Inhibitory Effects Of Soyasaponin Ba On Palmitic Acid-Induced Inflammation In Macrophages And Its Mechanism

BackgroundChronic non-communicable diseases (NCDs) are the leading causes for global death, which seriously threaten human health and social development. NCDs relate to lifestyle, behavior, occupation and environment. Currently the clinical treatment on NCDs is far from ideal. Fortunately, NCDs, to a large extent, are preventable. Inflammation is a key hazardous factor in the development and progression of NCDs. Nutrition epidemiological studies have shown that plant food-derived phytochemicals exhibit various physiological bioactivities such as anti-inflammation, anti-oxidant, anticancer etc. Phytochemicals have immense potential in the prevention and treatment of NCDs.Soyasaponins are major phytochemicals found in soybeans and soy products with the oleanane-type triterpenoid structure. In recent years, much attention has been paid to the anti-inflammatory activity and mechanism of soysaponins. It has been shown that crude extracts of soyasaponins and soyasaponin I (SS-I) could reduce the lipopolysaccharide (LPS)-stimulated expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) and production of nitric oxide (NO) and prostaglandin 2 (PGE2) by inhibiting nuclear factor kappa B (NF-κB) in RAW264.7 murine macrophages. By using primary murine peritoneal macrophages as inflammatory model, SSA1 has also been found to reduce the production of pro-inflammatory factors via inhibiting NF-κB activities. SSA1 and SS-I also alleviated the trinitrobenzene sulfonic acid-induced chronic colitis through suppressing NF-κB signaling pathway in mice. We previously screened the anti-inflammatory activities of five different structures of soyasaponins (SSA1、SSA2、 SS-I、SGA and SGB) by using in vitro RAW264.7 macrophage inflammatory model and found three of them (SSA1、SSA2 and SS-I) inhibited inflammatory enzymes and proinflammatory cytokines via blocking NF-κB activity in a dose-dependent manner. All these results show that soyasaponins may have anti-inflammatory bioactivities through regulating NF-κB signaling pathway. Currently, the anti-inflammatory activities and mechanisms of soyasaponins are still under tremendous investigation.ObjectivesPhytochemicals may inhibit inflammation (especially chronic inflammation) by targeting inflammatory signaling pathways. Soysaponins have huge potentials in the prevention of NCDs because of their multiple bioactivities such as anti-inflammation. SSBa is a newly extracted monomer from group B soyasaponins. Nothing is known about its anti-inflammatory activity and underlying mechanism. Therefore, in this study we first established palmitic acid (PA)-induced macrophage inflammatory model by refering to the classic lipopolysacchride (LPS)-stimulated macrophage inflammatory model. We then investigated the effect of SSBa on inflammatory enzymes, pro-inflammatory cytokines, and NF-κB inflammatory signaling pathways by comprehensively applying techniques such as western blot (WB), enzyme-linked immune sorbent assay (ELISA), fluorescence quantitative real time polymerase chain reaction (FQ-PCR), etc. We aimed to understand the anti-inflammatory activity of SSBa clarify the underlying mechanism. This study potentiall provided new revenues to prevent inflammation-mediated NCDs by using soyasaponins.Methods1. Cell proliferative toxicity by MTT assayRAW264.7 cells were treated with 25～500μmol/L of PA for different times (6, 12,24,48 or 72 h). Cell culture supernatants were discarded. MTT solution was then added to each well and incubated for further 4 h. Supernatants were discarded and DMSO was added. The absorbance at 490 nm was determined by using microplate reader. Experiment was repeated three times. The half inhibitory concentration (IC50) of cell proliferation was calculated by using PROBIT model of SPSS statistical software.2. Western BlotCell culture and treatments:(1) Establishment of LPS-stimulated cell inflammatory model:cells were treated with 0.2～2μg/mL LPS for 6 h or with 0.1～1 μg/mL LPS for 24 h. (2) Establishment of PA-stimulated cell inflammatory model: cells were treated with 50～250μmol/L PA for 6 h or 24 h. (3) SSBa treatment in PA-stimulated cell inflammatory model:cells were pretreated with 20～200μmol/L of SSBa for 2 h and then stimulated with 250μmol/L PA for 6 h or 24 h. (4) Detection of NF-κB signal molecules (p65/p-p65 and IκBa/p-IκBa):cells were treated with 50～500μmol/L PA, or preteated with 20～200μmol/L SSBa for 2 h followed by stimulation with 250μmol/L PA for 30 min. Cells were collected and lyzed. Total proteins were extracted and its concentrations were determined. All proteins were diluted with PBS and 5×SDS loading buffer, denaturated by heating, and then loaded for SDS-PAGE, transmembrane, antibody incubation and development. The protein bands were analyzed and quantified (molecular weight and net optical density value) by using Gel Imaging System.3. FQ-PCRCell culture and treatments:(1) Establishment of LPS-stimulated cell inflammatory model:cells were treated with 0.2-2μg/mL LPS for 6 h or with 0.1～1 μg/mL LPS for 24 h. (2) Establishment of PA-stimulated cell inflammatory model: cells were treated with 50～250μmol/L PA for 6 h or 24 h. (3) SSBa treatment in PA-stimulated cell inflammatory model:cells were pretreated with 20～200μmol/L of SSBa for 2 h and then stimulated with 250μmol/L