Betulinic acid-mediated inhibitory effect on hepatitis B virus by suppression of manganese superoxide dismutase expression Dachun Yao1,2, Huawen Li3, Yulan Gou1,4, Haimou Zhang5, Athanasios G Vlessidis2, Haiyan Zhou4, Nicholaos P Evmiridis2 and Zhengxiang Liu1 Internal Medicine of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Greece Department of Nutrition and Food Hygiene, Guangdong Medical College, China The First Hospital of Wuhan, China School of Life Sciences, Hubei University, Wuhan, China Keywords apoptosis; CREB; mitochondrial; Pulsatilla chinensis; reactive oxygen species Correspondence D Yao and Z Lin, Internal Medicine of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Fax: +86 27 83662622 Tel: +86 27 83662601 E-mail: dachun927@hotmail.com; zxliu_tjmu@yahoo.com (Received December 2008, revised 26 February 2009, accepted 27 February 2009) doi:10.1111/j.1742-4658.2009.06988.x The betulinic acid (BetA) purified from Pulsatilla chinensis (PC) has been found to have selective inhibitory effects on hepatitis B virus (HBV) In hepatocytes from HBV-transgenic mice, we showed that BetA substantially inhibited HBV replication by downregulation of manganese superoxide dismutase (SOD2) expression, with subsequent reactive oxygen species generation and mitochondrial dysfunction Also, the HBV X protein (HBx) is suppressed and translocated into the mitochondria followed by cytochrome c release Further investigation revealed that SOD2 expression was suppressed by BetA-induced cAMP-response element-binding protein dephosphorylation at Ser133, which subsequently prevented SOD2 transcription through the cAMP-response element-binding protein-binding motif on the SOD2 promoter SOD2 overexpression abolished the inhibitory effect of BetA on HBV replication, whereas SOD2 knockdown mimicked this effect, indicating that BetA-mediated HBV clearance was due to modulation of the mitochondrial redox balance This observation was further confirmed in HBV-transgenic mice, where both BetA and PC crude extracts suppressed SOD2 expression, with enhanced reactive oxygen species generation in liver tissues followed by substantial HBV clearance We conclude that BetA from PC could be a good candidate for anti-HBV drug development Hepatitis B virus (HBV) infection is a prevalent health problem, affecting 350 million people worldwide; it causes acute and chronic hepatitis, some cases of which may progress into cirrhosis and hepatocellular carcinoma [1] Chronic HBV patients are currently treated with interferon or some nucleotide analogs, including lamivudine and adefovir, but the poor success and frequent recurrence after cessation of therapy require new strategies for terminating this viral infection Some complementary and alternative medicines, Abbreviations BetA, betulinic acid; CREB, cAMP-response element-binding protein; DiOC6, 3,3¢-dihexiloxadicarbocyanine; HBeAg, hepatitis B external core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; MMP, mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PC, Pulsatilla chinensis; PKA, protein kinase A; PKD, protein kinase D; ROS, reactive oxygen species; siCREB, small interfering RNA for cAMP-response element-binding protein; siRNA, small interfering RNA; siSOD2, small interfering RNA for manganese superoxide dismutase; SOD2, manganese superoxide dismutase; TUNEL, deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2599 Betulinic acid inhibits hepatitis B virus D Yao et al including some herbs, have been used for centuries to treat viral hepatitis, but they are still not widely accepted by conventional medicine, owing to the lack of mechanisms and purity of herbs [2] Pulsatilla chinensis (PC) is a traditional Chinese herb used for the treatment of amoebic diseases, vaginal trichomoniasis, and bacterial infections, owing to its antiamoebic, antibacterial and antitrichomonal activities [3] Recently, this herb was used for the treatment of a hepatitis B patient, according to an old recipe in a specific area of China (Yichang, Hubei), with satisfactory results for HBV clearance In order to determine the mechanism of this, about 30 components from PC were isolated, and each of them was tested for HBV clearance The results revealed that the active components were betulinic acid (BetA) and its derivatives [4,5] BetA, identified as a pentacyclic triterpene, is widely available from common natural sources and possesses several biological properties, including antiinflammatory, antiviral, antimalarial, and antimicrobial, as well as impressive anticancer and anti-HIV activities [6–8], although the exact mechanism remains unclear [9,10] Manganese superoxide dismutase (SOD2) is an antioxidant enzyme located in mitochondria that can scavenge superoxide anions (O2·)) to form hydrogen peroxide Suppression of SOD2 expression may lead to the overgeneration of reactive oxygen species (ROS) from mitochondria, and this can subsequently trigger mitochondrial dysfunction and apoptosis Altered SOD2 expression is considered to be both beneficial and detrimental For instance, overexpression of SOD2 could be protective against ROS-mediated cell damage, but it may also increase the invasiveness of tumors and increase the possibility of infection [11,12] Several transcription factors, including specificity protein and nuclear factor-jB [13,14], as well as methylation [15,16], have been studied extensively for the regulation of SOD2 expression, whereas there are few reports on the role of cAMP-response element-binding protein (CREB) in SOD2 expression [17,18] CREB binds via its basic leucine zipper domain as a dimer to cAMP response elements containing the consensus motif 5¢-TGACGTCA-3¢; these are present in the promoters of many genes in which transcription rates are strongly regulated by cAMP CREB stimulates cellular gene transcription via the protein kinase A (PKA)-mediated phosphorylation of CREB at Ser133 [19] Ser133 phosphorylation of CREB, in turn, promotes recruitment of the coactivator paralogs CREB-binding protein and p300 via a kinase-inducible domain in CREB, which appears to be sufficient for the induction of cellular genes [20,21] On the other hand, inhibition of CREB 2600 phosphorylation or dephosphorylated CREB may be a negative regulator of CREB-responsive genes [22,23] In an effort to investigate the mechanism of the inhibitory effect of BetA on HBV, BetA was isolated from PC to treat hepatocytes from HBV-transgenic mice We found that SOD2 was downregulated by BetA-induced CREB dephosphorylation at Ser133 through the CREB-binding motif