Ionones from cigar tobacco leaves.

Phytochemical studies on cigar tobacco leaves led to the isolation of 18 ionone-type compounds, including previously undescribed cigatobanes E (1) and F (2). Additionally, compounds vomifoliol acetate (3), dehydrovomifoliol (4), 8,9-dihydromegastigmane-4,6-diene-3-one (5), 7α,8α-epoxyblumenol B (6), 3-oxoactinidol (12), and loliolide acetate (15), 4ß-hydroxy-dihydroactinidiolide (17), were found in tobacco leaves for the first time. The structural elucidation of all compounds was accomplished through rigorous spectral analysis.

Indole alkaloids isolated from the Nicotiana tabacum-derived Aspergillus fumigatus 0338 as potential inhibitors for tobacco powdery mildew and their mode of actions.

To explore active natural products against tobacco powdery mildew caused by Golovinomyces cichoracearum, an extract from the fermentation of endophytic Aspergillus fumigatus 0338 was investigated. The mechanisms of action for active compounds were also studied in detail. As a result, 14 indole alkaloid derivatives were isolated, with seven being newly discovered (1-7) and the remaining seven previously described (8-14). Notably, compounds 1-3 are rare linearly fused 6/6/5 tricyclic prenylated indole alkaloids, with asperversiamide J being the only known natural product of this kind. The isopentenyl substitutions at the 5-position in compounds 4 and 5 are also rare, with only compounds 1-(5-prenyl-1H-indol-3-yl)-propan-2-one (8) and 1-(6-methoxy-5-prenyl-1H-indol3-yl)-propan-2-one currently available. In addition, compounds 6 and 7 are new framework indole alkaloid derivatives bearing a 6-methyl-1,7-dihydro-2H-azepin-2-one ring. The purified compounds were evaluated for their activity against G. cichoracearum, and the results revealed that compounds 7 and 9 demonstrated obvious anti-G. cichoracearum activities with an inhibition rate of 82.6% and 85.2%, respectively, at a concentration of 250 µg/mL, these rates were better than that of the positive control agent, carbendazim (78.6%). The protective and curative effects of compounds 7 and 9 were also better than that of positive control, at the same concentration. Moreover, the mechanistic study showed that treatment with compound 9 significantly increased the structural tightness of tobacco leaves and directly affect the conidiospores of G. cichoracearum, thereby enhancing resistance. Compounds 7 and 9 could also induce systemic acquired resistance (SAR), directly regulating the expression of defense enzymes, defense genes, and plant semaphorins, which may further contribute to increased plant resistance. Based on the activity experiments and molecular dockings, the indole core structure may be the foundation of these compounds' anti-G. cichoracearum activity. Among them, the indole derivative parent structures of compounds 6, 7, and 9 exhibit strong effects. Moreover, the methoxy substitution in compound 7 can enhance their activity. By isolating and structurally identifying the above indole alkaloids, new candidates for anti-powdery mildew chemical screening were discovered, which could enhance the utilization of N. tabacum-derived fungi in pesticide development.

Sesquiterpenoids from cigar tobacco leaves.

Phytochemical investigation on cigar tobacco leaves led to four unknown sesquiterpenoids as well as nine reported ones. Among of them, 3-acetoxy-ß-damascone was first found in tobacco leaves. All the structures were elucidated by intensive spectroscopic analyses and X-ray diffraction. The relationship between the newly isolates and known ones was tried to describe.

Extraction and characterization of anti-virus anthraquinones from Nicotiana tabacum-derived Aspergillus oryzae YNCA1220.

