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:To explore the effects of IFN-γ on IL-8 secretion by human liver cancer cells and the impact on their malignant biological functions in vitro. Methods:HuH7 and Hep3B cells were treated with different concentrations of IFN-γ for 24 or 48 h. Changes in the cellular activity, IL-8 secretion, and the proportion of CD133 + liver cancer stem cells were evaluated using CCK8 kit and flow cytometry. Western blot was used to detect the effects of IFN-γ on the expression of several molecules such as phosphorylated protein kinase B (p-Akt), phosphorylated c-Jun N-terminal kinase (p-JNK), vimentin, and E-cadherin in the liver cancer cells. Effects of IFN-γ with or without IL-8 on the migration of liver cancer cells were detected by transwell assay. Additionally, effects of IFN-γ combined with IL-8 or IL-8 receptor inhibitor repertaxin on the differentiation of liver cancer stem cells were detected by flow cytometry. One-way analysis of variance and Tukey-Kramer test were used for statistical analysis. Results:HuH7 and Hep3B cells secreted significantly higher levels of IL-8 than normal hepatocytes LO2 ( P<0.01) and high expression level of IL-8 gene ( CXCL8) was closely correlated with the expression levels of vimentin gene ( VIMENTIN), CD133 gene ( PRCM1), PD-L1 gene ( CD274), PD-1 gene ( PDCD1), and CD163 gene ( CD163), as well as the poor prognosis of liver cancer patients ( P<0.01). IFN-γ (1-100 ng/ml) had no significant effect on the proliferative activity of HuH7 and Hep3B cells ( P>0.05), but could significantly inhibit IL-8 secretion, cell migration, CD133 + liver cancer stem cell differentiation and suspension tumor sphere formation through the Akt and JNK pathways ( P<0.01). IFN-γ combined with IL-8 could significantly reversed the inhibitory effects of IFN-γ on liver cancer cell migration, stem cell differentiation, and suspension tumor sphere formation ( P<0.01). IFN-γ in combination with repertaxin could synergistically inhibited the differentiation of CD133 + liver cancer stem cells ( P<0.01). Conclusion:IFN-γ inhibits the differentiation and migration of human liver cancer cells through the Akt/JNK-IL-8 signaling pathway, providing a new strategy for future clinical immunotherapy of liver cancer.

In recent years,as the survival time of liver transplant recipients continues to increase,serious complications after transplantation,including diabetes,which affects the long-term survival of patients,have attracted more and more attention.Diabetes after liver transplantation can increase the risk of infection and cardiovascular disease,which in turn affects the survival rate of grafts and patients,and therefore,identification and intervention for high-risk populations are of great importance for improving the prognosis of patients.This article reviews the risk factors for diabetes after liver transplantation,in order to deepen the understanding of diabetes after liver transplantation and provide a theoretical basis for disease prevention and treatment.

