognitive decline.Multivariable Logistic regression,propensity score correction,and propensity score matching were used to investigate the relationship between lifestyle and cognitive decline.Results A total of 1 348 participants were ultimately enrolled and 56(4.2％)of them met the criteria for marked cognitive decline(△MMSE≥4-points).Among them,386(28.6％)people had smoking history,184(13.6％)were drinkers,214(15.9％)lacked physical activity,and 400(29.7％)ate a diet high in oil and salt.Generally,304(22.6％)met the definition of the unhealthy lifestyle(comprehensive score＜6),which means more than one of the four subscales was unhealthy or more than two were relatively unhealthy.Multivariable Logistic regression analysis showed that unhealthy lifestyle was positively associated with marked cognitive decline(OR=2.838,95％CI:1.302-5.525,P=0.005).Propensity-score adjusted model yielded very similar results(OR=2.786,95％CI:1.371-5.661,P=0.005).Propensity score matching was performed to further balance the differences in covariates between the two groups.Multivariate Logistic regression analysis conducted in the matched population revealed that the risk of marked cognitive decline was still higher in those with unhealthy lifestyle(OR=3.994,95％CI:1.582-12.176,P=0.006).Conclusion Unhealthy lifestyle is associated with an increased risk of cognitive decline in cognitively normal people aged 40 years and older.

Objective To investigate the value of methylation levels of Discs large-associated protein5(DLGAP5)and Ras association domain family 1A(RASSF1A)in peripheral blood in the differential diagnosis of benign and malignant lung nodules.Methods 110 patients with pulmonary nodules admitted to the Seventh People's Hospital of Hebei Province from December 2022 to June 2024 were selected as study subjects.Based on the diagnosis confirmed by pathological examination,52 patients with malignant lung nodules were included in the malignant group and 58 patients with benign lung nodules were included in the benign group.The methylation levels of DLGAP5 and RASSF1A genes in peripheral blood were determined by quantitative real-time polymerase chain reaction(qRT-PCR).The diagnostic value of methylation of DLGAP5 and RASSF1A genes in peripheral blood on the benign and malignant nature of lung nodules and their relationship with clinicopathological characteristics of patients with malignant lung nodules were analyzed.Spearman's method was used to analyze the correlation between DLGAP5 and RASSF1A gene methylation levels and clinicopathological features.Results Compared with the benign group,the methylation levels of DLGAP5 and RASSF1A genes were higher in the peripheral blood of patients in the malignant group,and the differences were statistically significant(χ2=27.010,24.350,all P<0.001).The sensitivities of DLGAP5 methylation and RASSF1A methylation alone in the detection of malignant lung nodules were 61.54%,67.31%,and the specificity were 86.21%,79.31%,and the accuracies were 74.55%,73.63%,respectively.The sensitivity(84.62%)and accuracy(83.64%)of the combined two-gene methylation test for malignant lung nodules were better than that of single gene methylation(Z=2.816,2.497,all P<0.05).The concordance Kappa values of DLGAP5 methylation and RASSF1A methylation alone and in combination with the pathological results were 0.583,0.569 and 0.712,respectively.The nodules size,histological differentiation degree,lymph nodes and spiculation sign of patients with malignant pulmonary nodules were correlated with DLGAP5 and RASSF1A methylation levels,and the differences were statistically significant(χ2DLGAP5=4.644～14.981,χ2RASSF1A=4.293～12.629,all P<0.05).Correlation analysis showed that the methylation levels of DLGAP5 and RASSF1A genes were positively correlated with nodule size,histologic differentiation,lymph node metastasis and spiculation sign(rDLGAP5=0.512～0.683,rRASSF1A=0.527～0.691,all P<0.05).Conclusion The methylation levels of DLGAP5 and RASSF1A gene in peripheral blood were closely related to histological differentiation,lymph node metastasis and clinical staging.The combined detection of the two has high diagnostic value fro the benign and malignant nature of pulmonary nodules.

