Yubin Luo

Madecassoside isolated from Centella asiatica herbs facilitates burn wound healing in mice.

The current study was designed to investigate the effect of madecassoside, the major triterpene in CENTELLA ASIATICA, on burn wound healing and its possible mechanism of action. An oral administration of madecassoside (6, 12, 24 mg/kg) facilitated wound closure in a time-dependent manner and reached its peak effect, nearly completely wound closure, on day 20 in the group receiving the highest dose of 24 mg/kg of madecassoside. Further histopathological analysis revealed that madecassoside alleviated infiltration of inflammatory cells as well as enhanced epithelisation resulting from dermal proliferation of fibroblasts. Madecassoside at higher doses (12 and 24 mg/kg) decreased nitric oxide (NO) levels and malondialdehyde (MDA) content in the burn skin tissue. However, reduced glutathione (GSH) and hydroxyproline levels were increased in the same skin tissue. In addition, madecassoside promoted skin angiogenesis IN VIVO, correlating with our findings IN VITRO that it stimulated endothelial cell growth in a rat aortic ring assay. These data suggest that madecassoside has significant wound-healing activity and is one of the major reasons for the use of C. ASIATICA herbs in the successful treatment of burn injury. Moreover, the results from the present study indicate that the effect of madecassoside on wound healing may involve several mechanisms including antioxidative activity, collagen synthesis and angiogenesis.

Anti-rheumatoid arthritic effect of madecassoside on type II collagen-induced arthritis in mice.

Madecassoside is the highest amount of triterpene constituent in Centella asiatica herbs, a frequently prescribed crude drug in southeastern Asian and China for wound healing and scar management. The present study aimed to investigate the therapeutic potential and underlying mechanisms of madecassoside on collagen II (CII)-induced arthritis (CIA) in mice. Madecassoside (10, 20 and 40mg/kg), orally administered from the day of the antigen challenge for twenty consecutive days, dose-dependently alleviated the severity of the disease based on the reduced clinical scores, and elevated the body weights of mice. Histopathological examination indicated that madecassoside alleviated infiltration of inflammatory cells and synovial hyperplasia as well as protected joint destruction. Moreover, madecassoside reduced the serum level of anti-CII IgG, suppressed the delayed type hypersensitivity against CII in ears, and moderately suppress CII-stimulated proliferation of lymphocytes from popliteal lymph nodes in CIA mice. In vitro, madecassoside was ineffective in the activation of macrophages caused by lipopolysaccharide. It was concluded that madecassoside substantially prevented mouse CIA, and might be the major active constituent of C. asiatica herbs responsible for clinical uses for rheumatoid arthritis. The underlying mechanisms of action may be mainly through regulating the abnormal humoral and cellular immunity as well as protecting joint destruction.

Puerarin exerts antipyretic effect on lipopolysaccharide-induced fever in rats involving inhibition of pyrogen production from macrophages

ETHNOPHARMACOLOGICAL RELEVANCE Puerarin is the most abundant isoflavonoid in Radix Puerariae (Gegen), which has been prescribed as a medicinal herb for treating fever in China for a long history. AIM OF THE STUDY The present study aimed at evaluating the antipyretic effect of puerarin and revealing the related mechanisms. MATERIALS AND METHODS Lipopolysaccharide (LPS)-induced fever in rats was used to assess the antipyretic effect of puerarin. After an intraperitoneal injection of LPS (100μg/kg), body temperature was tested every 30min up to 8h. Different doses of puerarin (25, 50, 100mg/kg) were intraperitoneally administered 30min before LPS injection. In vitro, LPS-stimulated RAW 264.7 cells were treated with various concentrations of puerarin (25-200μM). The pyrogenic mediators, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E(2) (PGE(2)) and nitric oxide (NO), were examined on both transcription and expression levels. Furthermore, the influences of the activation of nuclear factor-kappa B (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs) by puerarin were assayed by western blot. RESULTS The intraperitoneal administration of puerarin at test doses clearly demonstrated apparent antipyretic effect through the declines in body temperature elevated by LPS in rats. The in vitro data showed that puerarin inhibited the production of IL-1β, TNF-α, IL-6, PGE(2) and NO; moreover, the RT-PCR analysis and the western blot analysis indicated that puerarin regulated the transcriptional level via suppression of NF-κB activation and blockade of MAPK signal pathway. CONCLUSIONS In summary, the antipyretic property of puerarin might result, at least in part, from an inhibition of endogenous pyrogen production and expression. Taken in this sense, our findings provide an explanation for puerarin acting as an important constituent in Gegen, thus, provide scientific basis for the wide use of Radix Puerariae in China as a traditional antipyretic.

