Effect of Gupi Xiaoji Decoction on Pyroptosis of HepG2.2.15 Cells Based on Network Pharmacology and Molecular Docking / 中国实验方剂学杂志

ObjectiveTo screen the active antitumor components of Gupi Xiaoji decoction by network pharmacology and molecular docking based on the pyroptosis mediated by cysteinyl aspartate-specific protease 1 （Caspase-1） and explore its molecular mechanism in intervening in the pyroptosis of HepG2.2.15 cells through in vitro experiments. MethodThe compounds and targets of Gupi Xiaoji decoction were screened out by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） to obtain the corresponding gene symbols. The targets of Caspase-1 were collected from GeneCards，online mendelian inheritance in man（OMIM），PharmGKB，and TTD，and the compound-gene target regulatory network was constructed by Cytoscape. The protein-protein interaction（PPI） network was established and analyzed by STRING. The mechanism of the effective components of Gupi Xiaoji decoction on Caspase-1 was predicted by gene ontology（GO） functional enrichment and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analyses. The molecular docking was verified with AutoDock Vina. The plasma medicated with Gupi Xiaoji Decoction was prepared and HepG2.2.15 cells were cultured in vitro. HepG2.2.15 cells were divided into a blank plasma group，a VX-765 group，a VX-765+medicated plasma group， and a medicated plasma group. After 48 hours of intervention with 15% medicated plasma， the expression and distribution of gasdermin D-N （GSDMD-N） on the surface of the cell membrane were detected by immunofluorescence staining. The release of lactic dehydrogenase （LDH）， interleukin（IL）-1β，and IL-18 in the cell supernatant was measured by enzyme-linked immunosorbent assay（ELISA） kits. The expression of Caspase-1 and GSDMD-N was measured by Western blot. ResultThe mitogen-activated protein kinase 14 （MAPK14），MAPK1，protein kinase B1 （Akt1）， MAPK8， V-Jun sarcoma virus oncogene homolog （JUN）， and TP53 screened by network pharmacology were the main targets. The compounds 7-hydroxy-5，8-dimethoxy-2-phenyl-chromone，wogonin，rhamnazin，moslosooflavone，isorhamnetin，7-O-methylisomucronulatol，formononetin，calycosin，luteolin，quercetin，kaempferol，β-sitosterol，and baicalein screened by network pharmacology were the main active components of Gupi Xiaoji decoction. Go enrichment analysis showed that multiple biological processes were involved， including responses to oxidative stress and metal ions，ubiquitin-like protein ligase binding，and phosphatase binding. KEGG pathway enrichment analysis showed MAPK pathway，nuclear factor（NF）-κB pathway，p53 pathway， and hypoxia-inducible factor-1（HIF-1） pathway were involved. Molecular docking showed that the targets had good binding with the components. In vitro experiments displayed that compared with the blank plasma group，the VX-765 group showed weakened GSDMD-N fluorescence signal，reduced release of LDH，IL-1β，and IL-18，and declining expression of Caspase-1 and GSDMD-N（P<0.01）， and the medicated plasma group showed increased GSDMD-N fluorescence signal， increased release of LDH，IL-1β，and IL-18，and up-regulated expression of Caspase-1 and GSDMD-N（P<0.01）. ConclusionGupi Xiaoji Decoction can induce the pyroptosis of HepG2.2.15 cells by regulating Caspase-1 through multiple targets and multiple pathways.

Preparation of herpetolide A nanosuspension lyophilized powder and evaluation of its anti-hepatitis B virus activity / 中国中药杂志

