Traditional Chinese medicine(TCM)tongue diagnosis has obvious advantages,but its strong subjectivity,susceptibility to environmental influences,and lack of a unified standard greatly affect the objectivity and accuracy of diagnostic results,and it is difficult to integrate into the modern scientific system.Therefore,developing tongue diagnosis equipment to objectively and normatively extract tongue image information,and simulating human brain thinking to construct an objective diagnostic model of TCM tongue image-disease syndrome,and promoting the quantitative and intelligent research of TCM tongue diagnosis have become the trend of future development.This article reviews the research status and application prospects of TCM tongue diagnosis equipment,in order to provide methodological references and research ideas for the research and application of this field.

Objective:Using methylation characteristics of human genes to construct machine learning predictive models for screening cervical cancer and precancerous lesions.Methods:Human DNA methylation detection was performed on 224 cervical exfoliated cell specimens from the Cancer Hospital of the Chinese Academy of Medical Sciences, Tianjin Central Hospital of Gynecology Obstetrics, Xinmi Maternal and Child Health Hospital of Henan Province, West China Second Affiliated Hospital of Sichuan University, and Heping Hospital Affiliated to Changzhi Medical College collected during April 2014 and March 2015. The hypermethylated gene fragments related to cervical cancer were selected by high-density, high-association, and hypermethylated gene fragment screening and the LASSO regression algorithm. Taking cervical intraepithelial neoplasia grade 2 (CIN2) or more severe lesions as the research outcome, machine learning predictive models based on the random forest (RF), naive Bayes (NB), and support vector machine (SVM) algorithm, respectively, were constructed. A total of 144 outpatient specimens were used as the training set and 80 cervical exfoliated cell specimens from women participating in the cervical cancer screening program were used as the test set to verify the predictive models. Using histological diagnosis results as the gold standard, the detection efficacy for CIN2 or more severe lesions of the three machine learning predictive models were compared with that of the human papilloma virus (HPV) detection and cytological diagnosis.Results:In the training set of 144 cases, there were 34 cases of HPV positivity, with a positive rate of 23.61%. Cytologically, there were 37 cases diagnosed as no intraepithelial lesion or malignancy (NILM), and 107 cases diagnosed as atypical squamous cells of undetermined significance (ASC-US) or above. Histologically, there were 28 cases without cervical intraepithelial neoplasia or benign cervical lesions, 31 cases of CIN1, 18 cases of CIN2, 31 cases of CIN3, and 36 cases of squamous cell carcinoma. Seven hypermethylated gene fragments were selected from 45 genes, and three machine learning prediction models based on the RF, NB, and SVM algorithm, respectively, were constructed. In the validation set of 80 cases, there were 28 cases of HPV positivity, with a positive rate of 35.00%. Cytologically, there were 65 cases diagnosed as NILM and 15 cases as ASC-US or above. Histologically, there were 39 cases without cervical intraepithelial neoplasia or benign cervical lesions, 10 cases of CIN1, 10 cases of CIN2, 11 cases of CIN3, and 10 cases of squamous cell carcinoma. In the validation set, the area under the curve (AUC) values of the RF model, NB model, SVM model, HPV detection, and cytological diagnosis of CIN2 or above were 0.90, 0.88, 0.82, 0.68, and 0.45, respectively. The DeLong test showed that there was no statistically significant difference in the AUC values between the RF, NB, and SVM models (all P＞0.05), and the AUC values of the RF and NB models were higher than that of HPV detection (both P＜0.01), and the AUC values of the RF, NB, and SVM models were higher than that of cytological diagnosis (all P＜0.01). Compared with the NB model, the sensitivity of the RF model was similar (80.65% vs. 77.42%), but the specificity of the NB model was much higher than that of the RF model (93.88% vs. 73.47%). Conclusion:Among the machine learning prediction models for cervical cancer and precancerous lesions constructed based on human DNA methylation, the NB model has good predictive performance for CIN2 and above lesions, and may be used for screening of cervical cancer and precancerous lesions.

