Scopoletin Stimulates Melanogenesis via cAMP/PKA Pathway and Partially p38 Activation.

Scopoletin was recently shown to stimulate melanogenesis through cAMP-response element-binding protein (CREB) phosphorylation. In this study, we investigated the molecular events of melanogenesis-induced by scopoletin. After exposure to scopoletin, the protein levels of tyrosinase and tyrosianse related protein-1 (TRP-1) were significantly increased in B16F10 cells. The mRNA levels of tyrosinase and microphthalmia-associated transcription factor (MITF) were also enhanced by scopoletin. cAMP production and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were increased by scopoletin treatment. Scopoletin-mediated increase of intracellular melanin and tyrosinase expression were significantly attenuated by protein kinase A (PKA) inhibitors (H-89 and KT5720), while a protein kinase C (PKC) inhibitor (Ro-32-0432) had no effect and a p38 MAPK inhibitor (SB203580) partially blocked the scopoletin-induced intracellular melanin and tyrosinase expression. Moreover, scopoletin synergistically with cell-permeable cAMP analog (dibutyryl cAMP) significantly induced tyrosinase activity and melanin content in B16F10 cells. The silencing of p38 MAPK by small interfering RNA (siRNA) decreased the scopoletin-induced tyrosinase expression in B16F10 cells. These results suggest that scopoletin could induce melanin synthesis through the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

EPA attenuates ultraviolet radiation-induced downregulation of aquaporin-3 in human keratinocytes.

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid (ω-3 PUFA) that protects against photodamage and photocarcinogenesis in mammals. Aquaporin-3 (AQP3) is a water/glycerol transport protein that is found in basal layer keratinocytes. In this study, we have investigated the protective effect of EPA against ultraviolet B (UVB)-induced AQP3 downregulation in human keratinocytes. EPA treatment was found to increase AQP3 gene and protein expression in human epidermal keratinocytes (HaCaT). Using a specific inhibitor, we observed that the effect of EPA on AQP3 expression was mediated by extracellular signal-regulated kinase (ERK) activation. UVB radiation induced AQP3 downregulation in HaCaT cells, and it was found that EPA treatment attenuated UVB-induced AQP3 reduction and the associated cell death. UVB-induced downregulation of AQP3 was blocked by EPA and p38 inhibitor SB203580. Collectively, the present results show that EPA increased AQP3 expression and that this led to a reduction UVB-induced photodamage.

Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway.

Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 µM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP) and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK)-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.

BBR induces apoptosis in HepG2 cell through an Akt-ASK1-ROS-p38MAPKs-linked cascade.

Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase-3. The expressions of Bcl-2 protein and pro-caspase-3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR-induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR-induced apoptosis effects via inhibition of Bax activation and Bcl-2 inactivation. BBR-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1-mediated activation of JNK and p38 pathways.

Antiinflammatory effect of Daesiho, a Korean traditional prescription for cerebral infarct patients.

Daesiho, a prescription composed of eight herbal mixtures, has been widely used in the treatment of cerebral infarct in Oriental medicine. However, the mechanisms by which the formula affects the production of pro-inflammatory cytokines in cerebral infarct patients remains unknown. The levels of secretory protein pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were significantly increased in both lipopolysaccharide (LPS) and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from cerebral infarct patients and LPS-stimulated THP-1 differentiated macrophage-like cells (THP-1/M). However, pretreatment with Daesiho significantly inhibited the secretion of pro-inflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, in stimulated PBMCs and THP-1/M cells. In addition, Daesiho significantly suppressed mRNA expression of pro-inflammatory cytokines. Therefore, these data indicate that Daesiho may be beneficial in the cessation of inflammatory processes of cerebral infarction through suppression of the production of pro-inflammatory cytokines via inhibition of mRNA expression.

Anti-inflammatory effect of So-Pung-Tang, a Korean traditional prescription for cerebral infarction patients.

So-Pung-Tang (Sopung), a prescription composed of 14 herbal mixtures, has been widely used in the treatment of cerebral infarction in Oriental Medicine. However, the mechanisms by which the formula affects on the production of pro-inflammatory cytokines in cerebral infarction patients remain unknown yet. The levels of secretory protein of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interlukin (IL)-1beta, and IL-6, were significantly increased in both THP-1 differentiated macrophage-like cells (THP-1/M) and peripheral blood mononuclear cells (PBMCs) from cerebral infarction patients after stimulation. However, pretreatment with Sopung markedly inhibited the secretion of TNF-alpha and IL-6, but not IL-1beta, in lipopolysaccharide (LPS)-stimulated THP-1/M cells and PBMCs treated with LPS and phytohemagglutinin (PHA). Furthermore, Sopung significantly inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 in THP-1/M cells. These data indicate that Sopung may be beneficial in the cessation of inflammatory processes of cerebral infarction through suppression of ERK1/2 and JNK activation.

