The Effect Of Extract A From Polygonatum Odoratum On Inflammatory Factor Produced By Mouse Peritoneal Macrophage

ObjectivePolygonatum Odoratum (Mill.) Druce is a Liliaceae Polygonatum Chinese herbal medicine, medicinal roots contain polysaccharides, flavonoids, alkaloids, saponins, sterols, tannins, phlegm,cardiac glycosides and variety of other ingredients. Modern pharmacodynamic studies have shown that Polygonatum have the inhibition of the immune function, the effection of anti-tumor, anticoagulant, anti-aging and the role of reducing blood sugar. Extract A from Polygonatum Odoratum (EA-PAOA)which is an alcohol-water extract has been found that it has the inhibition of cellular immune function and inflammatory responses. Early trials have demonstrated that a certain concentration of EA-PAOA can inhibit the inflammatory cells release IL-1, TNF-a and other proinflammatory cytokines.In view of EA-PAOA has low toxicity and safety features, further studies of its effection and mechanisms in inflammation is necessary.Nitric oxide (NO) involved in the process of inflammation and immune reactions in pathological cases, the synthesis of a large number NO may cause acute and chronic inflammatory reaction. Endogenous NO is generated from oxidation of L-arginine (L-Arg) by nitric oxide synthase (NOS). There are three NOS isoenzymes which are endothelial NOS (eNOS or NOS-Ⅲ), neural-type NOS (nNOS or NOS-Ⅰ) and inducible NOS (iNOS or NOS-Ⅱ). iNOS is only expressed in activated cells which is stimulated. iNOS is mainly expressed in the cytoplasm of white blood cells and other inflammatory cells.The induction of iNOS stimuli is including hemoglobin, LPS, cytokines IFN-γ, TNF-α, IL-1αand so on. Many experiments have confirmed that in case iNOS is activated in vivo, the release of a large number of NO associated with a variety of pathological conditions, including tissue damage and various inflammatory diseases, neurological diseases, autoimmune diseases etc. Therefore, selective inhibition of iNOS and inhibit excessive NO production, will have some prospects on the treatment of these diseases. iNOS inhibitor, inhibited the inflammatory role of information transmission channels, effectively inhibited iNOS not only in the initial stage of inflammation affecting the occurrence of inhibition and also end the role of inflammation.Early experiments have confirmed that EA-PAOA can inhibit LPS-induced macrophages to secrete proinflammatory cytokines (eg IL-1 and TNF-a), to suppress inflammation on certain extent. This study was designed to further confirm this result, wheather EA-PAOA could play an important role on the inflammatory injury in study of the inflammatory mediators NO and further reveal the inner mechanism. The research mainly studied the influence of LPS induced macrophage producing inflammatory mediators-NO, IL-6 and TNF-a by EA-PAOA, simultaneous detected of EA-PAOA on iNOS expression. Consequently further research the effect of EA-PAOA on immune regulation in mice.MethodsFour BALB/c mice (female,6-8 weeks) were used, mouse peritoneal cells were got under sterile conditions. Cultured in 37℃5% CO2 incubator for 1 h,then removed non adherent cells and digested other adherent cells by trypsin-EDTA solution. The cells were counted after being cleaned and centrifuged and adjusted the cell number to 2×106/ml. Trypan blue staining showed cell viability> 99%, while hold on culturing in 37℃5% C02 incubator. Adding the purification cells (2×106/ml) into the 96 well culture plates, the experimental groups were including control group (macrophages+ medium) and the multiple concentration of the drug groups (15.625-1000μg/ml EA-PAOA+macrophages cells+medium), quintuplicate in each group. So that the final volume of each hole was 200μl, the final density of cells was 1×106/ml. The cells were cultured in 37℃5% CO2 incubator for 24h. Macrophage survival was detected by assay of MTT. The same method to add the purified macrophages of density of 2×106/ml to 96 well plate.The experimental groups were including the control group (macrophages+medium), LPS control group (LPS+medium+macrophages)and the multiple concentration of the drug groups (250-1000μg/ml EA-PAOA+LPS+ macrophages+medium).After incubated in 37℃5% CO2 incubator for 6h, the IL-6 and TNF-a levels of supernate were detected by ELISA assay.The same grouping in another 96 well plate,after incubated in 37℃5% CO2 incubator for 24 h, the NO levels of supernate were detected by Griess assay. iNOS expression in the cells were also detected by Western blotting method. The data were presented as mean±standard deviation (X±s). Statistical significant difference was analyzed by Student's t test. A value ofp<0.05 was considered statistically significant.ResultsThe MTT assay results showed in the range of 15.625-1000μg/ml EA-PAOA had no significant effect on the activity of mouse peritoneal macrophage. Detection by ELISA proved dose range in 500-1000μg/ml of EA-PAOA significantly inhibit the LPS-activated mouse peritoneal macrophages secrete IL-6 and TNF-a (p<0.05), while has a dose-dependent. By the Griess assay, confirmed in range of 250-1000μg/ml EA-PAOA can inhibit the activation of mouse peritoneal macrophages release NO (p <0.05), and showed a dose dependent relationship. Detected by Western blotting method,results showed that the 1000μg/ml EA-PAOA can inhibited LPS-induced mouse peritoneal macrophages NO,maybe through declining the iNOS expression to achieve.DiscussionIn this research, we have studied the effect of EA-PAOA on the activation of mouse peritoneal macrophages cell proliferation, the results showed that in the range of 15.625 to 1000μg/ml EA-PAOA had no effect on mouse peritoneal macrophages viability. By detecting the EA-PAOA on the inhibition of actived-mouse peritoneal macrophages secreting proinflammatory cytokine, obtained in the dose range of 500-1000μg/ml EA-PAOA significantly inhibit the IL-6 and TNF-α,while has a dose-dependent.The study of EA-PAOA on mouse peritoneal macrophages confirmed that EA-PAOA in the dose range of 250-1000μg/ml can inhibit the activated-mouse peritoneal macrophages release NO (p<0.05), and showed a dose dependent relationship.Detected by Western blotting method,results showed that the high concentration EA-PAOA can inhibited LPS-induced mouse peritoneal macrophages NO,maybe through declining the iNOS expression to achieve.Macrophages are important antigen presenting cells in immune system, in specific and non-specific immunity played an important role. The immune response of macrophages is the basis of the immunity defense mechanism.When bacterial growth and were killed, a large number of internal toxins released into the body fluids, stimulated the activation of macrophages and released excessive inflammatory mediator such as NO, proinflammatory cytokines TNF-α, IL-6, etc., resulting in uncontrolled cascade of the inflammatory response to tissue damage and inflammation. Activated macrophages produce a large number of NO through the channels of iNOS.iNOS is one of three important enzymes on NO producing. Through the regulation of iNOS to control activation of macrophages on the release of NO, so that regulation of immune function is likely to be a new way to discover an effective anti-inflammatory drugs. By measuring the inhibitory effect on iNOS, low toxicity and safety of new anti-inflammatory drugs probably were screened from Chinese herb medicine.The traditional non-steroidal anti-inflammatory drugs have been reported, including increased cardiovascular risk,have more side effects.Therefore, a new research and development on security drug of inflammation inhibition is imperative. Has been shown that EA-PAOA has lower toxicity (LD50=14.5124 g/kg). Studying the value of EA-PAOA in inflammatory and immune diseases, a scientific theory have provided for treatment of inflammatory diseases on EA-PAOA and the further development and utilization of Polygonatum Odoratum.Conclusions1.The concentration of EA-PAOA in the range of 15.625 to 1000μg/ml had no significant effect on mouse peritoneal macrophage activity.2.EA-PAOA can inhibit LPS activation of murine peritoneal macrophages to release pro-inflammatory cytokines IL-6 and TNF-αand NO, and showed a dose dependent relationship.3.EA-PAOA can inhibit LPS activated mouse peritoneal macrophages secrete NO, achieved through dropping off the iNOS expression.