To prepare the herpetolide A nanosuspension lyophilized powder(HPA-NS-LP), in order to investigate its anti-hepatitis B virus(HBV) activity and the dissolution in vitro. Herpetolide A nanosuspension(HPA-NS) was prepared by ultrasonic precipitation method. The formulation and process of HPA-NS were optimized by the single factor experiment. Lyophilized powder(HPA-NS-LP) was prepared by freeze-drying method. Scanning electron microscopy was used to observe morphology of HPA-NS-LP. Paddle method was used to determinate the dissolution of HPT-NS-LP in vitro. The anti-HBV activity of herpetolide A coarse suspension lyophilized powder(HPA-CS-LP) and HPA-NS-LP was evaluated by HepG2.2.15 cell model. The mean particle size of optimized HPA-NS was(173.46±4.36) nm, with a polydispersity index of 0.110±0.012. After redispersion, the mean particle size and the polydispersity index of HPA-NS-LP increased, with changes within a rational range. Scanning electron microscopy showed that HPA-NS-LP was spherical in shape. Cumulative dissolution rate of HPA-NS-LP was more than 90% in 2 hours, which was higher than that of HPA-CS-LP. Both HPA-CS-LP and HPA-NS-LP could effectively inhibit the secretion of HepG2.2.15 cell antigens(HBsAg and HBeAg), and the inhibitory effect of HPA-NS-LP was significantly higher than that of HPA CS-LP(P<0.05). HBV-DNA test showed that high, medium and low-dose HPA-NS-LP(50, 25, 12.5 mg·kg~(-1)) significantly decreased the level of HBV-DNA(P<0.05), and the effect was better than that of the same dose of HPA-CS-LP(P<0.05). The results revealed that HPA-NS-LP exhibited anti-HBV activity in vitro, and its effect was superior to that of HPA-CS-LP.

Study on Cellular Uptake Mechanism of HepG2.2.15 Cells to Nanoparticles Co-loaded with Syringopicroside and Hydroxytyrosol / 中国中医药信息杂志

Objective To investigate the uptake mechanism of HepG2.2.15 cells to the nanoparticles co-loaded with syringopicroside and hydroxytyrosol (SH-NPs). Methods The nanoparticles were prepared by using a nanoprecipitation method with mPEG-PLGA as nano-carrier co-loaded with syringopicroside and hydroxytyrosol. The uptake mechanism of HepG2.2.15 cells to SH-NPs was studied by fluorescence microscopy and flow cytometry using fluoresceineisothiocyanate (FITC) as a fluorescent marker. Results With colchicine as the inhibitor, the incubation time ranged from 0.5 to 24 h, the percentage of positive cells increased from 1.9% to 56.4%; When the drug concentration was 125, 250 μg/mL and 500 μg/mL, the positive cell percentages were 4.9%, 3.4% and 3.9%. With chloroquine as the inhibitor; the incubation time ranged from 0.5 to 24 h, the percentage of positive cells increased from 7.4% to 55.4%; When the drug concentration was 125, 250 and 500 μg/mL, the percentage of positive cells was 19.5%, 22.5% and 27.6%. Conclusion Colchicine and chloroquine have an inhibitory effect on HepG2.2.15 cells uptake, and the uptake of SH-NPs in HepG2.2.15 cells was positively correlated with drug concentration and incubation time. It can be concluded that the uptake mechanism of HepG2.2.15 cells to SH-NPs was nonspecific adsorption endocytosis.

Classical drug resistance mutations of HepG2.2.15 cells persistently induced by appropriate concentrations of adefovir / 解放军医学杂志

Objective To observe whether the classic drug-resistant mutations can be induced in various concentrations of adefovir (ADV)-treated HepG2.2.15 cells persistently and explore the mechanism for emergence of drug resistance.Methods HepG2.2.15 cells were cultured continually in 12-well plates with medium containing 0,0.01,0.1,1.0μmol/L concentration of ADV,and passaged every 3 days up to the 110th generations.The intracellular and supernatant HBV DNA was extracted every 10 generations.Intracellular HBV cccDNA was amplified by plasmid-safe ATP dependent DNase (PSAD) digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR assay.And the RT region of supernatant HBV DNA was amplified by one-tube nested PCR assay.Then the classic drug-resistant mutations of the RT region of intracellular cccDNA and supernatant HBV DNA were analyzed using direct PCR sequencing combined with clonal sequencing (more than 20 clones/sample).Results HBV DNA stably replicated in ADV-untreated cells (control group).The intracellular total DNA and cccDNA levels,supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 μmol/L and 0.1μmol/L ADV group.Drug resistant mutations were not detected up to the 110th generation in 0.01 μmol/L ADV group;while rtA181V+N236T mutations were detected at the110th generation in 0.1μmol/L ADV group.The 1.0μmol/L ADV group was ceased of culture at the 15th generation due to inhibited cell growth.Conclusion HBV cccDNA exists in HepG2.2.15 cells,and the classical drug-resistant mutations of rtA181V+N236T could be induced by proper concentration of ADV.

