Seselin from Plumbago zeylanica inhibits phytohemagglutinin (PHA)-stimulated cell proliferation in human peripheral blood mononuclear cells.

Effects of seselin (C(14)H(12)O(3); MW 228) identified from Plumbago zeylanica on phytohemagglutinin (PHA)-stimulated cell proliferation were studied in human peripheral blood mononuclear cells (PBMC). The data demonstrated that seselin inhibited PBMC proliferation-activated with PHA with an IC(50) of 53.87+/-0.74 microM. Cell viability test indicated that inhibitory effects of seselin on PBMC proliferation were not through direct cytotoxicity. The action mechanisms of seselin may involve the regulation of cell cycle progression, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in PBMC. Since cell cycle analysis indicated that seselin arrested the cell cycle progression of activated PBMC from the G(1) transition to the S phase. Seselin suppressed IL-2 and IFN-gamma production in a concentration-dependent manner. Furthermore, seselin significantly decreased the IL-2 and IFN-gamma gene expression in PHA-activated PBMC. Therefore, results elucidated for the first time that seselin is likely an immunomodulatory agent for PBMC.

VE/VCO2 Slope and Functional Capacity in Patients Post-Heart Transplantation.

BACKGROUND: The ventilatory efficiency represented cardiovascular, pulmonary, and musculoskeletal performance into an integrate index has been used as long-term and short-term prognostic variables in congestive heart failure. The heart failure patients post heart transplantation, whether the ventilatory efficiency was also normalized is still unknown. METHODS: This was a cross-sectional study. We measured ventilation to carbon dioxide production slope and oxygen consumption in peak exercise (peak VO2) by cardiopulmonary exercise test, which represented ventilatory efficiency and functional capacity respectively. Strength of hand grip, the 30-second chair stand test, and 6-minute walking test were also evaluated. Patients with ventilation to carbon dioxide production slope <30 were defined as the normal group; others were defined as the abnormal group. Independent t tests and paired t tests were used when appropriate. The level of statistical significance was set at .05. RESULTS: There were 51 clinically stable post-heart transplantation patients (age 53 ± 12.4 years; 86.3% were male) at 65.14 ± 41.17 months after transplantation. The ventilation to carbon dioxide production slope was 29.2 ± 5.6, which significantly improved compared to that recorded 1 month after heart transplantation (32.6 ± 6.4). There were 20 patients in the abnormal group, characterized by lower 6-minute walking test distance (normal vs abnormal, 422.5 ± 97.8 vs 532.6 ± 87.6 m) and peak VO2 (normal vs abnormal, 14.9 ± 5.3 vs 18.8 ± 5.1 mL/kg/min). The abnormal ventilation to carbon dioxide production slope was significantly correlated with 6-minute walking test distances in multivariate analyses. CONCLUSION: Our findings indicate that the ventilation to carbon dioxide production slope is partially abnormal among patients post-heart transplantation. A ventilation to carbon dioxide production slope above the normal range is characterized by a lower peak VO2 during cardiopulmonary exercise test and lower 6-minute walking test distance. The ventilation to carbon dioxide production slope is also significantly negatively correlated with peak VO2, peak work rate, and 6-minute walking test distance. The prognostic utility of the ventilation to carbon dioxide production slope for patients post-heart transplantation requires further investigation.

Oxygen Consumption at Anaerobic Threshold Predicts Cardiac Events After Heart Transplantation.

OBJECTIVES: The ventilatory efficiency and functional capacity measured by the cardiopulmonary exercise test (CPET) have been used as important prognostic variables in congestive heart failure. This study sought to identify whether these predictors before heart transplantation (HTX) play a key role in predicting adverse events in patients with heart failure after HTX. METHODS: This was a retrospective cohort study design. HTX recipients were included for analysis. Ventilation to carbon dioxide production slope (VE/VCO2 slope) and oxygen consumption (VO2) during exercise were collected by CPET, which represented ventilator efficiency and functional capacity respectively. Cardiac-related events 2 years after HTX were recorded by chart review. We divided patients into 2 groups based on VE/VCO2 slope = 34, peak VO2 = 14 mL/kg/min and VO2 at aerobic threshold (AT) = 11 mL/kg/min. Kaplan-Meier survival curves was used to represent the events rate between groups and Log rank test was used to test significance. RESULTS: A total of 87 patients after HTX were included. Mean (SD) age was 48 (11) years and 73 were male; 28 subjects suffered from events, and 76 cardiac events were recorded. The mean (SD) data of peak VO2, VO2 at AT, and VE/VCO2 slope analyzed from CPET were 17.8 (5.6) mL/kg/min, 15.4 (4.4) mL/kg/min, and 33.1 (8.2) mL/kg/min, respectively. Lower VO2 at AT contributed to increase events rate (P < .05). CONCLUSION: Aerobic capacity may better predict 2-year cardiac events in patients after HTX. Strategies to improve aerobic capacity should be focused on in the cohort.

Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice.

BACKGROUND: The mechanisms responsible for thrombocytopenia associated with dengue fever (DF) and dengue hemorrhage fever (DHF) remain unclear. OBJECTIVE: In this study, we investigated the pathogenic effects of dengue virus (DENV) non-structural protein 1 (NS1) on the elicitation of platelet cross-reactive antibodies. RESULTS: The results showed that anti-DENV NS1 immunoglobulins (Igs) derived from both patients with DF/DHF and recombinant NS1-immunized rabbits could opsonize normal human platelets and enhance platelet-macrophage engagements in vitro. In addition, treatments with anti-NS1 Igs abnormally activated human platelets and induced thrombocytopenia in mice. These prothrombotic characteristics of anti-NS1 Ig might increase the disease burden of coagulant-aberrant DHF patients. To test this hypothesis, we injected anti-NS1 Igs into C57BL/6J mice that were preconditioned into a hypercoagulable state by warfarin treatments. When given before but not after platelet-lysate pre-adsorption, the anti-NS1 Igs injection treatments significantly increased mortality, fibrin deposition in lung, and plasma D-dimer levels, but significantly decreased anticoagulant proteins C, protein S and antithrombin III. CONCLUSIONS: These results suggest that the platelet-bound antibody fractions of anti-NS1 Ig are prothrombotic, which might exacerbate the severity of disease in hosts with an imbalanced coagulant system.

Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-kappaB.

BACKGROUND AND PURPOSE: Extracts of Plumbago zeylanica containing suberosin exhibit anti-inflammatory activity. We purified suberosin from such extracts and studied its effects on a set of key regulatory events in the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). EXPERIMENTAL APPROACH: Proliferation of PBMC in culture was measured by uptake of 3H-thymidine; production of cytokines and cyclins by Western blotting and RT-PCR. Transcription factors NF-AT and NF-kappaB were assayed by immunocytochemistry and EMSA. KEY RESULTS: Suberosin suppressed PHA-induced PBMC proliferation and arrested cell cycle progression from the G1 transition to the S phase. Suberosin suppressed, in activated PBMC, transcripts of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and cyclins D3, E, A, and B. DNA binding activity and nuclear translocation of NF-AT and NF-kappaB induced by PHA were blocked by suberosin. Suberosin decreased the rise in intracellular Ca2+ concentration ([Ca2+]i) in PBMC stimulated with PHA. Suberosin did not affect phosphorylation of p38 and JNK but did reduce activation of ERK in PHA-treated PBMC. Pharmacological inhibitors of NF-kappaB, NF-AT, and ERK decreased expression of mRNA for the cyclins, IL-2, and IFN-gamma and cell proliferation in PBMC activated by PHA. CONCLUSIONS AND IMPLICATIONS: The inhibitory effects of suberosin on PHA-induced PBMC proliferation, were mediated, at least in part, through reduction of [Ca2+]i, ERK, NF-AT, and NF-kappaB activation, and early gene expression in PBMC including cyclins and cytokines, and arrest of cell cycle progression in the cells. Our observations provide an explanation for the anti-inflammatory activity of P. zeylanica.

Tanshinlactone A from Salvia miltiorrhiza modulates interleukin-2 and interferon-gamma gene expression.

