HTPP-MDC inhibits replication and subcellular localization of hepatitis B virus in HepG2.2.15 cells

AIM：To investigate the antiviral effect of hepatocyte-targeting and cell-penetrating peptide and Musca domestica cecropin (HTPP-MDC) fusion polypeptide against hepatitis B virus (HBV) in vitro, and to observe the penetrating ability of HTPP-MDC in hepatocytes. METHODS： HepG2.2.15 cells and Chang liver cells were co-cultured in vitro with HTPP-MDC at different concentrations. MTT assay was used to detect the cytotoxicity of HTPP-MDC in vitro. The levels of HBsAg, HBeAg and HBV DNA in HepG2.2.15 cell culture supernatants were measured by ELISA and real-time fluorescence quantitative PCR. The penetrating ability of HTPP-MDC was detected by laser confocal microscopy. RESULTS：The levels of HBsAg, HBeAg and HBV DNA in the supernatants of HepG2.2.15 cells treated with HTPP-MDC were remarkably reduced. The inhibitory effect of HTPP-MDC depended on the dose and action time of the drug. FITC-labeled HTPP-MDC was observed inside the cells under laser confocal microscope. CONCLUSION：HTPP-MDC strongly inhibits HBV replication in HepG2.2.15 cells. The penetrating ability of HTPP-MDC into hepatocytes indicates that HTPP-MDC is useful in clinic therapy for chronic hepatitis B-related diseases in the future.     

Inhibitory effect of HMGN2 protein on human hepatitis B viral HBeAg and HBsAg expression and DNA replication in HBV-transformed HepG2. 2.15 cell line

AIM: We previously demonstrated that HMGN2 is an antibacterial effector molecule in human LAK cells. This study was to examine the antiviral activity of HMGN2 against human hepatitis virus.METHODS: The purification and identify of HMGN2 proteins including preparative acid-urea polyacymide gel electrophoresis elution, reverse-phase high-performance liquid chromatography, mass spectrum, Western blotting and antimicrobial assay were conducted. The cellular toxicity of HMGN2 to the HepG2.2.15 cells was detected by MTT assay. HBeAg and HBsAg expressions were measured by ELISA. HBV DNA copies were determined by real time quantitative PCR.RESULTS: A bulk of HMGN2 was isolated and purified from the acid soluble proteins of human lymph node, and identified. The HBV-transfected HepG2.2.15 cell line was used in the in vitro assay system.In the range of testing 1-100 mg/L of HMGN2, no cytotoxicity to HepG2.2.15 cells was detected by MTT assay.When incubated with HMGN2 at 1-5 mg/L for 72 h or 144 h, a significant reduction in HBeAg and HBsAg expression and in HBV DNA copies was observed in the supernatant of HepG2.2.15 cells. CONCLUSION: HMGN2 protein markedly inhibits HBV expression and replication in vitro.     

Effect of proprotein convertases on transforming growth factor β1-induced HBV replication inhibition

AIM: To study the effect of proprotein convertases (PCs) on the transforming growth factor (TGF) β1-induced inhibition of HBV replication.METHODS: HepG2.2.15 cells cultured regularly were exposed to recombinant TGFβ1 at concentration of 2 μg/L or 5 μg/L and/or PC inhibitor at concentration of 20 μmol/L for 18 h. The total RNA and HBV core particle DNA were extracted from these cells, and PC mRNA and core-associated HBV DNA were detected by real-time PCR technique. RESULTS: The mRNA expression levels of 7 PCs in HepG2.2.15 cells were observed with various degrees. Recombinant TGFβ1 significantly up-regulated the mRNA expression of all PCs except for the down-regulation of PC5/6, though PC1/3 and PC2 were up-regulated most obviously. Furin and PACE4 were the predominant PCs before and after TGFβ1 exposure when the basic mRNA expression was taken into account. Further study showed that TGFβ1-induced the inhibition of HBV replication was abrogated by PC inhibitor in HepG2.2.15 cells. CONCLUSION: TGFβ1-induced the inhibition of HBV replication is mediated by the up-regulation of PCs, which might be of many implications in efficient interferences of TGFβ1 on HBV replication.     

