The Anti-inflammatory Effect Of Viola Yedoensis And Bupleurum Polysaccharides

Viola yedoensis Makino is a small perennial herb with violet flowers distributed in China, Japan and Korea. The dried whole plant (including the roots) is an important Chinese traditional medicine named’ Zi Hua Di Ding’(Herba Violae), which is used as an antifebrile and detoxicant agent for the treatment of acute pyogenic infec-tions such as boils, furuncles and carbuncles. Another herb, Radix Bupleuri (dried roots of Bupleurum chinense or Bupleurum scorzonerifolium), known as ’Chai-Hu’, is one of the most frequently prescribed crude herbs in the prescriptions of traditional Chinese medicine for the treatment of inflammatory diseases, nephrosis syndrome and auto-immune diseases. Bupleurum smithii var. parvifolium is abundantly distributed in the northwest region of China and its roots are also used as Radix Bupleuri.Macrophages are originated in the bone marrow precursor cells and widely distributed in the body. They have tissue specificity, such as liver Kupffer cells, brain microglia, peritoneal macrophages, alveolar macrophages and osteoclast, which are located in different tissues. The phagocytosis of activated macrophages would be enhanced during inflammation, and they would kill pathgens directly through phagocytosis or indirectly by the secretion of inflammatory cytokines, reactive oxygen species, NO and any other mediators against pathogenic microorganisms in order to effectively defense the invasion that would cause inflammation as well as tissue injury. As the first line of host defense system against infections, macrophages play critical roles in the innnate immune system. Macrophages target the structurally conserved pathogen-associated molecule patterns (PAMPs) through the pattern recognition receptors (PRRs) on their surface, thus effectively perceive the invasion of pathogenic microorganisms and induce the immune response.Gut-associated lymphoid tissues (GALT) comprise Peyer’s patches (PPs), mesenteric lymph nodes and Isolated lymphoid tissue (ILF), which play a vital role in immune tolerance. The immune tolerance caused by the activation of intestinal mucosal immunity might be a possible way to treat some autoimmune diseases. Previous experiments in our lab confirmed that (1) PEVY has a therapeutic effect on LPS-induced lung injury;(2) Bps have beneficial effect on autoimmune disease induced by Campylobacter jejuni in BALB/c mice via inhibiting humoral immune hyperfunction and alleviating the activation of complement.Based on our previous studies, in the present research, we observed the anti-complementary activity of PEVY. Also we studied its effects on cell viability and secretory function of murine peritoneal macrophages and RAW264.7cells for further undertanding the anti-inflammatory and immunological mechanism of PEVY. In addition, in this study, we observed the effects of Bps on splenic cells and GALT, and induced systemic lupus erythematosus-like syndrome by Campylobacter jejuni, and then get the splenic cells to observe the effects of Bps in vitro.1. The anti-inflammatory effect of Viola yedoensisAim:To determine the anti-complementary activity of PEVY, and its effects on cell viability and secretory function of murine peritoneal macrophages and RAW264.7cells.Methods:(1) The anti-complementary activity of PEVY:Testing the anti-complementary activity of PEVY by the classical pathway-mediated complement activation. Complement C3c deposition was detected by immunohistochemistry;(2) The effect of PEVY on cell viability and secretory function of murine peritoneal macrophages and RAW264.7cells:Macrophages were randomly divided into5groups: control group, DMSO control group, LPS (1μg/ml) stimulated group, PEVY (1,10,50,100μg/ml) treated groups, LPS combined with PEVY (1,10,50,100μg/ml) treated groups. Macrophages were treated for24h. Cell viability was measured by the MTT assay. And the levels of nitric oxide (NO) and TNF-a were respectively quantified by Griess reaction and enzyme-linked immunosorbent assay (ELISA) to discover the effects of PEVY on the secretory functon of macrophages.Results:(1)PEVY had a significant anti-complementary activity (CH50=61±11μg/mL). It also improved and alleviated the complement depositon in lung of ALI mice in vivo.