ObjectiveTo investigate the mechanism of action of modified Shaofu Zhuyutang in antagonizing cellular pyroptosis and fibrosis in ectopic endometrial tissues of endometriosis through the Toll-like receptor 4/nuclear factor-κB/NOD-like receptor protein 3 （TRL4/NF-κB/NLPR3） signaling pathway. MethodsSeventy-two SPF-grade female SD rats were randomly divided into a sham-operated group （n = 12） and a modeling group （n = 60）. The rats in the sham-operated group underwent a caesarean section， while the rats in the modeling group were used to establish an endometriosis model through the auto-transplantation method. After successful modeling， the animals were randomly divided into the model group， progesterone group （0.25 mg·kg^-1）， and modified Shaofu Zhuyutang low-， medium-， and high-dose groups （7.5， 15， 30 g·kg^-1）， with 12 animals in each group. After 4 weeks of drug administration， voluntary activity and heat pain latency were observed. The rats were sacrificed for tissue collection， and Masson staining were used to observe histopathological changes in the endometrial tissues. Enzyme-linked immunosorbent assay （ELISA） was used to measure serum levels of interleukin-18 （IL-18）， interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and transforming growth factor-β （TGF-β）. Immunohistochemistry （IHC） was used to detect the protein expression area of tumor necrosis factor-related factor 6 （TRAF6） and NLPR3 in the endometrial tissues. Immunofluorescence （IF） was used to detect the relative fluorescence intensity of Caspase-1 and gasdermin D （GSDMD） in the endometrial tissues. Western blot was employed to measure the relative expression of TRL4， myeloid differentiation factor 88 （MyD88）， TRAF6， NF-κB p65， phosphorylated NF-κB p65 （p-NF-κB p65）， and NLPR3 proteins in endometrial tissues. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 in the endometrial tissues. ResultsCompared with the sham-operated group， rats in the model group showed reduced voluntary activity and shorter heat pain latency. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were elevated. The relative expression areas of TRAF6 and NLPR3 proteins were increased， and the relative fluorescence intensity of Caspase-1 and GSDMD was enhanced. The relative expression of TRL4， MyD88， TRAF6， NF-κB p65， p-NF-κB p65， and NLPR3 proteins， along with the expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA， were significantly increased （P<0.01）. Compared with the model group， rats in the progesterone group and the modified Shaofu Zhuyutang medium- and high-dose groups exhibited improved voluntary activity， longer heat pain latency， the fibrosis of endometrial tissue is alleviated. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were decreased. The relative expression areas of TRAF6 and NLPR3 proteins decreased， and the relative fluorescence intensity of Caspase-1 and GSDMD weakened. The relative expression of TRL4， MyD88， TRAF6， p-NF-κB p65， NLPR3 proteins， and TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA expression were reduced （P<0.05， P<0.01）. ConclusionModified Shaofu Zhuyutang may play a therapeutic role in endometriosis by interfering with key proteins in the TRL4/NF-κB/NLPR3 signaling pathway， reducing NLRP3 inflammasome-induced cellular pyroptosis， antagonizing the fibrosis process in ectopic endometrial tissues， improving the inflammatory microenvironment in the pelvic cavity， and alleviating pain.

Objective To establish a method for acquisition, perfusion, preservation and transportation of the genetically modified pig kidneys. Methods An eight genetically modified pig was utilized as experimental subject. Prior to kidneys procurement, the health status of the pig was assessed through hematology examination, and the vascular structure of the kidneys was examined using imaging techniques. Following kidneys acquisition, the pig kidneys were perfused and subsequently packaged into the cryogenic storage container labeled "For Organ Transportation Only" for interprovincial transport after communicating the transportation process with transportation department. To evaluate pathological damage to the pig kidneys, a serious of methods were employed such as hematoxylin-eosin (HE) staining, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorescence staining and enzyme-linked immune absorbent assay (ELISA). Results The preoperative examination of the eight genetically modified pig showed that the serum creatinine was 73.2 μmol/L, blood urea nitrogen was 2.8 mmol/L and hemoglobin was 116 g/L, all within the normal range, indicating normal renal function. CT angiography revealed no lesions in the pig kidneys, and no dilation, stenosis or premature branching of the blood vessels. The total time of obtaining the left and right kidneys from the eight genetically modified pig was (125 ± 10) min, with a blood loss of (20 ± 2) mL. The warm ischemia times were 3 min and 7 min, respectively. The perfusion and trimming times of the left and right kidneys were 36 min and 41 min, respectively. After perfusion, both kidneys were white and moist. The cold preservation and transportation time was 8 h. HE staining showed that some glomeruli were shrunk, and the lumens of the surrounding renal tubules were slightly depressed and swollen with partial inner membrane shedding and microvacuoles formed when the kidneys were preserved for 8 h. The level of cysteinyl aspartate-specific proteinase-3 messenger RNA in the kidneys tissue gradually increased with the extension of cold preservation time after 2 h (P<0.05). TUNEL fluorescence staining showed that only a small number of cells underwent apoptosis after 8 h of cold preservation, which was not significantly different from that at 0 h (P>0.05). ELISA results showed that the contents of lactate dehydrogenase (LDH) and creatinine in the preservation solution remained relatively stable, but the content of kidney injury molecule 1 (KIM-1) gradually increased with the extension of preservation time, suggesting that the pig kidneys had mild injury. Conclusions By establishing methods for acquisition, perfusion, preservation and transportation of the kidneys from genetically modified donor pig, it is possible to effectively and reliably use genetically modified pig kidneys for xenotransplantation.