Effects of xeroderma pigmentosum group D and p53 on replication of HBV

AIM:To investigate the effects of xeroderma pigmentosum D (XPD) and p53 on the replication of hepatitis B virus (HBV). METHODS:Recombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 cells by liposome. On the next day, these cells were incubated with pifithrin-α, a p53 inhibitor, at a concentration of 20 μmol/L for 24 h. The cells were divided into 5 groups: blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD+pifithrin-α group and pifithrin-α group. The mRNA expression of XPD, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus X protein (HBx) was detected by RT-PCR. The content of HBsAg and HBeAg in the supernatants of culture medium was measured by ELISA. The content of HBV-DNA in the supernatants of culture medium was examined by fluorescence quantitative PCR. Using the method of bDNA, the content of HBV-DNA in the core particles was assessed. RESULTS:The expression of XPD mRNA was elevated by the transfection of recombinant plasmid pEGFP-N2/XPD. The increase in XPD expression significantly down-regulated the mRNA expression of HBsAg, HBeAg and HBx. The content of HBsAg and HBeAg in the supernatants of culture medium was significantly decreased by the increase in XPD expression. The results of fluorescence quantitative PCR showed that the content of HBV-DNA in the supernatants of culture medium was significantly down-regulated by the increase in XPD expression. bDNA results showed that the content of HBV-DNA in the core particles was significantly decreased by the increase in XPD expression.Pifithrin-α abolished the above-mentioned effects of XPD (all P<0.01). CONCLUSION: XPD inhibits the replication of HBV through p53 pathway. Therefore, XPD and p53 may be the targets for antiviral therapy of hepatitis B.     

Effect of Paecilomyces cicadae polysaccharide on duplication of hepatitis B virus in HepG2.2.15 cell strain

AIM: To investigative the inhibitory effects of Paecilomyces cicadae polysaccharide (PcPS) against HBV in vitro, and the effects on the Toll like receptor 4 (TLR4) mRNA expression in HepG2.2.15 cell strain. METHODS: HepG2.2.15 cell strain was co-cultured in vitro with PcPS in different concentrations, and lamivudine (LMV) was applied as positive control. MTT assay was employed to detect the cytotoxicities of PcPS in vitro, when the HepG2.2.15 cells was used as target cells. The effects of PcPS on the secretion of HBsAg and HBeAg were assayed by ELISA method. Fluorescence quantitative-PCR (FQ-PCR) was used to detect the inhibitory effects of PcPS on the content of HBV-DNA and TLR4 mRNA in HepG2.2.15 cells. RESULTS: The inhibitory effects of PcPS on the HBsAg and HBeAg were observed and the maximum inhibitory ratio up to 44.8% and 31.0%, respectively. The same inhibitory effects of PcPS on the HBV-DNA replication and TLR4 mRNA expression in HepG2.2.15 cells were also found. CONCLUSION: A certain concentration of PcPS significantly inhibits HBV replication in vitro.     

Role of renal preS1/S2-Ag and other HBV antigen determination in diagnosis of HBV-associated glomerulonephritis

AIM：To detect the expression of preS1/S2 antigen (preS1/S2-Ag) and other antigens of hepatitis B virus (HBV) in renal tissues of patients with HBV-associated glomerulonephritis (HBV-GN), and to analyze their roles in the diagnosis of HBV-GN.
METHODS：Patients hospitalized in our department from January in 2003 to January in 2013 were retrospectively studied. A total of 49 patients with positive HBV surface antigen (HBsAg) serology, clinical manifestations of hematuria and/or proteinuria, and pathological diagnosis of glomerulonephritis, without systemic lupus erythematosus, anaphylactic purpura, diabetes or hepatitis C, were selected. PreS1/S2-Ag, HBV e antigen (HBeAg), HBsAg and HBV core antigen (HBcAg) in the renal tissues were examined. Five cases of glomerular minimal-change disease (MCD) with negative HBsAg and 5 cases of non-glomerulonephritis with positive HBsAg served as controls. RESULTS：The positive rates of preS1/S2-Ag, HBeAg, HBsAg and HBcAg in the renal tissues from the 49 patients of glomerulonephritis with HBV infection were 32.7% (16 cases), 38.8% (19 cases), 14.3% (7 cases) and 46.9% (23 cases), respectively. Total antigen positive rate was 70.2% (36 cases). The expression of preS1/S2-Ag was located in the cytoplasm of renal tubular epithelial cells, glomerular epithelial cells, endothelial cells and mesangial cells, and positively correlated with the expression of HBcAg (r=0.459, P<001). The 4 antigens were not detected in the 5 cases of HBsAg-negative patients with glomerular MCD. In the 5 cases of HBsAg-positive patients with non-glomerulonephritis, there were 2 cases expressing HBeAg and 1 case expressing HBcAg, but no cases expressing preS1/S2-Ag or HBsAg. CONCLUSION：The expression of preS1/S2-Ag in renal tissues suggests that HBV may invade the cells of renal tissue. Combined detection of the 4 antigens could elevate the rate of diagnosis of HBV-GN.     