Construction of human microRNA-21 eukaryotic overexpression vector and its up-regulation of c-myc gene expression in HepG2 .2 .15 cells / 重庆医学

Objective To construct the miRNA‐21 eukaryotic overexpression vector pmR‐21 and to explore its regulation effect on the expression of c‐myc gene in HepG2 .2 .15 cells .Methods The miRNA‐21 precursor gene fragment pre‐miRNA‐21 was amplified by PCR ,then connected to the pmR‐mCherry plasmid vector after double enzyme digestion ,the accuracy of the recombi‐nant vector was verified by double enzyme digestion and sequencing ;then the recombinant vector was transfected into HepG2 .2 .15 cells ,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was used to detect the cell proliferation in each group .Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;miRNA‐21 expression level of the pmR‐21 recombinant vector group was significantly increased;c‐myc gene expression was increased in the pmR‐21 recombinant vector group at 72 h after transfection ,the cell proliferation in the pmR‐21 recombinant group was faster than that in the control group(P<0 .05) .Conclusion The pmR‐21 eukaryotic overexpression vector is successfully con‐structed ,this recombinant vector can express miRNA‐21 stably ;miRNA‐21 can up‐regulate c‐myc gene expression ,c‐myc gene is one of miR‐21′s targets for playing a cancer‐promoting action .

Effect of siRNA on hepatitis B virus X gene to HepG2.2.15 cells / 实用医学杂志

Objective Using chemically synthesized small interfering RNA (siRNA) transfected HepG2.2.15 cells to construct a cell model in interfering hepatitis B virus (HBV) X gene, studying the inhibi-tion of HBV replication and antigen expression in vitro. Methods After transfection of HepG2.2.15 cell for 24 h, 48 h, 72 h, detecting the cell supernatant of HBsAg and HBeAg by chemiluminescence immunoassay, the cell supernatant HBxAg protein by ELISA , the HBx mRNA relative expression of transfected cell was detected by fluorescence quantitative polymerase chain reaction (PCR), the ability of cell proliferation was detected by CCK-8 assay. Results After HBx-siRNA transfected HepG2.2.15 cells, cell proliferation ability was inhibited. The cell of HBx mRNA and the cell supernatant of HBxAg expression decreased (P < 0.05); at the same time it in-hibited the expression of HBsAg and HBeAg. The suppressed peak and the inhibition rate were 66% and 58%respectively at 72 h. The fluorescence quantitative PCR confirmed that expression of HBV DNA in the super-natant was decreased. Conclusion The HepG2.2.15 cell interference model of HBV X gene has been success-fully constructed, which has an effect of inhibiting proliferation of HepG2.2.15 cells and replication and expres-sion of HBV gene in vitro.

Thymopolypeptides combined with matrine type alkaloids suppress HBV replication / 中国中药杂志

To investigate the antiviral effect of thymopolypeptides combined with 4 kinds of matrine type alkaloids on HepG2.2.15 cells, oxymatrine, sophocarpidine, sophocarpine, and sophoridine (at concentration of 0.2 mmol•L⁻¹ respectively) were respectively combined with thymopolypeptides (0.025, 0.1 g•L⁻¹), and after 48 h and 72 h treatment on HepG2.2.15 cells, the cells and supernatants were collected. The cells activity in various groups was determined by CCK-8 method to evaluate the toxic effects of the drugs on HepG2.2.15 cells. Enzyme linked immunosorbent assay (ELISA) was used to determine HBeAg and HBsAg levels in cellular supernatants. HBV DNA levels in cellular supernatants andcells were quantified with fluorogenic quantitative PCR method; and the expression level of IFN-α in supernatants was detected with CBA method. The results indicated that single thymopolypeptides at 0.025-0.4 g•L⁻¹ had no toxicity to cells. Thymopolypeptides in this concentration range combined with 0.2 mmol•L⁻¹ matrine type alkaloids also had no toxicity to cells. Anti-HBV activity of drug combination was better than that of alkali or thymopolypeptides alone. Thymopolypeptides at 0.025 g•L⁻¹ had better inhibitory effect than thymopolypeptides at 0.1 g•L⁻¹ on intracellular HBV DNA expression, but the inhibitory effect on supernatant HBeAg level was on the contrary. Anti-HBV activity was similar between alkaloids combined with 0.1 g•L⁻¹ and alkaloids combined with 0.025 g•L⁻¹. There was no statistical difference in anti-HBV effect between various combined groups (P<0.05). In general, 72 h anti-HBV effect was better than 48 h anti-HBV effect (P<0.05). The expression of IFN-α was increased after drug combination, with positive correlation to the changes of other four indicators (P<0.05). In conclusion, oxymatrine, sophocarpidine, sophocarpine and sophoridine combined with thymopolypeptides could inhibit HBsAg and HBeAg secretion in HepG2.2.15 cells and HBV DNA replication, and further promote the antiviral effect by promoting the expression of IFN-α.

