ObjectiveTo explore the molecular mechanism by which gypenoside L （Gyp-L） promotes apoptosis and inhibits ovarian cancer （OC） through the FK506-binding protein （FKBP） prolyl isomerase 8 （FKBP8）/B-cell lymphoma-2 （Bcl-2） axis， with the piR-hsa-2804461 pathway as a breakthrough point. MethodsThe effects of different concentrations of Gyp-L and cis-platinum on the proliferation of OVCAR3 cells were determined by the cell count kit-8 method to identify the appropriate intervention concentration for subsequent experiments. OVCAR3 cells were allocated into blank， low-dose Gyp-L （Gyp-L-L， 50 µmol·L^-1）， high-dose Gyp-L （Gyp-L-H， 100 µmol·L^-1）， and cis-platinum （15 µmol·L^-1） groups. The migration， colony formation， and apoptosis of OVCAR3 cells were detected by the cell scratch assay， colony formation assay， and flow cytometry， respectively. The mRNA levels of piR-hsa-2804461 and FKBP8/Bcl-2 axis-related genes in OVCAR3 cells were determined by Real-time PCR， and the expression levels of FKBP8/Bcl-2 axis-related proteins were determined by simple Western blot. Further， an OVCAR3 cell model with piR-hsa-2804461 knocked out was constructed. The cells were allocated into blank， NC-inhibitor， inhibitor， NC-inhibitor+Gyp-L， and inhibitor+Gyp-L groups. The colony formation of OVCAR3 cells was detected by the colony formation assay. The mRNA levels of piR-hsa-2804461 and FKBP8/Bcl-2 axis-related genes and the expression levels of FKBP8/Bcl-2 axis-related proteins were determined by Real-time PCR and simple Western blotting， respectively. ResultsGyp-L inhibited the migration and proliferation （P<0.01）， promoted the apoptosis （P<0.05）， up-regulated the mRNA level of piR-hsa-2804461 （P<0.05）， and down-regulated the mRNA and protein levels of FKBP8 and Bcl-2 （P<0.05） in OVCAR3 cells. Furthermore， Gyp-L increased the mRNA and protein levels of Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific proteinase （Caspase）-3， and Caspase-9， which are related to the FKBP8/Bcl-2 axis （P<0.05）. ConclusionGyp-L may promote apoptosis by regulating the piR-hsa-2804461/FKBP8/Bcl-2 axis， thus affecting the occurrence of ovarian cancer.

ObjectiveTo explore the molecular mechanism of gypenoside L （Gyp-L） in the treatment of ovarian cancer （OC） by taking the glycolytic pathway of OC as the key point. MethodsThe proliferation activity of OVCAR3 cells was measured by the cell counting kit-8 （CCK-8） assay to determine the appropriate intervention concentration for subsequent experiments. The cell clone formation assay and the scratch healing assay were employed to assess the proliferation and migration capabilities of OVCAR3 cells. OVCAR3 cells were divided into a blank group， a Gyp-L-L group （low concentration of Gyp-L， 50 µmol·L^-1）， a Gyp-L-H group （high concentration of Gyp-L， 100 µmol·L^-1）， and a cisplatin （15 µmol·L^-1） group. The glucose consumption in OC cells was determined using a kit， and the expression levels of related mRNAs in OVCAR3 cells were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expressions of related proteins were detected by Wes automatic Western blot quantitative analysis. ResultsValidated by the cell scratch assay， the scratch healing rate of OVCAR3 cells was decreased after Gyp-L intervention， reflecting that Gyp-L could significantly inhibit the migration of OVCAR3 cells （P<0.05）. According to the clone formation assay， the cell colony formation ability of the Gyp-L-L group， Gyp-L-H group， and cisplatin group was significantly lower than that of the control group （P<0.05）. In OVCAR3 cells， compared with those in the control group， the levels of glucose consumption and lactate generation in the Gyp-L-L group and Gyp-L-H group were significantly reduced （P<0.05）， indicating that Gyp-L could effectively inhibit the glycolysis level of OC cells. Meanwhile， Gyp-L could suppress the expression levels of glucokinase （GCK）， phosphofructokinase liver （PFKL）， signal transducer and activator of transcription 3 （STAT3）， lactate dehydrogenase D （LDHD）， phosphoglycerate mutase 1 （PGAM1）， mRNAs， and proteins related to the glycolytic pathway in OC cells. ConclusionGyp-L may inhibit the occurrence and development of OC by influencing the GCK-mediated glycolytic pathway.

