Objective To explore the mechanism of Yizhu Wendan Decoction in treating eczema through GEO database combined with network pharmacology and experimental verification.Methods TCMSP,BATMAN-TCM and ETCM databases were used to screen the active components of Yizhu Wendan Decoction.Disease target information related to eczema was collected through GEO database.The drug-component-target network and PPI network were constructed by intersections of active component targets and disease targets.GO and KEGG pathway enrichment analyses were performed using DAVID database.CCK-8 method was used to screen out the optimal intervention concentration of freeze-dried powder of Yizhu Wendan Decoction.HaCaT cells were divided into control group,model group,Yizhu Wendan Decoction low concentration group,Yizhu Wendan Decoction high concentration group,si-IL-17RA group,si-IL-17RA+Yizhu Wendan Decoction low concentration group,si-IL-17RA+Yizhu Wendan Decoction high concentration group,Dexamethasone group,si-IL-17RA+Dexamethasone group.Each group was given relevant intervention.The expressions of chemokines and inflammatory factors were detected by qPCR.EdU and Annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis.Western blot was performed to detect the expressions of proteins related to apoptosis,skin barrier and IL-17 signaling pathway.Results By using databases,180 active components of Yizhu Wendan Decoction were obtained.Combined with GEO database microarrays related to eczema(GSE6012 and GSE57225),8 potential targets of Yizhu Wendan Decoction in the treatment of eczema were obtained.KEGG enrichment pathway mainly involved IL-17 signaling pathway,lipid and atherosclerotic,TNF signaling pathway,fluid shear stress and atherosclerotic,etc.When Yizhu Wendan Decoction freeze-dried powder concentration was 100 μg/mL,cell viability was the strongest.Yizhu Wendan Decoction could significantly inhibit the mRNA expressions of chemokines and inflammatory factors CCL17,CCL22,IL-1β,TNF-α,IL-6,IFN-γ,and increase the mRNA expression of IL-4 in eczema.It promoted the proliferation of HaCaT cells,increased the protein expression of Bcl-2,and reduced the protein expressions of Bad and Cleaved Caspase-3,thus inhibiting HaCaT cells apoptosis;promoted the protein expressions of FLG and LOR,and reduced the expression of MMP9,MMP1,CCL2,FOSL1,IL-17RA proteins in IL-17 signaling pathway.Conclusion Yizhu Wendan Decoction can treat eczema with multiple components,multiple pathways and multiple targets,promote the proliferation of HaCaT cells,inhibit their apoptosis,and restore the skin barrier.Its mechanism may be related to inhibiting the activation of IL-17 signaling pathway.

Pseudopleuronectes yokohamae is an important marine economic fish species in northern China and is vulnerable to various pathogens during the breeding process.To investigate the viru-lence of Edwardsiella tarda on Pseudopleuronectes yokohamae and elucidate the role of miRNAs in their interaction,we conducted pathological analyses on various tissues of Pseudopleuronectes yokohamae larvae pre-and post-infection with Edwardsiella tarda.Subsequently,Illumina high-throughput sequencing was employed to identify and functionally characterize differentially ex-pressed miRNAs in liver and kidney tissues.The results demonstrated that infection with Ed-wardsiella tarda led to ulceration,hemorrhaging,significant hepatomegaly,nephromegaly,and as-cites in the abdominal cavity.Histopathological examination revealed swollen hepatocytes with vac-uolization,minor blood cell infiltration,and enlarged renal corpuscles with signs of cellular lysis.miRNA sequencing analysis indicated that 24 miRNAs were significantly upregulated and 45 miR-NAs were significantly downregulated in the liver,while in the kidney,6 miRNAs were upregulat-ed and 58 were downregulated following infection.Functional enrichment analysis revealed signifi-cant enrichment of differentially expressed miRNAs in Toll and IMD signaling pathways,HIF-1 signaling pathway,Rap1 signaling pathway,MAPK signaling pathway,and Apelin signaling path-way.These findings suggest the involvement of these miRNAs in host metabolism,pathogen rec-ognition,and immune response in the interaction between Pseudopleuronectes yokohamae and Ed-wardsiella tarda.This research lays a foundation for elucidating the mechanisms underlying bacte-rial disease susceptibility and resistance in Pseudopleuronectes yokohamae.

