ObjectiveTo investigate the mechanism by which Shenqi Yiliu prescription reverses cisplatin resistance in ovarian cancer cells by regulating the phosphatidylinositol-3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway-mediated glycolysis. MethodsThe human ovarian cancer A2780 cell line was intervened with progressively increasing doses of cisplatin （1 g·L^-1） to establish the cisplatin-resistant cell line A2780cisR， and the cell sensitivity to cisplatin was examined by the cell counting kit-8 （CCK-8） assay. High， medium， and low （39.9， 19.95， 9.98 g·kg^-1） doses of Shenqi Yiliu prescription-containing sera were used to treat A2780cisR cells for 48 h. Glucose consumption and lactate production were measured by the cuvette assay. Enzyme-linked immunosorbent assay （ELISA） was employed to determine the activities of glucose transporter （GLUT）， phosphofructokinase （PFK）， and pyruvate kinase （PK）. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） staining was used to detect apoptosis. Western blot was employed to quantify the protein levels of phosphorylated （p）-PI3K， p-Akt， p-mTOR， hexokinase 2 （HK2）， pyruvate kinase M2 （PKM2）， B-cell lymphoblastoma-2 （Bcl-2）， Bcl-2-associated X-protein （Bax）， and B-lymphoblastoma-2 gene-related promoter （Bad）. Real-time PCR was conducted to determine the mRNA levels of HK2， PKM2， Bax， Bcl-2， and Bad. ResultsThe median inhibitory concentration （IC₅₀） of cisplatin on A2780cisR cells was nearly 3 times that on A2780P cells. Compared with A2780P cells， A2780cisR cells showed increased glucose consumption， lactate production， GLUT， PFK， and PK activities， and mRNA and protein levels of p-PI3K， Akt， p-mTOR， HK2， PKM2， Bax （P<0.05）， and decreased apoptosis rate and Bcl-2 expression （P<0.05）. Compared with A2780cisR cells， medium- and high-dose Shenqi Yiliu prescription reduced the glucose consumption， lactate production， GLUT， PFK， and PK activities， and mRNA and protein levels of p-PI3K， Akt， p-mTOR， HK2， PKM2， Bax， and Bad （P<0.05）， while increasing the apoptosis rate and Bcl-2 expression （P<0.05）. ConclusionShenqi Yiliu prescription can inhibit glycolysis mediated by the PI3K/Akt/mTOR pathway to promote apoptosis， thereby reversing cisplatin resistance in ovarian cancer cells.

Objective To predict the molecular mechanism of Shenqi Yiliu Prescription for the treatment of ovarian cancer based on network pharmacology and molecular docking technology;To conduct experimental validation.Methods The active components and targets of Shenqi Yiliu Prescription were retrieved and screened through TCMSP database,and ovarian cancer disease targets were searched through GeneCards database.A protein-protein interaction network between drugs and disease was established using the STRING database,and GO and KEGG pathway enrichment analysis was performed.Molecular docking between key active components and core targets was conducted.Ovarian cancer A2780 cells were intervened with Shenqi Yiliu Prescription low-,medium-and high-dosages containing serum.ELISA was used to detect TNF-α and IL-17 contents in cell supernatant;TUNEL staining was used to detect cell apoptosis;Western blot was used to detect the protein expressions of p-PI3K,p-AKT,phosphorylated T218(MDM2),and tumor protein P53(P53);real-time fluorescence quantitative PCR was used to detect the mRNA expressions of PI3K,AKT,MDM2 and P53.Results Network pharmacology analysis showed that quercetin,β-sitosterol and kaempferol were the active components of Shenqi Yiliu Prescription to treat ovarian cancer,and TP53,AKT1 and TNF were the core targets.The molecular docking results showed that the key active components could bind well to the core targets.KEGG enrichment analysis showed that the PI3K/AKT signaling pathway may be the core pathway for Shenqi Yiliu Prescription to intervene in ovarian cancer.The cell experiment results showed that compared with the control group,the contents of TNF-α and IL-17 in cell supernatant decreased(P<0.05),the apoptosis rate increased(P<0.05),the expressions of p-PI3K,p-AKT,MDM2 protein and PI3K,AKT,MDM2 mRNA decreased(P<0.05),and the expressions of P53 protein and mRNA decreased(P<0.05)in each Shenqi Yiliu Prescription group.Moreover,the intervention effect of Shenqi Yiliu Prescription was dose-dependent.Conclusion Shenqi Yiliu Prescription can effectively inhibit the proliferation and promote apoptosis of ovarian cancer cells,and its possible mechanism maybe related to inhibition of the activation of AKT-MDM2-P53 signaling pathway and the reduction of IL-17 and TNF-α contents.

