Carnosic acid protects against acetaminophen-induced hepatotoxicity by potentiating Nrf2-mediated antioxidant capacity in mice.

Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure. The study aimed to investigate the protective effect of carnosic acid (CA) on APAP-induced acute hepatotoxicity and its underlying mechanism in mice. To induce hepatotoxicity, APAP solution (400 mg/kg) was administered into mice by intraperitoneal injection. Histological analysis revealed that CA treatment significantly ameliorated APAP-induced hepatic necrosis. The levels of both alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum were reduced by CA treatment. Moreover, CA treatment significantly inhibited APAP-induced hepatocytes necrosis and lactate dehydrogenase (LDH) releasing. Western blot analysis showed that CA abrogated APAP-induced cleaved caspase-3, Bax and phosphorylated JNK protein expression. Further results showed that CA treatment markedly inhibited APAP-induced pro-inflammatory cytokines TNF-α, IL-1ß, IL-6 and MCP-1 mRNA expression and the levels of phosphorylated IκBα and p65 protein in the liver. In addition, CA treatment reduced APAP- induced hepatic malondialdehyde (MDA) contents and reactive oxygen species (ROS) accumulation. Conversely, hepatic glutathione (GSH) level was increased by administration of CA in APAP-treated mice. Mechanistically, CA facilitated Nrf2 translocation into nuclear through blocking the interaction between Nrf2 and Keap1, which, in turn, upregulated anti-oxidant genes mRNA expression. Taken together, our results indicate that CA facilitates Nrf2 nuclear translocation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity.

Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity.

Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 µM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 µM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50]=25 to 79 µM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

Alphavbeta6 is required in maintaining the intestinal epithelial barrier function.

Epithelial barrier dysfunction is involved in a large number of diseases, but the pathogenesis is unclear. Integrin alphavbeta6 (avb6) in involved in the maintenance of the mucosal homeostasis. We have investigated the role of avb6 in maintaining the epithelial barrier function. Using T84 monolayers cultures, transepithelial electric resistance (TER) and permeability to ovalbumin (OVA) were measured as indicators of functioning. The antigenicity of OVA collected from the Transwell basal chambers was assessed using OVA-specific T cell proliferation. Knockdown of the avb6 genes increased the permeability of T84 monolayers to OVA, but did not affect TER. The deficiency of avb6-related hyperpermeability in T84 monolayers could be compensated by adding exogenous avb6 to the culture. The OVA samples collected from the basal chambers had strong antigenicity as it markedly induced the antigen specific T cell proliferation. Addition of recombinant avb6 blocked increases in permeability of T84 monolayers to OVA induced by tumor necrosis factor-α.

Spermidine mitigates ferroptosis in free fatty acid-induced AML-12 cells through the ATF4/SLC7A11/GCLM/GPX4 pathway.

Non-alcoholic fatty liver disease (NAFLD) is a prominent cause of chronic liver disease worldwide. Spermidine (SPD), a naturally occurring polyamine, has shown potential in alleviating the accumulation of hepatic lipids and reducing NAFLD symptoms in overweight mice. Nonetheless, the specific mechanisms through which SPD exerts its effects remain largely unknown. This study seeks to explore the protective effects of SPD on NAFLD and to clarify the underlying mechanisms. An in vitro model of NAFLD was established by inducing steatosis in AML-12 cells through the use of free fatty acids (FFAs). Our experimental results demonstrate that SPD significantly reduces NAFLD development induced by FFAs. This reduction is primarily achieved through the inhibition of cellular ferroptosis, as evidenced by decreased levels of Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS). Additionally, SPD was found to enhance cellular activity and ameliorate mitochondrial dysfunction and oxidative stress caused by FFA exposure. Further mechanistic studies have revealed that SPD upregulates the expression of solute transporter family 7a member 11 (SLC7A11), glutamate-cysteine ligase modifier subunit (GCLM), and glutathione peroxidase (GPX4). This upregulation is mediated by the activation of activating transcription factor 4 (ATF4). Knockdown experiments of ATF4 confirmed that its inhibition reverses the upregulation of SLC7A11, GCLM, and GPX4, thereby negating the protective effects of SPD. In conclusion, our findings suggest that SPD mitigates NAFLD by modulating the ATF4/SLC7A11/GCLM/GPX4 signaling pathway, resulting in the suppression of ferroptosis and the improvement of cellular health. These insights provide a novel molecular mechanism and identify potential therapeutic targets for the treatment of NAFLD.