Objective To explore the mechanism of Yizhu Wendan Decoction in treating eczema through GEO database combined with network pharmacology and experimental verification.Methods TCMSP,BATMAN-TCM and ETCM databases were used to screen the active components of Yizhu Wendan Decoction.Disease target information related to eczema was collected through GEO database.The drug-component-target network and PPI network were constructed by intersections of active component targets and disease targets.GO and KEGG pathway enrichment analyses were performed using DAVID database.CCK-8 method was used to screen out the optimal intervention concentration of freeze-dried powder of Yizhu Wendan Decoction.HaCaT cells were divided into control group,model group,Yizhu Wendan Decoction low concentration group,Yizhu Wendan Decoction high concentration group,si-IL-17RA group,si-IL-17RA+Yizhu Wendan Decoction low concentration group,si-IL-17RA+Yizhu Wendan Decoction high concentration group,Dexamethasone group,si-IL-17RA+Dexamethasone group.Each group was given relevant intervention.The expressions of chemokines and inflammatory factors were detected by qPCR.EdU and Annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis.Western blot was performed to detect the expressions of proteins related to apoptosis,skin barrier and IL-17 signaling pathway.Results By using databases,180 active components of Yizhu Wendan Decoction were obtained.Combined with GEO database microarrays related to eczema(GSE6012 and GSE57225),8 potential targets of Yizhu Wendan Decoction in the treatment of eczema were obtained.KEGG enrichment pathway mainly involved IL-17 signaling pathway,lipid and atherosclerotic,TNF signaling pathway,fluid shear stress and atherosclerotic,etc.When Yizhu Wendan Decoction freeze-dried powder concentration was 100 μg/mL,cell viability was the strongest.Yizhu Wendan Decoction could significantly inhibit the mRNA expressions of chemokines and inflammatory factors CCL17,CCL22,IL-1β,TNF-α,IL-6,IFN-γ,and increase the mRNA expression of IL-4 in eczema.It promoted the proliferation of HaCaT cells,increased the protein expression of Bcl-2,and reduced the protein expressions of Bad and Cleaved Caspase-3,thus inhibiting HaCaT cells apoptosis;promoted the protein expressions of FLG and LOR,and reduced the expression of MMP9,MMP1,CCL2,FOSL1,IL-17RA proteins in IL-17 signaling pathway.Conclusion Yizhu Wendan Decoction can treat eczema with multiple components,multiple pathways and multiple targets,promote the proliferation of HaCaT cells,inhibit their apoptosis,and restore the skin barrier.Its mechanism may be related to inhibiting the activation of IL-17 signaling pathway.

Pseudopleuronectes yokohamae is an important marine economic fish species in northern China and is vulnerable to various pathogens during the breeding process.To investigate the viru-lence of Edwardsiella tarda on Pseudopleuronectes yokohamae and elucidate the role of miRNAs in their interaction,we conducted pathological analyses on various tissues of Pseudopleuronectes yokohamae larvae pre-and post-infection with Edwardsiella tarda.Subsequently,Illumina high-throughput sequencing was employed to identify and functionally characterize differentially ex-pressed miRNAs in liver and kidney tissues.The results demonstrated that infection with Ed-wardsiella tarda led to ulceration,hemorrhaging,significant hepatomegaly,nephromegaly,and as-cites in the abdominal cavity.Histopathological examination revealed swollen hepatocytes with vac-uolization,minor blood cell infiltration,and enlarged renal corpuscles with signs of cellular lysis.miRNA sequencing analysis indicated that 24 miRNAs were significantly upregulated and 45 miR-NAs were significantly downregulated in the liver,while in the kidney,6 miRNAs were upregulat-ed and 58 were downregulated following infection.Functional enrichment analysis revealed signifi-cant enrichment of differentially expressed miRNAs in Toll and IMD signaling pathways,HIF-1 signaling pathway,Rap1 signaling pathway,MAPK signaling pathway,and Apelin signaling path-way.These findings suggest the involvement of these miRNAs in host metabolism,pathogen rec-ognition,and immune response in the interaction between Pseudopleuronectes yokohamae and Ed-wardsiella tarda.This research lays a foundation for elucidating the mechanisms underlying bacte-rial disease susceptibility and resistance in Pseudopleuronectes yokohamae.