Polysaccharide of Radix Pseudostellariae Improves Chronic Fatigue Syndrome Induced by Poly I:C in Mice

Radix Pseudostellariae is used as a tonic drug in traditional Chinese medicine with immunomodulating and anti-fatigue activities, and the polysaccharide is considered as the main active component. The purpose of this study is to examine the effect of the polysaccharide isolated from Radix Pseudostellariae (PRP) on mouse chronic fatigue syndrome (CFS) induced by intraperitoneal injection of polyriboinosinic:polyribocytidylic acid (poly I:C), a double-stranded synthetic RNA. It has shown that the fatigue symptom of mice lasted at least 1 week as evaluated by forced swimming time. PRP (100, 200, 400 mg kg−1), orally administered 3 days before poly I:C injection, showed dose-dependent anti-fatigue effects. In addition, poly I:C led to evident alternations in neuroendocrine and immune systems of mice, such as reduced spontaneous activity and learning ability, declined serum level of corticosterone, increased weight indexes and T lymphocyte numbers in thymuses and spleens, and increased CD4+/CD8+ ratio but decreased proliferation ability of T lymphocytes in spleens. PRP alleviated the abnormalities caused by poly I:C, and restored the function of hosts to normal conditions. The findings suggest that PRP is beneficial to CFS, and the underlying mechanisms of action involve neuroendocrine and immune systems.

Therapeutic effect of norisoboldine, an alkaloid isolated from Radix Linderae, on collagen-induced arthritis in mice.

The alkaloid fraction of Radix Linderae, the main active component of this herb drug, has been proven to exhibit anti-inflammatory, analgesic and antimicrobial activities. The present study was undertaken to investigate the therapeutic potential of norisoboldine, the major isoquinoline alkaloid present in Radix Linderae, in collagen II -induced arthritis (CIA) of mice as well as the possible mechanisms. CIA was induced in mice by immunization with chicken type II collagen (II). After boosted on day 21, mice were treated with norisoboldine (10, 20, 40 mg/kg) for twenty consecutive days. The clinical scores, body weight changes and joint histopathology were evaluated. Norisoboldine treatment significantly alleviated the severity of the disease, based on the reduced clinical scores and elevated the lowered body weights of model mice. Meanwhile, this alkaloid dose-dependently reduced the infiltration of inflammatory cells, synovial hyperplasia and protected joint from destruction. Additionally, the serum level of anti-CII IgG and the CII-stimulated lymphocyte proliferation were remarkably decreased in the groups administered with norisoboldine. An assessment of Th1 function using the delayed-type hypersensitivity model confirmed that norisoboldine also significantly suppressed the enhanced T cell responses in vivo. These findings suggest that norisoboldine might be a potential therapeutic agent for rheumatoid arthritis, and it functions through protecting joint destruction as well as regulating the abnormal immune responses.

