Zhengtao Wang

Astragaloside IV attenuates experimental autoimmune encephalomyelitis of mice by counteracting oxidative stress at multiple levels.

Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory disease found mostly in young adults in the western world. Oxidative stress induced neuronal apoptosis plays an important role in the pathogenesis of MS. In current study, astragaloside IV (ASI), a natural saponin molecule isolated from Astragalus membranceus, given at 20 mg/kg daily attenuated the severity of experimental autoimmune encephalomyelitis (EAE) in mice significantly. Further studies disclosed that ASI treatment inhibited the increase of ROS and pro-inflammatory cytokine levels, down-regulation of SOD and GSH-Px activities, and elevation of iNOS, p53 and phosphorylated tau in central nervous system (CNS) as well as the leakage of BBB of EAE mice. Meanwhile, the decreased ratio of Bcl-2/Bax was reversed by ASI. Moreover, ASI regulated T-cell differentiation and infiltration into CNS. In neuroblast SH-SY5Y cells, ASI dose-dependently reduced cellular ROS level and phosphorylation of tau in response to hydrogen peroxide challenge by modulation of Bcl-2/Bax ratio. ASI also inhibited activation of microglia both in vivo and in vitro. iNOS up-regulation induced by IFNγ stimulation was abolished by ASI dose-dependently in BV-2 cells. In summary, ASI prevented the severity of EAE progression possibly by counterbalancing oxidative stress and its effects via reduction of cellular ROS level, enhancement of antioxidant defense system, increase of anti-apoptotic and anti-inflammatory pathways, as well as modulation of T-cell differentiation and infiltration into CNS. The study suggested ASI may be effective for clinical therapy/prevention of MS.

Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 microM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 microM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 microM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.

Norisoboldine Suppresses Osteoclast Differentiation through Preventing the Accumulation of TRAF6-TAK1 Complexes and Activation of MAPKs/NF-κB/c-Fos/NFATc1 Pathways

Norisoboldine (NOR) is the main alkaloid constituent in the dry root of Lindera aggregata (Sims) Kosterm. (L. strychnifolia Vill.). As reported previously, orally administered NOR displayed a robust inhibition of joint bone destruction present in both mouse collagen-induced arthritis and rat adjuvant-induced arthritis with lower efficacious doses than that required for ameliorating systemic inflammation. This attracted us to assess the effects of NOR on differentiation and function of osteoclasts, primary effector cells for inflammatory bone destruction, to get insight into its anti-rheumatoid arthritis mechanisms. Both RAW264.7 cells and mouse bone marrow-derived macrophages (BMMs) were stimulated with RANKL (100 ng/mL) to establish osteoclast differentiation models. ELISA, RT-PCR, gelatin zymography, western blotting, immunoprecipitation and EMSA were used to reveal related signalling pathways. NOR (10 and 30 µM), without significant cytotoxicity, showed significant reduction of the number of osteoclasts and the resorption pit areas, and it targeted osteoclast differentiation at the early stage. In conjunction with the anti-resorption effect of NOR, mRNA levels of cathepsin K and MMP-9 were decreased, and the activity of MMP-9 was attenuated. Furthermore, our mechanistic studies indicated that NOR obviously suppressed the ubiquitination of TRAF6, the accumulation of TRAF6-TAK1 complexes and the activation of ERK and p38 MAPK, and reduced the nuclear translocation of NF-κB-p65 and DNA-binding activity of NF-κB. However, NOR had little effect on expressions of TRAF6 or the phosphorylation and degradation of IκBα. Moreover, NOR markedly inhibited expressions of transcription factor NFATc1, but not c-Fos. Intriguingly, the subsequent nuclear translocations of c-Fos and NFATc1 were substantially down-regulated. Hence, we demonstrated for the first time that preventing the differentiation and function of osteoclasts at the early stage was an important anti-bone destruction mechanism of NOR, which might be attributed to inhibition of ubiquitination of TRAF6, the accumulation of TRAF6-TAK1 complexes and the activation of MAPKs/NF-κB/c-Fos/NFATc1 pathways.

Protective mechanisms of N-acetyl-cysteine against pyrrolizidine alkaloid clivorine-induced hepatotoxicity.