Hepatocellular carcinoma （HCC） is the sixth most common cancer in the world. In recent years， the clinical early diagnosis and treatment protocols of HCC have been improved， whereas the prognosis of patients is still not satisfactory， which is due to the fact that the mechanism of HCC development has not been fully elucidated. Therefore， it is of great significance to explore the molecular mechanisms and key regulatory links of hepatocellular carcinoma development to further improve the diagnosis and treatment of HCC in China. Epigenetics has become a research hotspot because of its reversibility and easy regulation. According to relevant studies， HCC involves the accumulation of multiple genetic and epigenetic changes during the initiation， promotion， and progression stages. HCC is categorized as infantile malnutrition with accumulation， hypochondriac pain， tympan ites， and abdominal mass in traditional Chinese medicine （TCM）. In the treatment of HCC， TCM with low toxicity， multi-targets， and multi-mechanisms can inhibit tumor growth， alleviate the clinical symptoms， and enhance the quality of life of the patients. Chinese medicines and their active ingredients exert anti-HCC effects through epigenetic regulation of DNA methylation， histone modification， and non-coding RNA. Abnormal gene expression due to epigenetic regulation disorders is involved in all stages of HCC development. There are few studies on epigenetic regulation in TCM treatment of HCC， and there is still much room for development in basic and clinical trials. This paper reviews the mechanism of epigenetic regulation in HCC and summarizes the experimental results of TCM research on the related mechanism， with a view to providing a theoretical basis for future research on the mechanism of HCC development and clinical diagnosis and treatment of hepatocellular carcinoma with TCM.

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration.
Amyloidogenic Proteins/metabolism* ; Amyotrophic Lateral Sclerosis/genetics* ; Animals ; COVID-19 ; Cytoplasmic Granules/metabolism* ; Mammals ; SARS-CoV-2 ; Stress Granules

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with K _D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl₄-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

We report a case of fetal akinesia deformation sequence (FADS), which was prenatally suspected on ultrasound and confirmed by whole exome sequencing and Sanger sequencing after mid-term termination. Prenatal ultrasonography revealed multiple abnormalities in a fetus at 21 +4 weeks of gestation, consisting of fixed posture of limbs, narrow thorax, markedly shrunken gastric vacuole, and thickened nuchal fold. After genetic counseling, the pregnancy was terminated, and the appearance of the fetus was consistent with the ultrasound findings. Whole exome sequencing and Sanger sequencing of the fetal tissue verified a compound heterozygous variation of the RAPSN gene--c.149_153delins AGATGGGCCGCTACAAGGAGATGG (p.V50Efs*114) and c.227T>C (p.L76P), which were inherited from the father and mother, respectively, ultimately confirming the diagnosis of FADS.