(2) For peritoneal macrophages, accompanying dose increase, PEVY alone promoted cell viability (P＜0.001); for RAW264.7cells, PEVY alone suppressed cell activity at50,100μg/ml (P＜0.01); After LPS stimulation, PEVY furthermore promoted the viability of peritoneal macrophages (P＜0.01), but suppressed the viability of RAW264.7cells (P<0.001). Besides, PEVY obviously inhibited LPS-stimulated production of NO in both macrophages (P＜0.001). PEVY alone significantly promoted secretion of TNF-a (P<0.001), however, it had no significant effect on LPS-stimulated secretion of TNF-a. Conclusions:(1) In vitro test, we found that PEVY has a significant anti-complementary activity. In addition, PEVY by oral administration significantly alleviated the complement depositon in lung of ALI mice in vivo. This suggested that PEVY has great anti-complementary activity both in vitro and in vivo.(2) For these two types of macrophages, PEVY promoted its level of TNF-a, but decreased the secretion of NO. It suggested its potential anti-tumor activity.2. The anti-inflammatory effect of Bupleurum polysaccharidesAim:To determine the effects of Bps on splenic cells and GALT, and then study the effects of Bps on secretory function of splenic cells of systemic lupus erythematosus-like syndrome mice in vitro.Methods:(1) The effects of Bps on splenic cells and GALT in Balb/c mice:cells were randomly divided into4groups:control group, ConA (5μg/ml)-stimulated group, Bps treated groups, ConA combined with Bps treated groups.Splenic cells, MLN and PPs were treated respectively for48h. Cell viability was measured by the MTT assay. And the levels of IFN-y and IL-10were quantified by ELISA. Meanwhile, we measured the proportion of CD3+and CD4+cells in Balb/c mice by flow cytometry;(2) The effects of Bps on secretory function of splenic cells of systemic lupus erythematosus-like syndrome mice in vitro:systemic lupus erythematosus-like syndrome was induced by CJ-S131. Mice were sacrificed in day51for splenic cells. Cells were randomly divided into4groups:control group, CJ-S131-stimulated group, Bps treated groups, CJ-S131combined with Bps treated groups. Splenic cells were treated for48h. The levels of anti-dsDNA, total IgG, IFN-y, IL-10and IL-17were quantified by ELISA;(3) The effects of Bps on the proportion of lymphocytes of spleen and MLN in SLE-like mice: Mice were randomly divided into four groups:nil group, FCA group, model group and Bps group. Splenic cells and MLN were for flow cytomery detection.Results:(1) Accompanying dose increase, Bps alone promoted the proliferation of splenic cells and MLN (P<0.01); After ConA stimulation, compared with no stimulation, the lymphocyte proliferation significantly increased (P＜0.001), and Bps remarkably suppressed its proliferation (P<0.001), but had no impact on PPs. Bps alone had no significant effect on the secretion of IFN-y in splenic cells and MLN, however, Bps sharply decreased the level of IFN-y after ConA stimulation (P<0.001). For IL-10, Bps alone significantly increased IL-10; With ConA stimulation, Bps markably decreased the level of IL-10.(2) Bps significantly suppressed the high level of anti-dsDNA and total IgG of splenic cells in model group (P＜0.001), and decreased the secretion of IFN-y and IL-10in every group (P<0.001). However, Bps had no significant effect on the high level of IL-17in model group.(3) Compared with nil group, the proportion of T cells in model group showed descending trend, and B cells increased with no significant difference. And Bps has no significant effect on the proportion of lymphocytes of spleen and MLN in SLE-like mice.Conclusions:(1) The anti-inflammatory effect of Bps via oral administration may be not only through the central lymphoid organ spleen, but also by the MLN in intestinal mucosal immune system. And both reaction to Bps are similar. Bps has no significant effect on PPs, which suggests that the study of Bps on intestinal immunity should be focus on scattered lymphocyte in gut.(2) The splenic cells of SLE-like mice were restimulated by CJ-S131, which could simulate the pathological changes, such as high level of anti-dsDNA and total IgG, and SLE related cytokines. Bps significantly decreased the level of anti-dsDNA and total IgG in model group. It also remarkably suppressed the secretion of IFN-y and IL-10, but showed no effect on IL-17.(3) The tharapeatic effect of Bps may not be through the proportion of lymphocyte of spleen, but by affecting lymphocyte functions, such as seretion.