Naisheng Zhang

Geniposide, from Gardenia jasminoides Ellis, inhibits the inflammatory response in the primary mouse macrophages and mouse models

Geniposide, a main iridoid glucoside component of gardenia fruit, has been known to exhibit antibacterial, anti-inflammatory and other important therapeutic activities. The objective of this study was to investigate the protective effects of geniposide on inflammation in lipopolysaccharide (LPS) stimulated primary mouse macrophages in vitro and LPS induced lung injury model in vivo. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Toll-like receptor 4 (TLR4) were determined by Western blot. Further analysis was carried out in mTLR4 and mMD-2 co-transfected HEK293 cells. The results showed that geniposide markedly inhibited the LPS-induced TNF-α, IL-6 and IL-1β production both in vitro and in vivo. Geniposide blocked the phosphorylation of IκBα, p65, p38, ERK and JNK in LPS stimulated primary mouse macrophages. Furthermore, geniposide inhibited the expression of TLR4 in LPS stimulated primary mouse macrophages and inhibited the LPS-induced IL-8 production in HEK293-mTLR4/MD-2 cells. In vivo study, it was also observed that geniposide attenuated lung histopathologic changes in the mouse models. These results suggest that geniposide exerts an anti-inflammatory property by down-regulating the expression of TLR4 up-regulated by LPS. Geniposide is highly effective in inhibiting acute lung injury and may be a promising potential therapeutic reagent for acute lung injury treatment.

Magnolol inhibits lipopolysaccharide-induced inflammatory response by interfering with TLR4 mediated NF-κB and MAPKs signaling pathways

ETHNOPHARMACOLOGICAL RELEVANCE Magnolia officinalis as a traditional Chinese herb has long been used for the treatment of anxiety, cough, headache and allergic diseases, and also have been used in traditional Chinese medicine to treat a variety of mental disorders including depression. AIM OF THE STUDY Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been reported to have anti-inflammatory properties. However, the underlying molecular mechanisms are not well understood. The aim of this study was to investigate the molecular mechanism of magnolol in modifying lipopolysaccharide (LPS)-induced signal pathways in RAW264.7 cells. MATERIAL AND METHODS The purity of magnolol was determined by high performance liquid chromatography. RAW264.7 cells were stimulated with LPS in the presence or absence of magnolol. The expression of proinflammatory cytokines were determined by ELISA and reverse transcription-PCR. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Toll-like receptor 4 (TLR4) were determined by Western blot. Further analyses were performed on mTLR4 and mMD2 co-transfected HEK293 cells. RESULTS The result showed that the purity of magnolol used in this study was 100%. Magnolol inhibited the expression of TNF-α, IL-6 and IL-1β in LPS-stimulated RAW264.7 cells in a dose-dependent manner. Western blot analysis showed that magnolol suppressed LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK and P38. Magnolol could significantly down-regulated the expression of TLR4 stimulating by LPS. Furthermore, magnolol suppressed LPS-induced IL-8 production in HEK293-mTLR4/MD-2 cells. CONCLUSIONS Our results suggest that magnolol exerts an anti-inflammatory property by down-regulated the expression of TLR4 up-regulated by LPS, thereby attenuating TLR4 mediated the activation of NF-κB and MAPK signaling and the release of pro-inflammatory cytokines. These findings suggest that magnolol may be a therapeutic agent against inflammatory diseases.