Objective To investigate the effects of Huayu Xiaopi Decoction on the proliferation and migration of AGS cells;To explore its mechanism in treating precancerous lesions of gastric cancer.Methods AGS cells were divided into blank group,inhibitor group and Huayu Xiaopi Decoction high-,medium-and low-dosage groups.The blank group was cultured with 15%control serum,the inhibitor group was cultured with 10 μmol/L HIF-1α inhibitor,and the Huayu Xiaopi Decoction high-,medium-and low-dosage groups were cultured with 12%,6%and 3%drug containing serum,respectively.CCK-8 method was used to detect cell survival rate,cell migration ability was detected by scratch test,immunofluorescence was used to detect the expressions of proliferating cell nuclear antigen(PCNA),matrix metalloproteinase(MMP)-2 and MMP-9 in AGS cells,the expressions of HIF-1α,COX-2,VEGF,VEGFR2,MMP-2,MMP-9 mRNA and HIF-1α,COX-2,VEGF,VEGFR2 proteins in AGS cells were detected by RT-PCR and Western blot.Results Compared with the blank group,the cell survival rate and migration rate were significantly decreased in the inhibitor group and each dosage of Huayu Xiaopi Decoction groups(P<0.05),the expressions of PCNA,MMP-2 and MMP-9 significantly decreased in the inhibitor group and Huayu Xiaopi Decoction high-and medium-dosage groups(P<0.05),the mRNA expressions of HIF-1α,COX-2,VEGF,VEGFR2,MMP-2,MMP-9 and the protein expression of HIF-1α,COX-2,VEGF,VEGFR2 were significantly decreased(P<0.05).Conclusion Huayu Xiaopi Decoction can inhibit the proliferation and migration of AGS cells,and its mechanism is related to the regulation of the expression of key molecules in HIF-1α/VEGF signaling pathway.

Objective To predict the molecular mechanism of Shenqi Yiliu Prescription for the treatment of ovarian cancer based on network pharmacology and molecular docking technology;To conduct experimental validation.Methods The active components and targets of Shenqi Yiliu Prescription were retrieved and screened through TCMSP database,and ovarian cancer disease targets were searched through GeneCards database.A protein-protein interaction network between drugs and disease was established using the STRING database,and GO and KEGG pathway enrichment analysis was performed.Molecular docking between key active components and core targets was conducted.Ovarian cancer A2780 cells were intervened with Shenqi Yiliu Prescription low-,medium-and high-dosages containing serum.ELISA was used to detect TNF-α and IL-17 contents in cell supernatant;TUNEL staining was used to detect cell apoptosis;Western blot was used to detect the protein expressions of p-PI3K,p-AKT,phosphorylated T218(MDM2),and tumor protein P53(P53);real-time fluorescence quantitative PCR was used to detect the mRNA expressions of PI3K,AKT,MDM2 and P53.Results Network pharmacology analysis showed that quercetin,β-sitosterol and kaempferol were the active components of Shenqi Yiliu Prescription to treat ovarian cancer,and TP53,AKT1 and TNF were the core targets.The molecular docking results showed that the key active components could bind well to the core targets.KEGG enrichment analysis showed that the PI3K/AKT signaling pathway may be the core pathway for Shenqi Yiliu Prescription to intervene in ovarian cancer.The cell experiment results showed that compared with the control group,the contents of TNF-α and IL-17 in cell supernatant decreased(P<0.05),the apoptosis rate increased(P<0.05),the expressions of p-PI3K,p-AKT,MDM2 protein and PI3K,AKT,MDM2 mRNA decreased(P<0.05),and the expressions of P53 protein and mRNA decreased(P<0.05)in each Shenqi Yiliu Prescription group.Moreover,the intervention effect of Shenqi Yiliu Prescription was dose-dependent.Conclusion Shenqi Yiliu Prescription can effectively inhibit the proliferation and promote apoptosis of ovarian cancer cells,and its possible mechanism maybe related to inhibition of the activation of AKT-MDM2-P53 signaling pathway and the reduction of IL-17 and TNF-α contents.