Objective:To analyze the influencing factors of low fall alertness in elderly inpatients,construct a risk prediction model and validate it,providing a reference for clinical medical staff to identify elderly inpatients with low fall alertness in the early stage.Methods:A total of 605 elderly inpatients treated in Yijishan Hospital affiliated to Wannan Medical College from Oct 2023 to Mar 2024 were enrolled and randomly divided into the training group(n=423)and validation group(n=182)at a ratio of 7∶3.The patients were evaluated using a general information questionnaire,the Social Frailty Screening Tool(HALFT),the Tilburg Frailty Indicator(TFI),and the Self-Awareness of Falls in Elderly scale(SAFE).Multivariate logistic analysis was used to determine the influencing factors of low fall alertness in elderly inpatients.RStudio was used to construct a risk prediction model of low fall alertness.The discrimination,calibration,and clinical net benefit of the model were verified using the receiver operating characteristic(ROC)curves,calibration plots,and decision curve analysis(DCA).Results:Multivariate logistic analysis showed that the history of falls,monthly income,previous physical activity time,social frailty score and TFI score were independent risk factors for low fall alertness in elderly inpatients.The Hosmer-Lemeshow χ2 test showed that χ2=8.863,P=0.354,indicating good calibration of the prediction model.The area under the ROC curve of the training group and the validation group were 0.860(95%CI:0.815-0.904)and 0.937(95%CI:0.888-0.986),respectively,and the maximum Youden indices of the model was 0.576 and 0.788,respectively,indicating good discrimination of the model.The DCA decision curve showed that the model had good clinical effectiveness.Conclusion:The constructed model has a good prediction effect and can help clinical medical staff quickly and effectively screen out elderly inpatients at risk of low fall alertness.

This study was aimed atanalyzing the epidemic characteristics of dengue fever in Ruili City atthe China-Myanmar bor-der in late 2023,to provide evidence for local dengue fever prevention and control measures.Adult Aedes mosquitoes were collected from Ruili City with a backpack type mosquito sucking machine in October of 2023.Serum samples frompatients with suspected den-gue were collected in acutephase,in November of 2023.Detection ofdengue virus(DENV)nucleic acids in Aedesmosquitoes and acute phase serum samples from suspected dengue fever patients using reverse transcription-polymerase chain reaction,and nucleic acid positive samples were inoculated into Vero cells for viral culture.After three consecutive blind passage,samples with cytopathic effect(CPE)were collected for be sequencingand analysisof genetic and evolutionary information.Dengue case characteristics were analyzed through descriptive epidemiological methods.Among the 109 cases of dengue fever,the ratio of males to females was 1.27∶1.The youngest patient was 1 year old,the oldest patient was 84 years old,the age group of 20～59 years accounted for 73.39%,and the major-ity of occupations were freelancer(40.37%).A total of 827 female Aedes albopictus and 312 Aedes aegypti were collected,all of which tested negative for DENV nucleic acid.109 serum samples tested positive for DENV nucleic acid,including 49 DENV-1 and 60 DENV-2.Moreover,five DENV-1 and nine DENV-2 samples were obtainedthrough third-generation blind passaging with CPE.The E gene sequences of these five DENV-1 strains were detected,all were found to belong to DENV-1 genotype I,and had same evolu-tionary branch as the 2023 Guangzhou,China(PP563911),the 2019 Myanmar(MW793710),and 2019 Attapeu,Laos(MW559046).The nucleotide and amino acid sequence similarityamong the five DENV-1 genotype was 99.4%-99.9%and 99.8%-100.0%.Compared with the aforementioned epidemic strains,their nucleotide similarities were 99.5%-100.0%,99.4%-99.6%and 99.3%-99.5%,respec-tively,and amino acid similarity was 99.8%-100.0%.Nine DENV-2 E gene sequences were of Asian genotype I and belonged to the same evolutionary branch as the 2018 Myanmar(MW788982),2019 Hangzhou(OP684212)and 2019 Ruili(OQ928150).The nucleotide and amino acid similarities of the nine samples were 99.5%-100.0%and 99.8%-100.0%,respectively.Compared with the aforementioned epidemic strains,their nucleotide similarities were 99.7%-100.0%,99.3%-99.7%and 99.3%-99.7%,respectively,and the amino acid similarity was 99.8%-100%,99.8%-100.0%and 99.4%-99.6%,respectively.Two dengue vectors,Aedes albopictus and Aedes aegypti,were present in Ruili city,and the dengue outbreak was caused primarily by DENV-1 genotype I and DENV-2 Asian genotype I in later 2023.The sources of DENV-1 were probably the same as those of DENV-1 with Guangzhou(2023),and the sources of DENV-2 were probably from Myanmar.Dengue cases were found primarilyin the 20-59 year age group and freelancers,thus suggesting that relevant local departments should strengthen surveillance of dengue imported case and vector.