Salvia miltiorrhiza Bunge (Tanshen), a traditional Chinese herbal medicine, is popularly used to treat cardiovascular disorders. In the present study, effects of tanshinlactone A (C(16)H(12)O(4); M.W. 268), newly discovered from Salvia miltiorrhiza, on phytohemagglutinin (PHA)-stimulated cell proliferation were investigated in human peripheral blood mononuclear cells (PBMC). The results indicated that tanshinlactone A inhibited PBMC proliferation activated with PHA with an IC(50) of 15.6+/-1.9 microM. Cell viability test indicated that inhibitory effects of tanshinlactone A on PBMC proliferation were not through direct cytotoxicity. Furthermore, tanshinlactone A significantly decreased the interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression in PHA-activated PBMC. It reduced the phosphorylation of mitogen-activated protein kinases (MAPK) involving extracellular signal-regulated protein kinase (ERK), P38, and c-Jun NH(2)-terminal kinase (JNK) in PHA-treated PBMC. We suggested that the inhibitory effects of tanshinlactone A on PHA-induced PBMC proliferation, appeared to be mediated, at least in part, through reduction of MAPK activation and IL-2 and IFN-gamma production. Therefore, data demonstrate for the first time that tanshinlactone A is likely an immunomodulatory agent for PBMC.

Changes in both calcium pool size and morphology of human platelets incubated in various concentrations of calcium ion. Calcium-specific bleb formation on platelet-membrane surface.

In this study, the response of gel-filtered human platelets to extracellular Ca2+ at Ca2+ concentrations [Ca2+]o of 1-10 mM was investigated. The distribution of Ca2+ among various pools was studied using: (1) quin2, to estimate the cytosolic free Ca2+ concentration ([Ca2+]i); and (2) 45CaCl2 plus EGTA, to quantitate the sizes of the EGTA-releasable, EGTA-nonreleasable and surface-bound Ca2+ pools. The morphological changes were revealed by scanning electron-microscopy (scanning EM), and the effect on thrombin-stimulated aggregation was examined using an aggregometer. Platelets continuously sequestered Ca2+ into both EGTA-releasable and EGTA-nonreleasable pools to maintain a low [Ca2+]i level. The rate of sequestration to the EGTA-releasable pool was independent of [Ca2+]o, while that of the EGTA-nonreleasable pool exhibited first-order kinetics. The cell morphology changed gradually from discoid to the tadpole-like type, and finally to irregular forms. This morphological change correlated with the gradual increase in [Ca2+]i. The EGTA-nonreleasable pool saturated at about 3000 pmol/10(8) cells. This saturation resulted in a drastic increase in the EGTA-releasable pool size, and the cell was lysed concomitantly. The maximum safety capacity of the EGTA-releasable pool was estimated to be 1100 pmol/10(8) cells. The contribution of the cellular compartments to these two pool sizes is extensively discussed. The surface-bound pool size also increased continuously. When two different capacities were reached, i.e., 160 and 600 pmol/10(8) cells, the binding rate increased above the initial rate by 7- and 11-fold, respectively. Hence, the surface-binding capacity might be a critical factor which alters the membrane structure and exposes more binding sites. The cell surface appeared to have blebs, after the binding size had reached more than 600 pmol/10(8) cells. Bleb formation resulted in the inhibition of platelet function. Divalent cations, such as Mg2+, Sr2+ and Ba2+ did not cause bleb formation, which could mean that this formation is a Ca2+-specific phenomenon.

Estimation of the phospholipid distribution in the human platelet plasma membrane based on the effect of phospholipase A2 from Naja nigricollis.

Human platelets in three physiological states were prepared. These states were the gel-filtered, the thrombin-induced shape-changed, and the thrombin-activated platelets. The phospholipid distributions in these three types of membrane were probed by using the basic phospholipase A2 of Naja nigricollis. This enzyme could penetrate through these membranes to hydrolyze all of their accessible phospholipids and to cause cell lysis. The hydrolytic time-courses displayed three phases. The state of platelet in each lipid hydrolytic phase was examined by: (1) measuring the leakage of lactate dehydrogenase; (2) analyzing the morphology by both scanning and transmission electron microscopy (scanning EM and transmission EM); and (3) estimating the hydrolysis of the [32P]phosphate-labeled platelets. The existence of these three hydrolytic phases may signify that the phospholipase A2 sequentially hydrolyzed its substrates in the membrane outer leaflet, in the inner one, and in the cytosol. The content and the distribution of each phospholipid class in the plasma membranes of the resting and of the shape-changed platelets were similar. These membrane surfaces consisted mainly of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Phosphatidylserine (PS) was not exposed on the surface of the shape-changed platelet. The content of each lipid class in the activated platelet membrane was 10% more than that in the resting platelet. PS was found on the activated platelet cell surface. This implies that PS is exposed only during platelet secretion.