In this study, seven novel anthraquinones (1-7) and four described anthraquinones (8-11) were purified from Nicotiana tabacum-derived Aspergillus oryzae YNCA1220. It is worth noting that only analogs of 4 and 5 have been reported as natural products to date, while the nuclei of compounds 1-3, 6 and 7 were isolated for the first time in nature. Among them, compounds 1-3 bear an unusual anthra[2,3-b]furan-9,10-dione nucleus, 4 and 5 possess a rare 3-methyl-1H-pyrrol-2-yl substituent, and 6 and 7 are new framework anthraquinones bearing a 6-methyl-1,7-dihydro-2H-azepin-2-one ring. Interestingly, the in vivo assays indicated that 1, 4 and 5 had inactivation effects against tobacco mosaic virus (TMV) with inhibition rates of 41.6%, 55.4% and 38.6%, respectively, at a concentration of 50 µg/mL, which were better than that of the positive control agent, ningnanmycin (33.8%). Compounds 1, 4 and 5 also had protective effects with inhibition rates of 48.7%, 60.2% and 43.5% at the same concentration, while 4 had a better curative effect than ningnanmycin at a concentration of 100 µg/mL. In addition, mechanistic studies also revealed that a potent direct effect on TMV, the induction of SAR in tobacco plants, and the effective regulation of defense enzymes, defense genes, and defense hormones may be the reasons for the significant effects of 4 against TMV. At the same time, downregulation of the expression of total NtHsp70 protein by inhibiting the related Hsp70 genes may also be involved in tobacco resistance to TMV. To evaluate whether compounds have broader antiviral activities, the antirotavirus activities of new isolates were also evaluated and found to be highly effective with a therapeutic index (TI) value ranging from 11.6 to 17.7. This study suggests that the above anthraquinone compounds, particularly 4, have broad spectrum antiviral activities. The successful isolation and structure identification of the above anthraquinones provide new materials for the screening of anti-TMV agents and contribute to the improved utilization of N. tabacum-derived fungi.

Treg cells-derived exosomes promote blood-spinal cord barrier repair and motor function recovery after spinal cord injury by delivering miR-2861.

The blood-spinal cord barrier (BSCB) is a physical barrier between the blood and the spinal cord parenchyma. Current evidence suggests that the disruption of BSCB integrity after spinal cord injury can lead to secondary injuries such as spinal cord edema and excessive inflammatory response. Regulatory T (Treg) cells are effective anti-inflammatory cells that can inhibit neuroinflammation after spinal cord injury, and their infiltration after spinal cord injury exhibits the same temporal and spatial characteristics as the automatic repair of BSCB. However, few studies have assessed the relationship between Treg cells and spinal cord injury, emphasizing BSCB integrity. This study explored whether Treg affects the recovery of BSCB after SCI and the underlying mechanism. We confirmed that spinal cord angiogenesis and Treg cell infiltration occurred simultaneously after SCI. Furthermore, we observed significant effects on BSCB repair and motor function in mice by Treg cell knockout and overexpression. Subsequently, we demonstrated the presence and function of exosomes in vitro. In addition, we found that Treg cell-derived exosomes encapsulated miR-2861, and miR-2861 regulated the expression of vascular tight junction (TJs) proteins. The luciferase reporter assay confirmed the negative regulation of IRAK1 by miR-2861, and a series of rescue experiments validated the biological function of IRAKI in regulating BSCB. In summary, we demonstrated that Treg cell-derived exosomes could package and deliver miR-2861 and regulate the expression of IRAK1 to affect BSCB integrity and motor function after SCI in mice, which provides novel insights for functional repair and limiting inflammation after SCI.

Gasotransmitters in non-alcoholic fatty liver disease: just the tip of the iceberg.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by fatty lesions and fat accumulation in hepatic parenchymal cells, which is in the absence of excessive alcohol consumption or definite liver damage factors. The exact pathogenesis of NAFLD is not fully understood, but it is now recognized that oxidative stress, insulin resistance, and inflammation are essential mechanisms involved in the development and treatment of NAFLD. NAFLD therapy aims to stop, delay or reverse disease progressions, as well as improve the quality of life and clinical outcomes of patients with NAFLD. Gasotransmitters are produced by enzymatic reactions, regulated through metabolic pathways in vivo, which can freely penetrate cell membranes with specific physiological functions and targets. Three gasotransmitters, nitric oxide, carbon monoxide, and hydrogen sulfide have been discovered. Gasotransmitters exhibit the effects of anti-inflammatory, anti-oxidant, vasodilatory, and cardioprotective agents. Gasotransmitters and their donors can be used as new gas-derived drugs and provide new approaches to the clinical treatment of NAFLD. Gasotransmitters can modulate inflammation, oxidative stress, and numerous signaling pathways to protect against NAFLD. In this paper, we mainly review the status of gasotransmitters research on NAFLD. It provides clinical applications for the future use of exogenous and endogenous gasotransmitters for the treatment of NAFLD.