Emodin ameliorates lipopolysaccharide-induced mastitis in mice by inhibiting activation of NF-κB and MAPKs signal pathways

Emodin is an anthraquinone derivative from the Chinese herb Radix et Rhizoma Rhei. It has been reported that emodin possesses a number of biological properties, such as anti-inflammatory, anti-virus, anti-bacteria, anti-tumor, and immunosuppressive properties. However, the effect of emodin on mastitis is not yet known. The aim of this study was to investigate whether emodin has protective effect against lipopolysaccharide (LPS)-induced mastitis in a mouse model. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. Emodin was administered intraperitoneally with the dose of 1, 2, and 4 mg/kg respectively 1h before and 12h after induction of LPS. Emodin significantly reduced infiltration of neutrophilic granulocyte, activation of myeloperoxidase (MPO), concentration of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), mRNA expression levels of TNF-α, IL-1β and IL-6, which were increased in LPS-induced mouse mastitis. In addition, emodin influenced nuclear factor kappa-B signal transduction pathway by inhibiting activation of nuclear transcription factor-kappaB (NF-κB) p65 and degradation inhibitor of NF-κB α (IκBα), and emodin also influenced mitogen activated protein kinases signal transduction pathway by depression activation of p38, extracellular signal-regulated kinase (ERK), and c-jun NH2-terminal kinase (JNK). In conclusion, these results indicated that emodin could exert beneficial effects on experimental mastitis induced by LPS and may represent a novel treatment strategy for mastitis.

Baicalin plays an anti-inflammatory role through reducing nuclear factor-κB and p38 phosphorylation in S. aureus-induced mastitis.

Mastitis is an inflammatory disease caused by microbial infection. Staphylococcus aureus is the major etiological microorganism responsible for both clinical and subclinical mastitis in dairy cows. A mouse model of S. aureus mastitis is available. Baicalin is isolated from Scutellaria and is known to have anti-inflammatory properties. This study was designed to evaluate the effects of baicalin in S. aureus mastitis. In the present study, the mouse model was infected with S. aureus to cause mammary gland inflammation. Baicalin treatment was administered from 6h until 24h after infection. Baicalin significantly attenuated inflammatory cell infiltration and decreased levels of TNF-α, IL-β, and IL-6. Further studies revealed that baicalin downregulated phosphorylation of NF-κB and p38 in the mammary gland with S. aureus mastitis. Our results demonstrated that baicalin reduced the expression of the proinflammatory cytokines TNF-α, IL-β, and IL-6 by inhibiting NF-κB and p38 phosphorylation and mRNA expression.

Shikonin exerts anti-inflammatory effects in a murine model of lipopolysaccharide-induced acute lung injury by inhibiting the nuclear factor-kappaB signaling pathway

Shikonin, an analog of naphthoquinone pigments isolated from the root of Lithospermum erythrorhyzon, was recently reported to exert beneficial anti-inflammatory effects both in vivo and in vitro. The present study aimed to investigate the potential therapeutic effect of shikonin in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Dexamethasone was used as a positive control to evaluate the anti-inflammatory effect of shikonin in the study. Pretreatment with shikonin (intraperitoneal injection) significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, shikonin significantly reduced the concentrations of TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid induced by LPS. Compared with the LPS group, lung histopathologic changes were less pronounced in the shikonin-pretreated mice. Additionally, Western blotting results showed that shikonin efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that shikonin exerts anti-inflammatory properties in LPS-mediated ALI, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. Shikonin may be a potential agent for the prophylaxis of ALI.

Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts.

Saikosaponin a (SSa), the major triterpenoid saponin derivatives from Radix bupleuri (RB), has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effects of SSa on lipopolysaccharide (LPS)-induced oxidative stress and inflammatory response in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with LPS in the presence or absence of SSa. The levels of TNF-α and IL-8 were detected by ELISA. The expression of COX-2 and iNOS, NF-κB and IκB protein were determined by Western blotting. To investigate the protective mechanisms of SSa, TLR4 expression was detected by Western blotting and membrane lipid rafts were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The results showed that SSa dose-dependently inhibited the production of ROS, TNF-α, IL-8, COX-2 and iNOS in LPS-stimulated HUVECs. Western blot analysis showed that SSa suppressed LPS-induced NF-κB activation. SSa did not affect the expression of TLR4 induced by LPS. However, translocation of TLR4 into lipid rafts and oligomerization of TLR4 induce by LPS was inhibited by SSa. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol. Moreover, SSa activated LXRα-ABCA1 signaling pathway, which could induce cholesterol efflux from lipid rafts. Knockdown of LXRα abrogated the anti-inflammatory effects of SSa. In conclusion, the effects of SSa is associated with activating LXRα-ABCA1 signaling pathway which results in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts and oligomerization of TLR4, thereby attenuating LPS mediated oxidative and inflammatory responses.

