This paper summarizes Professor SHI Zaixiang's clinical experience in treating high fever caused by sepsis using Chaihu Guizhi Ganjiang Decoction （柴胡桂枝干姜汤）. He holds that the key pathogenesis of sepsis involves constrained heat in the shaoyang and internal accumulation of water and fluids. The clinical manifestations such as high fever， chills， and alternating sensations of cold and heat are attributed to pathogenic heat constrained in the shaoyang. Meanwhile， soft tissue edema and serous cavity effusions are due to shaoyang dysfunction and internal water retention. In clinical practice， treating sepsis-related high fever requires addressing both the shaoyang-constrained heat and the associated edema and effusions. The therapeutic approach focuses on harmonizing the shaoyang and resolving internal fluids， using Chaihu Guizhi Ganjiang Decoction as the base formula with flexible modifications. Professor SHI emphasizes that this formula shows a rapid antipyretic effect， particularly in cases where multiple anti-infective treatments have failed.

Objective : To investigate the mechanisms of bromodomain-containing protein 4(BRD4) in TGF-β1-induced epithelial-mesenchymal transition in alveolar type II epithelial cells. Methods : MLE-12 cells were stimulated with different concentrations(5 ng/ml, 10 ng/ml) of TGF-β1 for 48 h to establish an EMT cell model. The cells were pretreated with 50 nmol/L BRD4 inhibitor JQ-1, 100 μmol/L high mobility group box 1 protein(HMGB1)inhibitor glycyrrhizin acid(GA), and 3 μg/ml rHMGB1. The experimental groups were divided as follows: control group, TGF-β1 group, JQ-1 group, JQ-1+TGF-β1 group, GA group, GA+TGF-β1 group, and JQ-1+TGF-β1+rHMGB1 group. The effect of JQ-1 on cell viability was examined using cell counting kit-8(CCK-8). The protein expression levels of CDH1, ZO-1, Vimentin, α-SMA, BRD4, HMGB1, TGF-β1, Smad2/3 and p-Smad2/3 were detected by Western blot. The cell migration ability was detected by a scratch test. Results : Compared with the control group, the levels of Vimentin and α-SMA in the TGF-β1 group increased, and the levels of CDH1 and ZO-1 protein decreased, suggesting that the EMT model was successfully established. In this model, the expression of BRD4 and HMGB1 significantly increased. Different concentrations of JQ-1 could inhibit the cell viability of MLE-12 in a concentration-dependent manner. Both JQ-1 and GA could effectively alleviate TGF-β1-induced EMT, and reduce the increase in HMGB1 expression and the activation of TGF-β1/Smad2/3 pathway caused by TGF-β1. Moreover, rHMGB1 treatment could reduce the effects of JQ-1 on EMT and the TGF-β1/Smad2/3 pathway. Additionally, both JQ-1 and glycyrrhizin could effectively decrease TGF-β1-induced cell migration, whereas rHMGB1 could alleviate the inhibitory effect of JQ-1 on the rate of cell migration. Conclusion BRD4 can regulate epithelial-mesenchymal transition in alveolar type II epithelial cells via HMGB1/TGF-β1/Smad2/3 signaling cascade, and BRD4 may be a potential target for inhibition of pulmonary fibrosis.