ObjectiveTo explore the molecular mechanism by which gypenoside L （Gyp-L） promotes apoptosis and inhibits ovarian cancer （OC） through the FK506-binding protein （FKBP） prolyl isomerase 8 （FKBP8）/B-cell lymphoma-2 （Bcl-2） axis， with the piR-hsa-2804461 pathway as a breakthrough point. MethodsThe effects of different concentrations of Gyp-L and cis-platinum on the proliferation of OVCAR3 cells were determined by the cell count kit-8 method to identify the appropriate intervention concentration for subsequent experiments. OVCAR3 cells were allocated into blank， low-dose Gyp-L （Gyp-L-L， 50 µmol·L^-1）， high-dose Gyp-L （Gyp-L-H， 100 µmol·L^-1）， and cis-platinum （15 µmol·L^-1） groups. The migration， colony formation， and apoptosis of OVCAR3 cells were detected by the cell scratch assay， colony formation assay， and flow cytometry， respectively. The mRNA levels of piR-hsa-2804461 and FKBP8/Bcl-2 axis-related genes in OVCAR3 cells were determined by Real-time PCR， and the expression levels of FKBP8/Bcl-2 axis-related proteins were determined by simple Western blot. Further， an OVCAR3 cell model with piR-hsa-2804461 knocked out was constructed. The cells were allocated into blank， NC-inhibitor， inhibitor， NC-inhibitor+Gyp-L， and inhibitor+Gyp-L groups. The colony formation of OVCAR3 cells was detected by the colony formation assay. The mRNA levels of piR-hsa-2804461 and FKBP8/Bcl-2 axis-related genes and the expression levels of FKBP8/Bcl-2 axis-related proteins were determined by Real-time PCR and simple Western blotting， respectively. ResultsGyp-L inhibited the migration and proliferation （P<0.01）， promoted the apoptosis （P<0.05）， up-regulated the mRNA level of piR-hsa-2804461 （P<0.05）， and down-regulated the mRNA and protein levels of FKBP8 and Bcl-2 （P<0.05） in OVCAR3 cells. Furthermore， Gyp-L increased the mRNA and protein levels of Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific proteinase （Caspase）-3， and Caspase-9， which are related to the FKBP8/Bcl-2 axis （P<0.05）. ConclusionGyp-L may promote apoptosis by regulating the piR-hsa-2804461/FKBP8/Bcl-2 axis， thus affecting the occurrence of ovarian cancer.

ObjectiveTo explore the molecular mechanism of gypenoside L （Gyp-L） in the treatment of ovarian cancer （OC） by taking the glycolytic pathway of OC as the key point. MethodsThe proliferation activity of OVCAR3 cells was measured by the cell counting kit-8 （CCK-8） assay to determine the appropriate intervention concentration for subsequent experiments. The cell clone formation assay and the scratch healing assay were employed to assess the proliferation and migration capabilities of OVCAR3 cells. OVCAR3 cells were divided into a blank group， a Gyp-L-L group （low concentration of Gyp-L， 50 µmol·L^-1）， a Gyp-L-H group （high concentration of Gyp-L， 100 µmol·L^-1）， and a cisplatin （15 µmol·L^-1） group. The glucose consumption in OC cells was determined using a kit， and the expression levels of related mRNAs in OVCAR3 cells were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expressions of related proteins were detected by Wes automatic Western blot quantitative analysis. ResultsValidated by the cell scratch assay， the scratch healing rate of OVCAR3 cells was decreased after Gyp-L intervention， reflecting that Gyp-L could significantly inhibit the migration of OVCAR3 cells （P<0.05）. According to the clone formation assay， the cell colony formation ability of the Gyp-L-L group， Gyp-L-H group， and cisplatin group was significantly lower than that of the control group （P<0.05）. In OVCAR3 cells， compared with those in the control group， the levels of glucose consumption and lactate generation in the Gyp-L-L group and Gyp-L-H group were significantly reduced （P<0.05）， indicating that Gyp-L could effectively inhibit the glycolysis level of OC cells. Meanwhile， Gyp-L could suppress the expression levels of glucokinase （GCK）， phosphofructokinase liver （PFKL）， signal transducer and activator of transcription 3 （STAT3）， lactate dehydrogenase D （LDHD）， phosphoglycerate mutase 1 （PGAM1）， mRNAs， and proteins related to the glycolytic pathway in OC cells. ConclusionGyp-L may inhibit the occurrence and development of OC by influencing the GCK-mediated glycolytic pathway.