[Objective] To establish a cryopreservation protocol for small-volume (≤1 mL) red blood cells (RBCs) and to evaluate the reactivity and stability of blood group antigens after cryopreservation, so as to explore its potential application in immunohematology reference laboratories. [Methods] Small-volume RBCs were cryopreserved for 120 days, followed by thawing and deglycerolization to restore the RBC components. The quality of the RBCs was assessed. Serum antibodies were serially diluted and reacted with RBCs before and after cryopreservation, and agglutination scores were recorded to quantitatively evaluate the reactivity and stability of blood group antigens such as Rh, Duffy, Lewis, Kidd, M, and H. Flow cytometry was used to analyze the percentage and mean fluorescence intensity of ABO antigen expression on RBCs before and after cryopreservation to assess the usability of cryopreserved RBCs in flow immunophenotyping and blood group subtype studies. [Results] The hemolysis rate of thawed and deglycerolized RBCs was (0.27±0.10)%, with a supernatant free hemoglobin level of (0.52±0.14) g/L, and the RBC recovery rate was (69.12±7.91)%. The direct antiglobulin test (DAT) was negative for all thawed RBCs. There was no difference in the reactivity of blood group antigens before and after cryopreservation, and no difference in the percentage and mean fluorescence intensity of A and B antigen expression on RBCs before and after cryopreservation. [Conclusion] The small-volume RBC cryopreservation protocol can be applied to immunohematology analysis in reference laboratories and is expected to be widely used in blood group identification, antibody screening, identification, and blood group-related research.

Objective To establish and optimize a method for simultaneous determination for the contents of sodium,potassium,calci-um and magnesium ions in serum using microwave digestion combined with ion chromatography,and evaluate its performance.Methods Serum samples were pretreated by microwave digestion,and four cations were quantitatively determined using ion chromatography.The linearity,precision,recovery and accuracy of the established method were verified with reference to the EP6-A and EP15-A3 docu-ments issued by the Clinical and Laboratory Standards Institute(CLSI).Results The imprecisions of determinations for sodium,po-tassium,calcium and magnesium ions expressed as coefficient of variation(CV)were 0.10％to 0.36％,0.15％to 0.48％,0.22％to 0.87％and 0.21％to 0.73％,respectively.The recoveries with adding standard of the four cations were between 97.3％and 103.0％.Referred to RELA2020 and RELA2021,the deviations of the analysis results were-0.57％to-0.14％,-0.84％to-0.16％,-1.82％to-0.37％,and-0.60％to 0.34％for sodium,potassium,calcium,and magnesium,respectively,and all the comparisons were pas-sed.The limits of detection/quantification were 0.017 5/0.058 3 mmol/L,0.000 5/0.001 7 mmol/L,0.009 9/0.032 9 mmol/L and 0.001 1/0.003 5 mmol/L for the four cations,indicating that after pretreatment,no significantly interfere from other impurities for the results of determination was found.Conclusion A method for simultaneous determination of sodium,potassium,calcium and magnesi-um ions in serum was successfully established and optimized using microwave digestion combined with ion chromatography.The method showed good precision and accuracy,simple operation,short time consumption and low cost,thus it could be used for quality improve-ment of clinical electrolyte testing in serum.

Autophagy is a cellular self-degradation process essential for maintaining metabolic functions in cells and organisms.Dysfunc-tional autophagy has been linked to various diseases,including cancer.The m6A modification,a major RNA modification in eukaryotes,plays a crucial role in regulating autophagy in tumor cells by regulating the expression of autophagy-associated genes(ATGs)or interfering with autophagy-related signaling pathways.Aberrant m6A modification can lead to dysregulated autophagy and impact tumor progression.However,the specific role of m6A in regulating tumor autophagy remains to be explored.Therefore,in this review,we discuss the role of m6A modification in tumor cell autophagy and examine its relationship with tumor progression and drug resistance,aiming to provide a the-oretical foundation for developing new therapeutic strategies.