Objective: To explore the differential lipid metabolites in the plasma of mice with idiopathic pulmonary fibrosis(IPF). Methods : Thirty SPF C57BL/6 male mice were randomly divided into 2 groups with 15 mice in each group. The experimental groups were divided into control group and bleomycin(BLM) group. The model of idiopathic pulmonary fibrosis was induced by one-time intratracheal infusion of BLM(1 mg/kg). Hematoxylin-eosin(HE) staining was used to observe the lung histopathology. The collagen fiber deposition in lung tissue was observed by Sirius red staining. The differential lipid metabolites in plasma of IPF mice were screened and enriched by lipidomics. Results : HE staining showed that the pulmonary tissue structure was disordered, alveolar septum was broken and alveolar wall was destroyed in BLM group. Sirius red staining showed a large amount of collagen fiber deposition in the lung interstitium of BLM group. The results of lipidomics analysis showed that the lipid metabolism profile of BLM group changed, 15 differential lipid metabolites were screened out, of which 11 differential lipid metabolites were up-regulated, and 4 differential lipid metabolites were down-regulated, mainly concentrated in glycerophosphoglycerophosphates, glycerophosphocholines, steroid lactones, etc. Conclusion The lipid metabolism profile of BLM group mice changes, differential lipid metabolites such as phosphoglycolate phosphatase(PGP)(18:0/18:0), PGP(i-12:0/i-24:0), PGP(i-13:0/a-25:0), and phosphatidylcholine(PC)(18:0/14:0), PC(18:3/16:0), lysophosphatidylcholine(LPC)(16:1), and LPC(18:3) may play an important role in the progression of IPF. These findings provide a new reference for further study of the molecular mechanism of IPF, and also provide a potential new target for clinical treatment.

Objective To screen and analyze the differentially expressed genes (DEGs) related to oxidative stress in a silicosis mouse model using transcriptome sequencing technology. Methods i) A total of 30 workers without occupational dust-exposed history were selected as the control group and 17 patients with silicosis were selected as the silicosis group using a judgment sampling method. The levels of glutathione and malondialdehyde in the plasma of workers in the two groups were determined by enzyme-linked immunosorbent assay. ii) RAW264.7 cells in the logarithmic growth phase were randomly divided into the control group and the silica group, treated with 0 and 50 mg/L silica suspensions for 24 hours. Protein expression of superoxide dismutase 2 (SOD2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the cells was determined by Western blotting. iii) The specific pathogen free male C57BL/6 mice were randomly divided into the control group and the silicosis model group, with 10 mice in each group. Mice were exposed to 50 μL of 0.9% sodium chloride solution and silica suspension at a mass concentration of 100 g/L, respectively, using a single tracheal exposure method. After 28 days of exposure, the pathological changes of mouse lung tissues were observed. Transcriptome sequencing was used to screen DEGs in the lung tissues of the silicosis mouse model, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The expression of DEGs was verified using quantitative real-time polymerase chain reaction (qPCR). Results i) The level of malondialdehyde in the patients′ plasma was higher (P<0.01), while the level of glutathione was lower (P<0.01) in the silicosis group than that of the control group. ii) The relative expression of SOD2 protein decreased (P<0.05), while the relative expression of IL-6 and TNF-α proteins increased (all P<0.05) in the silica group of RAW264.7 cells compared with the control group. iii) The pathological results of lung tissues showed that the alveolar structure of mice was destroyed and silicotic nodules were formed in the silicosis model group. Transcriptome sequencing identified 3 703 DEGs, of which 3 199 were significantly down-regulated and 504 were significantly up-regulated. The GO enrichment analysis results showed that the DEGs were significantly enriched in biological processes such as oxidative stress, inflammation, immunity and hypoxia, with cellular components mainly located in membranes, cytoplasm, and nucleus. Molecular functions were enriched in oxidoreductase activity, protein binding, and adenosine triphosphate binding. The KEGG enrichment analysis results showed that the DEGs were mainly involved in the phosphatidylinositol 3-kinase-protein kinase B signaling pathway, cyclic adenosine monophosphate signaling pathway, chemokine signaling pathway, and apoptosis signaling pathway. A total of 28 DEGs involved in the "oxidative stress response" pathway were screened by GO enrichment analysis. The qPCR verification results showed that the relative expression of DEGs carbonic anhydrase 3 (Car3), matrix metalloproteinase 9 (Mmp9), and MutY DNA glycosylase (Mutyh) involved in the "oxidative stress response" of lung tissues in the silicosis model group were lower than those of the control group (all P<0.05). Conclusion Oxidative stress response exists in silicosis patients. The oxidative stress-related genes Car3, Mmp9, and Mutyh are altered in the mouse lung tissues of the silicosis model through the oxidative stress pathway, suggesting that they could be new targets for the treatment of silicosis.

