Elevated systemic levels of pro-inflammatory cytokines cause hypotension during septic shock and induce capillary leakage in acute lung injury. Manassantin B has anti-inflammatory and anti-plasmoidal properties. This study examined the effects of manassantin B on lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages.
RAW 264.7 macrophage cells were incubated without or with (1, 3 and 10 µM) manassantin B and without or with (100 ng/ml) LPS. Manassantin B dissolved in phosphate buffered saline was added to the medium 1 h prior to the addition of LPS. The degree of activation of mitogen-activated protein kinase (MAPK) including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino terminal kinases (JNK) and p38 MAPK, and the level of interleukin (IL)-1β were determined 30 min and 24 h after the addition of LPS respectively.
Manassantin B inhibited the production of IL-1β and attenuated the phosphorylations of ERK1/2 and p38 MAPK, but not that of JNK, in RAW 264.7 cells treated with LPS.
Manassantin B reduces LPS-induced IL-1β expression through effects on ERK1/2- and p38 MAPK-mediated pathways. Manassantin B has potential as a potent anti-inflammatory drug for use in pathological processes such as sepsis or acute lung injury.
Despite newly developed resuscitation therapies and the use of modern antibiotics, sepsis is still a leading cause of death in critically ill patients [
IL-1β is produced from the cleavage of the inactive pro-IL-1β by stimulated leukocytes and is a fundamental contributor to local and systemic inflammatory responses [
Nontoxic molecules that modulate macrophage-mediated inflammatory responses may provide a novel therapeutic strategy for the treatment of sepsis. Numerous studies have been performed to identify the new molecules. The manassantin group of structurally related and functionally unique dineolignans were recently isolated from active root extracts of Saururus cernuus L (Saururaceae), also referred to as "lizard tail" [
The purpose of this study was to investigate the effect of manassantin B on LPS-induced activation of the pro-inflammatory cytokine IL-1β and intracellular signaling pathway such as the MAPKs signaling system, and the possibility that manassantin B might be a promising agent for reducing inflammatory response.
Manassantin B was donated from the College of Pharmacy, Yeungnam University, Korea.
The RAW 264.7 murine macrophage cell line was purchased from the Korea Cell Line Bank (Seoul, Korea). The cells were grown in DMEM supplemented with heat-inactivated 10% FBS, penicillin (100 U/ml) and streptomycin sulfate (100 µg/ml) in a 5% CO2 incubator at 37℃. The cells were plated at a density of 2 × 106 cells/well in a 6-well plate or 5 × 105 cells/well in 24-well plates. For all experiments, the cells were incubated until they reached 80-90% confluence. The adherent macrophages were washed gently with Ca2+ and Mg2+-free Dulbecco's phosphate buffered saline (DPBS) and cultured in serum-free DMEM for 2 h. Then, the cells were incubated in the absence or presence (1, 3 and 10 µM) manassantin B without or with LPS (100 ng/ml). Manassantin B dissolved in phosphate-buffered saline (PBS) was added to the medium 1 h prior to the addition of LPS. The following groups were then isolated in separate wells: control (manassantin B, 0 µM), manassantin B (1, 3 and 10 µM), LPS (100 ng/ml) and LPS+ manassantin B (1, 3 and 10 µM). The degree of activation of ERK1/2, JNK and p38 MAPK were determined at 30 min and the level of IL-1β at 24 h after the addition of LPS, respectively.
To evaluate the effect of manassantin B on the viability of RAW 264.7 macrophages, the cells were treated with various extract concentrations for 24 h, and then cell viability was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Twenty-four h prior to culture termination, 10 µl of the MTT solution (5 mg/ml in PBS, pH 7.4) was added to each well, and the cells were continuously cultured for 4 h. Then 100 µl of dimethyl sulfoxide (DMSO) was added for solubilization. Absorbance was measured at 570 nm using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). Each assay was repeated at least three times.
Immunoreactive IL-1β were quantified using commercially available ELISA kits (R & D Systems, Minneapolis, MN, USA), according to manufacturer's instructions as described previously [
Western blots to detect levels of phosphorylated and total ERK, JNK and p38 MAPK were performed essentially as previously described [
Data are expressed as mean ± SD. A Kruskal-Wallis H test was used for group comparisons. A P value < 0.05 was considered significant.