Objective To explore the impact of HR-HPV infection on pregnancy outcomes and vaginal microbiota.Methods 128 pregnant women who underwent prenatal examinations at Mingshan Branch of the First Affiliated Hospital of Chengdu Medical College were selected as the research subjects.Both vaginal microbiota and HR-HPV testing were conducted,and single factor analysis and multiple linear regression analysis were used to identify the relevant factors of HR-HPV infection in pregnant women.The impact of HR-HPV infection on preg-nancy outcomes and vaginal microbiota was studied.Results Among the 49 cases in the infection group,30 cases contracted with type 16 HPV,accounting for 61.22%,13 type 18 HPV,accounting for 26.53%,and 1 type 31,33,51,52,53,and 58,respectively.Univariate analysis showed that HR-HPV infections were significantly correlated with age of first sexual activity,condom use,number of sexual partners,reproductive tract inflammation,and smoking history(P<0.05).Multivariate analysis showed that age of first sexual activity≤20 years,concurrent reproductive tract inflammation,multiple sexual partners,and smoking history were independent risk factors for HR-HPV infection,while condom use was an independent protective factor for HR-HPV infection.There were significant differences in the number of lactobacilli,candida infections,and vaginal cleanliness between the two groups(P<0.05).The incidence of spontaneous abortion and fetal growth restriction in the HR-HPV-infected group was significantly higher than that in the HR-HPV-uninfected group(P<0.05).Conclusions Pregnant women with HR-HPV infections are closely related to factors such as premature sexual conduct,number of sexual partners,reproductive tract inflammation,and smoking.Infections with HR-HPV during pregnancy can cause changes in the vaginal microenvironment and lead to adverse pregnancy outcomes.

Objective To study the quality comparability of human coagulation factor Ⅷ(FⅧ)produced before and after the change of factory site.Methods A comparative study was carried out on quality quantitative indexes,related im-purities and stability data of FⅧ produced before and after the change of factory site.Results The FⅧ quantitative quality before and after the change of factory site all met the quality standard,and the related impurities including aluminum resi-due,tributyl phosphate residue,polysorbate 80 residue and PEG residue all met the quality standard.Other impurities in-cluding human fibrinogen,fibronectin,plasminogen,IgA,IgM and IgG were extremely low in content and equivalent in quality.The content of VWF(von Willebrand factor)had no obvious change before and after the change of factory site,but was significantly higher than that of other domestic manufacturers'commercial products.The results of accelerated stability and long-term stability tests showed that the titer of FⅧ fluctuated within the methodological error range,and the results all met the quality standard.Conclusion The change of factory site of FⅧ has no effect on the quality.

Objective:To develop a candidate reference method for cortisol in human serum by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS).Methods:An isotope-labeled internal standard was added to samples, followed by alkalizing with sodium carbonate solution and liquid-liquid extraction with n-hexane/ethyl acetate. Isoelution with a methanol aqueous solution was employed for liquid chromatographic separation, while ESI in the positive ion and multiple reaction monitoring mode were used for mass spectrometry. The volume of sample, standard, and internal standard solutions were all controlled by weighing, and the results were calculated by bracketing method. The accuracy, precision, specificity, linearity, LOD and LOQ of this method were evaluated referring to the CLSI C62-A and C50-A guidelines and the domestic expert consensus documents.Results:The method demonstrated excellent accuracy and precision with the bias of ERM-DA192, ERM-DA193 and RELA 2021-2023 all being less than 2%. The recovery of added cortisol ranged from 98.2% to 101.1%. Both intra- and inter-assay imprecisions was <2%. The method was free from interference by structural analogue and showed a good linearity in the range of 25-1 600 nmol/L. The LOD and LOQ were 0.5 nmol/L and 1.0 nmol/L, respectively. A cortisol assay kit (chemiluminescence immunoassay) traced to this candidate reference method was used to determine 46 clinical serum samples concurrently, and the two methods exhibited good correlation.Conclusions:A candidate reference method for the determination of cortisol in serum was established, demonstrating high sensitivity, good repeatability and accuracy. This method can serve as a reference for the measurement traceability and accuracy evaluation of routine methods.