Anti-inflammatory effect of oyaksungisan in peripheral blood mononuclear cells from cerebral infarction patients.

Oyaksungisan, the herbal prescription composed of eleven herbs, has been widely used in treatment of cerebral infarct in Oriental Medicine. However, the mechanisms by which the herbal formula affects on the production of pro- and anti-inflammatory cytokines in cerebral infarction patients remain unknown yet. The secretory levels of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, and IL-10 were significantly increased in both LPS and PHA-stimulated peripheral blood mononuclear cells (PBMCs) from cerebral infarction patients. However, pretreatment with oyaksungisan significantly inhibited the secretion of pro- and anti-inflammatory in PBMCs. Also, oyaksungisan induced a significant increase of transforming growth factor (TGF)-beta1 in PBMCs. Thus, these data indicate that oyaksungisan may be beneficial in the cessation of inflammatory processes of cerebral infarct through suppression of TNF-alpha, IL-1beta, IL-6, and IL-10 and induction of TGF-beta1.

Anti-inflammatory effect of Sasim extracts in PHA-stimulated THP-1 and peripheral blood mononuclear cells from cerebral infarction patients.

Sasim, a prescription composed of seven herbal mixtures, has been widely used for the treatment of cerebral infarction as an oriental medicine in Korea. However, the mechanisms by which the formula affects on the production of pro-inflammatory cytokines in cerebral infarct patients remain unknown yet. The levels of secretory protein and mRNA of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interlukin (IL)-1beta, and IL-6, were significantly increased in both THP-1 differentiated macrophage-like cells (T/M) and peripheral blood mononuclear cells (PBMCs) from cerebral infarct patients at 24h after stimulation with phytohemagglutinin (PHA) (p<0.05). However, pretreatment of Sasim strongly suppressed the secretion of pro-inflammatory cytokines in PHA-stimulated T/M cells and PBMCs. Moreover, Sasim significantly suppressed the transcriptional levels of pro-inflammatory cytokines in PHA-stimulated THP-1/M cells. These data indicate that Sasim may be beneficial in the cessation of inflammatory processes of cerebral infarction through suppression on the production of pro-inflammatory cytokines via inhibition of mRNA expression.

Selaginella tamariscina induces apoptosis via a caspase-3-mediated mechanism in human promyelocytic leukemia cells.

Cell apoptosis is now known to play an important role in the maintenance of cellular homeostasis and anticarcinogenesis. Selaginella tamariscina (ST) is a traditional medicinal plant for treatment of advanced cancer in the Orient. In the present study, the anticancer effect of ST was investigated by analyzing its potential to induce apoptosis in human leukemia HL-60 cells. ST-induced cytotoxicity of HL-60 cells was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The apoptosis was determined by microscopic examination of apoptotic morphology, determination of DNA fragmentation by electrophoresis, activation of caspase-3, and protein expression of procaspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, Bcl-2, and Bax. ST was cytotoxic to HL-60 cells in a dose-dependent manner. However, ST-induced cytotoxicity was suppressed by reactive oxygen species scavengers, including superoxide dismutase (SOD) and catalase. ST caused DNA fragmentation and nuclear condensation, all characteristics of apoptosis. ST-induced apoptosis is accompanied by the activation of caspase-3 and the specific proteolytic cleavage of PARP. Concomitantly, ST treatments led to an increase in the proapoptotic Bax levels, while Bcl-2 expression was decreased. Moreover, this effect was attenuated by SOD and catalase. These results suggest that oxidative stress may be involved in the cytotoxicity of ST, and that ST-induced apoptosis of HL-60 cells is primarily mediated by the caspase activation pathway.

Antiasthmatic activity and selective inhibition of type 2 helper T cell response by aqueous extract of semen armeniacae amarum.