Overexpression of ARK5 is associated with poor prognosis in hepatocellular carcinoma.

ARK5 overexpression has been reported in a variety of human cancers. However, the role of ARK5 in hepatocellular carcinoma (HCC) remains unclear. The aim of the present study is to analyze the ARK5 protein expression in HCC tissue samples and to assess its prognostic significance for HCC. ARK5 mRNA and protein expression were determined by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blot in 20 pairs of fresh frozen HCC tissues and corresponding non-cancerous tissues. In addition, ARK5 expression was analyzed by immunohistochemistry in 130 clinicopathologically characterized HCC cases. The correlation of ARK5 expression with patients' survival rate was assessed by Kaplan-Meier and Cox regression. Our results showed that the expression levels of ARK5 mRNA and protein in HCC tissues were both significantly higher than those in non-cancerous tissues. Our results showed that the high expression of ARK5 in HCC was related to tumor size (p=0.005), histological differentiation (p=0.047), and tumor stage (p=0.005). Kaplan-Meier survival analysis showed that a high expression level of ARK5 resulted in a significantly poor prognosis of HCC patients. Multivariate analysis revealed that ARK5 expression level was an independent prognostic parameter for the overall survival rate of HCC patients. In conclusion, ARK5 might play a positive role in tumor development and could serve as an independent predictor of poor prognosis for HCC.

Down-regulation of HSP70 sensitizes gastric epithelial cells to apoptosis and growth retardation triggered by H. pylori.

BACKGROUND: H. pylori infection significantly attenuated the expression of HSP70 in gastric mucosal cells. However, the role of HSP70 cancellation in H. pylori-associated cell damages is largely unclear. METHODS: Small interfering RNA (siRNA) was used to down-regulate HSP70 in gastric epithelial cell lines AGS. The transfected cells were then incubated with H. pylori and the functions of HSP70 suppression were observed by viability assay, cell cycle analyses and TUNEL assay. HSP70 target apoptotic proteins were further identified by Western blot. RESULTS: The inhibition of HSP70 has further increased the effect of growth arrest and apoptosis activation triggered by H. pylori in gastric epithelial cells. The anti-proliferation function of HSP70 depletion was at least by up-regulating p21 and cell cycle modulation with S-phase accumulation. An increase of apoptosis-inducing factor (AIF) and cytosolic cytochrome C contributes to the activation of apoptosis following down-regulation of intracellular HSP70. Extracellular HSP70 increased cellular resistance to apoptosis by suppression the release of AIF and cytochrome c from mitochondria, as well as inhibition of p21 expression. CONCLUSIONS: The inhibition of HSP70 aggravated gastric cellular damages induced by H. pylori. Induction of HSP70 could be a potential therapeutic target for protection gastric mucosa from H. pylori-associated injury.

Oral Bifidobacterium modulates intestinal immune inflammation in mice with food allergy.