Different susceptibilities of platelet phospholipids to various phospholipases and modifications induced by thrombin. Possible evidence of rearrangement of lipid domains.

On the membrane surface of the human platelet, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed to different extents by the snake venom phospholipases A2 of varying pI values. The susceptibility of platelet phospholipids to basic phospholipase A2 of Naja nigricollis (pI 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244-258). The susceptibilities of platelet phospholipids to acidic phospholipase A2 of Naja naja atra (pI 5.2) and to neutral phospholipase A2 of Hemachatus haemachatus (pI 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: the PC and the PE domains exist on the platelet membrane surface; and the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.

Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae.

Effects of ergosterol peroxide (C28H44O3; Cpd 6A) from Cordyceps cicadae on phytohemagglutinin (PHA)-stimulated cell proliferation were studied in primary human T cells. The results showed that Cpd 6A suppressed T-cell proliferation for about 24 h after stimulation with PHA. Cell cycle analysis indicated that Cpd 6A arrested the cell cycle progression of activated T cells from the G1 transition to the S phase. To localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary, including the expression of cyclins D2, E, A1, and B1, interleukin (IL)-2, IL-4, interferon-gamma (IFN-gamma), and activating protein-1 (AP-1), was examined. Cpd 6A suppressed, in activated T lymphocytes, the production and mRNA expression of cyclin E, IL-2, IL-4, IL-10, and IFN-gamma in a dose-dependent manner. Expression of AP-1 proteins, consisting of c-Fos and c-Jun, in activated T lymphocytes was decreased by Cpd 6A. The kinetic study indicated that the inhibitory effects of Cpd 6A on IL-2 mRNA expressed in T cells might be related to blocking c-Fos protein synthesis. T-cell proliferation after Cpd 6A treatment was partially restored by addition of IL-2, IL-4, and IFN-gamma. These suppressant effects of Cpd 6A on T-cell proliferation, activated by PHA, appeared to be mediated, at least in part, through the inhibition of early gene transcripts, especially those of cyclin E, IFN-gamma, IL-2, and IL-4, and by arresting cell cycle progression in the cells.

Rhabdomyolysis: an unusual feature with mushroom poisoning.

Rhabdomyolysis resulting from mushroom poisoning previously has been unreported in the literature. We present an outbreak of Russula subnigricans poisoning with rhabdomyolysis. The most severely ill patient presented with rhabdomyolysis, severe electrolyte disturbance (hyperkalemia, hypocalcemia), respiratory failure, acute renal failure, pulmonary edema, ventricular tachycardia, and circulatory shock. Mycotoxin may be the cause of rhabdomyolysis. In areas where mushroom gathering is common, mushroom poisoning should be included in the differential diagnosis of rhabdomyolysis.

Regulation of herpes simplex virus type 1 replication in Vero cells by Psychotria serpens: relationship to gene expression, DNA replication, and protein synthesis.

Inhibitory effects of ethanolic extracts from seven Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. From a bioassay-guided fractionation procedure, PS-A-6 was isolated from Psychotria serpens (P. serpens), which suppressed HSV-1 multiplication in Vero cells without apparent cytotoxicity. Time-of-addition experiments suggested that the inhibitory action of PS-A-6 on HSV-1 replication was not through blocking of virus adsorption. In an attempt to further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to viral multiplication was examined, including viral gene expression, DNA replication, and structural protein synthesis. The results indicated that gB mRNA and protein expression in Vero cells were impeded by PS-A-6. Southern blot analysis showed that HSV-1 DNA replication in Vero cells was arrested by PS-A-6. In addition, PS-A-6 decreased thymidine kinase (tk) and ICP27 mRNA expression in the cells. The mechanisms of antiviral action of PS-A-6 seem to be mediated, at least in part, through inhibition of early transcripts of HSV-1, such as tk and ICP27 mRNAs, arresting HSV-1 DNA synthesis and gB gene expression in Vero cells. Plans are underway for the isolation of pure compounds from PS-A-6 and elucidation of their mechanism of action.

