AIM To compare total sugar,total protein,total phenol,total flavonoid,calycosin-7-O-β-D-glucoside,liquiritin,lobetyolin,quercetin,isoferulic acid,hesperidin,glycyrrhizic acid contents and their antioxidant activities,hypoglycemic activities of big honey pill,small honey pill,water pill,concentrated pill,granule,mixture and decoction of Buzhong Yiqi Recipe.METHODS Anthraquinone-sulfuric acid method,Coomassie brilliant blue method,Folin-phenol colorimetry method,sodium nitrite-aluminum nitrate method and HPLC were adopted in the content determination of total sugar,total protein,total phenol,total flavonoid and seven constituents,respectively,after which the scavenging capacities,reducing powers on DPPH·free radical,ABTS+free radical,hydroxyl free radical,and inhibition capacity on α-glucosidase activity were detected.Subsequently,correlation analysis was performed.RESULTS Total sugar,total protein,total phenol and total flavonoid contents demonstrated significant differences among different dosage forms(P＜0.05,P＜0.01).Calycosin-7-O-β-D-glucoside,glycyrrhizin,codonoside and quercetin displayed the highest contents in the decoction,while those of isoferulic acid,hesperidin and glycyrrhizin were observable in the mixture.The water pill exhibited the strongest antioxidant activity,while those of the concentrated pill and mixture were weak;the big honey pill exhibited the strongest hypoglycemic activity,while that of the decoction was the weakest.Total protein,total phenol,total flavonoid and liquiritin contents displayed significant positive correlations between antioxidant activity(P＜0.05,P＜0.01),while hesperidin content displayed significant negative correlation between the latter(P＜0.05);total protein,calycosin-7-O-β-D-glucoside,codonoside and quercetin contents displayed significant negative correlations between hypoglycemic activity(P＜0.05,P＜0.01).CONCLUSION Active constituent contents and their biological activities of Buzhong Yiqi Recipe with different dosage forms exist differences,total sugar,total protein,total flavonoids,calycosin-7-O-β-D-glucoside,licorice glycoside,hesperidin,codonoside and quercetin can be taken as quality control indices for this prescription.

Aim To study the role and mechanism of ML210 in glioma.Methods The cell viability was detected by CCK8 assay.The percentage of dead cells was detected by SYTOXstaining.The role of ferroptosis-signaling pathway in gliomas was detected bygenomics.Cell proliferation was observed by EdU staining and clone formation assay.Cell migration ability was detec-ted by scratch healing assay.The apoptosis was detec-ted by flow cytometry.Cell mitochondrial function was assesses by JC-1 staining.The mechanism of action of ML210 was detected by molecular docking coupled with immunoblotting assay(Western blot).The levels of ROS,MDA were observed by ELISA.Results Compared with the control group,ML210 treatment dose-dependently decreased glioma cell viability,in-hibited cell proliferation,migration,and increased cell apoptosis and mitochondrial dysfunction,which were reversed by ferroptosis antagonists.Gene microarray screening showed that 688 genes of the ferroptosissig-naling pathway were aberrant and 10 signaling path-ways were altered in gliomas.Molecular docking re-sults showed that ML210 binding to GPX4 significantly inhibited the protein expression level of GPX4 and pro-moted the elevation of ROS and MDA levels.Conclu-sions ML210 produces anti-glioma cells via GPX4-mediated ferroptosis pathway.

Objective To explore the mechanism of action of tanthine in the treatment of allergic rhinitis(AR)complicated with asthma in rats by regulating microRNA-27a-3p(miR-27a-3p)targeting thymic stromal lymphopoietin(TSLP).Methods The AR-asthma rat model was established using ovalbumin(OVA)sensitization and nasal drip attack method.Fifty rats were divided into control group(equal volume 0.9％NaCl),model group(AR-asthma model+equal volume 0.9％NaCl),experimental group(AR-asthma model+100 mg·kg-1 xanthortin)and miR-27a-3p inhibitor group(caudal vein injection of 0.5 nmol·μL-1 miR-27a-3p inhibitor 10 μL on the basis of the experimental group),si-TSLP group(0.5 nmol·μL-1 si-TSLP 10 μL intravenously injected on the basis of miR-27a-3p inhibitor group),10 rats in each group.Serum immunoglobulin E(IgE),interleukin-2(IL-2),IL-13 and tumor necrosis factor-α(TNF-α)levels of rats were detected by enzyme-linked immunosorbent assay.The level of superoxide dismutase(SOD)was detected by xanthoxine oxidase method.The level of malonaldehyde(MDA)was detected by thiobarbituric acid method(TBA).Results The levels of IgE in control group,model group,experimental group,miR-27a-3p inhibitor group and si-TSLP group were(18.33±3.53),(89.95±17.62),(55.70±10.08),(78.43±15.30)and(47.87±9.44)ng·mL-1,respectively;IL-2 levels were(8.01±1.36),(19.61±3.94),(14.12±2.51),(17.33±3.18)and(11.89±2.03)pg·mL-1,respectively;IL-13 levels were(6.79±1.33),(34.15±7.02),(24.70±5.13),(35.97±7.24)and(20.53±4.26)pg·mL-1,respectively;TNF-α levels were(94.08±19.07),(312.47±58.61),(209.78±41.49),(296.42±55.99)and(187.45±37.28)pg·mL-1,respectively;SOD levels were(29.14±5.04),(13.25±2.63),(24.19±4.89),(17.28±3.16)and(33.94±5.87)U·mg prot-1,respectively;MDA levels were(2.26±0.51),(4.43±0.72),(3.17±0.58),(3.94±0.69)and(2.62±0.45)nmol·m gprot-1,respectively.The above indicators were compared between control group and model group,model group and experimental group,experimental group and miR-27a-3p inhibitor group,miR-27a-3p inhibitor group and si-TSLP group.The differences were statistically significant(all P＜0.05).Conclusion Xanthine can improve oxidative stress and reduce inflammation of AR complicated with asthma in rats,and its mechanism may be related to the regulation of miR-27 a-3p targeting TSLP.