Objective:To establish the reference interval of 12 types of cytokines(IL-1β,IL-2,IL-4,IL-5,IL-6,IL-8,IL-10,IL-12p70,IL-17,IFN-γ,IFN-α,TNF-α)in adult plasma based on multiple microsphere flow immunofluorescence(MBFFI).Methods:A total of 140 healthy adult patients who were examined at Xuzhou Central Hospital between January 2022 and December 2023 were included in the study.Plasma cytokine levels were detected and reference intervals were established by the flow cytometer and the assay kits produced by Qingdao Raisecare Biotechnology Co.,Ltd and Jiangsu BioPredia Biotechnology Co.,Ltd.Results:All of the cytokines exhibited a non-normal distribution,and there was a discrepancy in the 95％reference interval between the two re-agents.The reference intervals for the 12 cytokine kits produced by Qingdao Raisecare Biotechnology Co.,Ltd.were as follows:IFN-α:<4.91 pg/ml,IL-12 p70:<1.95 pg/ml,IL-5:<12.72 pg/ml,IL-8:<60.68 pg/ml,IL-1β:<27.67 pg/ml,IL-2:<5.01 pg/ml,IL-4:<1.22 pg/ml,IL-6:<6.11 pg/ml,TNF-α:<2.92 pg/ml,IL-17:<10.27 pg/ml,IL-10:<6.88 pg/ml,IFN-γ:<17.68 pg/ml.The reference intervals of the 12 cytokines produced by Jiangsu BioPredia Biotechnology Co.,Ltd.were as follows:IFN-α:<4.05 pg/ml,IL-12 p70:<7.33 pg/ml,IL-5:<7.80 pg/ml,IL-8:<13.24 pg/ml,IL-1β:<19.24 pg/ml,IL-2:<2.42 pg/ml,IL-4:<0.99 pg/ml,IL-6:<2.10 pg/ml,TNF-α:<0.87 pg/ml,IL-17:<1.42 pg/ml,IL-10:<1.10 pg/ml,IFN-γ:<1.34 pg/ml.Conclusion:In this study,the ref-erence range of two reagents for the detection of 12 kinds of cytokines in plasma of healthy adults is established by MBFFI,which pro-vides a valuable reference for the diagnosis and treatment of clinical-related diseases.

In recent years,as the survival time of liver transplant recipients continues to increase,serious complications after transplantation,including diabetes,which affects the long-term survival of patients,have attracted more and more attention.Diabetes after liver transplantation can increase the risk of infection and cardiovascular disease,which in turn affects the survival rate of grafts and patients,and therefore,identification and intervention for high-risk populations are of great importance for improving the prognosis of patients.This article reviews the risk factors for diabetes after liver transplantation,in order to deepen the understanding of diabetes after liver transplantation and provide a theoretical basis for disease prevention and treatment.

Objective:To establish the reference interval of 12 types of cytokines(IL-1β,IL-2,IL-4,IL-5,IL-6,IL-8,IL-10,IL-12p70,IL-17,IFN-γ,IFN-α,TNF-α)in adult plasma based on multiple microsphere flow immunofluorescence(MBFFI).Methods:A total of 140 healthy adult patients who were examined at Xuzhou Central Hospital between January 2022 and December 2023 were included in the study.Plasma cytokine levels were detected and reference intervals were established by the flow cytometer and the assay kits produced by Qingdao Raisecare Biotechnology Co.,Ltd and Jiangsu BioPredia Biotechnology Co.,Ltd.Results:All of the cytokines exhibited a non-normal distribution,and there was a discrepancy in the 95％reference interval between the two re-agents.The reference intervals for the 12 cytokine kits produced by Qingdao Raisecare Biotechnology Co.,Ltd.were as follows:IFN-α:<4.91 pg/ml,IL-12 p70:<1.95 pg/ml,IL-5:<12.72 pg/ml,IL-8:<60.68 pg/ml,IL-1β:<27.67 pg/ml,IL-2:<5.01 pg/ml,IL-4:<1.22 pg/ml,IL-6:<6.11 pg/ml,TNF-α:<2.92 pg/ml,IL-17:<10.27 pg/ml,IL-10:<6.88 pg/ml,IFN-γ:<17.68 pg/ml.The reference intervals of the 12 cytokines produced by Jiangsu BioPredia Biotechnology Co.,Ltd.were as follows:IFN-α:<4.05 pg/ml,IL-12 p70:<7.33 pg/ml,IL-5:<7.80 pg/ml,IL-8:<13.24 pg/ml,IL-1β:<19.24 pg/ml,IL-2:<2.42 pg/ml,IL-4:<0.99 pg/ml,IL-6:<2.10 pg/ml,TNF-α:<0.87 pg/ml,IL-17:<1.42 pg/ml,IL-10:<1.10 pg/ml,IFN-γ:<1.34 pg/ml.Conclusion:In this study,the ref-erence range of two reagents for the detection of 12 kinds of cytokines in plasma of healthy adults is established by MBFFI,which pro-vides a valuable reference for the diagnosis and treatment of clinical-related diseases.