OBJECTIVE: To investigate the clinical characteristics and genetic etiology of a Chinese pedigree affected with Hereditary dentin dysplasia type II (DD-II) due to variant of dentin sialophosphoprotein (DSPP) gene. METHODS: A child diagnosed with DD- II at the Third Clinical Division of Peking University Hospital of Stomatology in December 2021 and her family members were selected as study subjects. Clinical data were retrospectively analyzed. Saliva samples were collected from the proband, her parents and sister for genomic DNA extraction. Whole exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing and TOPO-TA cloning sequencing. The candidate variant was also subjected to bioinformatics analysis using Mutation Taster v2021. Secondary and tertiary structures of the wild-type and variant DSPP proteins were predicted with psipred v4.0 and PyMOL v2.3 software, respectively. The pathogenicity of the variant was classified based on the guidelines from American College of Medical Genetics and Genomics (ACMG). This study was approved by the Medical Ethics Committee of Peking University Hospital of Stomatology (Ethics No.: PKUSSIRB-202162021). RESULTS: The proband and her mother and sister had all exhibited typical clinical manifestations of hereditary DD-II. The primary dentition of the proband displayed yellowish brown discoloration, wear, and obliteration in the chamber and root canal, while the permanent teeth of the proband's sister and mother appeared nearly normal in both color and appearance, though with obliteration in the chamber and root canal. Her father showed normal dentition. WES identified a heterozygous c.1915_1918delAAGT, p.(Lys639Glnfs*674) frameshift variant in the DSPP gene. Sanger sequencing and TOPO-TA cloning sequencing confirmed the presence of this variant in the proband, the proband's sister, and the mother, while the proband's father was negative for the variant, indicating an autosomal dominant inheritance pattern. The variant was predicted to be pathogenic by Mutation Taster v2021. Prediction of the secondary structure of the DSPP protein showed that the variant has changed it from coil to helix. The tertiary structure prediction of the DSPP protein showed change of the spatial structure of the variant DSPP, with the loops in the variant region replaced by helices at multiple sites. Based on the guidelines from the ACMG, the variant was classified as pathogenic (PVS1+PM2_Supporting+PP1+PP4). CONCLUSION Phenotypic analysis and genetic testing of this family has clarified the clinical diagnosis of hereditary DD- II. The c.1915_1918delAAGT variant probably underlay the pathogenesis of DD-II in this family. Above results have expanded the phenotypic spectrum of the disease and may contribute to further clinical and genetic research on this disease.
Humans ; Pedigree ; Female ; Extracellular Matrix Proteins/chemistry* ; Male ; Sialoglycoproteins/chemistry* ; Dentin Dysplasia/genetics* ; Asian People/genetics* ; Phosphoproteins/chemistry* ; Child ; Mutation ; China ; Exome Sequencing ; Adult ; East Asian People

Objective To investigate the molecular mechanism of Shema Zhichuan Liquid in the treatment of neutrophilic asthma(NA)based on network pharmacology and in vivo experiments.Methods(1)The TCMSP,literature search and Swiss ADME and Swiss Target Prediction databases were used to search and screen the active components and their targets of Shema Zhichuan Liquid.OMIM,GeneCards,DisGeNET and DrugBank databases were used to search and screen NA disease-related targets.The intersection of the active components and NA disease-related targets of Shema Zhichuan Liquid was obtained through the microbiology platform to obtain the potential targets of Shema Zhichuan Liquid for the treatment of NA(common targets).Cytoscape 3.8 software was used to construct the network of"Chinese medicinals-active components-potential targets".The PPI network of potential targets was established by STRING database,and the core targets were obtained by analysing the built-in Mcode plug-in.The Metascape platform was used to enrich the gene ontology(GO),Kyoto Encyclopaedia of Genes and Genomes(KEGG)pathways for the potential targets.