Objective To investigate the effects of Huayu Xiaopi Decoction on the proliferation and migration of AGS cells;To explore its mechanism in treating precancerous lesions of gastric cancer.Methods AGS cells were divided into blank group,inhibitor group and Huayu Xiaopi Decoction high-,medium-and low-dosage groups.The blank group was cultured with 15%control serum,the inhibitor group was cultured with 10 μmol/L HIF-1α inhibitor,and the Huayu Xiaopi Decoction high-,medium-and low-dosage groups were cultured with 12%,6%and 3%drug containing serum,respectively.CCK-8 method was used to detect cell survival rate,cell migration ability was detected by scratch test,immunofluorescence was used to detect the expressions of proliferating cell nuclear antigen(PCNA),matrix metalloproteinase(MMP)-2 and MMP-9 in AGS cells,the expressions of HIF-1α,COX-2,VEGF,VEGFR2,MMP-2,MMP-9 mRNA and HIF-1α,COX-2,VEGF,VEGFR2 proteins in AGS cells were detected by RT-PCR and Western blot.Results Compared with the blank group,the cell survival rate and migration rate were significantly decreased in the inhibitor group and each dosage of Huayu Xiaopi Decoction groups(P<0.05),the expressions of PCNA,MMP-2 and MMP-9 significantly decreased in the inhibitor group and Huayu Xiaopi Decoction high-and medium-dosage groups(P<0.05),the mRNA expressions of HIF-1α,COX-2,VEGF,VEGFR2,MMP-2,MMP-9 and the protein expression of HIF-1α,COX-2,VEGF,VEGFR2 were significantly decreased(P<0.05).Conclusion Huayu Xiaopi Decoction can inhibit the proliferation and migration of AGS cells,and its mechanism is related to the regulation of the expression of key molecules in HIF-1α/VEGF signaling pathway.

ObjectiveTo investigate the potential mechanism of Shenqi Yiliu Formula （参芪抑瘤方） in treating precancerous lesions of gastric cancer （PLGC） by the Wnt/β-catenin signaling pathway. MethodsThe CCK-8 assay was used to determine the optimal intervention time for Shenqi Yiliu Formula drug-containing serum and the concentration of the Wnt/β-catenin pathway inhibitor XAV939 depends on the survival rate of AGS gastric cancer cell line. AGS cells were divided into the gastric cancer cell group （15% blank serum）， inhibitor group （selected concentration of XAV939）， high-dose Shenqi Yiliu Formula group （12% Shenqi Yiliu Formula drug-containing serum + 3% blank serum）， medium-dose Shenqi Yiliu Formula group （6% Shenqi Yiliu Formula drug-containing serum + 9% blank serum）， and low-dose Shenqi Yiliu Formula group （3% Shenqi Yiliu Formula drug-containing serum + 12% blank serum）. Each group was tested in triplicate. After culturing for 24 and 48 hours， cell migration and invasion were assessed by scratch assays； after a selected intervention period （48 hours）， cell cycle distribution was analyzed using flow cytometry， Ki67 protein levels were detected by immunofluorescence， the protein levels of Wnt， β-catenin， GSK-3β， and intranuclear T-cell specific factor（TCF） were measured by the protein immunoblotting assay， and the mRNA expressions of these above factors were determined by quantitative real-time PCR. ResultsThe optimal intervention time for Shenqi Yiliu Formula drug-containing serum was determined to be 48 hours， and the effective concentration of XAV939 was 20 μmol/L. Compared with the gastric cancer cell group， Shenqi Yiliu Formula at all doses reduced the cell migration rate at 24 and 48 hours （P＜0.05）， except for the low-dose group at 24 hours. Compared to the low-dose group at corresponding time points， high- and medium-dose Shenqi Yiliu Formula groups showed significantly reduced migration rates， particularly the high-dose group at 48 hours （P＜0.05）. Compared with the gastric cancer cell group， the high-dose Shenqi Yiliu Formula and inhibitor groups exhibited reduced protein and mRNA levels of Wnt， β-catenin， and TCF， along with reduced Ki67 protein levels and a decreased proportion of cells in the S and G2 phases of the cell cycle， but GSK-3β protein levels， GSK-3β mRNA expression， and the proportion of cells in the G1 phase increased （P＜0.05）. Compared to the inhibitor group， the high-dose Shenqi Yiliu Formula group showed a decreased proportion of G1-phase cells and an increased proportion of G2-phase cells （P＜0.05）， although differences in Wnt and β-catenin protein levels and mRNA expressions were not statistically significant （P＞0.05）. ConclusionShenqi Yiliu Formula drug-containing serum inhibits the migration and invasion of gastric cancer AGS cells and block the cell cycle at G1 phase， and its underlying mechanism may be related to the regulation of the Wnt/β-catenin signaling pathway.