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:To explore the effects of IFN-γ on IL-8 secretion by human liver cancer cells and the impact on their malignant biological functions in vitro. Methods:HuH7 and Hep3B cells were treated with different concentrations of IFN-γ for 24 or 48 h. Changes in the cellular activity, IL-8 secretion, and the proportion of CD133 + liver cancer stem cells were evaluated using CCK8 kit and flow cytometry. Western blot was used to detect the effects of IFN-γ on the expression of several molecules such as phosphorylated protein kinase B (p-Akt), phosphorylated c-Jun N-terminal kinase (p-JNK), vimentin, and E-cadherin in the liver cancer cells. Effects of IFN-γ with or without IL-8 on the migration of liver cancer cells were detected by transwell assay. Additionally, effects of IFN-γ combined with IL-8 or IL-8 receptor inhibitor repertaxin on the differentiation of liver cancer stem cells were detected by flow cytometry. One-way analysis of variance and Tukey-Kramer test were used for statistical analysis. Results:HuH7 and Hep3B cells secreted significantly higher levels of IL-8 than normal hepatocytes LO2 ( P<0.01) and high expression level of IL-8 gene ( CXCL8) was closely correlated with the expression levels of vimentin gene ( VIMENTIN), CD133 gene ( PRCM1), PD-L1 gene ( CD274), PD-1 gene ( PDCD1), and CD163 gene ( CD163), as well as the poor prognosis of liver cancer patients ( P<0.01). IFN-γ (1-100 ng/ml) had no significant effect on the proliferative activity of HuH7 and Hep3B cells ( P>0.05), but could significantly inhibit IL-8 secretion, cell migration, CD133 + liver cancer stem cell differentiation and suspension tumor sphere formation through the Akt and JNK pathways ( P<0.01). IFN-γ combined with IL-8 could significantly reversed the inhibitory effects of IFN-γ on liver cancer cell migration, stem cell differentiation, and suspension tumor sphere formation ( P<0.01). IFN-γ in combination with repertaxin could synergistically inhibited the differentiation of CD133 + liver cancer stem cells ( P<0.01). Conclusion:IFN-γ inhibits the differentiation and migration of human liver cancer cells through the Akt/JNK-IL-8 signaling pathway, providing a new strategy for future clinical immunotherapy of liver cancer.

Objective:To investigate the tumor suppressing effect of Shenqi Yiliu decoction combined with cisplatin via ERK-mediated C-Myc/PD-L1 phase-coordinated pathway on H22 hepatocellular carcinoma tumor-bearing mice and its mechanism.Meth-ods:In 60 SPF-grade male Kunming mice,10 mice were taken as blank group by random number table method,and the other 50 mice were replicated as H22 hepatocellular carcinoma tumor-bearing mouse model.After successful replication of the model,the model mice were randomly divided into model group,cisplatin group[2.5×10-3 g/(kg·3 d)],Shenqi Yiliu decoction low[13.515 g/(kg·d)],me-dium[27.03 g/(kg·d-1)],and high dose[27.030 g/(kg·d)]combined with cisplatin group[2.5×10-3 g/(kg·3 d)],10 mice in each group were treated for 13 d.After 24 h of the last dose,the mice were anesthetized and sacrificed,and the tumor inhibition rate,spleen index and thymus index of each drug group were determined;HE staining was performed to observe the histopathological changes of tumor in mice;ELISA kit was used to detect the contents of EGF and IFN-γ in tumor tissue homogenate;p-ERK1/2,C-Myc and PD-L1 protein expression in tumor tissue were detected by IHC and Western blot;ERK,C-Myc and PD-L1 mRNA expression levels in tumor tissue were detected by RT-PCR.Results:Compared with blank group,the average body mass and spleen index of mice in model group were decreased(P<0.05).Compared with model group,the tumor inhibition effect of each treatment group was obvious,and Shenqi Yiliu decoction combined with cisplatin group inhibited tumor growth in liver cancer mice in a dose-dependent way,im-proved the average body mass,spleen index and thymus index of mice,promoted the necrosis of tumor cells and increased the necrotic area.EGF and IFN-γ contents,P-ERK1/2,C-Myc,PD-L1 protein expressions and ERK,C-Myc,PD-L1 mRNA expression levels were decreased in tumor tissues(P<0.05).Compared with cisplatin group,the therapeutic effect of Shenqi decoction combined with cisplatin in medium and high dose groups was significant,and the difference was statistically significant(P<0.05).Conclusion:Shenqi Yiliu decoction combined with cisplatin effectively inhibited the tumor growth of H22 liver cancer tumor-bearing mice and significantly reduces the expression of C-Myc and PD-L1 proteins in the tumor tissues,which may be through the regulation of ERK signaling path-way-related protein expression to exert tumor suppressive effect.