(2)BALB/C mice were acclimatised and fed for 1 week and randomly divided into a blank group,NA model group,low-dose group(2.5 g·kg-1)and high-dose group of Shema Zhichuan Liquid(10 g·kg-1),and control group of Dexamethasone(1 mg·kg-1);the NA mouse model was replicated by intraperitoneal injection of sensitizer(OVA+CFA)and nebulized inhalation excitation.OVA/CFA(20 μg OVA+75 μg CFA,0.3 mL)was injected intraperitoneally to sensitize on days 0,7 and 14 respectively,and 5%OVA suspension was nebulized on days 21-30(8 mL each time,40 minutes each time,once a day);1 hour before nebulisation,each group was administered by gastric gavage,and the Dexamethasone control group was administered by intraperitoneal injection once a day.The pathological changes of mouse lung tissue were observed by HE staining;IL-8 content in alveolar lavage fluid was detected by ELISA;mRNA expression levels of NLRP3 and CXCR2 were detected by RT-qPCR;and p-mTOR protein expression levels was detected by immunohistochemistry.Results(1)A total of 826 active component targets and 154 NA disease-related targets were obtained,and 51 potential targets(common targets)for the treatment of NA were obtained from the intersection of the active component and the NA disease-related targets of Shema Zhichuan Liquid.Through the network analysis of"Chinese medicinals-active components-potential targets",quercetin,lignocerotoxin,kaempferol,stigmasterol,naringenin and other key active components were obtained.The PPI network analysis of potential targets yielded 29 core targets,including AKT1,IL6,TNF,EGFR,NLRP3,RELA,MIF,CXCR2,VEGFA,etc..The GO functional enrichment analysis yielded 882 biological process entries,33 cellular component entries,and 61 molecular function entries;KEGG analysis yielded 142 signaling pathways,mainly involving TNF signaling pathway,influenza A signaling pathway,Toll-like receptor pathway,MAPK signaling pathway,mTOR signaling pathway and so on.(2)Results of animal experiments:compared with the blank group,mice in the NA model group showed obvious damage to the airway mucosa,structural disorders,a large number of inflammatory cells infiltration,mucosal congestion,oedema,obvious thickening of the alveolar wall,and narrowing of the alveolar lumen;the level of the inflammatory factor IL-8 in the alveolar lavage fluid was significantly elevated(P＜0.05);the mRNA expressions of NLRP3 and CXCR2 in the lung tissues of the mice were significantly up-regulated(P＜0.01),and the protein expression of p-mTOR was significantly increased.Compared with the NA model group,the structural arrangement of bronchial epithelial cells in the mice in the low-and high-dose groups of Shema Zhichuan Liquid was slightly disordered,with a small amount of inflammatory cell infiltration around the airways and blood vessels,and the congestion and edema of the bronchial mucosa were significantly reduced;the mRNA expression of CXCR2 in the lung tissues of the mice was significantly down-regulated(P＜0.01),and the level of expression of p-mTOR protein was significantly reduced.The IL-8 level in the vesicular lavage fluid of mice in the high-dose group was significantly reduced(P＜0.05);the mRNA expression of NLRP3 in the lung tissue of mice in the low-dose group was significantly down-regulated(P＜0.05).Conclusion The therapeutic effect of Shema Zhichuan Liquid on NA may be achieved through the key active components,such as quercetin,lignocerol and kaempferol,acting on the core targets,such as NLRP3 and CXCR2,and regulating the key signaling pathways,such as the TNF signaling pathway,the MAPK signaling pathway,the Toll-like signaling pathway,and the mTOR pathway.