Pyrrolizidine alkaloid (PA) clivorine, isolated from traditional Chinese medicinal plant Ligularia hodgsonii Hook, has been shown to induce apoptosis in hepatocytes via mitochondrial‐mediated apoptotic pathway in our previous research. The present study was designed to observe the protection of N‐acetyl‐cysteine (NAC) on clivorine‐induced hepatocytes apoptosis. Our results showed that 5 mM NAC significantly reversed clivorine‐induced cytotoxicity via MTT and Trypan Blue staining assay. DNA apoptotic fragmentation analysis and Western‐blot results showed that NAC decreased clivorine‐induced apoptotic DNA ladder and caspase‐3 activation. Further results showed that NAC inhibited clivorine‐induced Bcl‐xL decrease, mitochondrial cytochrome c release and caspase‐9 activation. Intracellular glutathione (GSH) is an important ubiquitous redox‐active reducing sulfhydryl (SH) tripeptide, and our results showed that clivorine (50 µM) decreased cellular GSH amounts and the ratio of GSH/GSSG in the time‐dependent manner, while 5 mM NAC obviously reversed this depletion. Further results showed that GSH synthesis inhibitor BSO augmented clivorine‐induced cytotoxicity, while exogenous GSH reversed its cytotoxicity on hepatocytes. Clivorine (50 µM) significantly induced cellular reactive oxygen species (ROS) generation. Further results showed that 50 µM Clivorine decreased glutathione peroxidase (GPx) activity and increased glutathione S transferase (GST) activity, which are both GSH‐related antioxidant enzymes. Thioredoxin‐1 (Trx) is also a ubiquitous redox‐active reducing (SH) protein, and clivorine (50 µM) decreased cellular expression of Trx in a time‐dependent manner, while 5 mM NAC reversed this decrease. Taken together, our results demonstrate that the protection of NAC is major via maintaining cellular reduced environment and thus prevents clivorine‐induced mitochondrial‐mediated hepatocytes apoptosis. J. Cell. Biochem. 108: 424–432, 2009.

Pyrrolizidine alkaloid isoline-induced oxidative injury in various mouse tissues

Isoline is a retronecine-type pyrrolizidine alkaloid (PA) isolated from the traditional Chinese medicinal herb Ligularia duciformis. The present investigation was carried out to evaluate isoline-induced oxidative injury in various important mouse organs. Various tissue samples were collected after mice were administrated with 100mg/kg isoline for 36h, and then lipid peroxidation (LPO) level, total antioxidant capacity, glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities were determined to evaluate the oxidative injury. Our results showed that the total antioxidant capacity of liver, brain and lung were all decreased after given isoline, and the LPO level was increased in liver and heart of isoline-treated mice. Further antioxidant-related enzyme activity assays showed that isoline (100mg/kg) decreased GPx activity in liver and heart, increased CAT activity in liver, brain and heart, and decreased the GST activity in lung. Taken together, our results demonstrate that isoline can induce different oxidative injury in various important mouse organs, and of which liver is the most sensitive organ.

Norisoboldine ameliorates collagen-induced arthritis through regulating the balance between Th17 and regulatory T cells in gut-associated lymphoid tissues

Norisoboldine (NOR), the main active ingredient of the dry root of Lindera aggregata, was previously proven to have substantial therapeutic effects on collagen-induced arthritis (CIA) in mice by oral administration. However, it exhibited a very poor bioavailability in normal rats. The pharmacokinetic-pharmacodynamics disconnection attracts us to explore its anti-arthritic mechanism in more detail. In this study, NOR, administered orally, markedly attenuated the pathological changes in CIA rats, which was accompanied by the down-regulation of pro-inflammatory cytokines and the up-regulation of anti-inflammatory cytokine IL-10. Pharmacokinetic studies demonstrated that the plasma concentration of NOR was moderately elevated in CIA rats compared with normal rats, but it was still far lower than the minimal effective concentration required for inhibiting the proliferation and activation of T lymphocytes in vitro. Interestingly, NOR was shown to regulate the balance between Th17 and regulatory T (Treg) cells in the intestinal lymph nodes more strikingly than in other tissues. It could increase the expression of Foxp3 mRNA in both gut and joints, and markedly up-regulate the number of integrin α4β7 (a marker of gut source)-positive Foxp3(+) cells in the joints of CIA rats. These results suggest that the gut might be the primary action site of NOR, and NOR exerts anti-arthritis effect through regulating the balance between Th17 and Treg cells in intestinal lymph nodes and yielding a trafficking of lymphocytes (especially Treg cells) from the gut to joint. The findings of the present study also provide a plausible explanation for the anti-arthritic effects of poorly absorbed compounds like NOR.