With the deepening of molecular biology and cell biology research,the regulatory mechanism of autophagy has been gradually revealed,providing new ideas for the treatment of numerous diseases.Autophagy may be closely related to pathological changes such as apoptosis resistance of fibroblast-like synoviocytes,disturbances in bone metabolic homeostasis,and antigen presentation,the regulation of autophagy homeostasis may be an important approach for the treatment of rheumatoid arthritis(RA).In this paper,we provide a review on the pathological mechanism of autophagy in RA,with a view to providing a theoretical basis for later studies.

In-fusion cloning technology,as a revolutionary and efficient molecular biology tool,has been applied in multiple research fields such as basic biology,biotechnology,and biomedicine.In this article,we introduce a teaching reform project suitable for undergraduate students in the course of"Molecular Bi-ology Laboratory",which utilizes in-Fusion cloning technology to construct a prokaryotic expression vector for alkaline phosphatase mutant genes.Through specific teaching cases,we systematically explored the design and implementation of experimental projects,and focused on analyzing the key and difficult points of the teaching content.Our teaching practice has found that the implementation of this educational re-form project has achieved very good results in enhancing students' core biological literacy,bioinformatics skills,research thinking,and innovation abilities.At the same time,the application of this technology can significantly improve the quality of experimental teaching,providing new ideas and practical refer-ences for promoting the reform and innovation of National First-Class Courses.

Aim To explore the role and mechanism of epimedokoreanin B(EKB)in HepaRG cell pyroptosis through endoplasmic reticulum stress and NLRP1-me-diated pyroptosis pathway.Methods The effect of EKB on the viability of HepaRG cells at different con-centrations was determined by MTT assay,and the cell growth status was recorded by Incucyte.Four groups of HepaRG cells were set up.The control group was cul-tured with complete medium for 24 h;the drug admin-istration group was cultured with three concentration gradients of 6.25,12.5 and 25 μmol·L-1 of EKB for 24 h.Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins and pyroptosis-related proteins in the cells of each group.Results HepaRG cells showed cytotoxicity at a concentration of 6.25 μmol·L-1 for 24 h,and the half maximal inhibitory concentration(IC50)was 12.41 μmol·L-1.Incucyte recordings of the cell growth status showed that the cells in the control group were in good growth status,and the vesicular pyropto-sis cells appeared in the different concentrations of EKB and the cells swelled and ruptured after 24 h.Western blot showed that the protein expression levels of endoplasmic reticulum stress-related proteins pERK,eIF-2α,ATF-4,GRP78,and CHOP significantly in-creased in HepaRG cells at 25 μmol·L-1 of EKB compared with the control group.The proteins of the classical pathway of cellular pyroptosis mediated by NLRP1,caspase-1,cleaved caspase-1,GSDMD,GS-DMD-N significantly increased in HepaRG cells.Con-clusion EKB administration induces HepaRG cell py-roptosis,and EKB activates HepaRG cells to undergo endoplasmic reticulum stress and activates the NLRP1/caspase-1/GSDMD-mediated pyroptosis pathway.

In-fusion cloning technology,as a revolutionary and efficient molecular biology tool,has been applied in multiple research fields such as basic biology,biotechnology,and biomedicine.In this article,we introduce a teaching reform project suitable for undergraduate students in the course of"Molecular Bi-ology Laboratory",which utilizes in-Fusion cloning technology to construct a prokaryotic expression vector for alkaline phosphatase mutant genes.Through specific teaching cases,we systematically explored the design and implementation of experimental projects,and focused on analyzing the key and difficult points of the teaching content.Our teaching practice has found that the implementation of this educational re-form project has achieved very good results in enhancing students' core biological literacy,bioinformatics skills,research thinking,and innovation abilities.At the same time,the application of this technology can significantly improve the quality of experimental teaching,providing new ideas and practical refer-ences for promoting the reform and innovation of National First-Class Courses.

