Betulin from Hedyotis hedyotidea ameliorates concanavalin A-induced and T cell-mediated autoimmune hepatitis in mice.

Hedyotis hedyotidea has been used in traditional Chinese medicine for the treatment of autoimmune diseases. However, the mechanisms underlying for the effect remain unknown. We previously showed that, among 11 compounds extracted from H hedyotidea, betulin produced the strongest suppressive effect on T cell activation. Here, we examined the hepatoprotective effects of betulin against acute autoimmune hepatitis in mice and the mechanisms underlying the effects. Freshly isolated mouse splenocytes were stimulated with concanavalin A (Con A, 5 µg/mL) in the presence of betulin, the cell proliferation was assessed with CSFE-dilution assay. Mice were injected with betulin (10, 20 mg·kg-1·d-1, ip) for 3 d. One hour after the last injection, the mice were injected with Con A (15 mg/kg, iv) to induce acute hepatitis. Blood samples and liver tissues were harvested at 10 h after Con A injection, and serum transaminase levels and liver histopathology were detected; serum levels of proinflammatory cytokines, hepatic T lymphocyte ratios, and functional statuses of conventional T and NKT cells were also analyzed. Betulin (16 and 32 µmol/L) dose-dependently suppressed the proliferation of Con A-stimulated mouse splenocytes in vitro. In Con A-challenged mice, preinjection with betulin (20 mg·kg-1·d-1) significantly decreased the levels of proinflammatory cytokines IFN-γ, TNF-α and IL-6, and ameliorated liver injury. Furthermore, pretreatment with betulin (20 mg·kg-1·d-1) significantly inhibited the Con A-induced activation of NKT and conventional T cells, and decreased production of proinflammatory cytokines IFN-γ, TNF-α and IL-6 in these two cell populations. Betulin has immunomodulatory effect on overly activated conventional T and NKT cells and exerts hepatoprotective action in mouse autoimmune hepatitis. The findings provide evidence for the use of H hedyotidea and its constituent betulin in the treatment of autoimmune diseases.

[Chemical constituents from stems of Hedyotis hedyotidea and their immunosuppressive activity].

Hedyotis hedyotidea has been traditionally used for the treatment of arthritis, cold, cough, gastro-enteritis, headstroke, etc. But few studies have screened the active compounds from extracts of H. hedyotidea. In this study, the structure of the chemical constituents from stems of H. hedyotidea were determined and the immunosuppressive activity of the compounds was evaluated. The compounds were separated and purified with silica gel, gel column chromatographies and preparative HPLC, and their structures were identified by spectral methods such as MS and NMR. Eleven compounds were obtained and identified as(6S,9S) -vomifoliol (1), betulonic acid (2), betulinic acid (3), betulin(4), 3-epi-betulinic acid (5), ursolic acid (6), ß-sitosterol (7), stigmast-4-en-3-one (8), 7ß-hydroxysitosterol (9), (3ß,7ß) -7-methoxystigmast-5-en-3-ol (10) and morindacin (11). This is the first report of compounds 1, 2, 4, 8, 9, 10 and 11 from H. hedyotidea. Compounds 1, 2 and 8-11 were firstly isolated from the genus Hedyotis, and compounds 9 and 10 were isolated from the family Rubiaceae for the first time. The immunosuppressive activity of these compounds was tested using the lymphocyte transsormationtest. Compounds 4, 6 and 9 showed significant immunosuppressive activity.

[Effect of centipede extract on cervical tumor of mice and its mechanism].

UNLABELLED: To study the anti-tumor activity of centipede extract on cervical tumor of mice and its mechanism. METHODS: The tumor-bearing mice were treated with centipede extract from two solvents [ether (CE) and alcohol (CA)] at different comcentration. The mice' life span, tumor inhibition rate and immune function were estimated. RESULTS: No mice died in CE and CA treatment groups and the tumor inhibition rate was 52.85% and 33.65% respectively. Observed the tumor tissue slices with light microscope and found infiltration of tumor cells in striated muscle in the control group but centipede treatment groups had massive necrosis and apoptosis. Karyopyknosis and apoptotic tumor cells were observed in the treatment groups under transmission electron microscopy. Compared with control group, the expression of Bax increased, the expressions of Bcl-2 and Survivin decreased, but the content of VEGF, the indexes of thymus and spleen had no significant change in treatment groups. The number of CD3+ T lymphocytes had no significant change while the ratio of CD4+ and CD8+, the number of CD19+ B lymphocytes decreaed in the CE group. The numbers of CD3+ and CD4+ lymphocytes decreased in the CA group. The pathological examine indicated no obvious change in the tissue slices of mice's liver and kidney, manifested the concentrations of CE and CA between the article's had no visible side effect. CONNCLUSION: The two extracts (CE and CA) can suppress the growth of cervical tumor and its mechanism may be related to Bax and Caspase-3 medicated the mitochondrial signal transit pathway.