Norisoboldine Inhibits the Production of Pro-inflammatory Cytokines in Lipopolysaccharide-Stimulated RAW 264.7 Cells by Down-Regulating the Activation of MAPKs but Not NF-κB

Norisoboldine is the main isoquinoline alkaloid occurring in Radix Linderae, the dry roots of Lindera aggregata (Lauraceae family). It has been previously implicated to be able to ameliorate the synovial inflammation and abnormal immune conditions in collagen-induced arthritis of mice. To get insight to the potential anti-inflammatory mechanisms of this alkaloid compound, the present study was undertaken to explore the effects of norisoboldine on the production of pro-inflammatory cytokines from macrophages stimulated by lipopolysaccharide. In vitro, norisoboldine substantially reduced the production of nitric oxide (NO), tumor necrosis factor (TNF)-α as well as interleukin (IL)-1β from RAW264.7 macrophage cells in a concentration-dependent manner, whereas it only slightly reduced the production of interleukin-6 (IL-6) at both protein and transcription levels. Of note, the preventive effects of norisoboldine on the release of pro-inflammatory cytokines were correlated with the inhibitory action on the phosphorylations of mitogen-activated protein (MAP) kinases including p38, extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK), but not on the activation and translocation of nuclear factor-κB (NF-κB). It can be therefore concluded that norisoboldine inhibits the macrophage activation and the resultant production of pro-inflammatory cytokines via down-regulating the activation of MAPKs signaling pathways rather than NF-κB.

Inhibition of monosodium urate crystal-induced inflammation by scopoletin and underlying mechanisms

The present study determined the anti-inflammatory activity of scopoletin in gout air pouch model and revealed the underlying mechanisms by in vitro assays. Monosodium urate (MSU) crystal-induced inflammation in mouse air pouch model, an experimental model for acute gout, was used to assess the efficacy of scopoletin. The neutrophil and mononuclear phagocyte numbers and MPO levels were increased significantly six hours after MSU crystal injection into the air pouch, whereas these changes were inhibited substantially upon scopoletin (100 and 200mg/kg, i.p.) treatment. To get insight into the underlying mechanisms, the in vitro studies were performed to investigate the effects of scopoletin on activation of macrophages and resultant production of inflammatory mediators. The secretions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E(2) (PGE(2)) and nitric oxide (NO) were elevated in MSU crystal-stimulated RAW 264.7 cells, and scopoletin (30-300 μM) suppressed the production of all mediators. Moreover, RT-PCR assay and western blot analysis indicated that scopoletin regulated the transcriptional level of these mediators via suppression of NF-κB activation and blockade of MAPK signal pathway. Thus, the results clearly indicated that scopoletin inhibited the monosodium urate crystal-induced inflammation both in vivo and in vitro. In combination with our previous findings that scopoletin shows hypouricemic, anti-angiogenesis and pro-apoptotic activities, this compound may be a potential agent for gout therapy and could serve as a structural base for developing new drugs.

Total alkaloids from Radix Linderae prevent the production of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells by suppressing NF-κB and MAPKs activation

Radix Linderae, the dry roots of Lindera aggregata (Sims) Kosterm (L. strychnifolia Vill), has been long-term used in traditional Chinese medicine for treating various diseases, and alkaloids are believed to be the main active components. Previously, we reported that the total alkaloids from Radix Linderae (TARL) could effectively alleviate inflammation and protect joints from destruction in mouse collagen-induced arthritis, an animal model of human rheumatoid arthritis (RA). To get insight into the underlying mechanisms of TARL, the present study was performed to investigate the effects of TARL on the activation of macrophages and resultant production of inflammatory mediators. In vitro, TARL concentration-dependently prevented the production of nitric oxide, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), as well as the expressions of iNOS, IL-1beta and TNF-alpha mRNA in RAW 264.7 cells stimulated by lipopolysaccharide (LPS). However, it showed little effect on the production of interleukin-6 (IL-6) and the expression of IL-6 mRNA. Signal transduction studies showed that TARL significantly down-regulated the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase rather than c-jun NH(2)-terminal kinase (JNK). Additionally, TARL prominently decreased LPS-induced activation of IKKalpha and phosphorylation of p65 on serine 276, but had little impact on the phosphorylation and degradation of IkappaBalpha. In summary, our results demonstrate that TARL exhibits inhibitory effects on the production of inflammatory mediators from macrophages via blocking NF-kappaB and MAPKs signaling pathways. The findings provide a plausible explanation for the therapeutic efficiency of TARL on the inflammation and joint destruction in RA.