Nodosin induces apoptosis of HepG2 cells by increasing Apaf-1 expression and activating caspase-3

AIM:To investigate the effects of nodosin on apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS:HepG2 cells were treated with nodosin at different concentrations (1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L and 20 μmol/L) for 24 h. The morphological changes of the HepG2 cells were observed by Hoechst 33258 staining and electron microscopy. The apoptotic rates were analyzed by flow cytometry. The mRNA expression of apoptotic protease-activating factor-1 (Apaf-1) was detected by RT-qPCR. The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:HepG2 cells showed obvious cell shrinkage and nucleus drift when treated with nodosin as the concentration was increased. Many apoptotic bodies were observed in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups. The mRNA expression of Apaf-1 was increased in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups as compared with control group (P<0.05). The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were increased with the increasing dose of nodosin (P<0.05). CONCLUSION:Nodosin induces the apoptosis of HepG2 cells. This effect was related to increasing Apaf-1 mRNA expression and subsequently promoting the activation of caspase-3.     

Two-unit ribozyme inhibit hepatitis B virus expressed in 2.2.15 cell

AIM: To observe the inhibition of HBc/HBeAg expression in the 2.2.15 cell transfected by two-unit ribozyme. METHODS: By use of subclone technique, two-unit ribozyme gene which was cutted from pGEM-Rz123 was ligated into the eukaryotic expression vector pBBS212. The recombinant plasmid was cotransfected into 2.2.15 cell using lipofectamine. Ribozyme was detected by dot-blot hybridization. The s and e/c antigen of HBV were detected by using ELISA, immunohistochemical technique, image analysis system and Western blot. RESULTS: After the transfected cell was selected two weeks by hygromycin B and G418. We found by dot-blot hybridization that ribozyme can express on 2.2.15 cell. HBeAg level can be reduced by 48.6% in the transfected 2.2.15 cell with two-ribozyme. Using immunohistochemical technique and image analysis system, Western blot, we observed that the level of HBcAg expressed in endocellular went down. CONCLUSION:Al these results strongly indicate that two-unit ribozyme can inhibit hepatitis B virus expression in the cell.     

Correlation between HBsAg level and HBV DNA titer during chronic hepatitis B treatment

AIM: Viral load is widely used as an indicator for the diagnosis and monitoring the treatment efficacy of chronic hepatitis B (CHB). Previous studies suggested that the quantity of hepatitis B surface antigen (HBsAg) in serum could be a surrogate marker of serum hepatitis B virus (HBV) DNA level. In this study, we aimed to investigate whether HBsAg level correlates with HBV DNA titer during CHB treatment. METHODS: Sera were collected from 47 CHB patients ［35 male, 12 female, mean age: (35±8) years］ treated for 48 weeks with a monotherapy (pegylated interferon alpha-2a, 18 patients; lamivudine, 15 patients) or a combination therapy (pegylated interferon alpha-2a and lamivudine, 14 patients). Serum samples were obtained at week 0 (just before the treatment), 4, 8, 24, 48 and week 72 (24 weeks after the treatment). HBV DNA was measured with real-time polymerase chain reaction (PCR). HBsAg was quantified with an automated chemiluminescent microparticle immunoassay. RESULTS: The titer of HBsAg correlated with the HBV DNA level in the 18 patients with monotherapy of pegylated interferon alpha-2a and the 14 patients with combination therapy (pegylated interferon alpha-2a and lamivudine). The significant correlation (canonical correlation=0.83) was found. However, no correlation in 15 patients with the monotherapy of lamivudine was observed. CONCLUSION: HBsAg titer correlates with HBV DNA level in CHB patients during the treatment with interferon or interferon and lamivudine, which can be a surrogate marker for monitoring the treatment efficacy.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0， 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