PA for 6 h or 24 h. Upon treatment, total RNA was extracted by using Trizol. RNA concentration and purity were then determined. Total RNA was reversely transcripted into cDNA. The concentration and purity of cDNA were then determined. The genes of COX-2, TNF-a, IL-1β, IL-6, iNOS and β-actin were amplified by FQ-PCR. The gene expression differences between treatment group and control group were calculated by using relative quantitative method (△△Ct).4. Determination of TNF-a by ELISACells were treated with 0.2-2μg/mL LPS for 6 h, or 50～250μmol/L PA for 6 h, or pretreated with 20～200μmol/L SSBa for 1 h followed by stimulation with 250μmol/L PA for 6 h. Upon treatment, culture medium were collected, centrifuged and determined by using TNF-a ELISA kit with the recommended method.5. Trypan blue dyeing countCells were treated with 20～200μmol/L SSBa for 6 h or 24 h. Trypan blue solution was mixed with cell suspension with the ratio 1:1 on slide. The dead cells and total cells were counted under microscope. The living cells rate and relative proliferative inhibition rate were calculated. the relative inhibition rate. The half inhibitory concentration (IC50) of cell proliferation was calculated by using PROBIT model of SPSS statistical software.6. Statistical AnalysisAll results were presented in mean±standard deviation (X±SD). Comparison between multiple samples means were analyzed by using One-way ANOVA of SPSS 19.0 software. LSD comparison was used when variance was homogeneous and Dunnett’s T3 comparison was used when variance was heterogeneous. The half inhibitory concentration (IC50) of cell proliferation was calculated by using PROBIT model of SPSS statistical software.Results1. Effect of PA on proliferative activities of RAW264.7 murine macrophagesPA inhibited the proliferation of RAW264.7 macrophages in a dose-and time dependent manner. The IC50 of RAW264.7 cells treated with PA for 6,12,24,48,72 h was 610.51,433.42,217.11,167.29 and 132.20μmol/L, respectively. After taking into account the results from WB,50～250μmol/L were chosen as the PA treatment concentration and 6 h and 24 h were chosen as the PA treatment time.2. Effect of SSBa on proliferative activities of RAW264.7 macrophagesSSBa inhibited the proliferation of RAW264.7 murine macrophages in a dose-and time-dependent manner. The IC50 of macrophages incubated with SSBa for 6 h and 24 h were 4139.03μmol/L and 4020.76μmol/L, respectively. The incubation concentration for SSBa was finally setted as 20,50,100 and 200μmol/L. The incubation time was setted as 1 h and 2 h.3. Establishment of LPS-induced RAW264.7 macrophages inflammatory modelThe mRNA and protein levels of inflammatory enzymes (COX-2 and iNOS) and the mRNA levels of proinflammatory cytokiens (TNF-a、IL-1β and IL-6) were increased (P<0.05) in a dose-dependent manner in RAW264.7 macrophages stimulated by LPS (0-2μg/mL) for 6 h or 24 h. The secretion of TNF-a in cell culture medium was increased after LPS stimulation for 6 h (P<0.01).4. Establishment of PA-induced RAW264.7 macrophages inflammatory modelThe mRNA and protein levels of inflammatory enzymes (COX-2 and iNOS) and the mRNA levels of proinflammatory cytokiens (TNF-a、IL-1β and IL-6) were increased (P<0.05) in a dose-dependent manner in RAW264.7 macrophages stimulated by PA (50～250μmol/L) for 6 h or 24 h. The secretion of TNF-a in cell culture medium was increased in a concentration-dependent way after LPS stimulation for 6 h (P<0.01).5. Effect of SSBa on PA-stimulated inflammation in RAW264.7 macrophagesSSBa pretreatment significantly inhibited the PA-induced increase of protein and mRNA levels of inflammatory enzymes (COX-2 and iNOS) (P<0.01) and mRNA levels of proinflammatory cytokines (TNF-a, IL-1β and IL-6) (P<0.01). SSBa pretreatment also significantly inhibited the PA-stimulated secretion of TNF-ain cell culture medium (P<0.01).6. Effect of SSBa on NF-κB inflammatory signaling pathways in PA-stimulated RAW264.7 macrophagesIn PA (250μmol/L)-stimulated cells, the phosphorylation of p65 (p-p65) started to increase at 5 min and reached the maximal expression level at 30 min. Total p65 levels were unchanged (P>0.05) and p-p65 levels were increased (P<0.01) in a dose-dependent manner in cells stimulated by different concentrations of PA for 30 min. In cells pretreated with SSBa for 1 h following by PA stimulation for 30 min, total p65 levels were unchanged (.P>0.05). The 100 and 200μmol/L of SSBa significantly suppressed PA-induced increase of p-p65 (P< 0.05). PA inhibited IκBa levels (P<0.01), whereas 20～100μmol/L of SSBa blocked PA-induced decrease of IκBα(P<0.01). PA increased p-IκBa levels, whereas 20～200μmol/L of SSBa inhibited PA-induced suppression of p-I「Ba levels (P<0.01).Conclusion1. Using inflammatory enzymes (iNOS and COX-2) and proinflammatory cytokines (TNF-a, IL-1β and IL-6) as indicators for characterizing inflammation, in vitro inflammatory model can be established by stimulation of PA in RAW264.7 murine macrophages similarly as the stimulation of classic stimulator LPS.2. In PA-stimulated RAW264.7 murine macrophage inflammatory model, SSBa can inhibit the mRNA and protein levels of inflammatory enzymes (COX-2 and iNOS), mRNA levels of (TNF-a, IL-1β and IL-6), and secretion of TNF-a.3. In PA-stimulated RAW264.7 murine macrophage inflammatory model, SSBa can inhibit the phosphorylation and degradation of IκBa and the phosphorylation of NF-κB p65.