OBJECTIVE：To discuss the inhibitory effect of lanthanum chloride on the calcification of vascular smooth muscle cells（VSMCs）induced by high phosphorus and its mechanism. METHODS ：On the basis of screening the action concentration and time of lanthanum chloride by MTT method ，human VSMCs were divided into control group （1 mmol/L phosphorus solution ）， lanthanum chloride high concentration control group （1 mmol/L phosphorus solution+ 60 μmol/L lanthanum chloride），model group （3 mmol/L phosphorus solution ），sodium chloride group （3 mmol/L phosphorus solution+ 180 μmol/L sodium chloride），nuclear factor κB（NF-κB）signaling pathway agonist+lanthanum chloride group （3 mmol/L phosphorus solution+ 1 μg/mL lipopolysaccharide+ 60 μmol/L lanthanum chloride），positive control group （3 mmol/L phosphorus solution+ 100 μmol/L sodium pyrophosphate），and lanthanum chloride low ，medium，and high concentration groups （3 mmol/L phosphorus solution+ 15，30，60 μmol/L lanthanum chloride）. Alizarin red S staining and Von Kossa staining were used to detect cell calcification in each group after treated with phosphorus solution for 6 d and relevant medicine for 2 d. Western blot assay was used to detect the protein expression of TNF-α receptor associated protein 6（TRAF6），nuclear factor κB inhibitor protein α（IκBα），NF-κB p65，bone morphogenetic protein 2 （BMP-2），smooth muscle 22 α（SM22α）and Runt related transcription factor 2（Runx2）. Real-time fluorescence quantitative polymerase chain reaction was used to detect mRNA expression of TRAF 6，IκBα，BMP-2，SM22α and Runx2. RESULTS ： Compared with control group ，no cell calcification was observed in the lanthanum chloride high concentration control group ，while obvious cell calcification and significant increase of OD value were observed in model group and sodium chloride group （P＜ 0.01）；protein and mRNA expression of TRAF 6 and BMP- 2 in cytoplasm as well as mRNA expression of Runx 2，protein expression of NF-κB p65 and Runx 2 in nucleus were significantly increased （P＜0.01）；protein and mRNA expression of IκBα and SM22α as well as protein expression of NF-κB p65 in cytoplasm were significantly decreased （P＜0.01）. Compared with model group，cell calcification was significantly improved in lanthanum chloride groups and positive control group ，while OD values were significantly reduced ；the expression levels of the above-mentioned protein and mRNA were reversed to varying degrees （P＜0.05 or P＜0.01）. Compared with lanthanum chloride high concentration group ，obvious cell calcification was observed in NF-κB signaling pathway agonist + lanthanum chloride group ，and OD value was significantly increased ；the above indexes were significantly reversed in cytoplasm and nucleus （P＜0.05 or P＜0.01）. CONCLUSIONS ：Lanthanum chloride can inhibit the calcification of VSMCs induced by high phosphorus ，and its mechanism may be related to the inhibition of NF-κB signaling pathway activation.

Tet2 (member 2 of the Tet family) plays an important role in DNA demethylation modification, epigenetic regulation, and hematopoiesis. In our previous study, we found that Tet2 knockout mice progressively developed lymphocytic leukemia and myeloid leukemia with aging. However，the role of Tet2 in bone marrow microenvironment is unclear. Here in this study, we found that more Tet2-/- mesenchymal stem cells (MSCs) from bone marrow were in the G2/M cell cycle stages. The division time of Tet2-/- MSCs was shorter than that of the control cells. The growth rate of Tet2-/- MSCs was accelerated. The cobblestone area-forming cells assay (CAFC) showed that Tet2 knockout MSCs supported the expansion of hematopoietic stem cells (HSCs) and the differentiation of HSCs was skewed towards myeloid cells. Through the dot blotting experiment, we found that the total methylation level was increased in Tet2-/- bone marrow cells (BM). We used the methylation-chip to analyze the methylation level of Tet2-/- bone marrow cells and found that the level of methylation was increased in the transcriptional starting area (TSS), exons (EXONS) and 3' untranslated region (3' UTR). Moreover, we found that the cytokines secreted by Tet2-/- MSCs, such as IL-8 and IL-18, were decreased. While the expressions of GM-CSF and CCL-3, which supported hematopoietic stem cells to differentiate to myeloid cells, were increased in Tet2-/- MSCs. Our results demonstrated that Tet2 regulates MSCs to support hematopoiesis.
Animals ; Bone Marrow Cells ; Cell Differentiation ; DNA-Binding Proteins ; Epigenesis, Genetic ; Hematopoiesis ; Hematopoietic Stem Cells ; Mesenchymal Stem Cells ; Mice ; Proto-Oncogene Proteins