Objective To construct a dynamic framework for bidirectional transitions of mild cognitive impairment(MCI),quantifying both rare reversion and high-risk progression trajectories in cognitive dynamics.Methods Patients diagnosed with MCI at baseline from 2005 to 2022 and completed at least two follow-up visits were selected from the Alzheimer's Disease Neuroimaging Initiative(ADNI),and a retrospective cohort was constructed.Demographic information,APOEε4 genotype,and neuropsychological scales data were collected.Longitudinal cognitive assessments were functionally reconstructed using multivariate functional principal component analysis(MFPCA),with functional principal components(FPCs)extracted based on cumulative variance contribution rate(PVE＞90％).Functional multi-state Markov models were developed to estimate inter-state transition intensities,year to year transition probabilities,and covariate effects.Results Among 1,019 MCI patients(4,657 follow-up visits),93(9.1％)reverted to normal cognition,while 359(35.2％)progressed to Alzheimer's disease(AD).Longitudinal trajectory analysis revealed significant heterogeneity:progressive MCI＞stable MCI＞reverted MCI in the first functional principal component(MFPC1)scores.The transition intensity for MCI reversion(0.020)was approximately one-fourth of the AD progression risk(0.086),but the post-reversion cognitive re-impairment intensity was 0.138.Reduced MFPC1(HR=0.993,95％Cl:0.991,0.995)and elevated MFPC2(HR=1.004,95％Cl:1.001,1.007)were closely associated with MCI reversion.Conclusion MCI exhibits marked heterogeneity in longitudinal cognitive trajectories.Although reversion is rare,reversed patients remain at high risk of cognitive re-impairment.

Objective:To investigate the effect of tolerogenic dendritic cells(tolDC)on autophagy of synovial cells in collagen-induced arthritis(CIA)rats.Methods:Bone marrow mononuclear cells of rats were extracted and induced into tolDC using IL-4,GM-CSF and NF-κB oligonucleotide decoy,and loaded with BⅡC to become BⅡC-tolDC.SD rats were randomly divided into normal control group,CIA model group and BⅡC-tolDC intervention group,with 3 rats in each group.Normal female SD rats were immunized with bovine type Ⅱ collagen solution to construct CIA model.Rats in BⅡC-tolDC intervention group were infused with BⅡC-tolDC via tail vein on the 21st day after initial immunization for two weeks,arthritis indexes were recorded weekly.On the 35th day,the rats were sacrificed,and synovial histopathology of ankle joint of rats in each group were observed by HE staining;the number of osteo-clasts in cartilage of rats in each group were observed by TRAP staining.The number of autophagic of ankle synovial cells of rats in each group were observed by transmission electron microscopy.Levels of serum TNF-α and IL-1β of rats in each group were detected by ELISA.LC3,Beclin-1 and ATG5 proteins of synovial cells of ankle joints of rats in each group were detected by Immunohistochemical staining.Results:CIA rats were constructed successfully by immunization with bovine type Ⅱ collagen.BⅡC-tolDC intervention re-duced the arthritis index of CIA rats,inhibited synovial inflammation and abnormal proliferation of synovial tissue,improved joint bone and cartilage injury,and reduced the number of osteoclasts in cartilage tissue and the number of autophagosomes in synovial cells.At the same time,reduced levels of serum TNF-α,IL-1β,and protein expressions of LC3,Beclin-1 and ATG5 of synovial cell of CIA rats.Conclusion:BⅡC-tolDC may alleviate arthritis lesions of CIA rats by inhibiting synovial cell autophagy of CIA rats.