BACKGROUND:Research has shown that vanillic acid has anti-inflammatory and anti-oxidative stress effects,but it is unclear whether it has a protective effect on endplate chondrocytes.OBJECTIVE:To explore the effect and mechanism of vanillic acid on endplate chondrocytes under inflammatory microenvironment.METHODS:(1)Primary endplate chondrocytes were isolated from the intervertebral disc of SD rats and identified by toluidine blue staining and collagenⅡ immunofluorescence.(2)The CCK-8 assay was employed to detect the effects of interleukin-1β and vanillic acid on the proliferation activity of endplate chondrocytes,in order to determine the concentration of vanillic acid for subsequent cell treatment.(3)An inflammatory microenvironment was simulated by adding 10 ng/mL interleukin-1β to the culture medium,and the endplate chondrocytes were treated with low,medium,and high mass concentrations of vanillic acid.The expression levels of inflammatory markers and extracellular matrix proteins were detected by western blot assay and immunofluorescence.(4)The expression of nuclear factor κB signaling pathway-related proteins was detected by western blot assay.RESULTS AND CONCLUSION:(1)The morphology of endplate chondrocytes in adherent culture was pike or triangular in shape,positive for toluidine blue staining and immunofluorescence for collagen Ⅱ,indicating that the experimentally extracted cells were endplate chondrocytes.(2)The CCK-8 assay results showed that treatment with 2.5,5,10,and 20 μg/mL vanillic acid for 24 hours did not significantly inhibit the proliferation of endplate chondrocytes.Compared with the interleukin-1β group,the viability of endplate chondrocytes treated with 5,10,and 20 μg/mL vanillic acid for 24 hours was significantly increased(P＜0.05).Therefore,5,10,and 20 μg/mL vanillic acid was selected as the low,medium,and high dose groups for subsequent treatment of endplate chondrocytes.(3)Compared with the model group(complete medium containing 10 ng/mL interleukin-1β),the expression of NOD-like receptor thermal domain associated protein 3(NLRP3),matrix metalloproteinase 13,matrix metalloproteinase 3,and tumor necrosis factor alpha protein in the endplate chondrocytes of the low,medium,and high doses of vanillic acid groups were significantly reduced(P＜0.05).(4)Compared with the model group,the protein expression of aggrecan and collagen Ⅱ in the endplate chondrocytes of the low,medium,and high dose groups of vanillic acid significantly increased(P＜0.05).(5)Compared with the model group,the protein expression of phospho-nuclear factor κB and phospho-inhibitor of nuclear factor κB in the endplate chondrocytes of the low,medium,and high dose groups of vanillic acid was significantly reduced(P＜0.05).(6)The above results indicate that vanillic acid may alleviate the inflammatory response and extracellular matrix degradation induced by interleukin-1β in rat endplate chondrocytes by inhibiting the activation of the nuclear factor κB signaling pathway.

Objective:To explore the potential role of mRNA m7G modification in the pathogenesis of human adenocarcinoma of esophagogastric junction (AEG).Methods:Pathological tissue specimens from four AEG patients who underwent surgical treatment at the People′s Hospital Affiliated to Jiangsu University between 2018 and 2019 were selected. Tumor tissues and adjacent normal tissues were collected from these patients. RNA was extracted from both tissue types and subjected to m7G methylated RNA immunoprecipitation sequencing (m7G-MeRIP-seq) to analyze the patterns of m7G modification, the characteristics of differential m7G modification sites, the differentially expressed mRNA, and the correlation between m7G modification and mRNA expression levels. Differential m7G-modified genes ( MSH6, BRCA1, and SOX9) were further validated using methylated RNA immunoprecipitation quantitative PCR (MeRIP-qPCR), while the expression of METTL1 and WDR4 genes was examined by real-time quantitative PCR (RT-qPCR). This study was approved by the Medical Ethics Committee of the People′s Hospital Affiliated to Jiangsu University (Ethics No. 20150083). Results:① m7G-MeRIP-seq analysis revealed that m7G modifications in both AEG and adjacent normal tissues were predominantly located in the GC-rich region surrounding the internal start codon of mRNA. Differential m7G modification sites between the two groups were closely associated with cancer-related genes. ② mRNA library analysis showed that differentially expressed mRNA were predominantly upregulated in AEG tissues and downregulated in adjacent normal tissues. ③ Cross-analysis indicated that genes with hypermethylation tended to exhibit upregulated expression, while genes with hypomethylation were typically downregulated in AEG tissues. ④ MeRIP-qPCR validation confirmed that the mRNA expression of MSH6, BRCA1, and SOX9 were significantly upregulated in AEG tissues compared to adjacent normal tissues (AEG vs. normal, P<0.05). ⑤ RT-qPCR results demonstrated that the mRNA expression levels of METTL1 and WDR4 were also upregulated in AEG tissues (AEG vs. normal, P<0.000 5). Conclusion:These findings suggest that mRNA m7G modification plays a significant role in the development of AEG. Furthermore, proteins as METTL1 and WDR4 may facilitate AEG progression by regulating mRNA m7G modification. These results provide valuable insights into the molecular mechanisms underlying AEG and may inform future therapeutic strategies for this malignancy.