OBJECTIVE To investigate the effect and mechanism of dihydroartemisinin(DHA)on lipo-polysaccharide(LPS)-induced acute lung injury(ALI)in mice using whole-genome sequencing.METHODS An ALI mouse model was established via intraperitoneal injection of 10 mg·kg-1 lipopolysaccharide.The mice were divided into normal control group(n=10),model group(n=10)and model+DHA group(n=10).The mice in the model+DHA group were injected intraperitoneally with 20 mg·kg-1 DHA,while those in the normal control group and LPS group were injected intraperitoneally with solvent of DHA,saline containing 1%Tween 80 and 10%Macrogol 400.The mice were executed 24 h after drug administration.The wet and dry weight ratio(W/D)of lung tissue was calculated.Hematoxylin-eosin(HE)staining was used to observe histopathological damage in the lung.Classified counts of inflamma-tory cells in alveolar lavage fluid were performed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-1β(IL-1β),IL-6,and tumor necrosis factor-α(TNF-α)in alveolar lavage fluid.Real-time quantitative PCR(RT-qPCR)was used to detect mRNA levels of placenta-specific 8(Plac8),Toll-like receptor 7(TLR7),IL-1β,IL-6 and TNF-αin lung tissue.The whole gene transcriptome was sequenced by RNA transcriptome sequencing(RNA-seq)using the Illumina HiSeq high-throughput sequencing platform before the function and signal pathway of differentially expressed gene mRNA between the groups were enriched and analyzed using GO and KEGG enrichment analysis methods.RESULTS Compared with the model group,the lung W/D values of mice,the pathological damage,inflammatory cells in alveolar lavage fluid,expression levels of IL-1β,IL-6 and TNF-α in alveolar lavage fluid(P<0.01,P<0.01,P<0.01),and the mRNA expression levels of IL-1β,IL-6 and TNF-α were significantly reduced in lung tissues in the model+DHA group(P<0.01,P<0.05,P<0.05).Whole gene transcriptome sequencing revealed that immune-related Plac8 and TLR7 genes were significantly upregu-lated(P<0.01)in mouse lung tissue of the model group but significantly downregulated(P<0.05)in mouse lung tissue of the model+DHA group.The results of RT-qPCR of Plac8 and TLR7 verified the results of whole gene transcriptome sequencing.GO and KEGG analysis showed that Plac8 and TLR7 were mainly related to the regulation of cytokine production,T/B cell activation and signal transduction,chemo-kine signal transduction and NF-κB signal transduction.CONCLUSION DHA might reduce LPS-induced lung damage and ameliorate the inflammatory condition in lungs of ALI mice.The mechanism of action may be that DHA negatively regulates the signaling pathways involved in TLR7 and Plac8 by decreasing the expressions of TLR7 and Plac8 mRNA before regulating a series of immune responses such as secretion of inflammation-related cytokines and activation of immune cells,thereby reducing inflam-matory damage in lungs.

OBJECTIVE To explain the material basis and biological mechanism of Astragali Radix’s “invigorating Qi” effect to regulate energy metabolism. METHODS The TCMSP database and literature search collected potential active components of Astragali Radix, the SEA database performed target prediction based on structural similarity, and the GeneCards, OMIM, and TTD databases obtained energy metabolism targets. Cytoscape software was used to construct protein-protein interaction network maps of Astragali Radix regulated energy metabolism targets, and GO and KEGG enrichment analyses were performed. Molecular docking and hierarchical cluster analysis were performed to evaluate the target-component affinity between the whole constituents of Astragali Radix and key targets, and the effects of representative compounds of Astragali Radix on the energy metabolism of H9C2 cardiomyocytes and GES-1 gastric epithelial cells were detected, and the binding mode analysis was conducted. RESULTS Network pharmacology results showed that there were 126 potential targets of Astragali Radix regulating energy metabolism. GO and KEGG enrichment analysis showed that Astragali Radix regulating energy metabolism might be related to gene expression of oxidation-reduction process, protein and enzyme synthesis. Among them, SIRT1 and PPARγ were key targets involved in the regulation of energy metabolism. Molecular docking and hierarchical clustering showed that Astragali Radix components had superior targeting to SIRT1 and PPARγ, and three representative compounds were selected for in vitro experimental verification in combination with molecular docking scores. Quercetin and kaempferol could promote energy metabolism in H9C2 cardiomyocytes and GES-1 gastric epithelial cells. The binding mode analysis showed that quercetin and kaempferol had preferable binding ability to SIRT1 and PPARγ. CONCLUSION In this study, the material basis and biological mechanism of Astragali Radix regulating energy metabolism are preliminarily explained by traditional Chinese medicine chemo-bio informatics methods, which provide a scientific basis for the connotation of Astragali Radix exerting the effect of stagnation and arthralgia through “invigorating Qi” in traditional Chinese medicine.