on the SOD2 promoter SOD2 suppression-mediated ROS generation subsequently inhibited HBV replication, decreased HBV X protein (HBx) total level, and translocated HBx to the mitochondria followed by cytochrome c release Overexpression of SOD2 totally abolished the BetA-mediated HBV-inhibitory effect, whereas SOD2 knockdown mimicked this effect, indicating that the BetA-induced HBV-inhibitory effect is due to SOD2 suppression and subsequent ROS generation Further in vivo experiments with HBV-transgenic mice confirmed our hypothesis; we found that BetA or PC crude extracts achieved significant HBV clearance, with decreased SOD2 expression and increased ROS generation in liver tissue This is the first time that suppression of SOD2 expression has been found to be the mechanism by which BetA inhibits HBV replication Results BetA-induced selective cytotoxicity in HBV-infected hepatocytes We first examined the cytotoxicity of BetA in wildtype (WT) and HBV-infected hepatocytes Different dosages of BetA were used to treat the cells for 48 h The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay results showed that there was little effect on WT cells, whereas BetA treatment caused significant cytotoxicity in HBV-infected cells (Fig 1A) Also, the time course results showed that WT cells were more resistant to BetA-mediated cytotoxicity than HBV-infected cells (Fig 1B) On the basis of the above observation, we further evaluated BetA-mediated cell proliferation; as shown in Fig 1C, HBV-infected cells showed a higher DNA synthesis rate than WT cells with a low dose (5 lgỈmL)1) of BetA, whereas with a high dose (15 lgỈmL)1), the DNA synthesis rate of HBV-infected cells was substantially decreased, but WT cells showed no significant decrease On the other hand, when the BetA dose was even higher (20 lgỈmL)1), the DNA synthesis rate of HBV-infected cells was substantially inhibited, whereas no difference was found in WT cells, indicating that BetA-induced cytotoxicity was specific to HBV-infected cells FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS D Yao et al A Betulinic acid inhibits hepatitis B virus C WT HBV 300 [3H]-thymidine incorporation (% control) Cell viability (A/106 cells) 3.5 2.5 * * Cell viability (A/106 cells) HBV 2.5 1.5 E 12 24 36 60 48 72 Betulinic acid exposing time (h) 84 CTL BetA 200 100 HBV 300 ¶ 200 * 100 HBV H 20 CTL BetA * 10 * HBV WT Caspase-3 activity (pmol–1·min–1·mg–1) Apoptosis rate (%) * CTL BetA * WT 15 ¶ * 100 G * 300 F 200 20 µg·mL–1 400 ¶ CTL BetA 15 µg·mL–1 WT 300 Intracellular ATP level (Arbitrary units) 96 ROS formation (Arbitrary units) D 10 µg·mL–1 µg·mL–1 WT ¶ 30 3.5 * ¶ 100 Relative ΔΨm B 10 15 20 25 Dosages of betulinic acid (µg·mL–1) * 200 1.5 WT HBV 300 CTL BetA * 200 100 * 0 WT HBV WT HBV Fig BetA-mediated selective effect on HBV-infected hepatocytes (A) WT or HBV-infected (HBV) hepatocytes were treated with different doses of BetA for 48 h, and cell viability was measured (B) Cells were treated with 15 lgỈmL)1 BetA for different times, and cell viability was measured (C) Cells were treated with different doses of BetA as indicated for 48 h, and then incubated with [3H]thymidine for h to measure the inhibitory effect of BetA on cell differentiation by the [3H]thymidine incorporation assay *P < 0.05 versus WT; –P < 0.05 versus lgỈmL)1 group (D–H) Cells were treated with 15 lgỈmL)1 BetA for 48 h, and the related parameters were measured (D) BetAinduced ROS generation (E) Intracellular ATP level (F) MMP (Dwm) (G) Apoptosis rate determined by TUNEL assay (H) Intracellular caspase-3 activity *P < 0.05 versus control (CTL); –P < 0.05 versus WT group FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2601 Betulinic acid inhibits hepatitis B virus D Yao et al BetA-mediated ROS generation and mitochondrial dysfunction was specific to HBV-infected hepatocytes BetA-mediated selective SOD2 suppression in HBV-infected hepatocytes In order to clarify the effect of BetA, a microarray assay after treatment with 15 lgỈmL)1 BetA for 48 h was conducted BetA specifically decreased SOD2 mRNA expression in HBV-infected cells, whereas little difference was seen in WT cells (data not shown) Real-time PCR was performed for confirmatory purposes, and suggested that the SOD2 mRNA level was decreased about  2.4-fold in HBV-infected cells treated with BetA as compared with the control, but showed no difference in WT cells (Fig 2A) Western blotting to measure the protein level (Fig 2B) showed a significant decrease in SOD2 protein in HBV-infected cells after BetA treatment, but no change in WT cells SOD2 enzyme activity (Fig 2C) decreased significantly in HBV-infected cells after BetA treatment, whereas little difference was found in WT cells The BetA-mediated SOD2 transcriptional response element was located at the CREB-binding site (nucleotide )1335) on the SOD2 promoter The mechanism of BetA-mediated SOD2 suppression was investigated further To localize the regulatory elements required for transcriptional suppression of 2602 400 CTL BetA ¶ 300 200 * 100 HBV WT SOD2 protein by Western blot (Arbitrary units) B 400 C 400 300 200 ¶ CTL BetA * 100 HBV WT SOD2 protein activity (Arbitrary units) ROS generation was then examined, and the results are shown in Fig 1D BetA substantially induced ROS generation in HBV-infected hepatocytes, as compared with WT cells As BetA inhibited HBV-infected cell growth with increased ROS generation, we hypothesized that BetA might also specifically affect mitochondrial function in those cells Measurement of intracellular ATP generation (Fig 1E) revealed that BetA treatment substantially decreased intracellular ATP generation in HBV-infected cells, but showed no effect on WT cells In addition, mitochondrial membrane protential (MMP, DWm) was substantially decreased in HBV-infected cells, but no difference was found in WT cells (Fig 1F) Finally, the apoptosis rates determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Fig 1G) and caspase-3 activity (Fig 1H) were assessed The results showed that BetA substantially increased the apoptosis rate and caspase-3 activity in HBV-infected cells as compared with WT cells SOD2 mRNA level by qPCR (Arbitrary units) A 300 CTL BetA 200 ¶ * 100 WT HBV Fig BetA-mediated selective SOD2 suppression in HBV-infected hepatocytes The 80% confluent WT or HBV-infected cells were treated with 15 lgỈmL)1 BetA for 48 h, and SOD2 expression and activity were measured (A) mRNA level (B) Protein level (C) SOD2 enzyme activity *P < 0.05 versus control (CTL); –P < 0.05 versus WT group the SOD2 gene