Objective:To investigate the effect of exosomes from lung cancer cells on the ability of lung cancer metastasis in-duced by M2 macrophages.Methods:The exosomes secreted by 16HBE,A549 and H522(16HBE exo,A549 exo,H522 exo)were extracted and identified.M0 macrophages co-incubated with 10 μg/ml 16HBE exo,A549 exo,H522 exo,PBS(16HBE exo group,A549 exo group,H522 exo group,PBS group),the expressions of Arginase-1,CD206,CD163,TGF-β,iNOS,and IL-1β in macro-phages in each group were detected by qRT-PCR,the proportion of CD206+CD163+macrophages was detected by flow cytometry,the phosphorylation levels of ERK1/2 and STAT3 in macrophages were detected by Western blot.A549 and H522 cells were co-incubated with macrophages in the above groups(16HBE exo+M0 group,A549 exo+M0 group,H522 exo+M0 group,PBS+M0 group),and the migration and invasive ability of A549 and H522 cells in each group were detected by Transwell chamber method.Results:Compared with PBS group,the expressions of Arginase-1,CD206,CD163 and TGF-β in macrophages in A549 exo group and H522 exo group were significantly up-regulated(P<0.001),and the expressions of iNOS and IL-1β were significantly down-regulated(P<0.001).The proportion of CD206+CD163+macrophages was increased significantly(P<0.001),and the phosphorylation levels of ERK1/2 and STAT3 were increased significantly(P<0.001).There was no significant difference in the above indicators between the 16HBE exo group and the PBS group(P>0.05).The migration ablility and invasion ablility of A549 and H522 cells in A549 exo+M0 group and H522 exo+M0 group were significantly increased(P<0.001).Conclusion:Lung cancer cell exosomes can induce the polarization of M2 macrophages by activating the ERK1/2/STAT3 signaling pathway to promote tumor migration and invasion.

BACKGROUND:Emodin has a variety of pharmacological activities such as anti-inflammatory,anti-viral and anti-oxidative stress,and also has a certain protective effect on sepsis-induced myocardial injury,but its mechanism of action is still unclear.OBJECTIVE:To investigate whether emodin can improve myocardial injury in septic mice by promoting autophagy.METHODS:Thirty-two male Kunming mice were divided into sham operation group(n=4),sham operation+emodin group(n=4),model group(n=8),model+emodin group(n=8),and emodin+3-methyladenine group(n=8).The myocardial injury model of septic mice was constructed by cecal ligation and puncture.3-methyladenine(10 mg/kg)was injected intraperitoneally 1 hour before modeling.Emodin(20 mg/kg)was injected intraperitoneally 30 minutes before modeling,and the other groups were injected with the same amount of normal saline at the same time point.Blood and myocardial samples were collected from all mice 24 hours after surgery.ELISA was used to detect the levels of brain natriuretic peptide and cardiac troponin Ⅰ in serum.Western blot assay was used to detect the protein expression of LC3B,Beclin-1,and p62 in myocardial tissue.Hematoxylin-eosin staining was used to observe the pathological changes in myocardial tissue.Ultrasound was used to evaluate the cardiac function of mice.RESULTS AND CONCLUSION:(1)Compared with the sham operation group,there was no significant difference in the levels of serum brain natriuretic peptide,cardiac troponin Ⅰ,and the protein expression of myocardial autophagy proteins LC3Ⅱ/LC3Ⅰ and p62 in the sham operation+emodin group(P＞0.05).(2)Compared with the sham operation+emodin group,the levels of serum brain natriuretic peptide and cardiac troponin I were significantly increased in the model group(P＜0.05).Compared with the model group,the levels of serum brain natriuretic peptide and cardiac troponin I were decreased in the model+emodin group(P＜0.05).(3)Compared with the model group,the expression of LC3Ⅱ/LC3Ⅰ and Beclin-1 protein was increased and the expression of p62 protein was decreased in the myocardial tissue of the model+emodin group(P＜0.05).Compared with the model+emodin group,the expression of LC3Ⅱ/LC3Ⅰ and Beclin-1 protein decreased and the expression of p62 protein increased in the emodin+3-methyladenine group(P＜0.05).(4)The myocardial fibers in the sham operation group were normal,the myocardial fibers in the model group were disordered with a large number of inflammatory cell infiltration,the myocardial fibers in the model+emodin group were slightly disordered,and some vacuolar changes were observed.The myocardial fibers were disordered,and more inflammatory cell infiltration was observed in the emodin+3-methyladenine group.(5)Compared with the sham operation group,the left ventricular short axis shortening rate and left ventricular ejection fraction were decreased in the model group(P＜0.05).Compared with the model group,the left ventricular short axis shortening rate and left ventricular ejection fraction were increased in the model+emodin group(P＜0.05).Compared with the model+emodin group,the left ventricular ejection fraction of emodin+3-methyladenine group was decreased(P＜0.05),and the left ventricular short axis shortening rate was reduced but not statistically significant(P＞0.05).(6)The above results indicate that emodin pretreatment can improve myocardial injury and myocardial dysfunction in septic mice by promoting autophagy.