Effect and mechanism of danshensu on hepatitis B virus reverse transcriptase and antigen expression / 中国中药杂志

MTT assay was used in this study to investigate the inhibitory effect of danshensu on the activity of 2.2.15 cells among human hepatoma cell line (HepG2); indirect fluorescence labeling method was used to measure the changes of reactive oxygen levels in the cells; ELISA method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in cellular supernatants; HBV DNA level was measured with fluorogenic quantitative PCR method. The inhibitory effect of danshensu on HBV RT(hepatitis B virus reverse transcriptase) was studied by using enzyme inhibition dynamics, and the effect of danshensu on secondary structure of HBV reverse transcriptase was monitored by using circular dichroism. The results showed that danshensu had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC₅₀) of (15.35±2.43) μmol•L⁻¹; danshensu could significantly inhibit HBsAg and HBeAg expressions, and showed an inhibitory effect on HBV DNA replication. In addition, danshensu was an effective inhibitor for HBV reverse transcriptase [IC₅₀ (21.32±2.43) μmol•L⁻¹]. The fluorescence labeling results showed that the reactive oxygen levels in the cells were increased with the increase of danshensu concentration. Circular dichroism analysis showed that danshensu could induce partial change of conformation of HBV reverse transcriptase and gradually increased α-helical content. These results indicated that danshensu could make the structure of the enzyme become closer by binding to HBV reverse transcriptase, which was not conducive to the formation of the active center, so it could finally decrease the activity of HBV reverse transcriptase. Such decrease in enzyme activity would directly affect the HBV DNA replication, and combined with the decrease of the antigen levels, the effect of danshensu on HBV was increased.

Effects of xeroderma pigmentosum group D and p53 on replication of HBV / 中国病理生理杂志

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 inhibi-tor, 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-αgroup and pifithrin-αgroup.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 con-tent 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 ele-vated by the transfection of recombinant plasmid pEGFP-N2/XPD.The increase in XPD expression significantly down-regu-lated 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-αabolished 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.

Interleukin-10 Is Expressed in HepG2.2.15 Cells and Regulated by STAT1 Pathway / 华中科技大学学报（医学）（英德文版）

This study investigated the expression profiles of IL-10 gene in three human hepatoma cell lines including Huh7,HepG2,and HepG2 transfected with a plasmid containing hepatitis B virus (HBV) named HepG2.2.15.RT-PCR analysis demonstrated that IL-10 message RNA was absent in HepG2 and Huh7 cells,whereas it was present in HepG2.2.15 cells,which was consistent with ELISA result.Furthermore,except for lamivudine other antiviral treatments did not significantly decrease the HBV DNA level in HepG2.2.15 cells,while they had different effects on the expression of IL-10 protein,although stimulation by LPS had no significant effect.In addition,except for poly(I:C),the other treatments decreased the expression of IL-10 protein to different degrees,but had no significant effects on the expression of NF-κB and MyD88.Meanwhile,all treatments we used had effect on the expression of STAT1.In conclusion,IL-10 was expressed in HepG2.2.15 cells and STAT1 pathway might be involved in the regulation of IL-10 expression in HepG2.2.15 cells,but it was not the sole pathway,the exact mechanism warrants further study.

Inhibition of HBV replication by small interference RNA in HepG2.2.15 cells / 中华肝胆外科杂志

Objective Virus-specific RNA interference (RNAi) is a powerful inhibitor of gene expression and replication of HBV. It is known to have high efficiency, specificity, and few side effects. We wanted to evaluate the effects of siRNA silencing HBV replication on the growth of hepatocellular carcinomatic(HCC) cells to find out an ideal method for treatment of HCC. Methods We transfected siRNA into HepG2.2. 15 cells (HCC cell inserting HBV gene) and detected the HBsAg and HBV DNA copies for evaluating the inhibitory effects of siRNA. Then we evaluated cell growth and self-renewal ability after transfection of siRNA by MTT. Results The HBsAg level and HBV DNA copies were reduced after the transfection of siRNA, the highest inhibition rate was 83.9%,while the inhibition rate of HBV DNA copies reached 73. 4%. The siRNA group's growth ability and self-renewal rate were lower than the control group in 5 days. Conclusion siRNA can effectively inhibit HBV replication and expression in HepG2.2.15 cells and silencing HBV replication can inhibit HepG2.2.15 cell's growth and self-renewal.