Expression of Toll-like receptor 4 on peripheral blood mononuclear cells and its effects on patients with acute myocardial infarction treated with thrombolysis.

BACKGROUND AND AIMS: TLR4 has been shown to mediate inflammation in animal models of myocardial ischemia/reperfusion injury (MI/RI). Here we hypothesized that TLR4 on peripheral blood mononuclear cells (PBMCs) may be involved in the inflammatory response in this type of clinical event. METHODS: Seventy two patients with acute myocardial infarction (AMI) who underwent thrombolysis were assigned into reperfusion group (n = 43) and non-reperfusion group (n = 29) according to recanalization of infarct-related artery (IRA) and 40 healthy volunteers were enrolled in this experiment. Eight mL of venous blood was taken from all patients 0 h before and 2, 6, 12, and 24 h after thrombolysis. Flow cytometry (FCM) was used to detect TLR4 protein expression and real-time quantitative RT-PCR was performed to determine TLR4 mRNA and myeloid differentiation protein-88 (Myd88) mRNA expression. The concentration of tumor necrosis factor-α (TNF-α) in plasma was evaluated using enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with controls, all detected indicators in AMI patients were upregulated before thrombolysis (p <0.01). After thrombolysis, they were further increased. In reperfusion group, all attained their peaks in earlier hours and the peak values were lower compared with non-reperfusion group. In both cases, either reperfusion or non-perfusion, TLR4 mRNA expression was positively correlated with the levels of Myd88 mRNA (r = 0.886 and 0.694, p <0.01), respectively. CONCLUSIONS: TLR4 expression on PBMCs was markedly elevated in AMI patients either reperfused or non-reperfused. Inflammatory reaction by activated TLR4 in MI/RI in patients may be through TLR4-Myd88-dependent signal pathway.

Highly expressed N1-acetylpolyamine oxidase detoxifies polyamine analogue N1-cyclopropylmethyl-N11-ethylnorspermine in human lung cancer cell line A549.

BACKGROUND: The critical roles of polyamines in cell growth and differentiation have made polyamine metabolic pathway a promising target for antitumor therapy. Recent studies have demonstrated in vitro that some antitumor polyamine analogues could be used as substrates and oxidized by purified recombinant human N(1)-acetylpolyamine oxidase (APAO, an enzyme that catabolizes natural polyamines), indicating a potential role of APAO in determining the sensitivity of cancer cells to specific antitumor analogues. This study evaluated, in vivo, the effect of APAO on cytotoxicity of antitumor polyamine analogue, N(1)-cyclopropylmethyl-N(11)-ethylnorspermine (CPENS) and its mechanism when highly expressed in human lung cancer line A549. METHODS: A clone with high expression of APAO was obtained by transfecting A549 lung cancer cell line with pcDNA3.1/APAO plasmid and selecting with quantitative realtime PCR and APAO activity assay. Cell proliferation was determined by MTT method and apoptosis related events were evaluated by DNA fragmentation, sub-G1/flow cytometric assay, western blotting (for cytochrome C and Bax) and colorimetric assay (for casapse-3 activity). RESULTS: A clone highly expressing APAO was obtained. High expression of APAO in A549 cells inhibited accumulation of CPENS, decreased their sensitivity to the toxicity of CPENS and prevented CPENS induced apoptosis. CONCLUSION: These results indicate a new drug resisting, mechanism in the tumor cells. High expression of APAO can greatly decrease the sensitivity of tumor cells to the specific polyamine analogues by detoxifying those analogues and prevent analogue induced apoptosis.