Objective To evaluate the immune responses of mice immunized with different doses of therapeutic tuberculosis DNA vaccine B21, in order to provide a reference for dose selection of quality control methods for the vaccine efficacy.Methods SPF female C57BL/6 mice were immunized by intramuscular injection of 25(LD), 50(MD), 100(HD) μg/100 μL B21 DNA vaccine through the medial thigh, combined with electroporation stimulation for a total of three times at an interval of two weeks. Three weeks after the last immunization, the spleen lymphocytes and peripheral blood serum were isolated. The cellular immunity and immune memory effects were detected by enzyme-linked immunosorbent spot(ELISpot) and flow cytometry(FCM), and the humoral immunity was detected by ELISA.Results Three weeks after the last immunization, the numbers of Ag85b-specific and Rv1738-specific IFNγ spot-forming cells(SFCs) in HD group were significantly higher than those in MD and LD groups(F = 11. 22 and 15. 89, respectively,each P < 0. 001); Rv2029c-specific IFNγ SFCs were significantly higher than that of MD group(F = 5. 761, P < 0. 05), but showed no significant difference with LD group(F = 5. 761,P > 0. 05). IL-2 SFCs of the three antigens in HD group were significantly higher than those in MD and LD groups(F =8. 044-33. 07, each P < 0. 05). The proportion of CD4~+ T cells secreting IFNγ in HD group was significantly higher than those in MD and LD groups(F = 5. 976, P < 0. 01), while there was no significant difference in the proportion of CD4~+ T cells secreting IL-2 among the three groups(F = 2. 836, P > 0. 05). The proportion of IFNγ~+ memory T cells(CD44~+ CD62L~+/~-) in HD group was significantly higher than those in MD and LD groups(F = 8. 793, P < 0. 05), and the proportion of activated memory B cells(CD19~+ CD27~+) in HD group was significantly higher than those in MD and LD groups(F = 7. 958, P < 0. 01).There was no significant difference in B21-specific IgG antibody level among the three groups(F = 5. 765, P > 0. 05).Conclusion B21DNA vaccine effectively induced cellular immunity, humoral immunity and immune memory responses in a dose-dependent manner. The dose of 100 μg/100 μL induced the best immunization effect, and ELISpot could detect IFNγ/IL-2 SFCs with good stability and reproducibility, which can be used as the recommended immune dose,evaluation indicator and detection method for quality control of B21 vaccine efficacy.

BACKGROUND: Epoxyeicosatrienoic acids (EETs), which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase, are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). Many studies have revealed that sEH gene deletion exerts protective effects against diabetes. Vascular calcification is a common complication of diabetes, but the potential effects of sEH on diabetic vascular calcification are still unknown. METHODS: The level of aortic calcification in wild-type and Ephx2-/- C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining, immunohistochemistry staining, and immunofluorescence staining. Mouse vascular smooth muscle cell lines (MOVAS cells) treated with β-glycerol phosphate (0.01 mol/L) plus advanced glycation end products (50 mg/L) were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells, which was detected by Western blotting, alizarin red staining, and Von Kossa staining. RESULTS: sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice. EETs (especially 11,12-EET and 14,15-EET) efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2 (NID2) expression. Interestingly, suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products. NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification. Moreover, NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2 (IGF2) and phospho-ERK1/2 expression in MOVAS cells. Overall, sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and, then, inactivating the downstream IGF2-ERK1/2 signaling pathway. CONCLUSIONS sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels, which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.
Animals ; Mice ; Vascular Calcification/metabolism* ; Mice, Inbred C57BL ; Epoxide Hydrolases/metabolism* ; Diabetes Mellitus, Experimental/genetics* ; Male ; Gene Deletion ; MAP Kinase Signaling System/genetics* ; Cell Line ; Immunohistochemistry ; Muscle, Smooth, Vascular/metabolism* ; Signal Transduction/genetics* ; Mice, Knockout