Objective This study was to investigate the effects of Ginseng Astragalus Tumor Suppressor Formula combined with cisplatin on relevant immune genes and immune functions in mice modeled with hepatocellular carcinoma based on bioinformatics research.Methods Gene expression profiles and clinical data of hepatocellular carcinoma patients were downloaded from TCGA and GEO databases and screened for immune-expressed genes by taking intersections with immune-related genes downloaded from ImmPort database.Immune-related gene pair coefficients(IRGPI)were calculated to construct IRGP prognostic models.The optimal cut-off value of IRGPI for 1-year overall survival of hepatocellular carcinoma patients was determined based on ROC curve analysis,and hepatocellular carcinoma patients were divided into high and low immune risk groups,and the survival status of patients in the two groups was analyzed using the Kaplan-Meier method and the Log-Rank test.Then,we screened the active ingredients and gene targets of Ginseng Astragali Tumor Suppressor Formula for the treatment of hepatocellular carcinoma by network pharmacology,and obtained the intersection genes between Ginseng Astragali Tumor Suppressor Formula and disease,and then extracted the intersection of hepatocellular carcinoma-related immune genes and the intersection genes between Ginseng Astragali Tumor Suppressor Formula and disease through the"VennDiagram"software,and verified it through the animal model.The effects of Astragalus tumor-suppressing formula combined with cisplatin on the immunity genes and immune functions in the tumor tissues of mice with hepatocellular carcinoma were verified in animal models.Results Among 2483 relevant immune genes,84 pairs of immune-related genes were significantly associated with OS in the experimental group(P<0.001),and among the patients categorized into high and low immune risk groups by cut-off value-0.258,the overall survival rate of the high immune risk group was significantly lower than that of the low immune risk group(P<0.001).Univariate Cox analysis showed that IRGP model risk values and clinical characteristics of tumor T-staging had an impact on prognosis.Multifactorial Cox analysis showed that IRGP model risk value and tumor T-stage could be used as independent prognostic factors.266 genes intersecting with hepatocellular carcinoma were screened by network pharmacology technique for Ginseng Astragalus Tumor Suppressor Formula,and further 8 genes were obtained by taking the intersection with 84 pairs of immune-related genes.The experimental results showed that compared with the model group,the tumor mass of mice in each treatment group decreased(P<0.05);the spleen index and thymus index of mice increased(P<0.05);the CD4+/CD8+ratio in serum and spleen tissues of mice decreased;ICAM1,FABP5,IGF2,CDK4,NR1,ADRB2,AR,NR3C2 in tumor tissue of mice mRNA expression were all decreased(P<0.05),and the therapeutic effect of the combined group was significant(P<0.01).Conclusion This study predicted the key immune genes related to hepatocellular carcinoma as well as the prognostic analysis of immune-related genes,and the experimental study verified that Ginseng and Astragalus Tumor Suppressor Formula could effectively reduce the expression of related immune genes in tumor tissues,and improve the proportion of related immune cells in the splenic tissues of mice with hepatocellular carcinoma model as well as improve the immune function of mice.