OBJECTIVE To explore the improvement effect and mechanism of salidroside on radiation-induced parotid gland injury in rats. METHODS Rats were randomly assigned into normal group， radiation group， salidroside low-dose （salidroside-L， 50 mg/kg） group， salidroside high-dose （salidroside-H， 100 mg/kg） group， and salidroside-H+inhibitor （100 mg/kg salidroside+0.1 µmol/kg H-89） group， with 10 rats in each group. Except for the normal group， rats in the other groups were subjected to radiation exposure to establish a model of radiation-induced parotid gland injury. Rats in each group were intraperitoneally injected with the corresponding drug or normal saline， once a day， for 40 consecutive days. After the last administration， the levels of reactive oxygen species （ROS）， cyclic adenosine monophosphate （cAMP）， superoxide dismutase （SOD）， and amylase in serum were detected； the pathological changes of parotid gland tissue were observed； the apoptosis rate of parotid gland tissue cells， the expression levels of B-cell lymphoma-2 （Bcl-2） and its associated X protein （Bax）， mRNA expression levels of interleukin-6 （IL- 6） and tumor necrosis factor-α （TNF-α）， the protein expression levels of type Ⅲ collagen （Col Ⅲ）， vasoactive intestinal peptide （VIP）， and the phosphorylation level of protein kinase A （PKA） in parotid gland tissue were determined. RESULTS Compared with normal group， the levels of ROS， amylase， apoptosis rate， Bax expression level， mRNA expression levels of IL-6 and TNF- α， and protein expression level of Col Ⅲ in the radiation group were significantly increased， while the levels of cAMP， SOD， Bcl-2 expression level， VIP protein expression level and PKA phosphorylation level were significantly decreased （P＜0.05）. Compared with radiation group， the above indicators in the salidroside-L group and salidroside-H group were significantly improved （P＜0.05）， and the improvement in the salidroside-H group was more significant （P＜0.05）； inhibitor H-89 significantly reversed the changes in the above indicators of the salidroside-H group （P＜0.05）. CONCLUSIONS Salidroside can effectively alleviate radiation-induced parotid gland injury in rats， and its mechanism may be related to the activation of the VIP-cAMP pathway.

OBJECTIVE: To explore the potential role of mRNA m7G modification in the pathogenesis of human adenocarcinoma of esophagogastric junction (AEG). METHODS: Pathological tissue specimens from four AEG patients who underwent surgical treatment at the People's Hospital Affiliated to Jiangsu University between 2018 and 2019 were selected. Tumor tissues and adjacent normal tissues were collected from these patients. RNA was extracted from both tissue types and subjected to m7G methylated RNA immunoprecipitation sequencing (m7G-MeRIP-seq) to analyze the patterns of m7G modification, the characteristics of differential m7G modification sites, the differentially expressed mRNA, and the correlation between m7G modification and mRNA expression levels. Differential m7G-modified genes (MSH6, BRCA1, and SOX9) were further validated using methylated RNA immunoprecipitation quantitative PCR (MeRIP-qPCR), while the expression of METTL1 and WDR4 genes was examined by real-time quantitative PCR (RT-qPCR). This study was approved by the Medical Ethics Committee of the People's Hospital Affiliated to Jiangsu University (Ethics No. 20150083). RESULTS: m7G-MeRIP-seq analysis revealed that m7G modifications in both AEG and adjacent normal tissues were predominantly located in the GC-rich region surrounding the internal start codon of mRNA. Differential m7G modification sites between the two groups were closely associated with cancer-related genes. mRNA library analysis showed that differentially expressed mRNA were predominantly upregulated in AEG tissues and downregulated in adjacent normal tissues. Cross-analysis indicated that genes with hypermethylation tended to exhibit upregulated expression, while genes with hypomethylation were typically downregulated in AEG tissues. MeRIP-qPCR validation confirmed that the mRNA expression of MSH6, BRCA1, and SOX9 were significantly upregulated in AEG tissues compared to adjacent normal tissues (AEG vs. normal, P < 0.05). RT-qPCR results demonstrated that the mRNA expression levels of METTL1 and WDR4 were also upregulated in AEG tissues (AEG vs. normal, P < 0.000 5). CONCLUSION These findings suggest that mRNA m7G modification plays a significant role in the development of AEG. Furthermore, proteins as METTL1 and WDR4 may facilitate AEG progression by regulating mRNA m7G modification. These results provide valuable insights into the molecular mechanisms underlying AEG and may inform future therapeutic strategies for this malignancy.