Semen armeniacae amarum (SAA) has long been used to control asthma in Korean traditional medicine. However, its antiasthmatic action still remains poorly understood. In the current study, effective mechanism of SAA was investigated in a mouse model of allergic asthma induced by repeated sensitization and intranasal challenge with OVA. Airway hyperreactivity (AHR) measured by beta-methacholine-induced airflow obstruction and airway recruitment of leukocytes including eosinophils were significantly reduced by oral treatment of SAA water extract. Level of interleukin (IL)-4, but not Interferon gamma (IFN-gamma), in the bronchoalveolar lavage fluid (BALF) also appeared considerably lower in SAA-treated mice than in controls. Collectively, these data show that SAA suppresses type 2 helper T cell (Th2), but not type 1 helper T cell (Th1), response. This hypothesis was supported further by the data of ex vivo cytokine production of peribronchial lymph node cells. Thus, oral administration of SAA attenuates asthmatic manifestations including AHR and airway inflammation, which possibly result from selective inhibition of Th2 response to allergen. Our data strongly suggest that SAA may be effectively applied to control other Th2-related diseases as well as allergic asthma.

Yukmijihwang-tang ameliorates ischemia/reperfusion-induced renal injury in rats.

The present study was designed to examine whether Yukmijihwang-tang (YJT), which is a Korean decoction for the treatment of renal disease, has an effect on renal functional parameters in association with the expression of aquaporin 2 (AQP 2), Na,K-ATPase, heme oxygenase-1 (HO-1) in rats with ischemia/reperfusion-induced acute renal failure (ARF). Polyuria caused by down-regulation of renal AQP 2 in the ischemia/reperfusion-induced ARF rats was markedly restored by administration of YJT (100 or 200 mg/kg, p.o.) with restoring expression of AQP 2 in the kidney. The expressions of Na,K-ATPase alpha1 and beta1 subunits in the renal medulla and cortex of the ARF rats were also restored in them by the administration of YJT. Administration of YJT lowered the expression of renal HO-1, which was up-regulated in rats with ischemia/reperfusion-induced ARF. The renal functional parameters including creatinine clearance, urinary sodium excretion, urinary osmolality, and solute-free reabsorption were also markedly restored in ischemia-ARF rats by administration of YJT. Histological study also showed that renal damages in the ARF rats were abrogated by administration of YJT. Taken together, these data indicate that YJT ameliorates renal defects in rats with ischemia/reperfusion-induced ARF.

Anti-atherogenic effects of the methanol extract of Sorbus cortex in atherogenic-diet rats.

The present study was designed to examine whether the methanol extract of Sorbus commixta cortex (MSC) could prevent the development of atherosclerosis through regulating the vascular nitric oxide (NO) and endothelin-1 (ET-1) systems in atherogenic-diet rats. Our findings show that aortic NO production as well as endothelial nitric oxide synthase (ecNOS) expression was significantly decreased in atherogenic-diet rats compared with those in the control group. Aortic ET-1 expression was augmented in rats fed an atherogenic-diet while NF-kappaB p65 was upregulated. Treatment of atherogenic-diet rats with either low (100 mg/kg/d) or high (200 mg/kg/d) doses of MSC led not only to significant increases in the aortic NOS/NO system, but also to decreases in aortic ET-1 expression. The aortic expression level of NF-kappaB p65 was also attenuated in atherogenic-diet rats by chronic treatment with low or high doses of MSC. Atherogenic-diet induced increases in the expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were markedly decreased by treatment with MSC. From the histopathological examination, MSC treatment was shown to lessen the thickening noted in the aortic intima and media of the atherogenic-diet rats. These results suggest that MSC affects the atherogenic process via the suppression of proinflammatory and adhesion molecules in atherogenic-diet rats, which may be, at least in part, causally related with the regulation of vasoactive systems such as the NO and ET-1 systems.

Effect of methanol extract of Sorbus cortex in a rat model of L-NAME-induced atherosclerosis.

Chronic inhibition of nitric oxide (NO) synthesis by administration of high dose of N(G)-nitro-L-arginine methylester (L-NAME) induces vascular inflammation and subsequent atherosclerosis. We aimed to investigate whether the methanol extract of Sorbus commixta cortex (MSC) is able to prevent inflammatory process in a rat model of L-NAME-induced atherosclerosis. Chronic treatment with low or high doses of MSC prevented the L-NAME-induced increase in monocyte chemoattractant protein-1 (MCP-1) and nuclear factor-kappaB (NF-kappaB) p65 expressions as well as adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in aorta. In addition, increased endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) expressions and decreased endothelial cell NO synthase (ecNOS) expression in aorta from L-NAME treated group was reversed by treatment with MSC. From the histological examination, aortic segment from the L-NAME-treated rats revealed a thickening of intima and media, which was ameliorated by treatment with MSC. In conclusion, our results indicate that MSC can prevent atherosclerosis by inhibiting vascular over-expressions of vasoactive materials, pro-inflammatory transcription factor, and adhesion molecules and by augmenting ecNOS in chronic L-NAME-treated rat model.