Effect of HSP70 on HBV replication

AIM: To investigate the role of heat shock protein 70(HSP70)in hepatitis B virus (HBV) replication. METHODS: The effect of HBV replication on the expression of HSP70 was analyzed by RT-qPCR. The overexpression efficiency of HSP70 was confirmed by Western blot. The effect of HSP70 overexpression on HBV DNA replicative intermediates was analyzed by RT-qPCR and Southern blot. The effects of HSP70 overexpression on the expression level of HBV 3.5 kb mRNA and HBV core protein were measured by RT-qPCR and Western blot, respectively. The Effect of HSP70 overexpression on HBV promoter activity was detected by dual luciferase reporter system. RESULTS: The mRNA levels of HSP70 were inhibited by HBV replication. Overexpression of HSP70 repressed the expression of HBV DNA replicative intermediates, 3.5 kb mRNA and core protein, as well as HBV core promoter activity. CONCLUSION: HBV replication inhibits the expression of HSP70. Overexpression of HSP70 represses HBV replication. These data suggest that HSP70 repressed HBV replication by inhibiting HBV core promoter activity.     

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.     

Mechanism of hepatitis B virus infection in renal tissues with IgA nephropathy

AIM: To identify the pathogenesis of renal lesions induced by hepatitis B virus (HBV) infection in IgA nephropathy (IgAN). METHODS: Forty-eight renal biopsy tissues of IgAN were selected, and were divided into five grades from Ⅰ to Ⅴ according to the classified standard of Meadow. HBsAg and HBcAg in renal tissues were detected by immunohistochemistry methods of Envision. Eighteen renal tissues with IgAN among 48 renal biopsy tissues were selected randomly, and then HBV DNA in these tissues was detected by direct in site polymerase chain reaction (IS-PCR) method. RESULTS: Thirty-six (36/48, 75.00%) and twenty-one (21/39, 53.85%) cases were positive with HBcAg and HBsAg in 48 cases renal tissues with IgAN, respectively. The positive rate of HBV DNA in 18 cases with IgAN was 61.11% (11/18). The positive rate of HBcAg， HBsAg and HBV DNA in renal tissues were all no significance betwen every grade (P＞0.05), but the positive rate of HBcAg, HBsAg and HBV DNA in renal tubule were all higher than that in glomeruli (P＜0.05). CONCLUSIONS: HBV really takes part in the occurrence of IgAN. HBV maybe induces the renal injury by cell-mediated immunity or a series of cytokines but not by virus direct damage. The renal tubule epithelium may be the targeting cells of HBV infection.     

Relationship between precore/basal core promoter mutations of hepatitis B virus and therapeutic effect of peginterferon α-2b,and changes of mutations before and after treatment

AIM:To investigate the relationship between therapeutic effect of peginterferon α-2b (Peg-IFNα-2b) and precore (PC) region G1896A and basal core promoter (BCP) region A1762T/G1764A mutations of hepatitis B virus (HBV), and the changes of the mutations before and after treatment. METHODS:The patients with HBeAg-positive chronic hepatitis B (CHB) (n=69) were treated with Peg-IFNα-2b for 48 weeks and followed up for 24 weeks. The PC and BCP sequences at baseline and the 72th week were determined using polymerase chain reaction (PCR) and direct sequencing. Serum HBsAg, HBeAg, alanine aminotransferase (ALT) and HBV DNA was quantified in the samples taken at baseline (week 0), during the treatment period (weeks 4, 8, 12, 24, 36 and 48), and during follow-up (weeks 60 and 72). RESULTS:Within the total cohort, wild-type (WT) virus was detectable in only 14 patients (20.29%), and mutants were detected in 55 patients (79.71%). The serum HBeAg level in the patients with mutant virus was significantly lower than that in the patients with WT virus (P=0.024). The proportion of WT, PC mutant, BCP mutant and PC+BCP mutant was significantly changed at baseline and week 72 (P=0.004). No significant difference of HBeAg seroconversion and combined response between patients with WT virus or mutants (PC, BCP and PC+BCP) was observed. CONCLUSION:PC and BCP mutations have no effect on the response of HBeAg-positive CHB patients to Peg-IFNα-2b. The proportion of each mutation was significantly changed before and after treatment.     