BACKGROUND AND AIMS: It is proposed that probiotics have a therapeutic effect on the treatment of immune disorders. However, the underlying mechanisms require clarification. The present study aimed to evaluate the effect of gavage-feeding Bifidobacteria on suppression of T helper 2 (Th2) pattern inflammation in the intestines of mice with food allergy. METHODS: Mice were sensitized to ovalbumin to induce the intestinal Th2 pattern inflammation. Allergic mice were treated with or without Bifidobacteria via gavage-feeding. Th2 response, number of regulatory T cells (Treg) in the spleen and intestine, intestinal epithelial barrier function and bifidobacterial translocation were examined. RESULTS: The results showed that serum-specific immunoglobulin E antibody and interleukin 4 (IL-4) were increased in allergic mice. Intestinal epithelial barrier function was impaired in allergic mice as shown by the increase in epithelial ion secretion and permeability to macromolecular protein horseradish peroxidase in Ussing chambers. Number of Treg was decreased in both spleen and intestines of allergic mice. Gavage-feeding Bifidobacteria significantly suppressed the skewed Th2 response and increased the number of Treg. Transient bifidobacterial translocation was observed in allergic mice. CONCLUSIONS: Oral administration of Bifidobacteria has the capacity to suppress the skewed Th2 response in allergic mice, increasing the number of Treg and IL-10-positive cells and improve the impaired intestinal epithelial barrier function.

Overexpression of MMP-2 and MMP-9 in esophageal squamous cell carcinoma.

Matrix metalloproteinases (MMPs) are known to play important roles in extracellular matrix remodeling during the process of tumor invasion and metastasis. However, little is known about their role in esophageal squamous cell carcinoma (ESCC). Expression of MMP-2 and MMP-9 in ESCC was detected in our research. Tissue microarray chip was prepared, consisting of 58 cases of ESCC and corresponding esophageal epithelium tissues. MMP-2 and MMP-9 were examined by immunohistochemistry. Overexpression of MMP-2 and MMP-9 was found in ESCC (42.1 and 60.3%, respectively), compared with paired distal normal esophageal tissues (22.9 and 8.9%, respectively). Expression of MMP-2 in ESCC was significantly associated with the tumor invasion depth, tumor-node-metastasis stages, and lymph node metastasis. MMP-2 and MMP-9 may play important roles in carcinogenesis, and MMP-2 may act as a biological marker of invasion and lymph node metastasis in ESCC.

Entecavir up-regulates dendritic cell function in patients with chronic hepatitis B.

AIM: To investigate the in vitro effect of entecavir (ETV) on the function of dendritic cells (DCs) derived from chronic hepatitis B (CHB) patients. METHODS: Mononuclear cells were isolated from peripheral blood of patients with CHB. DCs were incubated with RPMI-1640 medium supplemented with fetal bovine serum, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF). DCs were treated with or without ETV on the fourth day. Cell surface molecules, including CD1a, CD80, CD83 and HLA-DR, were assessed by flow cytometry. Concentrations of IL-6 and IL-12 in the supernatant were assayed by enzyme-linked immunosorbent assay (ELISA). The ability of the generated DCs to stimulate lymphocyte proliferation was observed. RESULTS: Compared with CHB control group, the expression levels of CD1a (29.07 +/- 3.20 vs 26.85 +/- 2.80), CD83 (25.66 +/- 3.19 vs 23.21 +/- 3.10), CD80 (28.00 +/- 2.76 vs 25.75 +/- 2.51) and HLA-DR (41.96 +/- 3.81 vs 32.20 +/- 3.04) in ETV-treated group were higher (P < 0.05). ETV-treated group secreted significantly more IL-12 (157.60 +/- 26.85 pg/mL vs 132.60 +/- 22.00 pg/mL (P < 0.05) and had a lower level of IL-6 in the culture supernatant (83.05 +/- 13.88 pg/mL vs 93.60 +/- 13.61 pg/mL, P < 0.05) than CHB control group. The ability of DCs to stimulate the proliferation of allogeneic lymphocytes was increased in ETV-treated group compared with CHB control group (1.53 +/- 0.09 vs 1.42 +/- 0.08, P < 0.05). CONCLUSION: Entecavir can enhance the biological activity of DCs derived from CHB patients.

Effects of lamivudine on the function of dendritic cells derived from patients with chronic hepatitis B virus infection.