The antiviral effect of interleukin 29 against hepatitis B virus in vitro / 中国生化药物杂志

Purpose To explore the antiviral effect of interleukin 29(IL-29) on hepatitis B virus in vitro.Methods To study the antiviral effect of IL-29 against hepatitis B virus by the amount of HBV mRNA detected .Through the quantity of mRNA translated from genes of MxA,2′,5′-OAS,PKR and RNase L as well as the signal pathway induced by IL-29,we used RT-PCR and Western blot to discuss the anti-hepatitis B virus mechanism which was stimulated by IL-29.Results The amount of HBV mRNA in HepG2.2.15 cells was reduced by stimulation of IL-29.The expression of MxA and 2′,5′-OAS was up-regulated,as well as P-ERK and P-AKT were activated by IL-29.Conclusion These findings showed that IL-29 had obvious antiviral activity towards HBV in HepG2.2.15 cells.

Chemiluminescent Southern blot System for Detection of HBV Replication in vitro and Its Application for Analysis the Drugs Inhibition to HBV / 中国生物工程杂志

Objective:To stablish of chemiluminescent Southern blot detection system for examining HBV DNA replication intermediates in HepG2.2.15,and analyse the inhibition of HBV replication with three kind of drugs with different targets.Methods:The HBV DNA replication intermediates were extracted and analyzed by Southern blot with HBV probe,which(pTHBV1047) was labelling with digoxigenin.The results of the hybridization were detected by chemiluminescent,and the condition of hybridization was optimized.After treated with lamivudine,Bay41-4109,?-Galcer in different concentration,the HBV DNA from the HepG2215 cells were detected with the system.Results:the sensitivity of the system was 1pg of pTHBV1047,and HBV specific positive signals was detected with the DNA from HepG2.2.15.The three kinds of drugs can inhibit the HBV replication obviously with chemiluminescent Southern blot detection system,the IC50 were 1.53?mol/L,0.41?mol/L,0.01?mol/L.Conclusion:The HBV replication intermediates from the cell of HepG2.2.15 can reflect the antiviral effect accurately with different targets drugs and this mothod would be used in the study of Chinese-midicine.

Depressant Effect of siRNA on the Expression and Replication of Hepatitis B Virus in HepG2.2.15 Cell / 实用儿科临床杂志

Objective To explore the siRNA as a new antiviral therapy,evaluate the inhibition effect of siRNA based on vector on the HBV of HepG2.2.15 cell,and observe the side effect and toxicity of siRNA vector on cells and the off-target effect of siRNA.Methods Three pairs of siRNA duplexes targeting HBV C gene were designed as double strands,and the duplex were annealed and ligated into the p-Silencer-Cmv 4.1-hygro vector.The ligation products were used to transform JM109 cells.The clones with shRNA were obtained,and the vectors were purified.After the initial identification of the vector with agarose gel and the size of the inserted sequence got examined by native polyacrylamide gel electrophoresis,furthermore the sequencing was further carried out.The recombinant plasmids were purified with ultrapure Midipreps DNA Purification System.Then HepG2.2.15 cells were transfected with the plasmid mixed with siPort XP-1.The expression of HBsAg and HBeAg were detected by immunofluorescence and Western blot,and the HBV RNA was investigated by RT-PCR.Furthermore the real-time quantitive PCR was carried out to detect the changes of HBV DNA.In order to evaluate the toxicity of the shRNA,MTT was used to examine the growth rate and curve of cells.The ELISA was performed to detect the changes of interferon-? (IFN-?).Results The Western blot showed that the HBsAg and HBeAg protein were suppressed with (81.15?0.69)%,(88.12?0.92)% respectively by vector p-C2 on the third day of post-transfection.It had the similar result indicated by immunofluorescence.And the RT-PCR showed that the specific siRNA targeting HBV C gene could markedly suppress the expression of HBV mRNA and the HBV C gene mRNA was inhibited with 96.9%.The real-time quantitive PCR showed that the specific functional siRNA could markedly suppress HBV DNA copy with two orders of magnitude,while the siRNA vector had no effect on the growth of cell showed by MTT detection.Compared with the non-transfected group and p-NC group,the IFN-? level was almost the same with siRNA p-C1,p-C2,p-C3 groups.Conclusions The siRNA based on the expression vector can suppress the expression and replication of HBV in HepG2.2.15 cell.The inhibition effect was specific and had a certain dependency on siRNA concentration.No toxicity effect was found in the study.And the drug resistance wouldn′t happen because the silence was based on the split of gene.