Characterization of the antiplatelet effects of (2S)-5-methoxy-6-methylflavan-7-ol from Draconis Resina.

(2S)-5-methoxy-6-methylflavan-7-ol (MMF) was purified from Draconis Resina and its in vitro effects on various aspects of platelet reactivity were examined. Results indicated that MMF dose dependently inhibited aggregation of washed rabbit platelets induced by collagen, arachidonic acid, ADP, U46619 or platelet-activating factor (PAF), with IC50) values of 17.2, 49.8, 179.8, 109.6, and 189.2 microM, respectively. Concomitantly, MMF also dose dependently suppressed ATP release by platelets activated by these stimulants. The increase in intracellular free calcium ([Ca2+]i), elicited by these activating agents, was inhibited by MMF as reflected by fura-2 fluorescence measurements. However, MMF had no effects on the cyclic AMP level of platelets. In addition, MMF inhibited the arachidonic acid-induced thromboxane B2 and prostaglandin D2 formation in intact platelet suspensions or homogenized platelet lysates. This study provided evidence that MMF is an antiplatelet agent whose activity is likely related to cyclooxygenase inhibition and suppression of [Ca2+]i increase.

Glutathione S-transferase-rhodostomin fusion protein inhibits platelet aggregation and induces platelet shape change.

Rhodostomin (RHO) from Agkistrodon rhodostoma venom, consisting of 68 amino acids with an arginine-glycine-aspartic acid (RGD) sequence and 12 cysteine residues, is a potent inhibitor of platelet aggregation. We previously demonstrated that cell culture plates coated with the bacterially produced fusion protein of glutathione S-transferase-RHO [GST-RHO(RGD)] can facilitate human hepatoma cell attachment via intergrin interaction within 15 min. In this study, we further characterized the effect of RHO fusion protein on platelet cells by creating two other related fusion proteins, GST-RHO(RGE) and GST-(PS)RHO. The former was a single amino acid-substituted mutant, in which the aspartic acid residue of RGD was replaced by glutamic acid, and the latter was an insertion mutant, in which a pentapeptide of protein kinase A phosphorylation site was inserted between GST and RHO. These two mutant proteins together with a wild-type of GST-RHO(RGD) and native form of RHO were used to study effects on the inhibition of ADP-induced platelet aggregation. Results indicated that GST-RHO(RGD) inhibited platelet aggregation as potently as the native RHO, while the two other mutants were inactive. Furthermore, when unactivated platelet cells attached on the GST-RHO(RGD)-coated plate, they became a flattened pancake shape. From the results of facilitation of cell attachment on fusion protein-coated plates, we concluded that: (1) the GST-RHO(RGD) fusion protein is equally functional in inhibition of platelet aggregation and facilitation of cell attachment, which is through the interaction of RGD and integrins on the cell membrane; (2) the GST-RHO(RGE) mutant protein is unable to bind with integrins and results in loss of function; (3) the insertion mutant of GST-(PS)RHO may disrupt a proper conformation of RHO and also results in loss of function; (4) the bacterially produced fusion protein GST-RHO(RGD) can be properly used as an antithrombotic agent and an extracellular matrix.

Acetylcholinesterase inhibition and the extrapyramidal syndrome: a review of the neurotoxicity of organophosphate.

Organophosphate poisonings are not uncommon, and are the leading cause of death in suicide patients in Taiwan. Acute cholinergic crisis caused by the inhibition of synaptic acetylcholinesterase is the major manifestation of organophosphate poisoning and may cause death within minutes. Delayed neurotoxicities include intermediate syndrome and delayed polyneuropathy have also been described. However, these symptoms may not characterize the complete picture of organophosphate poisoning. Among the 633 patients ever admitted to our hospital with organophosphate poisoning, three patients were found exhibiting impermanent neuromuscular dysfunction, including blepharoclonus, oculogyric crisis, intermittent dystonia, rigidity, and tremor, with two of them developing mask face, dyskinesia and akathisia later, following acute cholinergic crisis. The symptoms appeared within 4 days with the duration ranging from 25 days to 2 months. Other causes of the extrapyramidal syndrome noted on these patients have been excluded, and we consider the extrapyramidal syndrome a possible neurotoxic manifestation of organophosphate poisoning, which is transient, needs no treatment, and may be missed because of the critical condition, in a minority of patients. The mechanism remains to be identified, but may be related to the impediment of the function of acetylcholinesterase to modify nigrostriatal dopaminergic system, which is independent of hydrolyzing acetylcholine. More detailed observation for organophosphate poisoned patients and more studies for the biological functions of acetylcholinesterase including the influence on the nigrostriatal dopaminergic system are needed.