Objective This study was to investigate the effects of Ginseng Astragalus Tumor Suppressor Formula combined with cisplatin on relevant immune genes and immune functions in mice modeled with hepatocellular carcinoma based on bioinformatics research.Methods Gene expression profiles and clinical data of hepatocellular carcinoma patients were downloaded from TCGA and GEO databases and screened for immune-expressed genes by taking intersections with immune-related genes downloaded from ImmPort database.Immune-related gene pair coefficients(IRGPI)were calculated to construct IRGP prognostic models.The optimal cut-off value of IRGPI for 1-year overall survival of hepatocellular carcinoma patients was determined based on ROC curve analysis,and hepatocellular carcinoma patients were divided into high and low immune risk groups,and the survival status of patients in the two groups was analyzed using the Kaplan-Meier method and the Log-Rank test.Then,we screened the active ingredients and gene targets of Ginseng Astragali Tumor Suppressor Formula for the treatment of hepatocellular carcinoma by network pharmacology,and obtained the intersection genes between Ginseng Astragali Tumor Suppressor Formula and disease,and then extracted the intersection of hepatocellular carcinoma-related immune genes and the intersection genes between Ginseng Astragali Tumor Suppressor Formula and disease through the"VennDiagram"software,and verified it through the animal model.The effects of Astragalus tumor-suppressing formula combined with cisplatin on the immunity genes and immune functions in the tumor tissues of mice with hepatocellular carcinoma were verified in animal models.Results Among 2483 relevant immune genes,84 pairs of immune-related genes were significantly associated with OS in the experimental group(P<0.001),and among the patients categorized into high and low immune risk groups by cut-off value-0.258,the overall survival rate of the high immune risk group was significantly lower than that of the low immune risk group(P<0.001).Univariate Cox analysis showed that IRGP model risk values and clinical characteristics of tumor T-staging had an impact on prognosis.Multifactorial Cox analysis showed that IRGP model risk value and tumor T-stage could be used as independent prognostic factors.266 genes intersecting with hepatocellular carcinoma were screened by network pharmacology technique for Ginseng Astragalus Tumor Suppressor Formula,and further 8 genes were obtained by taking the intersection with 84 pairs of immune-related genes.The experimental results showed that compared with the model group,the tumor mass of mice in each treatment group decreased(P<0.05);the spleen index and thymus index of mice increased(P<0.05);the CD4+/CD8+ratio in serum and spleen tissues of mice decreased;ICAM1,FABP5,IGF2,CDK4,NR1,ADRB2,AR,NR3C2 in tumor tissue of mice mRNA expression were all decreased(P<0.05),and the therapeutic effect of the combined group was significant(P<0.01).Conclusion This study predicted the key immune genes related to hepatocellular carcinoma as well as the prognostic analysis of immune-related genes,and the experimental study verified that Ginseng and Astragalus Tumor Suppressor Formula could effectively reduce the expression of related immune genes in tumor tissues,and improve the proportion of related immune cells in the splenic tissues of mice with hepatocellular carcinoma model as well as improve the immune function of mice.