by BetA treatment, progressive 5¢promoter deletion constructs, including )2000, )1500, )1200, )1000, )500, )200, )100, and 0, were generated (numbered according to Ensembl Transcript ID: ENST00000337404) As shown in Fig 3A, the )2000 and )1500 constructs showed a decrease in activity of about 55%, whereas, with other deletions from )1200 to 0, the reporter activity showed no significant decrease after BetA treatment These data indicate that promoter elements between )1500 and )1200 are responsible for BetA-induced transcriptional suppression of the SOD2 promoter Comparison of these sequences with transcription factor databases (TFSEARCH) revealed several potential binding motifs, including GATA ()1488), c-Ets ()1377), CREB ()1335) and NRF2 ()1247) The FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS D Yao et al A Betulinic acid inhibits hepatitis B virus SOD2 transcriptional activity (Arbitrary units) 120 CTL Bet A 100 80 # # * * 60 * 40 * * * 20 B –1500 –1335 +308 Luc Luc CREB CREB + 308 Luc Luc 150 100 + 308 Luc uc CREB CREB * SOD2–1500 50 –1335 –1500 SOD2 transcriptional activity (Arbitrary units) TGACGTCT TGACGTCT SO D 2– SO D 2– 20 00 SO D 2– 15 00 SO D 2– 12 00 SO D 2– 10 00 SO D 2– 50 SO D 2– 20 SO D 2– 10 0 CTL T GACGT CT BetA TGATGTCT –1335 SOD2–1500 –1500 CREB CREB Mut–1335(T) As shown in Fig 3B, the SOD2 reporter with the CREB-binding motif single mutation at )1335 from nucleotides C to T totally abolished the BetAinduced SOD2 suppression, whereas the mutations in other motifs did not decrease the effect (data not shown) This indicates that the CREB motif at )1335 is required for BetA responsiveness of the SOD2 promoter As the CREB-binding motif was localized to the BetA-responsive element, the effect of CREB protein on SOD2 reporter activity was examined The SOD2 WT reporter (SOD2 )1500) showed suppression by BetA treatment, overexpression of CREB in the presence of BetA totally abolished the effect, and CREB knockdown alone [small interfering RNA (siRNA) for CREB (siCREB)] mimicked this effect (Fig 3C) On the other hand, the SOD2 mutation reporter [SOD2 )1500 ⁄ )1335(T)] showed no effect of either BetA, overexpression of CREB in the presence of BetA, or siCREB alone, further demonstrating that the BetA-induced SOD2 suppression is regulated by CREB BetA-mediated SOD2 suppression is due to BetA-induced CREB dephosphorylation 200 SOD2 transcriptional activity (Arbitrary units) C SOD2–1500 SOD2–1500/ – 1335 (T) 100 * * CTL BetA BetA / CREB si CREB Fig Mapping of the BetA-responsive element on the SOD2 promoter (A) HBV-infected hepatocytes were transfected with the indicated SOD2 reporter constructs, and then treated with either control (CTL) or 15 lgỈmL)1 BetA for 48 h; the SOD2 reporter activity was then measured *P < 0.05 versus CTL in the SOD2– 2000 group; –P < 0.05 versus CTL (B) The above cells were transfected with either SOD2–1500 reporter WT construct or SOD2–1500 single mutant )1335(T); after the treatment as indicated above, SOD2 reporter activity was measured *P < 0.05 versus CTL (C) HBV-infected hepatocytes were transfected with either SOD2 )1500 or SOD2 )1500 ⁄ )1335(T) single mutant reporters, and then treated with CTL, 15 lgỈmL)1 BetA, BetA with CREB overexpression (BetA ⁄ CREB›) or siCREB for 48 h, and SOD2 reporter activity was measured *P < 0.05 versus CTL in the SOD2 )1500 group possible involvement of these motifs in BetA-induced SOD2 transcriptional suppression was explored using a series of luciferase constructs with single mutations We have shown transcriptional activities of SOD2 that responsible to BetA treatment is due to the existence of CREB-binding elements on SOD2 promoter Here, we further confirmed the CREB-binding activity through chromatin immunoprecipitation analysis, as shown in Fig 4A After immunoprecipitation and reversal of the crosslinking, the endogenous SOD2 promoter was enriched by real-time PCR amplification, using specific primers that cover the CREBbinding motif The results showed that the PCR product was decreased to 47% after BetA treatment as compared with the control group, and that the effect was totally abolished by CREB overexpression in the presence of BetA, whereas CREB knockdown (siCREB) mimicked the effect As it is well known that CREB activity mainly depends on phosphorylation at Ser133, we next measured the levels of both CREB protein and CREB protein phosphorylated at Ser133 (pCREB) As shown in Fig 4B,C, the total CREB protein level did not change after BetA treatment as compared with control, whereas the pCREB level decreased by 42% On the other hand, overexpression of CREB in the presence of BetA increased the CREB level 1.7-fold, but did not increase the pCREB level, whereas knockdown of CREB (siCREB) decreased the levels of both CREB protein and pCREB Using the above treatment, we next measured the SOD2 mRNA level (Fig 4D) and FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2603 Betulinic acid inhibits hepatitis B virus D Yao et al D 200 100 * 50 100 BetA/CREB CTL E 300 (Arbitrary units) siCREB 200 * 100 * CTL BetA BetA/CREB siCREB 100 * * CTL BetA BetA/CREB siCREB F 200 * * (Arbitrary units) 100 pCREB level by Western blot (Arbitrary units) 200 pCREB (Ser133) level by Western blot BetA/CREB 200 siCREB C BetA 300 SOD2 protein level by Western blot (Arbitrary units) B BetA * * CTL CREB protein level by Western blot * SOD2 mRNA level by qPCR (Arbitrary units) 150 (Arbitrary units) qPCR on SOD2 promoter by CREB ChIP A * 100 *¶ CTL BetA BetA/CREB siCREB CREB PKA BetA + – – + + – + + + Fig BetA-mediated SOD2 suppression was due to direct inhibition of CREB phosphorylation (A) HBV-infected hepatocytes were treated with control (CTL), 15 lgỈmL)1 BetA, BetA with CREB overexpression (BetA ⁄ CREB›) or siRNA for CREB (siCREB) for 48 h; the chromatin from treated cells was immunoprecipitated with CREB antibody, and the SOD2 promoter that covers the CREB-binding motif was amplified by quantitative PCR (qPCR) (B–E) The cells treated as above were used for measurement of CREB protein level (B), pCREB protein level (C), SOD2 mRNA level (D), and SOD2 protein level (E) *P < 0.05 versus CTL for (A)–(E) (F) In vitro-purified proteins were phosphorylated by PKA in the presence or absence of BetA, and pCREB was measured by western blotting *P < 0.05 versus first panel; –P < 0.05 versus second panel protein level (Fig 4E) The results showed that both SOD2 protein expression and mRNA expression were decreased after BetA treatment, and that this effect was abolished by CREB overexpression in the