Norisoboldine, an alkaloid compound isolated from Radix Linderae, inhibits synovial angiogenesis in adjuvant-induced arthritis rats by moderating Notch1 pathway-related endothelial tip cell phenotype.

Synovial angiogenesis is well recognized as participating in the pathogenesis of rheumatoid arthritis (RA) and has been regarded as a potential target for RA therapy. Previously, we have shown that norisoboldine (NOR) can protect joints from destruction in mice with collagen II-induced arthritis (CIA). Here, we investigate the effect of NOR on synovial angiogenesis in adjuvant-induced arthritis (AA) rats, and clarify the mechanisms in vitro. NOR, administered orally, significantly reduced the number of blood vessels and expression of growth factors in the synovium of AA rats. In vitro, it markedly prevented the migration and sprouting of endothelial cells. Notably, the endothelial tip cell phenotype, which is essential for the migration of endothelial cells and subsequent angiogenesis, was significantly inhibited by NOR. This inhibitory effect was attenuated by pretreatment with N-{N-[2-(3,5-difluorophenyl) acetyl]-(S)-alanyl}-(S)-phenylglycine tert-butyl ester, a Notch1 inhibitor, suggesting that the action of NOR was related to the Notch1 pathway. A molecular docking study further confirmed that NOR was able to promote Notch1 activation by binding the Notch1 transcription complex. In conclusion, NOR was able to prevent synovial angiogenesis in AA rats, which is a putatively new mechanism responsible for its anti-rheumatoid effect. The anti-angiogenesis action of NOR was likely achieved by moderating the Notch1 pathway-related endothelial tip cell phenotype with a potential action target of the Notch1 transcription complex.

Norisoboldine suppresses VEGF-induced endothelial cell migration via the cAMP-PKA-NF-κB/Notch1 pathway.

The migration of endothelial cells has been regarded as a potential target for the treatment of angiogenesis-related diseases. Previously, we demonstrated that norisoboldine (NOR), an alkaloid compound isolated from Radix Linderae, can significantly suppress synovial angiogenesis by selectively inhibiting endothelial cell migration. In this study, we evaluated the importance of various pathways in VEGF-induced endothelial cell migration using specific inhibitor. VEGF-induced endothelial cell migration and sprouting were significantly inhibited by H-89 (an inhibitor of protein kinase A (PKA)) but not by inhibitors of other pathways. NOR markedly suppressed VEGF-induced intracytoplasmic cAMP production and PKA activation and thereby down-regulated the activation of downstream components of the PKA pathway, including enzymes (src, VASP and eNOS) and the transcription factor NF-κB. Moreover, the transcription activation potential of NF-κB, which is related to IκBα phosphorylation and the disruption of the p65/IκBα complex, was reduced by NOR. Meanwhile, NOR selectively inhibited the expression of p-p65 (ser276) but not p-p65 (ser536) or PKAc, indicating that PKAc participates in the regulation of NF-κB by NOR. Co-immunoprecipitation and immunofluorescence assays confirmed that NOR inhibited the formation of the PKAc/p65 complex and thereby decreased p65 (ser276) phosphorylation to prevent p65 binding to DNA. Docking models indicated that the affinity of NOR for PKA was higher than that of the original PKA ligand. Moreover, the fact that H-89 improved Notch1 activation, but DAPT (an inhibitor of Notch) failed to affect PKA activation, suggested that PKA may act on upstream of Notch1. In conclusion, the inhibitory effects of NOR on endothelial cell migration can be attributed to its modulation of the PKA pathway, especially on the processes of p65/IκBα complex disruption and PKAc/p65 complex formation. These results suggest that NOR inhibit VEGF-induced endothelial cell migration via a cAMP-PKA-NF-κB/Notch1 signaling pathway.