Dehydrocostus lactone alleviates irinotecan-induced intestinal mucositis by blocking TLR4/MD2 complex formation.

BACKGROUND: Irinotecan (CPT-11) is used as chemotherapeutic drug for treatment of colorectal cancer. However, without satisfactory treatments, its gastrointestinal toxicities such as diarrhea and intestinal inflammation severely restrained its clinical application. Roots of Aucklandia lappa Decne. are used as traditional Chinese medicine to relieve gastrointestinal dysfunction and dehydrocostus lactone (DHL) is one of its main active components. Nevertheless, the efficacy and mechanism of DHL against intestinal mucositis remains unclear. PURPOSE: The present study aimed to investigate the protective effects of DHL on CPT-11-induced intestinal mucositis and its underlying mechanisms. METHODS: The protective effect of DHL was investigated in CPT-11-induced mice and lipopolysaccharide (LPS)+CPT-11 induced THP-1 macrophages. Body weight, diarrhea score, survival rate, colon length, and histopathological changes in mice colon and jejunum were analyzed to evaluate the protective effect of DHL in vivo. And DHL on reducing inflammatory response and regulating TLR4/NF-κB/NLRP3 pathway in vivo and in vitro were explored. Moreover, DHL on the interaction between TLR4 and MD2 was investigated. And silencing TLR4 targeted by siRNA was performed to validate the mechanisms of DHL on regulating the inflammation. RESULTS: DHL prevented CPT-11-induced intestinal damage, represented by reducing weight loss, diarrhea score, mortality rate and the shortening of the colon. Histological analysis confirmed that DHL prevented intestinal epithelial injury and improved the intestinal barrier function in CPT-11 induced mice. Besides, DHL significantly downregulated the level of inflammatory cytokines by inhibiting TLR4/NF-κB/NLRP3 signaling pathway in CPT-11-induced mice and LPS+CPT-11-induced THP-1 macrophages. In addition, DHL blocked TLR4/MD2 complex formation. Molecular docking combined with SIP and DARTS assay showed that DHL could bind to TLR4/MD2 and occludes the hydrophobic pocket of MD2. Furthermore, Silencing TLR4 abrogated the effect of DHL on LPS+CPT-11 induced inflammatory response in THP-1 macrophages. Additionally, DHL ameliorate the CPT-11-induced intestinal mucositis without affecting the anti-tumor efficacy of CPT-11 in the tumor xenograft mice. CONCLUSION: This study found that DHL exhibited the anti-inflammatory effects in CPT-11-induced intestinal mucositis by inhibiting the formation of TLR4/MD2 complex and then regulation of NF-κB/NLRP3 signaling pathway. DHL is potentially served as a novel strategy of combined medication with CPT-11.

Oxybaphus himalaicus Mitigates Lipopolysaccharide-Induced Acute Kidney Injury by Inhibiting TLR4/MD2 Complex Formation.

Acute kidney injury (AKI) is described as the abrupt decrease in kidney function always accompanied by inflammation. The roots of Oxybaphus himalaicus Edgew. have long been used in Tibetan folk medicine for the treatment of nephritis. Nevertheless, modern pharmacological studies, especially about the underlying mechanism of O. himalaicus medications, are still lacking. Here, in lipopolysaccharide (LPS)-induced RAW264.7 macrophages, the O. himalaicus extract (OE) showed significant anti-inflammatory activity with the dose dependently reducing the LPS-stimulated release of nitric oxide and the mRNA level and protein expression of inflammatory cytokines and reversed the activation of nuclear factor kappa B (NF-κB). Co-immunoprecipitation assay indicated that OE inhibited Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2) complex formation and further suppressed both myeloid differentiation factor 88 (MyD88)-dependent and TIR-domain-containing adapter-inducing interferon-ß (TRIF)-dependent cascades activation. In addition, OE could restrain NADPH oxidase 2 (NOX2) endocytosis by blocking TLR4/MD2 complex formation to prevent reactive oxygen species production. In LPS-induced AKI mice, OE treatment mitigated renal injury and inflammatory infiltration by inhibiting TLR4/MD2 complex formation. UPLC-MS/MS analysis tentatively identified 41 components in OE. Our results indicated that OE presented significant anti-inflammatory activity by inhibiting TLR4/MD2 complex formation, which alleviated LPS-induced AKI in mice.