Humans ; RNA, Messenger/metabolism* ; Adenocarcinoma/pathology* ; Esophagogastric Junction/metabolism* ; Esophageal Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic ; Female ; Male ; Middle Aged ; DNA Methylation ; Methyltransferases/metabolism* ; Stomach Neoplasms/genetics*

Lymphoma is a highly heterogeneous malignancy characterized by complex molecular regulatory mechanisms that result in significant differences in aggressiveness and prognosis across its subtypes.Gene copy number alteration(CNA)analysis,an emerging technology,has become a pivotal tool in the precision re-search and management of lymphoma.By detecting DNA deletions,amplifications,and chromosomal copy number changes,CNA analysis addresses the limitations of traditional cytogenetic techniques,enhances the ac-curacy of subtype classification,and aids in evaluating tumor heterogeneity and disease progression.This re-view provides a comprehensive summary of CNA detection methods and their applications in lymphoma,with a focus on recent advancements in the field.It offers a comparative analysis of CNA detection techniques and discusses their role in precision diagnosis,subtype classification,monitoring disease progression,predicting therapeutic resistance,and assessing prognosis.Additionally,the review explores the potential applications of CNA analysis in uncovering molecular regulatory mechanisms,optimizing therapeutic strategies,and impro-ving patient survival outcomes.

ObjectiveThis study aims to explore the mechanism of action of Huangqi Guizhi Wuwutang in treating rheumatoid arthritis （RA）-associated sarcopenia by regulating autophagy and improving skeletal muscle homeostasis based on network pharmacology，bioinformatics，machine learning，and animal experiments. MethodsActive ingredients and targets of Huangqi Guizhi Wuwutang were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP），PubChem，and SwissTargetPrediction databases. RA-related datasets were retrieved from the GEO database，and differential genes were screened. Sarcopenia-related targets were searched through GeneCards and the Comparative Toxicology Database （CTD），and autophagy-related gene sets were downloaded from the Human Autophagy Database （HADb）. Their intersection was analyzed to identify autophagy-related therapeutic targets，followed by enrichment analysis. A protein-protein interaction （PPI） network was constructed using the STRING database，and key targets were selected using multiple methods. Machine learning was applied to predict models based on the expression profiles of intersecting targets，and nomogram models were constructed based on key targets. Molecular docking of the top four active ingredients with key targets was performed using AutoDockVina. A collagen-induced arthritis （CIA） rat model was established using bovine type Ⅱ collagen，with SD rats divided into groups including a blank group，a model group，and low-，medium-，and high-dose groups of Huangqi Guizhi Wuwutang （2.44，4.88，and 9.76 g·kg^-1） and administered for five consecutive weeks. Joint scores and gastrocnemius muscle mass were recorded and analyzed after modeling. Hematoxylin and eosin （HE） staining and Masson's staining were used to observe pathological changes in muscle tissue. Immunofluorescence staining was applied to observe the protein expression levels of myosin heavy chain （MYHC） and insulin-like growth factor-1 （IGF-1） in skeletal muscle. Western blot was used to detect the protein expression levels of autophagy-related proteins ATG5，Beclin1，LC3B，muscle-specific proteins （MuRF1），MaFbx，and MYHC. Real-time quantitative reverse transcription PCR （Real-time PCR） was performed to measure the mRNA expression levels of ATG5，Beclin1，LC3B，MuRF1，MaFbx，and MYHC in muscle tissue. ResultsNetwork pharmacology revealed that Huangqi Guizhi Wuwutang shared 25 common targets with autophagy genes related to RA-associated sarcopenia. The PPI network and machine learning identified six key targets，which were primarily involved in autophagy and inflammatory pathways. Animal experiments showed that compared to the blank group，the model group had significantly higher joint scores （P<0.01） and lower gastrocnemius muscle index （P<0.01）. HE staining indicated a significant reduction in the cross-sectional area of gastrocnemius muscle fibers，with notable inflammatory cell infiltration and muscle atrophy in