Wenlong Zhang

Liver X receptor agonist prevents LPS-induced mastitis in mice.

Liver X receptor-α (LXR-α) which belongs to the nuclear receptor superfamily, is a ligand-activated transcription factor. Best known for its ability to regulate lipid metabolism and transport, LXRs have recently also been implicated in regulation of inflammatory response. The aim of this study was to investigate the preventive effects of synthetic LXR-α agonist T0901317 on LPS-induced mastitis in mice. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. T0901317 was injected 1h before and 12h after induction of LPS intraperitoneally. The results showed that T0901317 significantly attenuated the infiltration of neutrophilic granulocytes, and the activation of myeloperoxidase (MPO); down-regulated the level of pro-inflammatory mediators including TNF-α, IL-1β, IL-6, COX-2 and PEG2; inhibited the phosphorylation of IκB-α and NF-κB p65, caused by LPS. Moreover, we report for the first time that LXR-α activation impaired LPS-induced mastitis. Taken together, these data indicated that T0901317 had protective effect on mastitis and the anti-inflammatory mechanism of T0901317 on LPS induced mastitis in mice may be due to its ability to inhibit NF-κB signaling pathway. LXR-α activation can be used as a therapeutic approach to treat mastitis.

Inhibitory effects of astragalin on lipopolysaccharide-induced inflammatory response in mouse mammary epithelial cells

BACKGROUND Tea brewed from the leaves of persimmon or Rosa agrestis have several medical functions including treating allergy, antiatopic dermatitis, and anti-inflammatory effects. The objective of this study was to investigate the molecular mechanisms of astragalin, a main flavonoid component isolated from these herbs, in modifying lipopolysaccharide (LPS)-induced signaling pathways in primary cultured mouse mammary epithelial cells (mMECs). MATERIALS AND METHODS The mMECs were treated with LPS in the absence or presence of different concentrations of astragalin. The expression of proinflammatory cytokines tumor necrosis factor α, and interleukin 6, as well as nitric oxide production were determined by enzyme-linked immunosorbent assay and Griess reaction, respectively. Cyclooxygenase-2, inducible nitric oxide synthase, toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), inhibitor protein of NF-κB (IκBα), P38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were measured by Western blot. RESULTS The results showed that astragalin suppressed the expression of tumor necrosis factor α, interleukin 6, and nitric oxide in a dose-dependent manner in mMECs. Western blot results showed that the expression of inducible nitric oxide synthase and cyclooxygenase-2 was inhibited by astragalin. Besides, astragalin efficiently decreased LPS-induced TLR4 expression, NF-κB activation, IκBα degradation, and the phosphorylation of p38, extracellular signal-regulated kinase in BMECs. CONCLUSIONS Our results indicated that astragalin exerts anti-inflammatory properties possibly via the inactivation of TLR4-mediated NF-κB and mitogen-activated protein kinases signaling pathways in LPS-stimulated mMECs. Thus, astragalin may be a potential therapeutic agent for bovine mastitis.

Inhibitory Effects of Emodin, Thymol, and Astragalin on Leptospira interrogans-Induced Inflammatory Response in the Uterine and Endometrium Epithelial Cells of Mice

Leptospirosis is a systemic infection that causes, among others, acute kidney injury, acute liver disease, muscle pain, vasculitis, bleeding disorders, and reproductive loss. In an effort to reduce uterine inflammatory responses induced by Leptospira, we evaluated the anti-inflammation effects of emodin, thymol, and astragalin in a mouse model. Our results showed that treatment with emodin, thymol, and astragalin alleviated uterine inflammation induced by leptospira infection via suppression of pro-inflammatory cytokine expression and prevented tissue damage. Furthermore, we used primary endometrium epithelial cells to show that treatment with these chemicals inhibited the expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Western blot results showed that these chemicals suppressed the phosphorylation of p38, p65, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. These results indicate that treatment with emodin, thymol, and astragalin suppressed inflammatory response by regulating NF-κB and mitogen-activated protein kinase signaling pathways in leptospira-infected uterine and endometrium epithelial cells of mice.