A tumor cell growth inhibitor from Polygonum hypoleucum Ohwi.

Polygonum hypoleucum Ohwi (P. hypoleucum Ohwi) has been used as a Chinese medicine for a long time. In the present study, four anthraquinones, emodin, emodin 1-O-beta-D-glucoside (49A), physcion (62A), and physcion 1-O-beta-D-glucoside (50A) were identified from P. hypoleucum Ohwi and their inhibitory effects on various tumor cells proliferation were investigated. On a percentage basis, emodin had the highest suppressing activity on the various tumor cells proliferation. At 10 microg/ml, the percentage inhibition on K562 cells proliferation for emodin, 49A, 62A, and 50A were 97+/-3.4%, 18+7.3%, 24+/-3.6%, and 31+/-8.9%, respectively. However, inhibitory activities of 10 microg/ml of emodin, 49A, 62A, or 50A on Raji cells proliferation were 98+/-5.0%, 25+/-5.0%, 22+/-3.2%, and 28+/-4.3%, respectively. It was also found that the both C1 and C3 positions of emodin were important for antitumor action. The IC50s of emodin, 49A, 62A, and 50A on various tumor cells were also calculated. The IC50 of emodin on K562 cells was significantly lower than on Raji, HeLa, Calu-1, Wish, and Vero cells (1.5+/-0.2 vs. 2.8+/-0.4 microg/ml, P < 0.01 ;1.5+/-0.2 vs. 8.4+/-1.6 microg/ml; 1.5+/-0.2 vs. 8.9+/-1.0 microg/ml; 1.5+/-0.2 vs. 8.7+/-0.5 microg/ml; 1.5/-0.2 vs. 3.5+/-0.12 microg/ml; P < 0.001). The results indicated that K562 and Raji cells were more sensitive to emodin treatment. Cell viability test indicated that inhibitory effect of emodin on various tumor cell lines was not through direct cytotoxicity. It suggested P. hypoleucum Ohwi included a tumor cell growth inhibitor.

Immune reponses in human mesangial cells regulated by emodin from Polygonum hypoleucum Ohwi.

In the hope of identifying agents of therapeutic value in glomerulonephritis from Chinese herbs, we found that methanolic extracts of Polygonum hypoleucum Ohwi (P. hypoleucum Ohwi) inhibit human mesangial cells proliferation activated with interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) previously. This study was designed to identify bioactive components from P. hypoleucum Ohwi and elucidate their action mechanisms. We tested four anthraquinones emodin, emodin 1-O-beta-D-glucoside (49A), physcion (62A), and physcion 1-O-beta-D-glucoside (50A) purified from P. hypoleucum Ohwi for their effects on human mesangial cell proliferation and cytokines production in vitro. On a percentage basis, emodin had the highest suppressing activity on the human mesangial cells proliferation activated by IL-1beta and IL-6. The IC50 of emodin on human mesangial cells proliferation were 17.9+/-1.2 microM. In contrast to 49A, 50A, and 62A, emodin also decreased IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) production in human mesangial cells activated with IL-1beta and IL-6. The IC50 of emodin on IL-1beta, IL-6 and TNF-alpha production in activated human mesangial cells were 16.6+/-1.8 microM, 8.2+/-1.3 microM, and 9.5+/-1.6 microM, respectively. Moreover, IL-1beta and TNF-alpha mRNA expression in activated human mesangial cells was impaired by emodin. The intracellular free Ca2+ concentration ([Ca2+]i) in IL-1beta and IL-6 activated human mesangial cells was decreased by emodin. It is unlikely that cytotoxicity was involved because no cell deaths were observable. We hypothesize that the inhibitory mechanisms of emodin on activated human mesangial cells proliferation may be related to the impairments of gene expression and production of cytokines and [Ca2+]i in the cells.

Regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from Cordyceps sinensis.