presence of BetA, but was mimicked by siCREB This indicates that SOD2 expression is regulated by CREB phosphorylation at Ser133 As we had already shown that BetA treatment decreased CREB 2604 phosphorylation at Ser133 (Fig 4C), we next performed in vitro experiments to determine whether BetA could inhibit CREB phosphorylation directly As shown in Fig 4F, the purified CREB was substantially phosphorylated at Ser133 in the presence of PKA, whereas phosphorylation was markedly inhibited by BetA, indicating that BetA could directly inhibit CREB phosphorylation, and this FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS Betulinic acid inhibits hepatitis B virus 200 BetA siCREB * * 100 * * * * Total lysate Mitochondria Cytosol –1 BetA siCREB * * 200 100 * * Total lysate Mitochondria Cytosol 20 CTL BetA 15 * 10 * * *¶ * * SOD2 mRNA level by qPCR (Arbitrary units) E D Apoptosis rate (%) CTL F 400 CTL BetA * 300 * 200 100 0 WT WT-HBx HBV WT WT-HBx HBV 300 CTL BetA –1 CTL C 300 Caspase-3 activity (pmol·min ·mg ) 300 * 200 100 * * * * HBx protein in mitochondria by Western blot (Arbitrary units) B HBx protein level by Western blot (Arbitrary units) A CytC protein level by Western blot (Arbitrary units) D Yao et al WT WT-HBx *¶ HBV 400 CTL BetA 300 * 200 ? 100 HBV Fig BetA reduces the level of HBx and translocates HBx to mitochondria through SOD2 suppression and subsequent ROS generation (A, B) HBV-infected hepatocytes were treated with 15 lgỈmL)1 BetA for 48 h, the cells were separated as mitochondrial and cytosolic fractions, and the protein levels were measured by western blotting (A) HBx protein (B) Cytochrome c (CytC) protein *P < 0.05 versus control (CTL) group (C–E) WT hepatocytes, WT cells overexpressing HBX (WT-HBx cells) or HBV-infected hepatocytes were treated with either CTL, 15 lgỈmL)1BetA or BetA with CREB overexpression (BetA ⁄ CREB›) for 48 h (C) Intracellular caspase-3 activity *P < 0.05 versus CTL; –P < 0.05 versus BetA in the HBV-infected group (D) Apoptosis rate determined by TUNEL assay *P < 0.05 versus CTL; –P < 0.05 versus BetA in the HBV-infected group (E) SOD2 mRNA level *P < 0.05 versus CTL in the WT group (F) The mitochondrial fraction from the HBVinfected hepatocytes treated as above was used for analysis of HBx by western blotting The WT HBx group shows no detectable bands in mitochondria (data not shown) *P < 0.05 versus CTL decreased amount of phosphorylated CREB (or decreased CREB activity) downregulates SOD2 expression through the CREB-binding motif on the SOD2 promoter BetA suppresses HBx and translocates HBx to mitochondria We further examined the effect of BetA on HBx from HBV-infected hepatocytes The cells treated with control, BetA or siCREB were isolated into mitochondrial and cytosolic fractions for western blotting analysis As shown in Fig 5A, the level of HBx protein was decreased in total lysates and cytosolic fractions but increased in mitochondrial fractions after BetA treatment, and siCREB mimicked the effect of BetA, indicating that BetA treatment not only suppressed HBx expression, but also translocated HBx into mitochondria We further measured cytochrome c release for the treated cells As shown in Fig 5B, the cytochrome c level was substantially increased in cytosolic fractions after BetA or siCREB treatment as compared with control, was decreased in mitochondria, but was unchanged in total lysates This suggests that BetA-mediated cytochrome c release and apoptosis may be associated with HBx translocation to mitochondria The BetA-mediated proapoptotic effect depends on BetA-induced SOD2 suppression in HBV-infected cells In order to further determine the mechanisms of BetA-induced HBx translocation and proapoptotic effects, we measured the BetA-induced cytotoxicity in different cells; as shown in Fig 5C,D, BetA slightly increased caspase-3 activity (Fig 5C) and the apoptosis rate (Fig 5D) in WT cells, and a similar effect was observed in WT hepatocytes overexpressing HBx; overexpression of CREB could not abolish this effect, suggesting that the BetA-induced basal toxic effect in WT cells is not due to BetA-induced SOD2 suppression, and HBx alone is not directly involved in BetA-induced basal toxicity On the other hand, the BetA-induced toxicity was substantially increased in HBV-infected hepatocytes as compared with WT cells, and this effect was mostly abolished by overexpression of CREB, suggesting that, in HBV-infected cells, BetA-induced toxicity is due to SOD suppression We next measured SOD2 expression in different FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2605 Betulinic acid inhibits hepatitis B virus D Yao et al We previously found that BetA suppresses SOD2 expression by inhibiting CREB phosphorylation, with subsequent ROS overgeneration Here, we further investigated the potential effect of SOD2 on HBV replication The HBV-infected hepatocytes were treated with BetA, or BetA with SOD2 overexpression, or siRNA for SOD2 (siSOD2) alone, and the related biomedical parameters were measured As shown in Fig 6, the levels of SOD2 mRNA (Fig 6A) and SOD2 protein (Fig 6B) decreased to 33% and 46%, respectively, after BetA treatment, BetA treatment with SOD2 overexpression caused no difference in SOD2 level, and siSOD2 mimicked the effect of BetA We also measured ROS formation (Fig 6C) 100 * ? * HBsAg level (Arbitrary units) 200 100 * * F 200 200 * * 200 * * 200 100 * * 20 10 HBx protein level (Arbitrary units) H 30 Apoptosis rate by TINEL assay (%) * 100 * * * * 0 D * 0 300 ROS formation (Arbitrary units) 100 G 100 HBeAg level (Arbitrary units) C The BetA-mediated inhibitory effect on HBV is due to SOD2 suppression and subsequent ROS generation E 200 HBV DNA level by qPCR (Arbitrary units) B SOD2 mRNA level by Western blot (Arbitrary units) A SOD2 mRNA level by qPCR (Arbitrary units) cells; as shown in Fig 5E, the basal level of SOD2 was not changed in WT cells or WT hepatocytes overexpressing HBx, whereas the SOD2 level was substantially increased in HBV-infected cells as compared with WT cells; this increase was totally normalized by BetA, and overexpression of CREB minimized the effect of BetA, suggesting that BetAinduced toxicity in HBV-infected cells is due to BetA-mediated SOD suppression Finally, we measured HBx translocation to mitochondria in different cells, as shown in Fig 5F In WT hepatocytes overexpressing HBx, HBx was not found in mitochondria at all in the presence of BetA (data not shown), whereas in HBV-infected cells, BetA-induced HBx translocation was totally abolished by CREB overexpression, suggesting