Protective effect of TM6 on LPS-induced acute lung injury in mice

Acute lung injury (ALI) is an acute failure of the respiratory system for which effective treatment is urgently necessary. Previous studies found that several peptides potently inhibited the production of cytokines induced by lipopolysaccharide (LPS). In this study, we synthetized a cell-permeable TIR domain-derived decoy peptide (TM6) and examined its substance for the ability to inhibit TLR signaling in the model of ALI induced by LPS. We demonstrated that TM6 (2.5, 5 and 10 nmol/g) alleviated the histological changes in the lung tissues as well as myeloperoxtidase (MPO) activity, lung W/D ratio, the production of TNF-α, IL-1β and IL-6 induced by LPS. Furthermore, the numbers of total cells, neutrophils and macrophages in the BALF were suppressed by TM6. In vitro, TM6 (5, 10 and 20 µM) inhibited the production of TNF-α, IL-1β and IL-6 in LPS-stimulated alveolar macrophages. Moreover, the activation of Nuclear factor-kappaB (NF-κB) and Mitogen activated protein kinases (MAPK) signaling pathways induced by LPS were also inhibited by TM6. Collectively, our results suggested that TM6 was an effective inhibitor of ALI induced by LPS, and this peptide may very well serve as a future treatment for ALI.

Toll-Like Receptor 2 Agonist Pam3CSK4 Alleviates the Pathology of Leptospirosis in Hamster

ABSTRACT Leptospirosis, caused by pathogenic spirochetes, is a zoonotic disease of global importance. The detailed pathogenesis of leptospirosis is still unclear, which limits the ideal treatment of leptospirosis. In this study, we analyzed the expression of Toll-like receptor 2 (TLR2) and TLR4 in target organs of both resistant mice and susceptible hamsters after Leptospira interrogans serovar Autumnalis infection. TLR2 but not TLR4 transcripts in mouse organs contrasted with delayed induction and overexpression in hamster organs. Coinjection of leptospires and the TLR2 agonist Pam3CSK4 into hamsters improved their survival rate, alleviated tissue injury, and decreased the abundance of leptospires in target organs. The production of interleukin-10 (IL-10) from tissues was enhanced in hamsters of the group coinjected with leptospires and Pam3CSK4 compared with the leptospira-injected group. Similarly, IL-10 levels in TLR2-deficient mice were lower than those in wild-type mice. A high ratio of IL-10/tumor necrosis factor alpha (TNF-α) levels was found in both infected wild-type mice and hamsters coinjected with leptospires and Pam3CSK4. Moreover, TLR2-dependent IL-10 expression was detected in peritoneal macrophages after leptospira infection. Our data demonstrate that coinjection of leptospires and Pam3CSK4 alleviates the pathology of leptospirosis in hamsters; this effect may result from the enhanced expression of TLR2-dependent IL-10.

Protective Effects of Platycodin D on Lipopolysaccharide-Induced Acute Lung Injury by Activating LXRα–ABCA1 Signaling Pathway

The purpose of this study was to investigate the protective effects of platycodin D (PLD) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and clarify the possible mechanism. An LPS-induced ALI model was used to confirm the anti-inflammatory activity of PLD in vivo. The A549 lung epithelial cells were used to investigate the molecular mechanism and targets of PLD in vitro. In vivo, the results showed that PLD significantly attenuated lung histopathologic changes, myeloperoxidase activity, and pro-inflammatory cytokines levels, including TNF-α, IL-1β, and IL-6. In vitro, PLD inhibited LPS-induced IL-6 and IL-8 production in LPS-stimulated A549 lung epithelial cells. Western blot analysis showed that PLD suppressed LPS-induced NF-κB and IRF3 activation. Moreover, PLD did not act though affecting the expression of TLR4. We also showed that PLD disrupted the formation of lipid rafts by depleting cholesterol and prevented LPS-induced TLR4 trafficking to lipid rafts, thereby blocking LPS-induced inflammatory response. Finally, PLD activated LXRα–ABCA1-dependent cholesterol efflux. Knockdown of LXRα abrogated the anti-inflammatory effects of PLD. The anti-inflammatory effects of PLD was associated with upregulation of the LXRα–ABCA1 pathway, which resulted in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts.