Effect of RNAi on HBV replication and expression / 吉林大学学报(医学版)

Objective To observe the suppression of special shRNA producing plasmid to hepatitis B virus(HBV) S gene and C gene on HBV replication and expression in HepG2.2.15 cells.Methods pSilenceCircle-U6 including pol Ⅲ promoter was used to construct HBV special shRNA producing plasmid as SC-S and SC-C.The experimental groups included SC-S group,SC-C group,unrelated control SC-N group and blank control group.With different dosages and at different time,shRNA producing plasmid was transfected into HepG2.2.15 cells.HBeAg and HBsAg in the culture media was detected by ELISA assay and HBV DNA in the culture media was measured by dot blotting assay. Results The recombinant shRNA producing plasmid with target sequence was constructed successfully.The inhibitory rates of HBeAg and HBsAg expressions by SC-S were much higher than those by SC-C.The inhibitory effects of HBeAg and HBsAg expressions were increased when the dose of SC-S was greater.The inhibitory effects of HBeAg and HBsAg expressions by SC-S were significant on the 3rd day after transfection and the inhibitory effect was the strongest on the 6th day.The inhibitory rate was still higher on the 9th day after transfection.Dot blotting assay showed the inhibitory effect of HBV replication by SC-S was greater than that by SC-C.Conclusion The shRNA producing plasmid with HBV S gene and C gene can be highly effective to inhibit the replication and expression of HBV.

Effect of Glycyrrhizin on Heptitis B Virus Infection in HepG2.2.15 Cell Line / 中国中医药信息杂志

Objective To investigate the effect of glycyrrhizin(GL) on the expression of hepatitis B virus e antigen(HBeAg),hepatitis B virus surface antigen(HBsAg),HBV DNA levels and cell proliferation in HepG2.2.15 cell line.Methods HBV DNA level was examined by Real-time PCR.HBsAg and HBeAg levels were examined by ELASA,and cell proliferation was examined by MTT before and after stimulated with GL.The GL groups were compared with the blank control group.Results HBsAg levels in the GL groups were inhibited in dose-dependent manner compared with the blank control group(P

HBcAg-specific CD8~+T Cells Inhibit HBV Replication in vitro / 医学研究杂志

Objective To investigate the effects of HBcAg-specific CD8+T cells on inhibiting HBV replication in vitro,and to search the cytokine of noncytolytic mechanisms in viral clearance. Methods By the method of coculture of HepG2.2.15 cell (target cells) with HLA-A2 matched HBcAg-specific CD8+T cell clone (effector cells) at E:T ratios of 1:50,and monitoring HBV production (HBsAg,HBeAg,and HBV-DNA)in coculture supernatants at 24h,48h and 72h,the percentage of decrease in HBV replication level was observed. Furthermore,blocking experiment with neutralizing mAbs to IFN-? was performed to evaluate the effect of this cytokine. Results CD8+T clone produced high levels of IFN-?following coculture with 2.2.15 cells. HBsAg,HBeAg and HBV-DNA in coculture supernatants were significantly reduced,and the greatest effect was observed at 72h by 54.55%,50.36% and 74.55%,respectively. The reduction of HBV DNA was decreased followed by using neutralizing mAbs to IFN-?. The maximum activity of cytotoxicity of target cells was at 24h by 15.66%. Conclusion ①HBV-specific CD8+T cells inhibit HBV replication by cytolytic and noncytolytic mechanisms.②The effect of noncytolytic mechanisms is mainly mediated by IFN-?.