that BetA-induced SOD2 suppression and subsequent ROS generation is the driving force for HBx transcloation to mitochondria 200 100 2606 SO D si tA Be TL C SO D si tA Be C TL 2 Fig BetA-mediated inhibitory effect on HBV through SOD2 suppression and ROS generation HBV-infected hepatocytes were treated with control (CTL), 15 lgỈmL)1 BetA, BetA with CREB overexpression (BetA ⁄ CREB›) or siCREB for 48 h, and the cells were used for measurement of the indicated parameters (A) SOD2 mRNA level (B) SOD2 protein level (C) ROS generation (D) Apoptosis rate determined by TUNEL assay (E) HBsAg secreted from cell culture medium (F) HBeAg secreted from cell culture medium (G) HBV DNA from treated cells was measured by real-time quantitative PCR (H) HBx protein level was measured by western blotting and quantitated *P < 0.05 versus the CTL group FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS D Yao et al and apoptosis (Fig 6D); BetA treatment substantially increased ROS generation and the apoptosis rate, SOD2 overexpression in the presence of BetA minimized the effect, and siSOD2 mimicked the effect of BetA We next measured the effect of these treatments with different SOD2 expression levels on HBV replication; the results showed that BetA alone substantially inhibited HBV replication, including hepatitis B surface antigen level (HBsAg) level (Fig 6E), hepatitis B external core antigen (HBeAg) level (Fig 6F), HBV DNA (Fig 6G), and HBx protein expression (Fig 6H), whereas a combination of BetA and SOD2 overexpression totally abolished the BetAmediated HBV-inhibitory effect On the other hand, SOD2 knockdown (siSOD2) mimicked the BetAinduced inhibitory effect This suggests that BetAinduced ROS generation plays an important role in HBV inhibition; scavenging of ROS by overexpression of the antioxidant enzyme SOD2 might not be beneficial, but worsen the HBV infection, whereas the increase in ROS generation caused by direct SOD2 knockdown could achieve similar HBV-inhibitory effects BetA mimics the PC-induced inhibitory effect on HBV in mice In order to verify that BetA or PC extract does not alter general liver function and has no toxic effects in healthy liver, nontransgenic mice were employed to evaluate the proapoptotic effect Thirty male nonHBV-transgenic mice were randomly separated into three groups (10 in each) Experimental groups received either purified BetA (2 mgỈkg)1) or PC crude extracts (50 mgỈkg)1), whereas the control group received only vehicle Drugs or vehicle were added to the normal food and mixed for feeding After months, mice were killed by decapitation The liver tissues were collected for measurement of biomedical parameters: (a) SOD2 mRNA level; (b) enzymatic activities of caspase-3 and SOD2; (c) superoxide release; and (d) enzymatic activities of alanine aminotranferase and aspartate aminotransferase We found that neither BetA nor PC extract had significant cytotoxic effects on hepatocytes from mice (data not shown) In addition, we have previously found that BetA isolated from PC inhibits HBV replication in vitro by SOD2 suppression, which is similar to the effect that PC had in hepatitis B patients in our preliminary observation (data not shown) Here, we used HBV-transgenic mice to determine whether BetA could achieve the same inhibitory effect As shown in Fig 7A, both BetA Betulinic acid inhibits hepatitis B virus and PC significantly reduced HBsAg and HBeAg serum levels and HBV DNA replication Also, both BetA and PC substantially decreased SOD2 mRNA expression, whereas CREB mRNA showed no changes (Fig 7B) In addition, protein levels of SOD2 and pCREB were substantially reduced after BetA and PC treatment, whereas no changes were found in CREB total protein level (Fig 7C) We also examined the enzymatic activities, and showed that both BetA and PC not only decreased SOD2 activity, but also increased caspase-3 activity, indicating increased cytotoxicity with apoptosis rate (Fig 6D) Finally, we examined the levels of superoxide release in different tissues (Fig 6E,F); both BetA and PC specifically increased superoxide anion generation in liver tissue, but had little effect in aorta, and no effect at all in kidney and brain, indicating that both BetA-mediated and PC-mediated HBV inhibition are due to specifically decreased SOD2 expression with subsequent ROS generation in liver tissue Discussion This study demonstrates that BetA inhibits HBV replication by suppression of SOD2 expression with subsequent mitochondrial ROS overgeneration, with promising HBV clearance in both in vitro and in vivo mouse experiments This is the first time that we have shown the potential effects and possible mechanism of HBV inhibition by BetA BetA-mediated selective cytotoxicity in HBV-infected hepatocytes We have found that BetA has little cytotoxic effect on WT hepatocytes, but shows a selective cytotoxic effect on HBV-infected hepatocytes In addition, our data showed that the basal level of SOD2 was not changed in WT or WT hepatocytes overexpressing HBx, whereas the SOD2 level was substantially increased in HBV-infected cells as compared with WT cells, that this increase was totally normalized by BetA, and that overexpression of CREB could minimize the effect of BetA (Fig 5E), suggesting that BetA-induced toxicity in HBV-infected cells is due to BetA-mediated SOD suppression This indicates that HBV infection in HBV-infected cells specifically increases SOD2 expression, even though the detailed mechanisms are still unknown Furthermore, the basal SOD2 protein level in HBV-infected cells is much higher than in WT cells, and it is reasonable that the HBV-infected cells with high levels of SOD2 expression should be more FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2607 Betulinic acid inhibits hepatitis B virus B 150 CTL BetA Enzyme activity (Arbitrary units) HBV expression (Arbitrary units) A D Yao et al PC 100 * 50 * * * * * HBsAg HBeAg HBV-DNA 400 * 300 CTL BetA PC * 200 100 150 CTL BetA PC 100 Protein level by Western blot (Arbitrary units) * * 50 CREB E SOD2 D Superoxide anions release (nM) mRNA expression (Arbitrary units) Caspase-3 C * * SOD2 200 CTL * 150 100 BetA PC * * * 50 Liver Aorta Kidney Brain F 200 CLT BAte PC 100 * * * * 16.00 µm CTL 16.00 µm 16.00 µm BetA PC CREB pCREB SOD2 Fig BetA-mediated HBV inhibitory effect in mice through SOD2 suppression and ROS generation HBV-transgenic mice were treated with either vehicle [control (CTL)], BetA or PC crude extracts for months, the mice were killed, and the medical parameters from blood or different