The detrimental effects of any compound or plant extract on the cell metabolism need to be determined before the biological activity can be examined. Cell viability was not significantly changed by the presence of manassantin B up to 10 µM (
Manassantin B alone did not affect the production of IL-1β in RAW 264.7 cells over the range of concentrations examined. However, it attenuated an increase in IL-1β levels induced by LPS in a dose-dependent manner (
The effects of manassantin B on the phosphorylation of ERK1/2, JNK and p38 MAPK were examined to determine its effect on MAPKs in LPS-stimulated RAW 264.7 cells. In a preliminary study, the maximum MAPKs phosphorylation occurred 30 min after the LPS (100 ng/ml) treatment in RAW 264.7 cells (data not shown). Therefore, the cells were incubated with manassantin B (1, 3, 10 uM) for 1 h and then with LPS (100 ng/ml) for 30 min. Manassantin B attenuated the phosphorylations of ERK1/2 and p38 MAPK in LPS-stimulated RAW 264.7 cells, but had no effect on JNK (
In this study, we investigated the effects of manassantin B, an active root extract of Saururus cernuus L, on proinflammatory cytokine production and activation of MAPKs in murine macrophage RAW 264.7 cells stimulated with LPS. Manassantin B attenuated the LPS-induced increase of inflammatory cytokine IL-1β and phosphorylations of signaling molecules ERK 1/2 and p38 MAPK. These findings suggest that manassantin B has an anti-inflammatory property.
RAW 264.7 murine macrophages are a popular macrophage cell model to examine a variety of inflammatory processes [
The MAPK signaling pathways consist of a series of kinases that are activated sequentially and consequently phosphorylate the downstream kinases and transduce extracellular stimuli into intracellular responses. The MAPK family includes ERK, JNK and p38 MAPK. One of the major functions of MAPK is the activation of transcription factors, several of which bind to the promoters of pro-inflammatory cytokines [
There are some limitations to this study. First, the study is limited in its in vitro character of the data, therefore much remains to be done in vivo studies in terms of safety and optimal dosage. Second, we measured only IL-1β as a pro-inflammatory cytokine. Potential interactions with other cytokines related with inflammation must be considered. Third, we measured the effects of manassantin B on the phosphorylation of MAPK without inhibitors due to the generalized results of inhibitor study.
In summary, manassantin B inhibits the production of the proinflammatory cytokine IL-1β and the phosphorylations of ERK 1/2 and p38 MAPK in LPS-stimulated RAW 264.7 murine macrophages. These findings suggest that manassantin B reduces LPS-induced IL-1β expression through effects on the ERK1/2- and p38 MAPK-mediated pathways in vitro. Thusm manassantin B might be a valuable therapeutic agent in reducing inflammatory response due to systemic inflammatory response syndrome involving sepsis in the clinical setting.
This study was supported by a grant (CRI09005-1) Chonnam National University Hospital Research Institute of Clinical Medicine.
Effect of manassantin B on the viability of RAW 264.7 macrophages. The cells were treated with various extract concentrations for 24 h, and the cell viability was measured using a methylthiazolyldiphenyl-tetrazolium bromide assay. The data are reported as mean ± SD (n = 4).
Effects of manassantin B on LPS-induced ERK1/2, JNK and p38 MAPK phosphorylation in RAW 264.7 cells. The cells were incubated with media only (CON) or the indicated concentrations of manassantin B for 1 h and then incubated with LPS (100 ng/ml) for 30 min. P: phosphorylated, T: total.
The Effects of Manassantin B on LPS Induced IL-1β Production and MAPKs Phosphorylation in RAW 264.7 Cells
Each score represents mean ± SD. Control: The cells were incubated with manassantin B (0, 1, 3 and 10 µM) only for IL-1β and medium only for MAPKs. P/T: relative increase of phosphorylated- to total-MAPKs. *P < 0.05 versus control, †P < 0.05 versus LPS 100 ng/ml + manassantin B 0 uM, ‡P < 0.05 versus LPS 100 ng/ml + manassantin B 1 uM.