Efficacy of the Rabbit Polyclonal Anti-leptospira Antibody against Homotype or Heterotype Leptospira Infection in Hamster

Leptospirosis, caused by Leptospira, is one of the most important of neglected emerging zoonotic diseases that has important impacts on public health worldwide. Polyclonal antibody (pcAb) therapy is a potential method to process a series of pathogens for which there are limited determination of treatment, such as leptospirosis. First, we evaluated the efficacy of pcAb, derived from the sera of rabbits inoculated with Leptospira, against homotype (Leptospira interrogans serovar Lai) or heterotype (Leptospira interrogans serovar Autumnalis) Leptospira infection in a lethal hamster model. The pcAb treatment improved survival compared to the controls. The histopathology’s of the infected kidney, liver and lung were also examined by hematoxylin and eosin staining. Using real-time quantitative PCR, we determined that most of the leptospires in the primary organs were almost completely removed by pcAb. In the second experiment, treatments, including antibiotic, pcAb, and combination, were started immediately after occurrence of the first serious sickness mouse in any group. No significant difference in survival rate between pcAb group and antibiotic group was found, but the combination therapy group significantly improved survival rate compared to the others (P<0.05). We conclude that the rabbit pcAb treatment may cure both the homotype and the heterotype lethal Leptospira infections in hamster, and combination therapy improved survival compared to antibiotic group in the late treatment of homotype leptospirosis.

Low-dose Norfloxacin-treated leptospires induce less IL-1β release in J774A.1 cells following discrepant leptospiral gene expression

Currently, accumulating evidence is challenging subtherapeutic therapy. Low-dose Norfloxacin (Nor) has been reported to suppress the immune response and worsen leptospirosis. In this study, we investigated the influence of low-dose Nor (0.03 μg/ml, 0.06 μg/ml, 0.125 μg/ml) on leptospiral gene expression and analyzed the immunomodulatory effects of low-dose Nor-treated leptospires in J774A.1 cells. To study the expression profiles of low-dose Nor-treated leptospires, we chose LipL71/LipL21 as reference genes determined by the geNorm applet in this experiment. The results showed that low-dose Nor up-regulated the expression of FlaB and inhibited the expression of 16S rRNA, LipL32, LipL41, Loa22, KdpA, and KdpB compared with the untreated leptospires. These results indicated that low-dose Nor could regulate leptospiral gene expression. Using RT-PCR, the gene expression of IL-1β and TNF-α in J774A.1 cells was detected. Nor-treated leptospires induced higher expression levels of both IL-1β and TNF-α. However, when analyzed by ELISA, the release of mature IL-1β was reduced compared with that observed in cells induced with no Nor-treated leptospires, although the TNF-α protein level showed no significant change. Our study indicated that the gene expression of leptospires could be modulated by low-dose Nor, which induced less IL-1β release in J774A.1 cells.

Doxycycline Attenuates Leptospira-Induced IL-1β by Suppressing NLRP3 Inflammasome Priming

Doxycycline (Dox), a semisynthetic antibiotic, has been reported to exert multiple immunomodulatory effects. Treatment with Dox has a satisfactory curative effect against leptospirosis. In addition to its antibacterial action, we supposed that Dox also modulated immune response in controlling leptospira infection. Using J774A.1 mouse macrophages, the effects of Dox on protein and mRNA levels of IL-1β and TNF-α were investigated after infection with live or sonicated Leptospira interrogans serovar Lai strain Lai (56601). Specifically, the level of IL-1β but not TNF-α was sharply decreased when treated with Dox in leptospira-infected macrophages. Western blot analysis showed that Dox suppressed the activation of leptospira-induced MAPK and NF-κB signaling pathways. Using NLRP3-deficient and NLRC4-deficient mice, the data showed that the expression of leptospira-induced IL-1β was mainly dependent on the presence of NLRP3 inflammasome in macrophages. Meanwhile, Dox suppressed leptospira-induced NLRP3 inflammasome priming with the upregulation of the Na/K-ATPase Pump β1 subunit. The inhibition effect of Dox on IL-1β was also conspicuous in cells with lipopolysaccharide and ATP stimulation. These results were confirmed in vivo, as peritoneal fluids of mice and organs of hamsters expressed less IL-1β after treatment of leptospiral infection with Dox. Our results indicated that Dox also modulated immune response to attenuate leptospira-induced IL-1β by suppressing p38, JNK, p65, and NLRP3 inflammasome priming.