Intestinal fibrosis is a significant clinical challenge in inflammatory bowel diseases, but no effective anti-fibrotic therapy is currently available. Glucagon receptor (GCGR) and glucagon-like peptide 1 receptor (GLP1R) are both peptide hormone receptors involved in energy metabolism of epithelial cells. However, their role in intestinal fibrosis and the underlying mechanisms remain largely unexplored. Herein GCGR and GLP1R were found to be reduced in the stenotic ileum of patients with Crohn's disease as well as in the fibrotic colon of mice with chronic colitis. The downregulation of GCGR and GLP1R led to the accumulation of the metabolic byproduct lactate, resulting in histone H3K9 lactylation and exacerbated intestinal fibrosis through epithelial-to-mesenchymal transition (EMT). Dual activating GCGR and GLP1R by peptide 1907B reduced the H3K9 lactylation in epithelial cells and ameliorated intestinal fibrosis in vivo. We uncovered the role of GCGR/GLP1R in regulating EMT involved in intestinal fibrosis via histone lactylation. Simultaneously activating GCGR/GLP1R with the novel dual agonist peptide 1907B holds promise as a treatment strategy for alleviating intestinal fibrosis.

A chemical investigation of secondary metabolites (SMs) from Aspergillus nidulans resulted in the identification of five novel dioxopiperazine (DKP)-diphenyl ether hybrids, designated as diphenylemestrins A-E (1-5). These compounds 1-5 represent the first known dimers combining DKP and diphenyl ether structures, with compound 4 featuring an uncommon dibenzofuran as the diphenyl ether component. The structural elucidation and determination of absolute stereochemistry were accomplished through spectroscopic analysis and electronic circular dichroism (ECD) calculations. Notably, diphenylemestrin C (3) exhibited moderate cytostatic activity against NB4 cells, with a half maximal inhibitory concentration (IC₅₀) value of 21.99 μmol·L^-1, and induced apoptosis at higher concentrations.
Aspergillus nidulans/metabolism* ; Diketopiperazines/pharmacology* ; Molecular Structure ; Phenyl Ethers/pharmacology* ; Humans ; Apoptosis/drug effects* ; Cell Line, Tumor

Objective:A genome-wide molecular characterization of FJ21351116, a strain of G3P[8]-E2 2021 collected in Fujian, China, was performed.Methods:Whole genome sequencing of FJ21351116 was performed using a high-sensitivity group A rotavirus whole genome sequencing method. Genomic characteriza-tion of the virus was assessed by nucleic acid sequence analysis using MEGA 11.0, Geneious 9.0.2 and DNASTAR software. Neutralization epitopes of VP7 and VP4 (VP8*) were analyzed using BioEdit v. 7.0.9.0 and PyMOL v. 2.5.2.Results:In this study, FJ21351116 was shown to be a G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genotype, and the result of phylogenetic tree showed that the VP7, VP4, VP3, and NSP2-NSP5 genes of the FJ21351116 strain were related to the equine-like DS-1-like G3P[8] genes that have been detected in Japan in recent years. VP6, VP1, VP2, and NSP1 genes are closely related to G2P[4] in most countries, especially in Singapore, suggesting that this strain was formed by genetic reassortment during the evolution of equine-like G3P[8] and G2P[4]. Evolutionary relationships between the VP7/VP4 genes of FJ21351116 and Rotarix and RotaTeq vaccines suggest that the multiple mutations in both VP7 and VP4 (VP8*) neutralizing antigenic epitopes and vaccine amino acid sites. It is hypothesized that the Rotarix and RotaTeq vaccines may be less effective against equine DS-1-like G3P[8] RVA, and the sequence differences with Rotarix are higher than those with RotaTeq.Conclusions:In this study, we found a rare case of DS-1-like G3P [8] RVA strain in China. Currently, horse-like DS-1-like G3P [8] RVA is relatively rare in China and may be poorly protected by vaccine strains, emphasizing the importance of continuous monitoring of RVA strains and the development of efficient and full-coverage RVA vaccines.