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells.

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.

Selenium Inhibits LPS-Induced Pro-inflammatory Gene Expression by Modulating MAPK and NF-κB Signaling Pathways in Mouse Mammary Epithelial Cells in Primary Culture

Mastitis is characterized by an inflammation of the mammary gland of dairy animals and humans; this condition is one of the major causes of economic losses in dairy industries. Selenium (Se), a biological trace element, modulates the functions of many regulatory proteins in signal transduction and provides advantages for animals with inflammatory diseases, including mastitis. The current study aimed to assess the protective effects and the active mechanism of Na2SeO3 against lipopolysaccharide (LPS)-induced inflammation in mouse mammary epithelial cells (MMECs). Our results showed that LPS-induced expressions of cyclooxygenase-2 and tumor necrosis factor-α significantly decreased after Se was supplemented to Se-deficient MMECs. Na2SeO3 also suppressed LPS-induced nuclear factor-κB activation, inhibitory kappa B degradation, and ERK, JNK, and P38 phosphorylation in a dose-dependent manner. These results suggested that Se functions as an anti-inflammatory agent in mastitis.

Curcumin attenuates inflammatory responses by suppressing TLR4-mediated NF-κB signaling pathway in lipopolysaccharide-induced mastitis in mice.

Curcumin, the main constituent of the spice turmeric, has been reported to have potent anti-inflammatory properties. However, the effect of curcumin on lipopolysaccharide (LPS)-induced mice mastitis has not been investigated. The aim of this study was to investigate whether curcumin could ameliorate the inflammation response in LPS-induced mice mastitis and to clarify the possible mechanism. The mouse model of mastitis was induced by injection of LPS through the duct of the mammary gland. Curcumin was applied 1h before and 12h after LPS treatment. The results showed that curcumin attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in a dose-dependent manner. Additionally, Western blotting results showed that curcumin inhibited the phosphorylation of IκB-α and NF-κB p65 and the expression of TLR4. These results indicated that curcumin has protective effect on mice mastitis and the anti-inflammatory mechanism of curcumin on LPS-induced mastitis in mice may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathways. Curcumin may be a potential therapeutic agent against mastitis.

Geniposide Plays an Anti-inflammatory Role via Regulating TLR4 and Downstream Signaling Pathways in Lipopolysaccharide-Induced Mastitis in Mice

Geniposide is a medicine isolated from Gardenia jasminoides Ellis, which is a traditional Chinese herb that is widely used in Asia for the treatment of inflammation, brain diseases, and hepatic disorders. Mastitis is a highly prevalent and important infectious disease. In this study, we used a lipopolysaccharide (LPS)-induced mouse mastitis model and LPS-stimulated primary mouse mammary epithelial cells (mMECs) to explore the anti-inflammatory effect and the mechanism of action of geniposide. Using intraductal injection of LPS as a mouse model of mastitis, we found that geniposide significantly reduced the infiltration of inflammatory cells and downregulated the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). To further investigate the anti-inflammatory mechanism, we used LPS-stimulated mMECs as an in vitro mastitis model. The results of enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) showed that geniposide inhibited the expression of TNF-α, IL-1β, and IL-6 in a dose-dependent manner. Western blot analysis demonstrated that geniposide could suppress the phosphorylation of inhibitory kappa B (IκBα), nuclear factor-κB (NF-κB), p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Geniposide also inhibited the expression of toll-like receptor 4 (TLR4) in the LPS-stimulated mMECs. In conclusion, geniposide exerted its anti-inflammatory effect by regulating TLR4 expression, which affected the downstream NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Thus, geniposide may be a potential drug for mastitis therapy.