tissues were measured (A) HBsAg, HBeAg and HBV DNA were measured from blood (B) mRNA expression for CREB and SOD2 was measured by quantitative PCR from liver tissue (C) Protein levels for CREB, pCREB and SOD2 were measured by western blotting from liver tissue and quantitated (D) Enzyme activities for caspase-3 and SOD2 from liver tissue were measured and expressed as arbitrary units (E) Superoxide anion release from different tissues was measured (F) Representative images for in vivo superoxide staining from liver tissue *P < 0.05 versus CTL group susceptible to BetA-mediated SOD2 suppression, and, subsequently, the SOD2 suppression-mediated large increase in mitochondrial ROS generation may further induce mitochondrial dysfunction and apoptosis [24] Given the fact that HBV-infected cells are more susceptible to BetA-induced SOD2 suppression, and BetA-induced SOD2 suppression could directly inhibit HBV replication, as shown in Fig 6, we conclude that BetA could be a good candidate for anti-HBV drug development 2608 BetA-mediated CREB dephosphorylation As BetA could cause CREB dephosphorylation at Ser133 both in vivo and in vitro, and a mutated form of CREB with an Ala substitution for Ser133 has been reported to be a negative transcriptional regulator, BetA-induced dephosphorylation of CREB could act as a repressor of SOD2 gene transcription directly [25] CREB, as a direct substrate of both PKA [21,26] and protein kinase D (PKD) [27], could be phosphorylated FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS D Yao et al with the recruitment of coactivator CBP ⁄ p300 [21,28]; our results showed that PKA-induced in vitro CREB phosphorylation was directly inhibited by BetA (Fig 4F), whereas no effect on PKD-induced phosphorylation was observed (data not shown), and the expression of neither PKA nor PKD was changed after BetA treatment (data not shown), suggesting that BetA-induced CREB dephosphorylation is due to direct inhibition of PKA by BetA BetA-induced HBx suppression and translocation HBx protein, which is encoded by the X gene of the HBV genome, could stimulate many different signal transduction pathways, or directly interact with transcription factors, resulting in several different biological activities in HBV-associated liver disease [29,30] Also, HBx could alter mitochondrial function through direct association with mitochondria [31,32] In BetAtreated HBV-infected hepatocytes, we found that the total HBx protein level was substantially decreased, indicating that BetA may inhibit HBV-associated hepatocarcinogenesis through suppression of HBx expression; this may partly explain its potential antitumor effect Furthermore, this is the first time that HBx has been found to be translocated into mitochondria from the cytosol in BetA-treated HBV-infected hepatocytes; given the fact that HBx could induce apoptosis [33,34] and alter mitochondrial function by inhibiting the mitochondrial electron transport chain and oxidative phosphorylation (complexes I, III, IV, and V) [31], we suppose that BetA-mediated HBx suppression and translocation worsens the mitochondrial dysfunction, which may further trigger apoptosis As SOD2 expression could totally abolish the BetAinduced HBx suppression (Fig 5A), we suppose that HBx translocation to mitochondria could be associated with BetA-induced SOD2 suppression In order to determine the possible role and effect of HBx in this procedure, as shown in Fig 5C–F, HBx protein was overexpressed in WT hepatocytes overexpressing HBx, and the related proapoptotic effect was measured We found that HBx alone (instead of full HBV infection) showed a similar effect in WT cells after BetA treatment, and caused small increases in caspase-3 activity (Fig 5C) and apoptosis rate (Fig 5D), no increase in SOD2 expression (Fig 5E), and no HBx translocation into mitochondria (data not shown) On the other hand, in HBV-infected hepatocytes, SOD2 expression was substantially increased, and became sensitive to the BetA-induced proapoptotic effect, the HBx was translocated into mitochondria, and the effect was normalized by CREB overexpression (Fig 5F), suggesting Betulinic acid inhibits hepatitis B virus that HBx alone does not directly contribute to BetAinduced SOD2 suppression and HBV inhibition, whereas HBx translocation to mitochondria could be a consequence of BetA-induced SOD2 suppression and subsequent ROS generation BetA-mediated mitochondrial redox balance We found that BetA has a strong redox effect through suppression of SOD2 expression with subsequent mitochondrial ROS generation; even though ROS generation could cause cell damage, our results show that BetA-mediated ROS generation is favorable for HBV clearance, as SOD2 overexpression could abolish BetA-induced HBV inhibition, whereas siSOD2 mimicked BetA-induced HBV inhibition, suggesting that the BetA-mediated redox effect could be both beneficial and detrimental to cells; the most important thing is to maintain a balance that achieves the best redox environment for living organisms [24,35] BetA-mediated selective superoxide release in liver tissue An interesting result was found from the mouse experiments with specifically increased superoxide anion release in liver tissue under BetA treatment, instead of in other tissues As superoxide formation may directly correspond to SOD2 expression, we measured SOD2 protein levels in some other tissues, and found that liver tissue has much higher basal SOD2 levels than other tissues, including aorta, kidney, heart, and lung, with the exception of a higher SOD2 level in brain (data not shown) Given the fact that the liver accepts most of the circulating BetA under dietary conditions for subsequent deposition and detoxification as the first target organ for xenobiotics, the high susceptibility of the liver could due to its higher SOD2 basal expression and higher accessibility to BetA On the other hand, in brain tissue, the circulating BetA can hardly gain access, owing to the protection of the blood–brain barrier, so the brain is insensitive to BetA treatment, even with higher SOD2 expression levels Taken together, these findings demonstrate that BetA could achieve an impressive HBV-inhibitory effect by specific suppression of SOD2 expression and modulation of the mitochondrial redox balance The BetA used in this work was isolated and purified from PC, a traditional Chinese herb that has been succesfully used for the treatment of hepatitis B patients, according to a secret recipe According to the Chinese traditional medicine theory: ‘Everything