Background Silicosis is an occupational disease mainly characterized by pulmonary progressive fibrosis induced by the accumulation of free silica (SiO₂) in the lungs due to long-term exposure to SiO₂ dust. It has been shown that neutrophil extracellular traps (NETs) are increased in the lung tissues of silicotic mice after 28 d SiO₂ exposure, but it is unclear how the levels of NETs change throughout entire progression of silicosis in mice. Objective To observe the levels of NETs and pathological changes in the lungs of silicotic mice after different duration of SiO₂ exposure, and to confirm the possible role and significance of NETsin the development of SiO₂-induced pulmonary fibrosis. Methods A total of 28 SPF male C57BL/6J mice were randomly divided into a control group, and a model group, and the model group was subdivided into, a 2 d model group, a 7 d model group, and a 28 d model group, with 7 mice in each group. The mice in the model groups were given intratracheal instillation with 10 mg SiO₂ suspension (50 μL), and the mice in the control group were received same volume of saline. Mice were sacrificed and samples were collected at designed time points. The pathological changes of lung tissues of mice were observed after hematoxylin-eosin (HE) and Van Gieson (VG) staining. Immunofluorescence was used to observe the NETs markers citrullination histone H3 (CitH3) and myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF), and the percentage of NETs-positive cells was calculated. PicoGreen fluorescent dye kit was used to detect the content of extracelluar DNA (ex-DNA) in mouse BALF, and the expression levels of fibrosis-related proteins α-smooth muscle actin (α-SMA) and fibronectin (FN) and NETs marker CitH3 in lung tissues of mice were detected by Western blot (WB). Results Compared with the control group, inflammatory cells accumulation, alveolar wall thickening, and collagen deposition were obviously observed in the lungs of the silicosis model groups, and a large number of silicone nodules were recorded in the lung tissues in the 28 d group. Compared with the control group, the expressions of α-SMA and FN in the lung tissue of the 28 d group were significantly increased (P<0.05). The percentages of NETs in BALF increased significantly in the 2 d and the 7 d model group, then decreased in the 28 d model group (P<0.05). Compared with the control group (7.434±0.258) ng·mL⁻¹, the ex-DNA levels in BALF of mice in the 2 d [(35.110±6.331) ng·mL⁻¹], the 7 d [(39.491±6.948) ng·mL⁻¹], and the 28 d [(23.360±4.809) ng·mL⁻¹] model groups were increased (P<0.05), and the increase of ex-DNA in the 2 d and the 7 d model groups were statistically significant (P<0.05). In comparison with the control group, the protein level of CitH3 was significantly increased in the lung tissues of mice in the 7 d model group (P<0.05). Conclusion The content of NETs increases significantly and reaches a peak in the early inflammatory stage of silicosis, and decreases as the disease progresses to the fibrotic stage, suggesting that NETs may play a role in early stage of silicosis.

Objective To investigate the correlation between the anatomical structure of patent foramen ovale(PFO)observed by transesophageal echocardiography(TEE)and the right to left shunt(RLS)grade of contrast-enhanced transthoracic echocardiography(c-TTE).Methods Ninety cases in which the presence of PFO was suggested by TEE examination as a diagnostic criterion from November 2021 to December 2022 in the First Hospital of Nanchang were retrospectively analysed.According to the c-TTE results of patients,the RLS was divided into 4 levels,and the correlation between PFO structural characteristics and RLS grading was analyzed.Results There was a positive correlation between PFO diameter size and RLS grading in resting state(r=0.381,P<0.05);The PFO diameter of patients with hypermobile interatrial septum(HIS)was larger and the difference was statistically significant(P<0.05);The PFO diameter of patients with persistent RLS was larger than that of excited phase patients,and the difference was statistically significant(P<0.05);There was no significant difference in RLS shunt degree between patients with long tunnel and those without long tunnel;There was no significant difference in RLS grade and PFO diameter size under Valsalva state.Conclusion Research has shown that certain anatomical structures of PFO interact with RLS grading,and PFO anatomical structures can also interact with each other(the opening diameter of the foramen ovale with HIS is larger);At the same time,TEE can clearly show the morphological characteristics of PFO and predict the degree of RLS,so as to further evaluate the possibility of ischemic stroke in patients with PFO,and provide more evidence for the indications for foramen ovale closure.