Objective:To discuss the protective effect of ginsenoside Rh1(G-Rh1)on kidney injury in the diabetic mellitus(DM)mice,and to clarify its mechanism.Methods:The diabetic kidney disease(DKD)model was prepared by using the high-fat,high-sugar diet combined with intraperitoneal injection of streptozotocin(STZ).A total of 48 C57/BL6 model mice were randomly divided into model group,nuclear factor erythroid 2-related factor 2(Nrf2)inhibitor ML385 group(ML385 group)(30 mg·kg-1),G-Rh1 group(30 mg·kg-1),and G-Rh1+ML385 group(30 mg·kg-1 G-Rh1+30 mg·kg-1 ML385),and there were 12 mice in each group.Additionally,12 C57/BL6 mice were selected as control group.After treated for 8 weeks,automatic analyzer was used to detect the levels of fasting blood glucose(FBG),blood urea nitrogen(BUN),and serum creatinine(Scr)in serum of the mice in various groups,as well as 24 h urinary protein(24 h UP)levels in urine,and the kidney index was calculated;kits were used to detect the activities of superoxide dismutase(SOD)and lactate dehydrogenase(LDH),and the levels of malondialdehyde(MDA)in kidney tissue of the mice in various groups;Western blotting method was used to detect the expression levels of Nrf2 and heme oxygenase-1(HO-1)proteins in kidney tissue of the mice in various groups.Results:Compared with control group,the levels of FBG and kidney indexes in serum of the mice in model group,ML385 group,and G-Rh1+ML385 group were significantly increased(P<0.01),and the level of FBG in serum of the mice in G-Rh1 group was significantly increased(P<0.01);compared with model group,the kidney index of the mice in ML385 group was significantly increased(P<0.05),while the levels of FBG and kidney index of the mice in G-Rh1 group were significantly decreased(P<0.05 or P<0.01);compared with G-Rh1 group,the level of FBG and kidney index of the mice in G-Rh1+ML385 group were significantly increased(P<0.01).Compared with control group,the levels of BUN and Scr in serum,and 24 h UP in urine of the mice in model group,ML385 group,G-Rh1 group,and G-Rh1+ML385 group were significantly increased(P<0.01);compared with model group,the level of BUN in serum and 24 h UP in urine of the mice in ML385 group were significantly increased(P<0.05),while the levels of BUN and Scr in serum,and 24 h UP in urine of the mice in G-Rh1 group were significantly decreased(P<0.01);compared with G-Rh1 group,the levels of BUN and Scr in serum,and 24 h UP in urine of the mice in G-Rh1+ML385 group were significantly increased(P<0.01).Compared with control group,the activities of SOD in kidney tissue of the mice in model group,ML385 group,G-Rh1 group,and G-Rh1+ML385 group were significantly decreased(P<0.01),while the levels of MDA and LDH activities were significantly increased(P<0.01);compared with model group,the activity of SOD in kidney tissue of the mice in ML385 group was significantly decreased(P<0.05),and the level of MDA was significantly increased(P<0.05);the activity of SOD in kidney tissue of the mice in of G-Rh1 group was significantly increased(P<0.01),and the level of MDA and activity of LDH were significantly decreased(P<0.01);compared with G-Rh1 group,the activity of SOD in kidney tissue of the mice in G-Rh1+ML385 group was significantly decreased(P<0.01),and the level of MDA and activity of LDH were significantly increased(P<0.01).Compared with control group,the expression levels of Nrf2 and HO-1 proteins in kidney tissue of the mice in model group,ML385 group,G-Rh1 group,and G-Rh1+ML385 group were significantly decreased(P<0.05 or P<0.01);compared with model group,the expression levels of Nrf2 and HO-1 proteins in kidney tissue of the mice in ML385 group and G-Rh1+ML385 group were significantly decreased(P<0.05),while the expression levels of Nrf2 and HO-1 proteins in kidney tissue of the mice in G-Rh1 group were significantly increased(P<0.01);compared with G-Rh1 group,the expression levels of Nrf2 and HO-1 proteins in kidney tissue of the mice in G-Rh1+ML385 group were significantly decreased(P<0.01).Conclusion:Ginsenoside Rh1 reduces the oxidative stress and improves the kidney function,providing protective effects on kidney injury in the DM mice,and its mechanism may be related to the activation of the Nrf2/HO-1 signaling pathway.