Objective To observe the effects of Yiqi Huoxue Tuodu Prescription on Keap1Nrf2/HO-1 signaling pathway in rats with chronic nonbacterial prostatitis(CNP);To explore its mechanism for the treatment of CNP.Methods CNP rat model was prepared using castration combined with estrogen induction method.Totally 48 SD rats were divided into blank group,model group,celecoxib group and Yiqi Huoxue Tuodu Prescription group according to the random number table method,with 12 rats in each group.In the celecoxib group,celecoxib suspension was instilled 0.035 g/kg,and in the Yiqi Huoxue Tuodu Prescription group,Yiqi Huoxue Tuodu Prescription water decoction was instilled 8.64 g/kg,and the blank group and the model group were instilled with equal volume of normal saline for 28 days.Mechanical pain threshold in rats was measured using Von Frey fiber optic pain gauge,HE staining was used to observe pathological changes in prostate tissue and pathological scoring,the content of reactive oxygen species(ROS)in prostate tissue were detected by chemical fluorescence method and the glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)content in prostate tissue were detected by colorimetric method,Western blot was used to detect the expressions of Kelch like ECH related protein 1(Keap1),nuclear factor E2 related factor 2(Nrf2),and heme oxygenase-1(HO-1)protein in prostate tissue.Results Compared with the blank group,rats in the model group had significantly lower mechanical pain threshold and significantly decreased prostate index(P<0.01);the size of the glandular cavity in prostate tissue varied,with the disappearance of secretions in the cavity,interstitial looseness and edema,a large amount of fibrous tissue hyperplasia and inflammatory cell infiltration,and a significant increase in pathological scores(P<0.01);the contents of ROS and MDA in prostate tissue significantly increased,the activity of GSH-Px significantly decreased(P<0.01),the expression of Keap1 and Nrf2 proteins significantly decreased,and the expression of HO-1 protein significantly increased(P<0.01,P<0.05).Compared with the model group,the mechanical pain threshold of the rats in the Yiqi Huoxue Tuodu Prescription group was significantly higher(P<0.01);there was mild damage to prostate tissue,with a small amount of fibrous hyperplasia and inflammatory cell infiltration,and a significant decrease in pathological scores(P<0.01,P<0.05);the contents of ROS and MDA in prostate tissue significantly decreased,and the GSH-Px activity significantly increased(P<0.01),the Keap1 and Nrf2 protein expressions significantly increased and HO-1 protein expression significantly decreased in prostate tissue(P<0.01,P<0.05).Conclusion Yiqi Huoxue Tuodu Prescription can effectively improve the histopathological morphology and increase the pain threshold of the prostate gland in CNP rats,and its mechanism of action may be related to the regulation of Keap1/Nrf2/HO-1 signaling pathway and reduction of oxidative stress damage in prostate tissue of rats.