ObjectiveTo explore the distribution of pharyngeal microbiota in coal miners exposed to dust. Methods Eight coal miners who had been engaged in occupational dust exposure for more than 20 years were selected as the dust-exposed group, and four coal miners who were not exposed to dust at work were selected as the control group using the judgment sampling method. Pharyngeal secretions of the coal miners were collected with throat swabs, and its pharyngeal microbiota was analyzed. The diversity, abundance and evenness of the microbiota were analyzed by gene sequencing using the 16sRNA gene high-throughput sequencing technology. Results A total of 254 operational taxonomic units of pharyngeal microbiota were detected in the coal miners in the control group, which was 210 more than that in the dust-exposed group. The Chao1 index, Shannon index, PD-tree index and Pielou index of pharyngeal microbiota in the dust-exposed group decreased compared with the control group (all P<0.01). The abundance of Bacteroidetes and Clostridum, at the phylum level, in the pharynx of coal miners in the dust-exposed group was higher than that in the control group (all P<0.05). The abundance of Prevotella, Neisseria, and Monas, at the genus level, in the pharynx of coal miners in the dust-exposed group was higher than that in the control group(all P<0.05), while the abundance of Lactobacillus decreased (P<0.05). The analysis results of the receiver operating characteristic curve showed that Lactobacillus, Fusobacterium and Rothia may play a role for pharyngeal microbiota imbalance prediction in dust-exposed workers, and the area under the curves were all 1.00±0.00. Conclusion The species diversity and evenness of pharyngeal microbiota in coal miners exposed to dust are decreased, which may be related to the continuous inhalation of coal dust that disrupts the microbial environment of the throat.

Objective To screen mitochondria-targeting differential genes in alveolar macrophages of silicosis pa-tients and explore the role of mitochondrial homeostasis in alveolar macrophages of silicosis patients.Methods High-throughput sequencing dataset GSE174725 was downloaded,and differentially expressed genes were screened with R software and P＜0.05,|LogFC|＞1,and then intermixed with mitochondrial gene bank MitoCarta3.0 to obtain mitochondria-targeted differentially expressed genes.Then enrichment analysis was carried out to obtain the biological processes and pathways involved in differential genes,and the protein-protein interaction network was constructed.In addition,alveolar macrophages from silicosis patients and healthy controls were collected,the ex-pression levels of differential genes were detected by RT-qPCR,and the expressions of mitochondria-related factors mitochondrial fusion protein 1(MFN1),optic atrophy 1(OPA1)and dynamin-related protein 1(DRP1)in alveolar macrophages of silicosis patients were investigated by Western blot.Results A total of 204 differentially expressed genes were screened in silicosis patients,among which 62 differentially expressed genes were up-regulated,142 dif-ferentially expressed genes were down-regulated,and 22 differentially expressed genes were mitochondria-targeted.The concentration analysis of differentially expressed genes targeted by mitochondria showed that the cell compo-nents mainly enriched included mitochondrial membrane,endoplasmic membrane side components,etc.The bio-logical processes mainly enriched included mitochondrial electron transfer from NADH to ubiquinone,inflammatory response,immune response,etc.The main molecular functions enriched included the rotation mechanism of proton transport ATP synthase activity,NADH dehydrogenase activity,chemokine activity,etc.KEGG enrichment analy-sis mainly focused on the involvement in chemical carcinogenesis-ROS,IL-17 signaling pathway,toll-like receptor signaling pathway,chemokine signaling pathway,TNF signaling pathway,etc.In addition,RT-qPCR results showed that the expressions of mitochondrial cytochrome coxidase 1,mitochondrial cytochrome coxidase 2,mito-chondrial cytochrome coxidase 3,mitochondrially encoded NADH dehydrogenase 1,mitochondrially encoded NADH dehydrogenase 3,mitochondrially encoded NADH dehydrogenase 5,superoxide dismutase and mitochondri-ally encoded ATP synthase 6 gene were down-regulated in silicosis patients(P＜0.05).Western blot and RT-qPCR results showed that,in silicosis patients,the expression of MFN1 and OPA1 decreased(P＜0.05),while the expression of DRP1 increased(P＜0.05).Conclusion Bioinformatics analysis and validation,eight mito-chondrial targeted differential genes(MT-CO1,MT-C02,MT-CO3,MT-ND1,MT-ND3,MT-ND5,SOD and MT-ATP6)were finally obtained,which were enriched in mitochondrial respiratory chain and oxidative stress pathways and might play an important role in the process of silicosis.