has its own enemy from nature,’ which essentially means FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2609 Betulinic acid inhibits hepatitis B virus D Yao et al that everything within the world not only has its own method of survival, but also has its own method of destruction, thus preserving the balance of nature In fact, we can derive elements for good health from nature if we utilize it properly Given the fact that PC is well known and relatively nontoxic, with the promising effects presented by its component BetA, PC scould be a good candidate for anti-HBV drug development Experimental procedures Materials and methods HBV-transgenic mice (male, 6–8 weeks old, 18–22 g) were obtained from the Transgenic Engineering Laboratory in the Infectious Disease Center (Guangzhou, China) [36,37] The mouse genotypes were identified from both positive HBsAg and HBeAg; the serum HBV virus load in these mice was 104–105 copies per mL; HBV DNA, HBV mRNA and hepatitis B core antigen in hepatocytes were shown to be positive by Southern blotting, northern blotting, and immunohistochemistry, respectively Animals showed no evident cytopathological changes in the liver, with normal serum alanine aminotransferase levels Animals were housed in a pathogen-free room under strict barrier conditions All procedures used in dealing with the experimental animals were approved by our institutional animal care and use committee Primary hepatocytes from WT or HBV-transgenic mice were prepared from residual liver tissue from related mice by enzymatic dissociation; the isolation protocol was approved by the Ethics Committee, Tongji Medical School Cells were kept in William’s E medium supplemented with 5% fetal bovine serum and antibiotics at 37 °C in 5% CO2 [38] HBx DNA amplified by PCR from HBV genomic DNA, CREB-1 cDNA and SOD2 cDNA were subcloned into pcDNA3.1 for overexpression of HBx, CREB-1, and SOD2, respectively The CREB-1 cDNA was further subcloned into the pGEX-4T vector (no 27-4580-01; GE Healthcare, Shanghai, China) for expression of CREB protein in Escherichia coli BL21 cells, and this was followed by glutathione S-transferase protein purification with the MagneGST Protein Purification System (no V8600; Promega, Beijing, China) and thrombin protease digestion The SOD2 promoter was amplified from human genomic DNA and subcloned into the pGL3-basic vector (no E1751; Promega) to construct the SOD2 reporter plasmid For mapping of the SOD2 promoter response element, the related deletion or point mutation constructs were generated by PCR methods or a sitedirected mutagenesis kit (no Q9280; Promega) Detailed information on those plasmids is available upon request Antibody against HBx (no RD981038100) was obtained from BioVendor Laboratories Ltd (Guangzhou, China), 2610 and rabbit voltage-dependent anion selective channel ⁄ porin antibody (V2139) was obtained from Sigma Antibodies against b-actin (sc-47778), CREB-1 (sc-58), pCREB-1 (Ser133 phosphorylation, sc-7978) and SOD2 (sc-30080) were obtained from Santa Cruz (Beijing, China) The mitochondrial and cytosolic fractions were prepared and characterized using differential centrifugation as previously described [39] Protein concentrations were measured with the BioRad protein assay kit (Bradford method), according to the manufacturer’s instructions Small interfering RNAs for CREB-1 (no s3491), SOD2 (no 9052) and negative control (no AM4636) were obtained from Ambion (Shanghai, China) The SOD2 activity was measured with the SOD Detection Kit (no CSOD100-2; Cell Technology Inc., Shanghai, China) The plasmid DNA and siRNA were transfected with Lipofectamine Reagent (Invitrogen, Beijing, China) Luciferase activity assays were carried out using the Dual-Luciferase Assay System (Promega), and transfection efficiencies were normalized using a cotransfected Renilla plasmid BetA isolation and purification BetA was isolated from PC collected from a special area of China (Yichang, Hubei), in order to achieve reliable and repeatable clinical effects The root of PC was washed, dried at room temperature (avoiding direct sunlight), and cut into small pieces Five hundred grams of PC was then added to 495 mL of distilled water and mL of fish oil (Sigma), and incubated at 37 °C for no less than h, with continuous stirring The temperature was then increased slowly up to boiling point, and maintained for 10 for decoction The solution was sterile filtered, concentrated and lyophilized into powder for PC crude extracts; the powder was mixed with a standard powdered rodent chow for animal treatment, or the PC crude extracts were further dissolved in : acetonitrile ⁄ methanol, purified, and characterized with an HPLC system coupled to a negative IES mass spectrometer [40] Cell viability and MTT assay Cells were pooled in 12-well plates, following exposure to different treatments as indicated, at 80% confluence Cell viability was analyzed by the MTT reduction assay Briefly, cells in each well were aspirated and washed with NaCl ⁄ Pi, and 0.2 mL of 0.3 mgỈmL)1 MTT solution was then added at 25 °C for h Thereafter, the precipitated blue formazan product was extracted by incubating samples with 0.1 mL of 10% SDS (dissolved in 0.01 m HCl) overnight at 37 °C The absorbances of formazan concentrations were determined at 570 nm, then normalized by cell numbers and expressed as A ⁄ 106 cells FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS D Yao et al DNA synthesis evaluated by [3H]thymidine incorporation Cell proliferation was evaluated as the rate of DNA synthesis by [3H]methylthymidine incorporation [41] Cells were pooled in 24-well plates up to 80% confluence, and different concentrations of BetA were then added and incubated for 48 h At the end of the treatment, cells were incubated with serum-free medium containing [3H]methylthymidine (0.5 lCi per well) for h, and then washed twice with NaCl ⁄ Pi Cellular DNA was precipitated with 10% trichloroacetic acid and solubilized with 0.4 m NaOH (0.5 mL per well) Incorporation of [3H]methylthymidine into DNA was measured in a scintillation counter and was normalized to protein concentration Reverse transcription reaction and real-time PCR Total RNA from treated cells was extracted with an RNeasy Mini Kit (Qiagen, Shanghai, China), and the RNA was reverse transcribed with an Omniscript RT kit (Qiagen) Real-time quantitative PCR was performed on an iCycler iQ (Bio-Rad, Shanghai, China) with the Quantitect SYBR Green PCR kit (Qiagen) Two-step PCR was performed at 95 °C for min, followed by 45 cycles of