Aim To explore the role and mechanism of epimedokoreanin B(EKB)in HepaRG cell pyroptosis through endoplasmic reticulum stress and NLRP1-me-diated pyroptosis pathway.Methods The effect of EKB on the viability of HepaRG cells at different con-centrations was determined by MTT assay,and the cell growth status was recorded by Incucyte.Four groups of HepaRG cells were set up.The control group was cul-tured with complete medium for 24 h;the drug admin-istration group was cultured with three concentration gradients of 6.25,12.5 and 25 μmol·L-1 of EKB for 24 h.Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins and pyroptosis-related proteins in the cells of each group.Results HepaRG cells showed cytotoxicity at a concentration of 6.25 μmol·L-1 for 24 h,and the half maximal inhibitory concentration(IC50)was 12.41 μmol·L-1.Incucyte recordings of the cell growth status showed that the cells in the control group were in good growth status,and the vesicular pyropto-sis cells appeared in the different concentrations of EKB and the cells swelled and ruptured after 24 h.Western blot showed that the protein expression levels of endoplasmic reticulum stress-related proteins pERK,eIF-2α,ATF-4,GRP78,and CHOP significantly in-creased in HepaRG cells at 25 μmol·L-1 of EKB compared with the control group.The proteins of the classical pathway of cellular pyroptosis mediated by NLRP1,caspase-1,cleaved caspase-1,GSDMD,GS-DMD-N significantly increased in HepaRG cells.Con-clusion EKB administration induces HepaRG cell py-roptosis,and EKB activates HepaRG cells to undergo endoplasmic reticulum stress and activates the NLRP1/caspase-1/GSDMD-mediated pyroptosis pathway.

AIM To study the chemical constituents from the bark of Toona sinensis(A.Juss.)Roem and their anti-inflammatory activity.METHODS Silica gel,RP-18 reverse phase silica gel and HPLC were used for isolation and purification,then the structures of obtained compounds were identified by physicochemical properties and spectral data.Their anti-inflammatory activity were evaluated by RAW264.7 model.RESULTS Sixteen compounds were isolated and identified as(9Z)-18-hydroxyo-ctadec-9-en-4,6-diyn-3-one(1),toonapolyyne C(2),(9S,10E,16R)-octadec-10-ene-12,14-diyne-1,9,16-triol(3),(9S,10E,16R)-9,16-dihydroxyoctadec-10-ene-12,14-diy-n-1-yl acetate(4),toonapolyyne A(5),toonasindiyne B(6),scopoletin(7),sco-parone(8),3-O-acetyl(-)-epicatechin(9),p-ethoxyacetanilide(10),kulonic acid(11),β-sitosterol-3-O-β-D-glucopyranoside(12),vanillic acid(13),cleomiscosin A(14),(-)-isolariciresinol(15),resveratrol(16).The IC50 values of compounds 3-4,6,11-13 for NO were 6.90,10.49,20.03,9.49,18.34,24.36 μmol/L,respectively.CONCLUSION Compound 1 is a new polyacetylene,8-16 are isolated from T.sinensis for the first time.Compounds 3-4,6,11-13 have good anti-inflammatory activity.

Objective:It is of great significance to analyze the expression characteristics of immune-related genes in pancreatic cancer and their relationship with prognosis,construct and verify a reliable prognostic model,and explore prognostic methods of pancreatic cancer from the perspective of immune microenvironment.Methods:GSEA enrich-ment analysis of differentially expressed genes in pancreatic cancer was performed to identify key immune-related pathways and genes.The genes involved in the immune pathway were screened through the STRING database and combined with univariate Cox regression and LASSO regression analysis.Three key genes,RIPK2,IRAK2 and CXCL11,were finally identified to construct the prognostic model.The accuracy of the model was evaluated using ROC curves and calibration curves,and verified in an independent verification set(GSE57495).At the same time,the expression pat-terns of key genes in the immune microenvironment were analyzed by single-cell RNA sequencing,and the expression levels of these genes were verified in pancreatic cancer cell lines by RT-qPCR.Results:The expressions of RIPK2,IRAK2 and CXCL11 in pancreatic cancer cell lines were higher than those in normal pancreatic cancer cells(P<0.05).The model based on these three genes divided the patients into a high-risk group(n=87)and a low-risk group(n=89),and the difference in survival time between the high-risk group and the low-risk group was statistically significant(P<0.001).Risk score was correlated with G stage,N stage and tumor residue(P<0.01).Single-cell analysis showed that the ex-pression of these genes was highest in tumor-associated macrophages(mean>0.5)and correlated with regulatory T cells and macrophage infiltration(P<0.05).Multivariate analysis showed that risk score was correlated with overall sur-vival after adjusting for clinical factors(P=0.0014).Conclusion:Based on three key immune-related genes(RIPK2,IRAK2 and CXCL11),we successfully constructed a model to accurately predict the prognosis of pancreatic cancer pa-tients,revealing the important role of these genes in the tumor immune microenvironment,and providing new insights and theoretical basis for pancreatic cancer prognosis assessment and immunotherapy research.