ObjectiveTo establish the quality standards for Sennae Folium（Cassia angustifolia） dispensing granules based on standard decoctions. MethodsHigh performance liquid chromatography（HPLC） specific chromatograms were established for 15 batches of Sennae Folium（C. angustifolia） standard decoctions and 10 of Sennae Folium（C. angustifolia） dispensing granules from different manufacturers， and the similarity evaluation， hierarchical cluster analysis（HCA） and principal component analysis（PCA） were performed. Linear calibration with two reference substances（LCTRS） and quantitative analysis of multi-components by single-marker（QAMS） were established for the common peaks in the specific chromatograms to determine the contents of main components in the decoction pieces， standard decoctions and dispensing granules， and to calculate their transfer rates from decoction pieces to standard decoctions and dispensing granules. ResultsThe similarities of specific chromatograms of 15 batches of Sennae Folium（C. angustifolia） standard decoctions and 10 batches of Sennae Folium（C. angustifolia） dispensing granules were all greater than 0.95， and a total of 8 characteristic peaks were calibrated， and five of them were identified， including kaempferol-3，7-O-diglucoside， apigenin-6，8-di-C-glucoside， quercetin-3-O-gentianoside， sennoside B and sennoside A. HCA and PCA results showed that there were certain differences in the composition of different batches of standard decoctions， but no clustering was observed in the production area. As the standard decoctions， the extract rate of 15 batches of samples was 26.54%-45.38%， the contents of kaempferol-3，7-O-diglucoside， apigenin-6，8-di-C-glucoside， quercetin-3-O-gentianoside， sennoside B and sennoside A were 12.16-19.26， 2.57-4.94， 3.27-5.11， 6.75-11.39， 4.69-7.79 mg·g^-1， and their transfer rates from decoction pieces to standard decoctions were 45.41%-79.02%， 29.12%-55.07%， 40.52%-67.90%， 24.72%-49.12%， 27.54%-49.34%， respectively. The extract rates of Sennae Folium（C. angustifolia） dispensing granules（C8-C10） were 38.10%-39.50%， the transfer rates of the above five components from decoction pieces to dispensing granules were 72.85%-73.58%， 53.43%-53.94%， 40.19%-40.74%， 24.62%-25.00%， 28.65%-29.11%， respectively， which were generally consistent with the transfer rates from decoction pieces to standard decoctions. ConclusionCompared with the relative retention time method， LCTRS has higher prediction accuracy and is more suitable for chromatographic columns. The established quality control standard of Sennae Folium（C. angustifolia） dispensing granules based on standard decoction is reasonable and reliable， and all indicators of samples from different manufacturers are within the range specified based on the standard decoction， which can provide reference for the quality control and process research of this dispensing granules.

ObjectiveTo establish the specific chromatogram of Chuanxiong Rhizoma dispensing granules（CRdg）， and to evaluate its quality by chemometrics and two reference substances for determination of multiple components（TRSDMC）. MethodsHigh performance liquid chromatography（HPLC） specific chromatograms were established using 13 batches of CRdg from 7 manufacturers， and preliminary quality evaluation was performed by similarity evaluation and chemometrics analysis. Eight characteristic peaks in the specific chromatogram of CRdg were measured on 22 different types of C₁₈ columns， and the actual retention times were recorded. Taking chlorogenic acid（peak 1） and senkyunolide A（peak 8） as double standard compounds， the retention times of the eight characteristic peaks were predicted by linear calibration using two reference substances（LCTRS）， and the method was validated on three other columns of different brands. Taking chlorogenic acid as reference peak， the relative correction factor method（RCFM） was used to quantify cryptochlorogenic acid， caffeic acid， ferulic acid， senkyunolide I and senkyunolide A， and the results were compared with the external standard method（ESM）. ResultsThe similarities of specific chromatograms of 13 batches of CRdg were all >0.90， and a total of 8 characteristic peaks were calibrated， and six of them were identified， including chlorogenic acid（peak 1）， cryptochlorogenic acid（peak 2）， caffeic acid（peak 3）， ferulic acid（peak 5）， senkyunolide I（peak 6） and senkyunolide A（peak 8）. Through chemometric analysis， it was found that ferulic acid， chlorogenic acid， senkyunolide I and cryptochlorogenic acid were the main components causing quality difference in CRdg， and the accuracy of LCTRS in predicting the retention time of 8 characteristic peaks was superior to that of the relative retention time method（RRT）. Further comparison of the results obtained from RCFM and ESM showed that there was no statistically significant difference between the two methods. ConclusionA quality evaluation method for CRdg based on HPLC specific chromatogram and TRSDMC is established， its qualitative accuracy is better than that of RRT， the quantitative accuracy is similar to that of ESM， and 4 quality-differentiated components among different manufacturers are found. This method is stable and reliable， and has reference value for the quality evaluation of other dispensing granules.