AIM: To investigate if the nucleoside analogue lamivudine (LAM), a potent inhibitor of HBV replication, could restore the function of dendritic cells derived from patients with chronic hepatitis B (CHB) in an Asian population. METHODS: Dendritic cells (DCs) derived from mononuclearcytes of patients with chronic HBV infection were cultured in the presence of IL-4, granulocyte-macrophage colony-stimulating factors (GM-CSF) and gradient concentrations of LAM (0-2 mmol/L). Cell morphology was observed under light microscopy. Cell surface molecules, including HLA-DR, CD80, CD83, and CD1alpha, were analyzed with flow cytometry. The concentrations of IL-6 and IL-12 in the supernatant were assayed by ELISA. T cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT). RESULTS: The expression of CD1alpha on DC treated with 0.5 mmol/L LAM (LAM-DC 0.5 mmol/L) was significantly higher than that of DC untreated with LAM (54.1 +/- 4.21 vs 33.57 +/- 3.14, P < 0.05), and so was the expression of CD83 (20.24 +/- 2.51 vs 12.83 +/- 2.12, P < 0.05) as well as the expression of HLA-DR (74.5 +/- 5.16 vs 52.8 +/- 2.51, P < 0.05). Compared with control group, LAM-DC group (0.5 mmol/L) secreted significantly more IL-12 (910 +/- 91.5 vs 268 +/- 34.3 pg/mL, P < 0.05), had lower levels of IL-6 in the culture supernatant (28 +/- 2.6 vs 55 +/- 7.36 pg/mL, P < 0.05), markedly enhanced the stimulatory capacity in the allogeneic mixed leukocyte reaction (MLR) (1.87 +/- 0.6 vs 1.24 +/- 0.51, P < 0.05). CONCLUSION: The lower expression of phenotypic molecules and impaired allogeneic mixed lymphocyte reaction function of dendritic cells derived from patients with HBV infection could be restored in vitro by incubation with LAM.

Calycosin attenuates dextran sulfate sodium (DSS)-induced experimental colitis.

OBJECTIVES: Inflammatory bowel disease (IBD) results from dysregulation of intestinal mucosal immunity. It is an incurable disease that affects millions of people worldwide. Developing new strategies for the treatment of colitis has been a major challenge. Here, we report the effect of calycosin, a plant-derived flavonoid, in successfully managing colitis in murine model. MATERIAL AND METHODS: In vivo model of colitis was induced using 2.5% (w/v) dextran sodium sulfate (DSS, 36,000 to 50,000 Mw). Body weight and disease activity index (DAI) were evaluated every day. Hematoxylin-Eosin (H&E) staining was used to estimate the effect of calycosin on DSS-induced colon damage. The levels of proinflammatory genes and mRNA expression were determined using real-time PCR, whereas the proinflammatory cytokines were assessed with ELISA. The content of other parameters including myeloperoxidase (MPO), glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were also evaluated. Western blot assay was further used to determine the effect of calycosin on both NF-κB and mitogen activated protein kinases (MAPK) pathways. RESULTS: The results showed that calycosin prevented weight loss and shortening of the colon length, maintained an intact mucosa, increased GSH and SOD activities, and decreased MDA levels. The drug also significantly inhibited proinflammatory cytokine mRNA expression and decreased MPO activity. Additionally, it remarkably inhibited NF-κB pathway and c-Jun N-terminal kinase (JNK) phosphorylation with no effect on p38 and extracellular signal-regulated kinase (ERK1/2) phosphorylation levels in colon tissue. CONCLUSION: These findings revealed that calycosin successfully ameliorated the effect of DSS-induced colitis in mice, which could be associated with NF-κB and JNK pathway modulations.

Hepatitis B virus genetic diversity has minimal impact on sensitivity of the viral ribonuclease H to inhibitors.