Treg cell-derived exosomes miR-709 attenuates microglia pyroptosis and promotes motor function recovery after spinal cord injury.

Neuroinflammation is an important cause of poor prognosis in patients with spinal cord injury. pyroptosis is a new type of inflammatory cell death. Treg cells has been shown to play an anti-inflammatory role in a variety of inflammatory diseases, including inflammatory bowel disease, amyotrophic lateral sclerosis, and arthritis. However, little is known about Treg cells' potential role in pyroptosis following spinal cord injury. The aim of this research was to look into the effect of Treg cells to motor function recovery, pyroptosis and the mechanism behind it after SCI. Here, we found that pyroptosis mainly occurred in microglia on the seventh day after spinal cord injury. Konckout Treg cells resulted in widely pyroptosis and poor motor recovery after SCI. In conversely, over-infiltration of Treg cell in mice by tail vein injection had beneficial effects following SCI.Treg cell-derived exosomes promote functional recovery by inhibiting microglia pyroptosis in vivo. Bioinformatic analysis revealed that miRNA-709 was significantly enriched in Treg cells and Treg cell-secreted exosomes. NKAP has been identified as a miRNA-709 target gene. Moreover, experiments confirmed that Treg cells targeted the NKAP via exosomal miR-709 to reduce microglia pyroptosis and promote motor function recovery after SCI. More importantly, The miR-709 overexpressed exosomes we constructed significantly reduced the inflammatory response and improved motor recovery after spinal cord injury. In brief, our findings indicate a possible mechanism for communication between Treg cells and microglia, which opens up a new perspective for alleviating neuroinflammation after SCI.

Role of peripheral immune cells in spinal cord injury.

Secondary spinal cord injury is caused by an inflammatory response cascade, and the process is irreversible. The immune system, as a mediator of inflammation, plays an important role in spinal cord injury. The spinal cord retains its immune privilege in a physiological state. Hence, elucidating the mechanisms by which peripheral immune cells are recruited to the lesion site and function after spinal cord injury is meaningful for the exploration of clinical therapeutic targets. In this review, we provide an overview of the multifaceted roles of peripheral immune cells in spinal cord injury.

Paclitaxel-incorporated nanoparticles improve functional recovery after spinal cord injury.

As a worldwide medical problem, spinal cord injury has no clear and effective treatment to improve its prognosis. Hence, new treatment strategies for spinal cord injury with good therapeutic efficacy have been actively pursued. As a new drug loading system, acetal dextran nanoparticles (SAD) have good biocompatibility and biodegradability. Therefore, we designed spermine-functionalized acetal-dextran (SAD) nanoparticles and encapsulated paclitaxel (PCL) into them. This design can ensure the sustained release of paclitaxel in the injured area for 4 days and promote the extension of nerve processes in vitro. In our experiment, we found that paclitaxel-loaded SAD nanoparticles (PCL@SAD) decreased the level of chondroitin sulfate proteoglycan in the rat spinal cord injury model, which reduced the scar repair of the injured site and changed the inhibitory environment after spinal cord injury. This reveals that PCL@SAD can effectively protect the injured spinal cord and ultimately improve the functional recovery of the injured spinal cord. One single injection of PCL@SAD shows better therapeutic effect than that of PCL. This study opens an exciting perspective toward the application of neuroprotective PCL@SAD for the treatment of severe neurological diseases.

NAC Transcription Factor TwNAC01 Positively Regulates Drought Stress Responses in Arabidopsis and Triticale.