The interaction of MD-2 with small molecules in huanglian jiedu decoction play a critical role in the treatment of sepsis.

Huanglian Jiedu Decoction (HJD) is used for treating sepsis in China. Active components from HJD refer to various active ingredients of HJD, while active component formulation (ACF) refers to the combination of palmatine, berberine, baicalin, and geniposide from HJD according to the quantity of HJD. The detailed mechanisms of the active components from HJD and ACF in sepsis treatment are unclear. Molecular docking, surface plasmon resonance (SPR), ELISA, RT-qPCR, and Western blotting were used to assay the possible mechanism in vitro. The efficacy and mechanism of ACF and HJD were assessed by pharmacodynamics and metabolomics analyses, respectively. The results revealed that palmatine, berberine, baicalin, and geniposide showed good binding capacity to MD-2; decreased the release of NO, TNF-α, IL-6, and IL-1ß; inhibited the mRNA expression of iNOS, TNF-α, IL-6, IL-1ß, and COX-2; and downregulated the protein expressions of MD-2, MyD88, p-p65, and iNOS induced by LPS; which indicated that they can inactivate the LPS-TLR4/MD-2-NF-κB pathway. Thus, ACF was formed, and the pharmacodynamics assay suggested that ACF can reduce inflammatory cell infiltration and organ damage in accordance with HJD. Furthermore, 39 metabolites were selected and identified and the regulatory effect of these metabolites by ACF and HJD was almost consistent, but ACF might alleviate physical damage caused by HJD through regulating metabolites, such as 3-hydroxyanthranilic acid. ACF could represent HJD as a new formulation to treat sepsis.

Astilbin prevents osteoarthritis development through the TLR4/MD-2 pathway.

Osteoarthritis has become one of the main diseases affecting the life of many elderly people with high incidence of disability, and local chronic inflammation in the joint cavity is the most crucial pathological feature of osteoarthritis. Astilbin is the main active component in a variety of natural plants such as Hypericum perforatum and Sarcandra glabra, which possess antioxidant and anti-inflammatory effects. At present, there is no study about the protective effect of Astilbin for osteoarthritis. The purpose of this study was to investigate the effect of Astilbin in human OA chondrocytes and mouse OA model, which was established by surgery-mediated destabilization of the medial meniscus (DMM). In vitro, we found that Astilbin pre-treatment inhibited lipopolysaccharide (LPS)-induced overproduction of inflammation-correlated cytokines such as nitric oxide (NO), prostaglandin E2 (PGE2), tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6), and suppressed overexpression of inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Astilbin, on the other hand, prevented the LPS-induced degradation of extracellular matrix (ECM) by down-regulating MMP13 (matrix metalloproteinases 13) and ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5). Moreover, by inhibiting the formation of the TLR4/MD-2/LPS complex, Astilbin blocked LPS-induced activation of TLR4/NF-κB signalling cascade. In vivo, Astilbin showed the chondro-protective effect in the surgical-induced OA mouse models. In conclusion, our findings provided evidence that develops Astilbin as a potential therapeutic drug for OA patients.

Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-κB signaling pathway.

BACKGROUND: Hibiscus syriacus L. has been used as a medicinal plant in many Asian countries. However, anti-inflammatory activity of H. syriacus L. remains unknown. PURPOSE: This study was aimed to investigating the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. variety Pulsae (PS) on the lipopolysaccharide (LPS)-induced inflammation and endotoxic shock. STUDY DESIGN AND METHODS: MTT assay and flow cytometry analysis were performed to determine cytotoxicity of PS. RT-PCR, western blotting, and ELISA were conducted to evaluate the expression of proinflammatory mediators and cytokines. Molecular docking study predicted the binding scores and sites of PS to TLR4/MD2 complex. Immunohistochemical assay was conducted to evaluate the binding capability of PS to TLR4/MD2 and nuclear translocation of NF-κB p65. A zebrafish endotoxic shock model was used to evaluate anti-inflammatory activity of PS in vivo. RESULTS: PS suppressed LPS-induced nitric oxide and prostaglandin E2 secretion concomitant with the downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in LPS-stimulated RAW 264.7 macrophages. Additionally, molecular docking data showed that PS mostly fit into the hydrophobic pocket of MD2 and bound to TLR4. In particular, apigenin-7-O-glucoside powerfully bound to MD2 and TLR4 via hydrogen bonding. Additionally, immunohistochemistry assay revealed that PS inhibited LPS-induced TLR4 dimerization or expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation, resulting in the inhibition of NF-κB activity. PS also attenuated LPS-mediated mortality and abnormality in zebrafish larvae and diminished the recruitment of neutrophils and macrophages at the inflammatory site accompanied by the low levels of proinflammatory mediators and cytokines. CONCLUSION: PS might be a novel immunomodulator for the effective treatment of LPS-mediated inflammatory diseases.