Effects of Korean traditional herbal remedy on heart rate variability: linear and nonlinear analysis.

Heart rate variability was compared in 20 subjects taking traditional herbal remedy (Ondamtanggamibang) or placebo control pill, in placebo controlled design experiment. Herbal remedy group reported more pleasant and clam emotions than did the placebo group. Herbal remedy reduced the heart rate and increased heart rate variability (HRV) as indicated by a reduced low frequency/high frequency power ratio of heart rate variability. With nonlinear analysis, the Poincaré plot index of HRV and approximate entropy was greater in the herbal remedy group than in the control group. These findings suggest that herbal remedy stabilizes the sympathovagal function and cardiac autonomic nervous system by inducing more positive emotions than does placebo therapy. In conclusion, herbal remedy may act by stabilizing both the autonomic nervous system and the emotional state.

Catalposide, a compound isolated from catalpa ovata, attenuates induction of intestinal epithelial proinflammatory gene expression and reduces the severity of trinitrobenzene sulfonic Acid-induced colitis in mice.

Certain irinoid-producing plants have been used as herbal anti-inflammatory remedies. Here we evaluated whether catalposide (CATP), a single compound isolated from irinoid-producing plant Catalpa ovata, has a potential for preventing or ameliorating diseases characterized by mucosal inflammation. Preliminary microarray-based gene expression test revealed that CATP, which alone did not significantly affect expression of any of the >8,000 genes analyzed, attenuated the expression of tumor necrosis factor-alpha (TNF-alpha)-induced proinflammatory genes including interleukin-8 (IL-8) in human intestinal epithelial HT-29 cells. Down-regulation of IL-8 mRNA accumulation was also reflected by the decreased IL-8 secretion in CATP-treated HT-29 cells. The signal transduction study revealed that CATP significantly attenuates TNF-alpha-mediated p38 and extracellular signal-regulated kinase (ERK) phosphorylation. Further, CATP reduced NF-kappaB-mediated transcriptional activation as well as Ikappa-Balpha degradation. To establish the in vivo relevance of these findings, we examined whether CATP could affect intestinal inflammation in vivo using the mouse model of trinitrobenzene sulfonic acid (TNBS)-induced inflammatory colitis. Intrarectal administration of CATP dramatically reduced the weight loss, colonic damage, and mucosal ulceration that characterize TNBS colitis. Moreover, CATP suppressed the expression of TNF-alpha, interleukin-1beta, and intercellular adhesion molecule-1 along with the inhibition of NF-kappa B p65 translocation into nucleus in TNBS colitis. Collectively, current results demonstrate that CATP may be an effective agent for the treatment of diseases characterized by mucosal inflammation.

External Qi therapy to treat symptoms of Agent Orange Sequelae in Korean combat veterans of the Vietnam War.

We investigated the efficacy of Qi therapy as a non-pharmacological treatment for various symptoms presented by Korean combat veterans of the Vietnam War with Agent Orange Sequelae. Nine subjects volunteered to receive 30 minutes of Qi therapy, twice per day for 7 days. There was marked improvement in 89% of the patients with impaired physical activity, 86% of those with psychological disorder, 78% of those with heavy drug use, and 67% of those with fatigue, indigestion and high blood glucose levels. This data suggests that Qi therapy combined with conventional treatment has positive effects in reducing and managing the pain, psychosomatic disorders, and substance abuse in patients with Agent Orange Sequelae. We cannot completely discount the possible influence of the placebo effect, and more objective, clinical measures are needed to study the long-term effects of Qi therapy.

Antitumor activity of Soamsan, a traditional Korean medicine, via suppressing angiogenesis and growth factor transcription.

Antiangiogenic and antitumor activities of Soamsan known as an anticancer remedy in traditional Korean medicine were examined. In contrast to the normal branching of vascular vessels in chorioallantoic membrane (CAM), blood vessels in CAM treated with Soamsan (50 microg per egg) were run parallel to each other with less branching. Oral administration of Soamsan (20 mg/kg per day) for 4 weeks significantly inhibited the rat corneal neovascularization induced by suture, and the length of blood vessels in Soamsan-treated rat cornea was conspicuously low compared to control. When HT1080 cells, human fibrosarcoma, were treated with 2.18 mg/ml of Soamsan up to 24 h, mRNA transcription of VEGF, TGF-beta and bFGF genes was dramatically reduced in a time-dependent manner. Soamsan showed a prolongation of life span and a reduction of tumor volume in CT-26 cell (colon adenocarcinoma)-bearing mice. These results suggest that antitumor activity of Soamsan may be mediated, at least in part, by antiangiogenic mechanism.