The NAC transcription factors play important roles in regulating plant growth, development, and senescence, and responding to biotic and abiotic stressors in plants. A novel coding sequence (1,059 bp) was cloned from hexaploid triticale in this study. The putative protein (352 amino acids) encoded by this sequence was over 95% similar to the amino acid sequence of a NAC protein from Aegilops tauschii (XP020161331), and it formed a clade with Ae. tauschii, durum wheat, and barley. The putative protein contained a conserved nature actomyosin (NAM) domain (129 consecutive amino acids) between the 20th and 148th amino acids at the N-terminus and three transcription activation regions at the C-terminus. The novel gene was identified as a triticale NAC gene localized in the nucleus and designated as TwNAC01 (GenBank accession MG736919). The expression levels of TwNAC01 were the highest in roots, followed by leaves and stems when triticale lines were exposed to drought, polyethylene glycol 6,000 (PEG6000), NaCl, cold, methyl jasmonate (MeJA), and abscisic acid (ABA). Transgenic Arabidopsis thaliana overexpressing TwNAC01 had significantly lower leaf water loss rates and longer roots than wild-type (WT) A. thaliana. Virus-induced silencing of the TwNAC01 gene in triticale delayed root development and decreased length of primary root. Under drought stress, leaves of TwNAC01-silenced triticale had higher levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2), but lower relative water content (RWC), net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate than the leaves of the WT. Gene overexpression and silencing experiments suggested that TwNAC01 improves plant stress tolerance by increasing root length, regulating the water content of plant leaves by reducing MDA and H2O2 content, and adjusting respiration rate. The results suggest that TwNAC01 is a novel NAC transcription factor gene that can be exploited for triticale and cereal improvement.

Glioblastoma: two immune subtypes under the surface of the cold tumor.

Glioblastoma is classified as an immunocompromised tumor. The immune pattern beneath the cold tumor surface, however, has yet to be confirmed. Understanding the immune pattern of glioblastoma will aid in the development of effective treatment strategies. We performed weighted gene co-expression network analysis on all immune-related genes in TCGA-GBM transcriptional data and screened 35 prognosis-related immune genes. Unsupervised consistent clustering of these genes was used to analyze the immunological pattern of GBM. A glioblastoma immune prognostic score was developed by using 13 genes discovered by cox regression methods and verified with the GEO dataset to assess the immune profile, prognosis, and immunotherapy effects in individual patients. Glioblastoma has two immune modalities, immune tolerance and immunodeficiency, with distinct immune microenvironments, tumor-associated macrophages being one of the most promising new therapeutic targets. GIPS is a promising biomarker for assessing immune evasion mechanisms, immunotherapy responses, and prognosis in patients.

Spleen contributes to restraint stress induced hepatocellular carcinoma progression.

The spleen is the largest secondary immune organ and plays a critical role in the progression of tumor. Psychological stress promotes tumor progression through inhibiting antitumor immune. However, the role of spleen in tumor progression induced by stress is unclear. Here, we showed that restraint stress promoted tumor growth, increased the percentage of CD11b+Gr-1+ MDSC while decreased the percentages of CD3-NK1.1+ NK and CD3+NK1.1+ NKT in the tumor tissues. Restraint stress decreased the percentages of CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes while increased the percentage of CD11b+Gr-1+ MDSC in the blood of tumor-bearing mice. Restraint stress increased the percentages of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, CD4+PD1+ T lymphocytes and CD8+PD1+ T lymphocytes while decreased the percentage of CD11b+Gr-1+ MDSC in the spleen of tumor-bearing mice. Interestingly, splenectomy inhibited tumor growth and attenuated the changes of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, and CD11b+Gr-1+ MDSC in blood induced by chronic restraint stress. Finally, splenectomy blocked the increases of CD11b+Gr-1+ MDSC but did not attenuate the decreases of CD3-NK1.1+ NK and CD3+NK1.1+ NKT in tumor tissue induced by chronic stress. Together, these data indicate that chronic restraint stress promotes hepatocellular carcinoma growth and suppresses the antitumor immunity of tumor-bearing mice. Splenectomy could inhibit tumor growth and partly block the decrease of antitumor immune activity induced by stress.

Chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells through activating ß-adrenergic signaling.