the model group. Masson's staining revealed obvious collagen fiber proliferation and deposition，with significant muscle fibrosis in the model group. The protein and mRNA expression levels of ATG5，Beclin1，LC3B，MuRF1，and MaFbx were significantly increased （P<0.01），while the protein expression of MYHC and IGF1 was significantly downregulated （P<0.01）. Compared with the model group，the high-dose group of Huangqi Guizhi Wuwutang showed significantly reduced protein and mRNA expression levels of ATG5，Beclin1，LC3B，MuRF1，and MaFbx （P<0.01） and increased protein expression levels of MYHC and IGF1 （P<0.01）. The cross-sectional area of muscle fibers increased，and the muscle cell morphology approached normal. Moreover，pathological abnormalities in the gastrocnemius muscle were significantly improved，with reduced collagen fiber proliferation （P<0.01）. ConclusionHuangqi Guizhi Wuwutang can mediate autophagy by regulating the expression of ATG5，Beclin1，LC3B，and IGF1，thereby reducing skeletal muscle catabolism and improving skeletal muscle homeostasis，which contributes to the treatment of RA-associated sarcopenia. The findings provide insight into the mechanisms underlying the effects of Huangqi Guizhi Wuwutang in the treatment of RA-related sarcopenia and offer a reference for its enhanced clinical application.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

BACKGROUND:Research has shown that vanillic acid has anti-inflammatory and anti-oxidative stress effects,but it is unclear whether it has a protective effect on endplate chondrocytes.OBJECTIVE:To explore the effect and mechanism of vanillic acid on endplate chondrocytes under inflammatory microenvironment.METHODS:(1)Primary endplate chondrocytes were isolated from the intervertebral disc of SD rats and identified by toluidine blue staining and collagenⅡ immunofluorescence.(2)The CCK-8 assay was employed to detect the effects of interleukin-1β and vanillic acid on the proliferation activity of endplate chondrocytes,in order to determine the concentration of vanillic acid for subsequent cell treatment.(3)An inflammatory microenvironment was simulated by adding 10 ng/mL interleukin-1β to the culture medium,and the endplate chondrocytes were treated with low,medium,and high mass concentrations of vanillic acid.The expression levels of inflammatory markers and extracellular matrix proteins were detected by western blot assay and immunofluorescence.(4)The expression of nuclear factor κB signaling pathway-related proteins was detected by western blot assay.RESULTS AND CONCLUSION:(1)The morphology of endplate chondrocytes in adherent culture was pike or triangular in shape,positive for toluidine blue staining and immunofluorescence for collagen Ⅱ,indicating that the experimentally extracted cells were endplate chondrocytes.(2)The CCK-8 assay results showed that treatment with 2.5,5,10,and 20 μg/mL vanillic acid for 24 hours did not significantly inhibit the proliferation of endplate chondrocytes.Compared with the interleukin-1β group,the viability of endplate chondrocytes treated with 5,10,and 20 μg/mL vanillic acid for 24 hours was significantly increased(P＜0.05).Therefore,5,10,and 20 μg/mL vanillic acid was selected as the low,medium,and high dose groups for subsequent treatment of endplate chondrocytes.(3)Compared with the model group(complete medium containing 10 ng/mL interleukin-1β),the expression of NOD-like receptor thermal domain associated protein 3(NLRP3),matrix metalloproteinase 13,matrix metalloproteinase 3,and tumor necrosis factor alpha protein in the endplate chondrocytes of the low,medium,and high doses of vanillic acid groups were significantly reduced(P＜0.05).(4)Compared with the model group,the protein expression of aggrecan and collagen Ⅱ in the endplate chondrocytes of the low,medium,and high dose groups of vanillic acid significantly increased(P＜0.05).(5)Compared with the model group,the protein expression of phospho-nuclear factor κB and phospho-inhibitor of nuclear factor κB in the endplate chondrocytes of the low,medium,and high dose groups of vanillic acid was significantly reduced(P＜0.05).(6)The above results indicate that vanillic acid may alleviate the inflammatory response and extracellular matrix degradation induced by interleukin-1β in rat endplate chondrocytes by inhibiting the activation of the nuclear factor κB signaling pathway.