Inhibitory effect of miconazole on melanogenesis.

Miconazole (MIC), a regional antifungal agent, has been used worldwide in the treatment of superficial mycosis. However, the effect of MIC on skin pigmentation is not known. In this study, we investigated the inhibitory effect of MIC on melanogenesis in B16 melanoma cells. Tyrosinase activity and melanin content were dose dependently decreased by MIC as compared with untreated cells. The level of tyrosinase protein expression was reduced with treatment MIC. A decrease in cell proliferation was observed in B16 cells treated with MIC 30 microM, indicating that the MIC-induced depigmenting effect was caused by inhibition of melanin synthesis and not by destruction of B16 cells. Furthermore, MIC markedly suppressed alpha-melanocyte stimulating hormone or forskolin-induced tyrosinase activity in B16 cells. Therefore the depigmenting effect of MIC might be due to the inhibition of tyrosinase activity and tyrosinase expression, which eventually slows melanin biosynthesis. These results indicate that MIC may be a useful inhibitor of melanogenesis in B16 cells and suggest that it may have beneficial effects in the treatment of hyperpigmentation disorders such as ephelis and melasma.

Butein ameliorates renal concentrating ability in cisplatin-induced acute renal failure in rats.

The present study examined whether the cisplatin-induced nephropathy could be ameliorated by administration of butein isolated from the stems of Rhus verniciflua STOCKS. The present study showed that polyuric profile was revealed in cisplatin-induced acute renal failure (ARF) rats associated with decreases in urinary sodium, potassium, chloride, and creatinine excretion, and osmolality. Among these renal functional parameters, urinary volume and osmolality were partially restored by administration of butein (10 mg/kg, i.p.), but electrolytes and creatinine excretion were not restored. Both solute-free water reabsorption and creatinine clearance were also significantly decreased in rats subjected to cisplatin. When butein was administered in rats with cisplatin-induced ARF for 4 d, solute-free water reabsorption was improved by 91% compared with that of cisplatin-induced ARF rats, but creatinine clearance was not restored. The expression levels of aquaporin 2 (AQP 2) in the inner, outer medulla, and cortex were significantly decreased in the kidney of ARF, which were partially reverted by administration of butein. In histological examination of the kidney, butein treatment partially prevented the lesions at tubules of renal cortex in cisplatin-induced ARF rats, while the lesions at glomeruli were not ameliorated. Taken together, butein ameliorates renal concentrating ability via up-regulation of renal AQP 2 water channel in rats with cisplatin-induced ARF without ameliorating effect on renal filtration defect.

In vitro immunomodulatory activity of Bo-yang-hwan-o-tang.

Bo-yang-hwan-o-tang (BHT), an herbal decoction has been mainly used for improvement of blood flow in oriental medicine. Its in vivo immunomodulation was recently demonstrated but the effective mechanisms have not been described. This study was carried out to evaluate in vitro immunomodulatory activity of BHT. Water extract of BHT significantly promoted in vitro proliferative responses of mouse spleen cells (SPC) and also further enhanced the proliferation of SPC stimulated with anti-CD3 antibody. Unexpectedly, addition of BHT extract did not affect proliferation of both resting and CD3-activated T cells, whereas it showed a strong mitogenic activity on B cells. Flow cytometric analysis of CFSE-stained SPC showed that BHT-mediated enhancement of CD3-activated SPC proliferation is due to T cell, but not B cell, division. Mixed culture experiment combining T and mitomycin C-treated B cells demonstrated that BHT-mediated enhancement of CD3-activated T cell proliferation was dependent on the presence of B cells. However, B cell-derived factors were not involved in BHT effect on T cell proliferation. In the presence of B cells, BHT treatment resulted in a great enhancement in IL-2 production of CD3-activated T cells, and BHT effect on T cell proliferation was completely abrogated by addition of exogenous IL-2, indicating that IL-2 plays a critical role in BHT-mediated enhancement of CD3-activated T cell proliferation. Taken together, our data revealed that BHT possesses a potent B cell mitogenic activity and also can enhance activated T cell response through B cell regulation.