Psychological stress promotes tumor progression and has a large impact on the immune system, particularly the spleen. The spleen plays an important role in tumor behavior. However, the role and mechanism of the spleen in hepatocellular carcinoma progression induced by stress is unclear. Here, we showed that the spleen plays a critical role in hepatocellular carcinoma growth induced by restraint stress. Our results demonstrated that restraint stress promoted hepatocellular carcinoma growth, changed the spleen structure, and redistributed splenic myeloid cells to tumor tissues. Interestingly, we found that splenectomy could inhibit hepatocellular carcinoma growth and prevent increases in myeloid cells and macrophages in tumor tissues in stressed mice. Restraint stress significantly elevated the concentration of norepinephrine in the spleen, serum and tumor tissues. Meanwhile, propranolol, an inhibitor of ß-adrenergic signaling, could inhibit hepatocellular carcinoma growth and prevent the redistribution of splenic myeloid cells induced by restraint stress, suggesting that restraint stress promotes hepatocellular carcinoma growth and redistributes splenic myeloid cells through ß-adrenergic signaling. Mechanistic studies revealed that restraint stress upregulated the expressions of CXCL2/CXCL3 in tumor tissues and changed the expression of CXCR2 in myeloid cells. SB225002, an inhibitor of CXCR2, could prevent the recruitment of myeloid cells in tumor tissues and inhibit tumor growth in stressed mice. Together, these data indicate that chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells to tumor tissues via activating ß-adrenergic signaling. The CXCR2-CXCL2/CXCL3 axis contributed to the recruitment of myeloid cells in tumor tissues induced by restraint stress.

Repetitive restraint stress changes spleen immune cell subsets through glucocorticoid receptor or ß-adrenergic receptor in a stage dependent manner.

Immune system is sensitive to stress. Spleen is the largest peripheral immune organ innervated with sympathetic nerves and controlled by adrenomedullary system in the body. However, the alterations and mechanism of spleen immune cell subsets caused by repetitive restraint stress (RRS) is poorly understood. In this study, we found that RRS reduced spleen index in mice, and induced an expansion of white pulp and involution of the red pulp. Meanwhile, the percentage of CD3+CD8+ T lymphocytes, CD11b+F4/80+ macrophages, CD11b+Ly-6G-Ly-6Chi monocytic myeloid derived suppressor cells (mMDSCs) and CD11b+Ly-6G+Ly-6Cint granulocytic myeloid derived suppressor cells (gMDSCs) in spleen were significantly changed by RRS. Mechanistically, we found that the expression of norepinephrine (NE) and ß-adrenergic receptor (ß-AR) in spleen were up-regulated after 21 days of RRS, but not 7 days. The expression of corticosterone (CORT) and glucocorticoid receptor (GR) in spleen were up-regulated after 7 days of RRS but were lower after 21 days of RRS, even though they were still higher than that in mice without stress. By treating the stressed mice with RU486 (antagonist of GR) or propranolol (antagonist of ß-AR), we demonstrated that GR was responsible for the changes of spleen induced by 7 days of RRS and ß-AR was for 21 days of RRS. Our data suggest that RRS changes spleen immune cell subsets through GR or ß-AR in a stage dependent manner.

Spleen contributes to restraint stress induced changes in blood leukocytes distribution.

Psychological stress has great impacts on the immune system, particularly the leukocytes distribution. Although the impacts of acute stress on blood leukocytes distribution are well studied, however, it remains unclear how chronic stress affects leukocytes distribution in peripheral circulation. Furthermore, there is no report about the role of spleen in the blood leukocytes distribution induced by stress. Here we show that spleen contributes to the alteration of restraint stress induced blood leukocytes distribution. Our data confirmed that restraint stress induced anxiety-like behavior in mice. Furthermore, we found that restraint stress decreased the CD4/CD8 ratio and elevated the percentages of natural killer cells, monocytes and polymorphonuclear myeloid-derived suppressor cell. We demonstrated that activation of hypothalamic-pituitary-adrenal axis (HPA) and sympathetic nervous system (SNS) contributes to restraint stress induced alteration of blood leukocyte distribution. Interestingly, we found that splenectomy could reverse the change of CD4/CD8 ratio induced by restraint stress. Together, our findings suggest that activation of HPA axis and SNS was responsible for the blood leukocyte subsets changes induced by restraint stress. Spleen, at least in part, contributed to the alteration in peripheral circulation induced by restraint stress.

Cytoprotection of methionine on hyperthermia-induced damage in bovine mammary epithelial cells.

The protective effects of methionine against hyperthermia-induced damage in bovine mammary epithelial cells (BMEC) were studied. We have investigated the effects of methionine on proliferation, antioxidant activity, and apoptosis of the mammary epithelial cells of dairy cow after heat treatment. The structure of BMEC membrane was damaged by hyperthermia. Methionine (30 and 60 mg/L) efficiently increased cell viability and attenuated morphological damages in hyperthermia-treated BMEC. It significantly reduced lactate dehydrogenase leakage and malondialdehyde formation, whereas superoxide dismutase activity increased significantly. It also increased cell survival and decreased early apoptosis. Methionine therefore is cytoprotective on hyperthermia-induced damage in BMEC by increasing intracellular antioxidant levels and decreasing lipid peroxidation.