Hepatitis B virus (HBV) causes hepatitis, cirrhosis, liver failure, and liver cancer, but the current therapies that employ either nucelos(t)ide analogs or (pegylated)interferon α do not clear the infection in the large majority of patients. Inhibitors of the HBV ribonuclease H (RNaseH) that are being developed with the goal of producing anti-HBV drugs are promising candidates for use in combination with the nucleos(t)ide analogs to improve therapeutic efficacy. HBV is genetically very diverse, with at least 8 genotypes that differ by ≥8% at the sequence level. This diversity is reflected in the viral RNaseH enzyme, raising the possibility that divergent HBV genotypes or isolates may have varying sensitivity to RNaseH inhibitors. To evaluate this possibility, we expressed and purified 18 patient-derived RNaseHs from genotypes B, C, and D. Basal RNaseH activity and sensitivity to three novel RNaseH inhibitors from three different chemotypes were assessed. We also evaluated four consensus HBV RNaseHs to determine if such sequences would be suitable for use in antiviral drug screening. The patient-derived enzymes varied by over 10-fold in their basal RNaseH activities, but they were equivalently sensitive to each of the three inhibitors. Similarly, all four consensus HBV RNaseH enzymes were active and were equally sensitive to an RNaseH inhibitor. These data indicate that a wide range of RNaseH sequences would be suitable for use in antiviral drug screening, and that genotype- or isolate-specific genetic variations are unlikely to present a barrier during antiviral drug development against the HBV RNaseH.

Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors.

Hepatitis B virus (HBV) reverse transcription requires coordinated function of the reverse transcriptase and ribonuclease H (RNaseH) activities of the viral polymerase protein. The reverse transcriptase has been biochemically characterized, but technical difficulties have prevented both assessment of the RNaseH and development of high throughput inhibitor screens against the RNaseH. Expressing the HBV RNaseH domain with both maltose binding protein and hexahistidine tags led to stable, high-level accumulation of the RNaseH in bacteria. Nickel-affinity purification in the presence of Mg(2+) and ATP removed co-purifying bacterial chaperones and yielded nearly pure monomeric recombinant enzyme. The endonucleolytic RNaseH activity required an DNA:RNA duplex ≥14 nt, could not tolerate a stem-loop in either the RNA or DNA strands, and could tolerate a nick in the DNA strand but not a gap. The RNaseH had no obvious sequence specificity or positional dependence within the RNA, and it cut the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3'-5' exoribonuclease activity that is slower than the endonucleolytic reaction. These results are consistent with the HBV reverse transcription mechanism that features an initial endoribonucleolytic cut, 3'-5' degradation of RNA, and a sequence-independent terminal RNA cleavage. These data provide support for ongoing anti-RNaseH drug discovery efforts.

Distinct compartmental distribution of mature and immature dendritic cells in esophageal squamous cell carcinoma.

Dendritic cells (DCs) play a critical role in generating anti-tumor immunity. DC functional defect has been related to the growth and progression of various human cancers. In esophageal squamous cell carcinoma (ESCC), the examination of DCs using immunohistochemistry (IHC) with anti-S100 antibody has demonstrated an increased infiltration of DCs into the tumor mass, however, the distribution patterns of DCs at different maturation states in ESCC are not fully evaluated. In this study, we immunohistochemically analyzed the DC maturation status by examining the S100-positive DCs, CD1alpha-positive immature DCs (iDCs), and CD208-positive mature DCs (mDCs) and their distribution patterns in 45 ESCCs and 10 control tissues. The IHC analysis showed that the number of S100-positive DCs was increased in both the cancer epithelium and tumor stroma. Further phenotypic analyses revealed that intraepithelial DCs in the cancer mass were predominantly CD1alpha-positive iDCs. Whereas DCs presented in the tumor stroma were exclusively CD208-positive mDCs, CD208-positive mDCs were particularly dense in the margin of cancerous lesions and formed clusters with CD3-positive lymphocytes. The number of CD208-positive mDCs in the tumor mass was significantly lower than the number of CD1alpha-positive iDCs. The current results suggest that ESCC tissue comprises a high frequency of iDCs in the cancerous epithelium and a low density of mDCs in the tumor stroma. Such a distinct distribution pattern may reflect the ongoing DC tracking in ESCCs.

Localization of indoleamine 2,3-dioxygenase in human esophageal squamous cell carcinomas.