Cordyceps sinensis (C. sinensis) is one of the well known fungi used in traditional Chinese medicine for treatment asthma and bronchial and lung inflammation. In this study, effects of C. sinensis methanolic extracts on bronchoalveolar lavage fluids (BALF) cells proliferation, inflammatory cytokines production, and genes expression were evaluated. The proliferative response of BALF cells to lipopolysaccharide (LPS) was determined by the tritiated thymidine uptake method. The cell-free supernatants were harvested then tested for interlukin-1beta (IL-1beta), interlukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), and interferon-gamma (IFN-gamma) by the enzyme immunoassay. The results indicated that the CS-19-22 fraction dose dependently suppressed BALF cells proliferation activated by LPS. The CS-19-22 fraction also reduced IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha production in LPS activated BALF cell cultures. Furthermore, the IL-12 and IFN-gamma production in activated BALF cells were enhanced by CS-19-22 treatment. The CS-19-22 fraction did not affect IL-1beta, IL-6, TNF-alpha, and IL-8 mRNAs expression in BALF cells detected by reverse transcription-polymerase chain reaction (RT-PCR). By contrast, the CS-19-22 fraction increased IL-12 and IFN-gamma mRNAs expression and decreased IL-10 mRNA expression in the BALF cells activated with LPS. These results indicated the CS-19-22 fraction suppressed IL-1beta, IL-6, TNF-alpha, and IL-8 cytokines production in BALF cells through other than inhibition of mRNAs expression pathway. These results also demonstrate that the therapeutic activity of C. sinensis in Chinese medicine may be related to modulation of TH1 and TH2 cells functions in bronchial airway.

Blocking of cell proliferation, cytokines production and genes expression following administration of Chinese herbs in the human mesangial cells.

In the hope of identifying agents of therapeutic value in immuoglobulin A nephropathy (IgA-N), we tested crude methanol extracts of 15 Chinese herbs for their effect on human mesangial cell proliferation. The results indicated that 4 out of the 15 crude extracts inhibited human cells proliferation activated by IL-1beta and IL-6. The extracts and their median inhibitory concentrations were as follows (in microg/ml): Ludwiga octovalvis (MLS-052), 49.9 +/- 1.8; Rhus semialata (MLS-053), 31.2 +/- 1.6; Tabernaemontana divaricata (MLS-054), 50.0 +/- 2.1; Amepelopsis brevipedunculata (MLS-059), 42.9 +/- 1.1. These findings indicate that human mesangial cells were most sensitive to MLS-053 treatment. These herbs also decreased interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) production. Moreover, IL- 1beta mRNA expression was inhibited by Rhus semialata (R. semialata; MLS-053). It is unlikely that cytotoxicity was involved, because no cell deaths were observable. We hypothesize that the inhibitory mechanisms of these Chinese herbs may be related to the impairments of gene expression and production of cytokines in human mesangial cells. Plans are underway for the isolation of pure compounds from these Chinese herbs and the elucidation of their mechanisms of action.

Effect of acute skin thermal injury on subcutaneous glutathione, ascorbic acid and hydroxyl radical concentrations in anesthetized rats.

The effect of acute thermal injury on subcutaneous oxidative stress, in anesthetized rats, was evaluated. A microdialysis probe was implanted in the subcutaneous tissue for continuous sampling of interstitial fluids, and the microdialysates were injected onto either an on-line or an off-line high performance liquid chromatography system. Hydroxyl radicals, glutathione, and ascorbic acid concentrations in the microdialysates were analyzed. Acute thermal injury was induced by skin contact of a hot (90°C) iron bar for 30 or 15 s. Subcutaneous hydroxyl radical production, represented as the increased formation of 2,3 and 2,5 dihydroxybenzoic acid (DHBA), did not increase significantly after thermal contact. Interestingly, both ascorbic acid and glutathione, two major physiological antioxidants, were significantly elevated in the subcutaneous interstitial fluids immediately after thermal contacts. The elevated subcutaneous glutathione levels rapidly decreased and returned to basal values 60 min after thermal contact. Ascorbic acid concentrations did not fully return to basal values even 3 h after thermal contact. The increase in ascorbic acid and glutathione may be responsible for scavenging of hydroxyl radicals that may form following thermal injury.