Epimedium sagittatum inhibits TLR4/MD-2 mediated NF-κB signaling pathway with anti-inflammatory activity.

BACKGROUND: Epimedium sagittatum (Sieb.et Zucc.) Maxim., Ying-Yang-Huo in Chinese has been used as a traditional Chinese medicine and is deemed to "reinforce the kidney Yang". Previous studies showed that E. sagittatum could modulate the immune system and treat some chronic disease such as rheumatic arthritis, cardiovascular diseases and osteoporosis. The aim of this study is to evaluate the anti-inflammatory effects of ethyl acetate extracts (YYHs) of E. sagittatum and its mechanisms of action. METHODS: In order to explore the composition of YYHs, YYHs was analyzed using high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS/MS) and in comparison with reference standards. Anti-inflammatory model was established in LPS-induced RAW264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Production of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were measured by enzyme-linked immunosorbent assays (ELISA). In addition, expression of p-p65 protein and TLR4/MD-2 complex was detected by western blots and flow cytometric, respectively. Nuclear factor kappa B (NF-κB) nuclear translocation was observed by fluorescence microscope. RESULTS: A total of eight compounds were identified, of which icariside II was the most abundant compound. YYHs (12.5-50 µg/mL) had no obvious cytotoxic effect on cells, and remarkably inhibited LPS-induced production of NO, TNF-α and IL-2 with a dose-dependent manner. Additionally, YYHs up-regulated expression of p-p65 and TLR4/MD-2 complex. Further research showed that YYHs significantly suppressed NF-κB p65 nuclear translocation. CONCLUSION: In brief, YYHs contributed to the inhibition of LPS-induced inflammatory response through the TLR4/MD-2-mediated NF-κB pathway and may be a potential choice to combat inflammation diseases. It includes a schema of pathways at the end of the paper.

Lycium barbarum polysaccharide LBPF4-OL may be a new Toll-like receptor 4/MD2-MAPK signaling pathway activator and inducer.

Recognition of the utility of the traditional Chinese medicine Lycium barbarum L. has been gradually increasing in Europe and the Americas. Many immunoregulation and antitumor effects of L. barbarum polysaccharides (LBP) have been reported, but its molecular mechanism is not yet clear. In this study, we reported that the activity of the polysaccharide LBPF4-OL, which was purified from LBP, is closely associated with the TLR4-MAPK signaling pathway. We found that LBPF4-OL can significantly induce TNF-α and IL-1ß production in peritoneal macrophages isolated from wild-type (C3H/HeN) but not TLR4-deficient mice (C3H/HeJ). We also determined that the proliferation of LBPF4-OL-stimulated lymphocytes from C3H/HeJ mice is significantly weaker than that of lymphocytes from C3H/HeN mice. Furthermore, through a bio-layer interferometry assay, we found that LPS but not LBPF4-OL can directly associate with the TLR4/MD2 molecular complex. Flow cytometry analysis indicated that LBPF4-OL markedly upregulates TLR4/MD2 expression in both peritoneal macrophages and Raw264.7 cells. As its mechanism of action, LBPF4-OL increases the phosphorylation of p38-MAPK and inhibits the phosphorylation of JNK and ERK1/2, as was observed through Western blot analysis. These data suggest that the L. barbarum polysaccharide LBPF4-OL is a new Toll-like receptor 4/MD2-MAPK signaling pathway activator and inducer.