Experimental study on effects of Chinese medicine ICA on the inhibition of cell proliferation and reversion of immune escape in hepatocarcinoma cell line HepG2.2.15 cells / 中国免疫学杂志

Objective:To study the effects of ICA on HepG2.2.15 cell proliferation, their sensitivity to the lysis by CD3AK effector cell, to investigate the reversal action of ICA on hepatocarcinoma cells from immune escape through Fas/FasL pathway.To provide the theoretical and experimental bases for ICA development.Methods:MTT assay was used to detect cell proliferation and CD3AK cells cytotoxicity activity;flow cytometry assay was used to examine expression of surface molecules and apoptosis rate of HepG2.2.15 cells.Results:When HepG2.2.15 cells line was treated with 50 ?g/ml ICA,a significant reduction of the rate of cell proliferation was observed. Inhibition rate at 48h was 22.04%,and 29.68% at 72h.Kinetic study showed that inhibition of cell proliferation was time dependent (P0.05).ICA could inhibit apoptosis of Jurkat cells induced by HepG2.2.15 cells. In the co-culture system of HepG2.2.15 cell and Jurkat T cell, apoptosis ratio of Jurkat cell was reduced from 46.66% to 18.20% by ICA (P

Mycophenolate acid inhibits hepatitis B virus replication in HepG2.2.15 cells / 中国病理生理杂志

AIM: To investigate the effect of mycophenolate acid(MPA) on hepatitis B virus(HBV) replication in vitro.METHODS: In the presence or absence of guanosine,the HepG2.2.15 cells were treated with different concentrations of MPA(1-20 mg/L) for 4 days.Hepatitis B surface antigen(HBsAg) and hepatitis Be antigen(HBeAg) in supernatant were detected by ELISA.Intracellular HBV core mRNA and HBV DNA were analyzed by reverse transcription-polymerase chain reaction(RT-PCR) and slot blot hybridization,respectively.RESULTS: MPA suppressed the expression of HBsAg and HBeAg,and inhibited the replication of HBV DNA.The effect of MPA on HBV replication was reversed by addition of exogenous guanosine.CONCLUSION: MPA suppresses the expression of HBsAg,HBeAg and replication of HBV DNA in HepG2.2.15 cells.Reducing the synthesis of guanosine nucleotides may be involved in the mechanism of the inhibitory activity of MPA on HBV replication.

Association of Hepatitis B virus infection and the expression of Toll-like receptors 2 and 4 in HepG2 cells / 中华传染病杂志

Objective In order to explore the roles of TLR2 and TLR4 in the hepatocyte dam- age caused hy hepatitis B virus infection,and to find whether LPS can affect the damage of hepato- cytes pre-and pos-HBV infection,we detected the changes of TLR2 and TLR4 expressions in hu- man hepatocyte lines HepG2 cells and 2.2.15 cells.Methods HepG2 ceils are most similar to normal human hepatocytes and 2.2.15 ceils are HepG2 cells infected with HBV.We selected these two cell lines to study the differences of TLR2 and TLR4 expression between HepG2 cells before and after HBV infection.In this research,both HepG2 and 2.2.15 cells were stimulated with 0?g/ml, 1?g/ml,10?g/ml,100?g/ml,1 mg/ml and 10 mg/ml LPS.Then the expression of protein of TLR2 and TLR4 were examined by immuno-histochemistry(IHC).The cnRNA of HepG2 and 2.2.15 ceils stimulated with 0?g/ml and 10 mg/ml LPS were examined by reversal transcription-pol- ymerase chain reaction(RT-PCR).Thereafter,the apoptosis of HepG2 and HepG2.2.15 cells were examined by flow cytometry(FC),and the expressions of HBsAg and HBeAg of HepG2.2.15 cells tested with Abbott kits.Results IHC and RT-PCR analysis revealed that TLR2 and TLR4 expres- sions could he detected in both HepG2 and 2.2.15 cells.Moreover,without immune activation, TLR2 and TLR4 expressions were higher in the presence of higher concentrations of LPS.FC analy sis revealed that no apoptosis detected in HepG2 ceils stimulated with LPS in this research,but apop- tosis could be detected in 2.2.15 cells when treated with the same factors.Furthermore,the apoptosis ratios increased with the increase of LPS concentrations.When concentrations of LPS were 1?g/ml, 10?g/ml,100?g/ml,1 mg/ml and 10 mg/ml,the apoptosis ratios were 1.94%,3.03%,3.50%, 3.72%,5.30%,respectively.Abbott analysis revealed that expressions of HBsAg and HBeAg of 2.2.15 cells stimulated with LPS were lower than those not stimulated with LPS.Conclusion HBV can affect the expressions of TLR2 and TLR4 in HepG2 cell lines.LPS can lead 2.2.15 cells to apop- tosis but not HepG2 cells.Although LPS cannot damage normal hepatocytes,it might aggravate hep- atocytes damage when their microenvironment was changed by HBV infection.