Three novel xanthones from Garcinia paucinervis and their anti-TMV activity.

Phytochemical investigations of the leaves of Garcinia paucinervis resulted in the isolation of three new xanthones 1-3 and five known analogues 4-8. Structural elucidations of 1-3 were performed by spectral methods such as 1D and 2D (HMQC, HMBC, and ROESY) NMR spectroscopy, in addition to high resolution mass spectrometry. Compounds 1-3 showed anti-TMV activities, with inhibition rates above 20%, especially for 1, which had a lower IC50 value of 21.4 µM.

Effect of icarisid II on diabetic rats with erectile dysfunction and its potential mechanism via assessment of AGEs, autophagy, mTOR and the NO-cGMP pathway.

Erectile dysfunction (ED) is a major complication of diabetes mellitus. Icariin has been shown to enhance erectile function through its bioactive form, icarisid II. This study investigates the effects of icarisid II on diabetic rats with ED and its potential mechanism via the assessment of advanced glycosylation end products (AGEs), autophagy, mTOR and the NO-cGMP pathway. Icarisid II was extracted from icariin by an enzymatic method. In the control and diabetic ED groups, rats were administered normal saline; in the icarisid II group, rats were administered icarisid II intragastrically. Erectile function was evaluated by measuring intracavernosal pressure/mean arterial pressure (ICP/MAP). AGE concentrations, nitric oxide synthase (NOS) activity and cGMP concentration were assessed by enzyme immunoassay. Cell proliferation was analysed using methyl thiazolyl tetrazolium assay and flow cytometry. Autophagosomes were observed by transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 localisation. The expression of NOS isoforms and key proteins in autophagy were examined by western blot. Our results have shown that Icarisid II increased ICP/MAP values, the smooth muscle cell (SMC) growth curve, S phase and SMC/collagen fibril (SMC/CF) proportions and decreased Beclin 1 (P<0.05). Icarisid II significantly increased the proliferative index and p-p70S6K(Thr389) levels and decreased the numbers of autophagosomes and the levels of LC3-II (P<0.01). Icarisid II decreased AGE concentrations and increased cGMP concentration, NOS activity (P<0.05) and cNOS levels (P<0.01) in the diabetic ED group. Therefore, Icarisid II constitutes a promising compound for diabetic ED and might be involved in the upregulation of SMC proliferation and the NO-cGMP pathway and the downregulation of AGEs, autophagy and the mTOR pathway.

Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2.

AIM: To investigate the mechanism underlying the increase of hepatoma cell proliferation by hepatitis B virus X protein (HBx). METHODS: HepG2, H7402 and HepG2.2.15 cells, which constitutively replicated hepatitis B virus were used. The effects of HBx on hepatoma cell proliferation were examined using 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and MTT assay. The expression level of MEKK2 was measured using RT-PCR, Western blot and luciferase reporter gene assay. The activity of activator protein 1 (AP-1) was detected using luciferase reporter gene assay. The phosphorylation levels of JNK and c-Jun were measured using Western blot. The expression levels of HBx and MEKK2 in 11 clinical hepatocellular carcinoma (HCC) tissues were measured using real time PCR and Western blot. In addition, the expression of MEKK2 in 95 clinical HCC tissues was examined using immunohistochemistry. RESULTS: HBx significantly enhanced HepG2-X cell proliferation. In HepG2-X, H7402-X and HepG2.2.15 cells, the expression level of MEKK2 was remarkably increased. In HepG2.2.15 cells, HBx was found to activate JNK and AP-1, which were the downstream effectors of MEKK2 in HepG2-X and HepG2.2.15 cells. In 11 clinical HCC tissues, both HBx and MEKK2 expression levels were remarkably increased, as compared to those in the corresponding peritumor tissues. In 95 clinical HCC tissues, the rate of detection of MEKK2 was 85.3%. CONCLUSION: HBx promotes hepatoma cell proliferation via upregulating MEKK2, which may be involved in hepatocarcinogenesis.