denaturation at 95 °C for 30 s, and annealing and extension at 60 °C for 60 s The fluorescence was detected at the end of each extension step The results are normalized to b-actin Western blotting Cells were lysed in ice-cold lysis buffer (0.137 m NaCl, mm EDTA, 10% glycerol, 1% NP-40, 20 mm Tris base, pH 8.0) plus protease inhibitor cocktail (Sigma) The proteins were separated by 10% SDS ⁄ PAGE and transferred to a poly (vinylidene difluoride) membrane The membrane was then incubated with appropriate antibodies, washed, and incubated with horseradish peroxidase-labeled secondary antibodies The blots were visualized with the ECL+plus Western Blotting Detection System (Amersham, Shanghai, China), and quantitated by imagequant The final results were normalized to b-actin or porin (for mitochondrial protein) Chromatin immunoprecipitation Treated cells were crosslinked using 1% formaldehyde for 20 and terminating with 0.1 m glycine Cell lysates were sonicated and centrifuged at 18 000 g for 12 to get supernatant Five hundred micrograms of protein was precleared by BSA ⁄ salmon sperm DNA plus preimmune IgG and a slurry of protein A–agarose beads, as previously described [42] Immunoprecipitations were performed with the indicated antibodies, BSA ⁄ salmon sperm DNA, and a 50% slurry of protein A–agarose beads The immunoprecipitates were washed and eluted, and then incubated with Betulinic acid inhibits hepatitis B virus 0.2 mgỈmL)1 proteinase K for h at 42 °C, and then for h at 65 °C, to reverse the formaldehyde crosslinking DNA fragments were recovered by phenol ⁄ chloroform extraction and ethanol precipitation A 150 bp fragment from the human SOD2 promoter was amplified by real-time quantitative PCR In vitro phosphorylation of CREB at Ser133 Purified CREB was phosphorylated by the catalytic subunit of PKA (no P2645; Sigma) by incubating 2.0 lm CREB in a reaction mixture containing 4.0 lm ATP (or UTP for mock reactions), mm MgCl2 and 100 U of PKA in 25 mm NaCl ⁄ Pi (pH 6.6) at 30 °C for h in the presence or absence of 15 lgỈmL)1 BetA The PKA was heat-inactivated at 75 °C for 10 The level of pCREB was determined by western blotting [43] Measurement of mitochondrial function The intracellular ATP level was determined by the luciferin ⁄ luciferase-induced bioluminescence system An ATP standard curve was generated at concentrations of 10)12– 10)3 m Intracellular ATP levels were calculated and expressed as nmol ⁄ mg protein MMP (DWm) was measured by the 3,3¢-dihexiloxadicarbocyanine (DiOC6) method Cells were trypsinized, resuspended, and incubated with 0.2 lm DiOC6 for 20 at 37 °C, and cells were then treated with lm propidium iodide for 30 The DiOC6 fluorescence was measured by fluorescence activated cell sorting at an excitation ⁄ emission wavelength of 485 ⁄ 500 nm [39] Evaluation of apoptosis Apoptosis was evaluated by TUNEL assay using an In Situ Cell Death Detection Kit (Roche, Shanghai, China) Cells were fixed in 4% paraformaldehyde, and labeled with TUNEL reagents Stained cells were photographed with a fluorescence microscope, and further quantified by fluorescence activated cell sorting analysis Caspase-3 activity was determined with an ApoAlert caspase assay kit (Clontech, Beijing, China) Treated cells were harvested, and 50 lg of protein was incubated with the fluorogenic peptide substrate Ac-DEVD-7-amino-4-trifluoromethyl coumarin The initial rate of free Ac-DEVD-7-amino-4-trifluoromethyl coumarin release was measured using an FLx800 microplate reader (Bio-Tek, Guangzhou, China) at excitation ⁄ emission wavelengths of 380 ⁄ 505 nm, and enzyme activity was calculated as pmol ⁄ ⁄ mg or as arbitrary units [39] Measurement of oxidative stress Intracellular ROS generation was determined by using oxidation of 2¢,7¢-dihydrochlorofluorescein-diacetate Treated FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2611 Betulinic acid inhibits hepatitis B virus D Yao et al cells were washed and incubated with 0.1 mL of 10 lm 2¢,7¢-dihydrochlorofluorescein-diacetate, and the fluorescence was measured at excitation ⁄ emission wavelengths of 485 ⁄ 530 nm, using an FLx800 microplate fluorescence reader (Bio-Tek) The data were normalized to arbitrary units [39] Measurement of HBV replication HBV-infected hepatocytes were seeded in 24-well plates, and then subjected to BetA treatment for 48 h Cell numbers were determined by Trypan blue exclusion Secretion of HBsAg and HBeAg in cultured medium or serum from mice was assayed by using commercially available kits (Abbott, Shanghai, China) as previously described [44] The DNA from cells or serum samples was extracted with phenol ⁄ chloroform ⁄ isoamyl alcohol (25 : 24 : 1), and precipitated with ethanol after digestion at 37 °C for h with proteinase K (0.5 mgỈmL)1) in the presence of SDS (0.5%) The HBV DNA level was measured by real time-PCR, using human b-actin as control [45] Effect of BetA on HBV-transgenic mice Sixty male HBV-transgenic mice were randomly separated into three groups (20 in each) Experimental groups received either purified BetA (2 mgỈkg)1) or PC crude extracts (50 mgỈkg)1), whereas the control group received only vehicle Drugs or vehicle were added to the normal food and mixed for feeding to the experimental mice After months, mice were killed by decapitation Blood (with EDTA as anticoagulant) and other tissues, including brain, heart, aorta, and kidney, were collected for serum HBV replications or other biomedical parameters as indicated Serum HBsAg, HBeAg and HBV DNA were analyzed as described above, gene expression was measured by quantitative PCR and western blotting, and superoxide release from tissues was measured as previously described [39] The in vitro staining of superoxide anions (O2·)) was performed with the oxidative fluorescent dye dihydroethidium [46,47] Briefly, fresh and unfixed liver tissues were frozen and cut in a cryostat into 30 lm sections and placed on glass 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BetA BetA/CREB siCREB F 200 * * (Arbitrary units) 100 pCREB level by Western blot (Arbitrary units) 200 pCREB (Ser133) level by Western blot BetA/CREB 200 siCREB C BetA 300 SOD2 protein level by. .. that HBx alone does not directly contribute to BetAinduced SOD2 suppression and HBV inhibition, whereas HBx translocation to mitochondria could be a consequence of BetA-induced SOD2 suppression. .. BetA-induced CREB dephosphorylation is due to direct inhibition of PKA by BetA BetA-induced HBx suppression and translocation HBx protein, which is encoded by the X gene of the HBV genome, could