Norisoboldine attenuates inflammatory pain via the adenosine A1 receptor

Norisoboldine (NOR) is a benzylisoquinoline alkaloid isolated from Radix Linderae, a traditional Chinese medicine. Our previous studies have demonstrated that it produces anti‐inflammatory and anti‐rheumatoid arthritis effects.

Astragalosides from Radix Astragali benefits experimental autoimmune encephalomyelitis in C57BL /6 mice at multiple levels

BackgroundRadix Astragali is famous for its beneficial effect on inflammation associated diseases. This study was to assess the efficacy of astragalosides (AST) extracted from Radix Astragali, on the progression of experimental autoimmune encephalomyelitis (EAE), and explore its possible underlying molecular mechanisms.MethodsEAE was induced by subcutaneous immunization of MOG35–55. Infiltration of inflammatory cells was examined by HE staining. ROS level was detected by measuring infiltrated hydroethidine. Leakage of blood brain barrier (BBB) was assessed using Evan’s blue dye extravasation method. Levels of inflammatory cytokines were measured using ELISA kits. Activities of total-SOD, GSH-Px, and iNOS and MDA concentration were measured using biochemical analytic kits. Gene expression was detected using real-time PCR method. Protein expression was assayed using western blotting approach.ResultsAST administration attenuated the progression of EAE in mice remarkably. Further studies manifested that AST treatment inhibited infiltration of inflammatory cells, lessened ROS production and decreased BBB leakage. In peripheral immune-systems, AST up-regulated mRNA expression of transcriptional factors T-bet and Foxp3 but decreased that of RORγt to modulate T cell differentiation. In CNS, AST stopped BBB leakage, reduced ROS production by up-regulation of T-SOD, and reduced neuroinflammation by inhibition of iNOS and other inflammatory cytokines. Moreover, AST inhibited production of p53 and phosphorylation of tau by modulation of the Bcl-2/Bax ratio.ConclusionsAST orchestrated multiple pathways, including immuno-regulation, anti-oxidative stress, anti-neuroinflammation and anti-neuroapoptosis involved in the MS pathogenesis, to prevent the deterioration of EAE, which paves the way for the application of it in clinical prevention/therapy of MS.

Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes.

Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated clivorine-induced oxidative stress injury on primary cultured rat hepatocytes. Rat hepatocytes were treated with various concentrations of clivorine (1-100 microM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 microM and 100 microM. Further results showed that clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that clivorine increased CAT activity at the low concentration of 5 muM and decreased cellular SOD activity at all concentrations. Taken together, our results demonstrated that clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated clivorine-induced oxidative stress injury on primary cultured rat hepatocytes. Rat hepatocytes were treated with various concentrations of clivorine (1—100 μM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 μM and 100 μM. Further results showed that clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that clivorine increased CAT activity at the low concentration of 5 μM and decreased cellular SOD activity at all concentrations. Taken together, our results demonstrated that clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.

Protection of epidermal growth factor against clivorine-induced mitochondrial-mediated apoptosis in hepatocytes.

Pyrrolizidine alkaloids (PAs) are well‐known natural hepatotoxins. In this study, we investigated the protection of epidermal growth factor (EGF) against the hepatotoxicity of clivorine, which is an otonecine‐type PA from traditional Chinese medicine Ligularia hodgsonii Hook. Cell viability assay and cell morphology observation showed that EGF (1 ng/mL) reversed clivorine‐induced cytotoxicity on human normal liver L‐02 cells. EGF (1 ng/mL) also inhibited clivorine‐induced DNA fragmentation and caspase‐3 cleavage. Our previous study has showed that antiapoptotic Bcl‐xL degradation and mitochondrial‐mediated apoptosis was involved in clivorine‐induced hepatotoxicity. In this study, we found that EGF (1 ng/mL) inhibited clivorine‐induced antiapoptotic Bcl‐xL protein decrease, caspase‐9 activation, and release of cytosolic cytochrome C. We further investigated the effects of vascular epidermal growth factor, basic fibroblast growth factor, and insulin‐like growth factor‐1 on clivorine‐induced cytotoxicity, and there is no significant protection observed. Our results suggest that EGF exerts its protective effects against clivorine‐induced hepatotoxicity probably by modulating mitochondrial‐mediated apoptotic signal pathway.