Immunosuppressive factors derived from the tumor and nontumor cells present in the tumor microenvironment contribute to tumor escape from host immune attack. Recently, the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) derived from both the tumor cells and surrounding nontumor cells was found to function as a critical immunosuppressive factor. While the expression of IDO is intensively under investigation in many types of cancers, little information is available in esophageal squamous cell carcinomas (ESCC) thus far. In this study, we have therefore investigated the cellular localization of IDO in 45 ESSCs and ten morphologically normal esophageal tissues; the correlation of IDO with clinicopathological parameters was also analyzed. Immunohistochemistry (IHC) analysis revealed that the density of IDO-positive cells was increased in ESCCs relative to controls (P < 0.01). These cells were distributed as clusters and formed a patchy pattern in both the cancerous epithelium and the surrounding noncancerous cells. Double IHC further confirmed that many IDO-positive cells in the tumor stroma were smooth-muscle-actin-alpha-positive myofibroblasts, CD68-positive macrophages, and S100-positive dendritic cells. Statistical analysis showed that the densities of IDO-positive cells were not significantly correlated with tumor clinical parameters (tumor invasion depth, node metastasis, and TNM stages) and lymphocytic infiltration. Our current findings suggested that the increased IDO expression in ESCCs is from a mixed cellular source (both cancer cells and noncancerous cells). Further studies on immune cell functional analysis are required in the future.

[In vivo antitumor effect of canstatin gene on human esophageal carcinoma xenografts in nude mice].

BACKGROUND AND OBJECTIVE: Canstatin is a newly discovered endogenous inhibitor of angiogenesis. Previous study has shown that canstatin can efficiently suppress the growth of human cancers, even more potent than endostatin. This study was to investigate the antitumor effects of canstatin gene on human esophageal carcinoma xenografts. METHODS: Tumor xenografts were induced with KYSE150 cells in BALB/c nude mice, and randomized into three groups: PBS, adenovirus green fluorescent protein (Ad-GFP), and Ad-GFP-canstatin groups. During treatment, tumor size was measured. The mice were killed 30 days later to observe tumor morphology. The expression of vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), caspase-3 and microvessel density (MVD) were detected by immunohistochemistry. RESULTS: Compared with that in Ad-GFP and PBS groups, tumor growth in Ad-GFP-canstatin group was significantly suppressed in the first week after gene transfection. The inhibition rate of tumor growth was up to 61% at the sixth day. Necrotic regions were observed in all groups, especially in Ad-GFP-canstatin group. Compared with those in Ad-GFP and PBS groups, caspase-3 expression in Ad-GFP-canstatin group was higher (P<0.05), while Flk-1 expression and MVD was lower (P<0.05). There was no obvious difference in VEGF expression among the three groups. CONCLUSION: Canstatin can inhibit the growth of human esophageal carcinoma by suppressing angiogenesis via down-regulating Flk-1 expression.

[Pseudotyping of murine leukemia virus particles with porcine reproductive and respiratory syndrome virus M protein-mediated E protein].

For constructing pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6, the ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified by RT-PCR, and transiently transfected into 293T cells by calciumphosphate co-precipitation. After 48 h, 293T cells were collected and surveyed by flow cytometry examination. The result indicated that the expression level of the E protein that mediated by the M protein was higher than that of the E protein expressed independently. Then pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6 were respectivly co-transfected into 293T cells with pHIT60 (include MuLV structural genes,namely gag and pol) and pHIT111 (retroviral genome, containing LacZ as a reporter). The supernatants were harvested 48 h post-transfection,and the analysis of the characteristic of the pseudotyping virions was performed by Western blot and infection test. The result indicated that the E proteins were expressed on the virions, and incorporated into the retroviral virions. Infection test were performed on Marc-145 and PAM, all the cells infected were Lac Z positive. These results indicated the pseudotype virions of MuLV-E and MuLV-E/M were infectious, and higher infectivity was achieved by MuLV-E/M.