Effects of PAR-2 agonist peptide on proliferation and cytosolic calcium level in hepatoma cells

AIM: To investigate the effects of PAR-2 agonist peptide on the proliferation and cytosolic calcium concentration (［Ca2+］c) in human hepatoma cells HepG2. METHODS: Human hepatoma cell line HepG2 was cultured. The cells were treated with PAR-2 agonist peptide SLIGKV-NH2 and the reverse PAR-2 agonist peptide VKGILS-NH2, respectively. The ［Ca2+］c of hepatoma cells were measured by microfluorimetric techniques based on calcium indicator fura-2/AM. The influences on proliferation of hepatoma cells were determined by MTT method. The changes of cell cycle were evaluated by flow cytometry, and the changes of cyclin D1 mRNA expression were detected by RT-PCR. RESULTS: After treated with 50 μmol/L SLIGKV-NH2, a rapid rise of ［Ca2+］c in HepG2 cells was induced (P<0.01), percent S phase, G2/M phase and proliferation index (PI) of HepG2 cells were elevated (P<0.01), and cyclin D1 mRNA expression was significantly upregulated (P<0.01). The proliferation rates of HepG2 cells treated with 1-50 μmol/L SLIGKV-NH2 were significantly increased, and the effect was in a dose-dependent manner (P<0.01 or P<0.05). No statistical significance of the difference between VKGILS-NH2 and control group was observed (P>0.05). CONCLUSION: PAR-2 agonist peptide induces the rise of ［Ca2+］c in HepG2 cells, upregulates the expression of cyclin D1 mRNA, accelerates the progress of cell cycle, promotes the synthesis of DNA and the proliferation of hepatoma cells via activating PAR-2 in vitro.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Suberoylanilide hydroxamic acid induces apoptosis of HepG2 cells by endoplasmic reticulum stress apoptotic pathway

AIM: To investigate the effect of suberoylanilide hydroxamic acid (SAHA) on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells and to explore its possible mechanism. METHODS: HepG2 cells were treated with SAHA at different concentrations for 48 h. The proliferation of HepG2 cells was detected by real-time cellular analysis. The protein levels of acetylated histones H3K9 and H3K27, glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK) and p-PERK were determined by Western blot. The cell apoptosis was analyzed by flow cytometry. RESULTS: Compared with control group, treatment with SAHA at 0.1 μmol/L and 1 μmol/L for 48 h showed no significant inhibitory effect on the proliferation of HepG2 cells, while SAHA at 6 μmol/L and 12 μmol/L significantly inhibited the proliferation of HepG2 cells (P<0.05). The results of Western blot showed that the protein levels of acH3K9, acH3K27, GRP78 and p-PERK increased significantly after treated with SAHA at diffe-rent concentrations for 48 h, while the protein level of PERK was decreased significantly (P<0.05). The results of flow cytometry analysis showed that the apoptotic rates of the HepG2 cells increased with the increase in SAHA concentration. CONCLUSION: SAHA up-regulates the acetylation of H3K9 and H3K27 in the HepG2 cells and induces apoptosis of HepG2 cells by activating the endoplasmic reticulum stress-related apoptosis pathway.     

Effects of leptin on BSEP protein expression and signaling pathway in HepG2 cells

AIM: To investigate the effects of leptin on the expression of bile salt export pump (BSEP) and signaling pathway in human hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were cultured in vitro. Leptin at concentrations of 10^-8, 10^-7 and 10^-6 mol/L was used as a stimulating factor. The protein levels of adenosine monophosphate-activated protein kinase alpha subunit (AMPKa), phosphorylated AMPKa (p-AMPKa) and BSEP in the HepG2 cells at 24 h, 48 h and 72 h were detected by Western blotting. The optimal culture time and leptin concentration were selected, and compound C at concentration of 10 μmol/L was added to this group. The protein expression of BSEP was detected by Western blotting. RESULTS: Intervention of HepG2 cells with leptin for 72 h increased the protein expression of AMPKa gradually in a concentration-dependent manner, and leptin at concentration of 10^-6 mol/L induced the strongest AMPKa expression (P<0.01). Intervention of HepG2 cells with leptin for 24 h increased the phosphorylation level of AMPKa gradually in a dose-dependent manner (P<0.01). The effect of leptin on the increase in the protein expression of p-AMPKa was also in a time-dependent manner (P<0.01). After intervention with different concentrations of leptin for 24 h, the protein expression of BSEP in the HepG2 cells was gradually increased by the stimulation of leptin in a concentration- and time-dependent manner (P<0.01). Compared with NC group, the protein expression of BSEP in 10^-6 mol/L leptin group and 10^-6 mol/L leptin+10 μmol/L compound C group was increased at 72 h (P<0.01), and that in 10^-6 mol/L leptin+10 μmol/L compound C group was lower than that in 10^-6 mol/L leptin group at 72 h (P<0.01). CONCLUSION: Leptin promotes the protein expression of BSEP in HepG2 cells by leptin-AMPK-BSEP signaling pathway. Leptin promotes the increases in AMPKa protein and the level of phosphorylation of AMPKa in HepG2 cells.