| Ginsenosides, as one of the major biologically ingredients in ginseng (Panax ginseng C.A.Meyer), are found to have potent immunemodulation. Rgl and Re are two major ginsenosides in the root of ginseng, and they are both categorized into protopanaxatriol. Rgl and Re both can act as adjuvant to promote host immune responses to antigen. Toll-like receptor-4 (TLR4) is an important receptor in the immune modulation, and also is a major receptor for endotoxin (LPS) to induce acute inflammatory responses. As a component in the wall of gram-negative bacteria, LPS could be released to the host after the death of bacteria, and subsequently activate TLR4 signaling pathway, over express pro-inflammatory mediators, cause acute sepsis and damage to organs, even lead to septic shock and death. In the present study, we investigated the involvement of TLR4 in the adjuvant effect of Rgl and Re, and tested the inhibitory effect of Rgl and Re on LPS binding to TLR4 and inducing expression of pro-inflammatory mediators, and also evaluated the potential of Rgl and Re in the protective effect against endotoxin and E.coli infection.1. Ginsenoside Rgl and Re act as adjuvant via TL4 signaling pathwayObjective To investigate whether TLR4 is involved in the adjuvant effect of Rgl and Re. Methods 1) Either C3H/HeB (TLR4+/+) or C3H/HeJ (TLR4-/-) mice were subcutaneously immunized with OVA (10μg) plus Rgl or Re (50μg). Antigen-specific immune responses were detected; 2) RAW-Blue cells were stimulated by LPS after TLR4 receptor being blocked with TLR4 neutralizing antibody. The supernatants were collected for secreted embryonic alkaline phosphatase (SEAP) secretion assay, which indirectly indicate the activation of nuclear transcription factor-κB (NF-κB); 3) Primary mouse macrophages harvested from the peritoneal cavity of C3H/HeB or C3H/HeJ mice were treated with Rgl respectively. Phosphorylation of NF-κB was detected by Western blot.4) We used docking to evaluate the possibility of interation between Rgl/Re and TLR4-MD-2 molecule. Results 1) Both Rg1 and Re could promote anti-OVA specific IgG antibody level and proliferation responses of splenocytes, and mRNA expression of interleukin-12, interferon-y, interleukin-4 and IL-10 cytokines in wide-type mice (C3H/HeB), but not in TLR4 mutant mice (C3H/HeJ); 2) Rg1 and Re could activate NF-κB in mouse macrophages. But after blocking TLR4 on the membrane by a neutralizing antibody, the activation of NF-κB was inhibited in Re, but not Rg1; 3) Rg1 activated the phosphorylation of NF-κB immediately after stimulation in the macrophages from wide-type mice, while Rgl failed to induce the phosphorylation of NF-κB in TLR4 mutant mice; 4) Docking results suggested that Rg1 and Re could mimic LPS to bind to TLR4-MD-2 and act as a "bridge"between TLR4 and MD-2.2. Rgl and Re down-regulate LPS singaling pathwayObjective To find whether Rgl and Re would down-regulate the activity of LPS in the TLR4 signaling pathway. Methods 1) RAW264.7 cells were incubated with Rg1 or Re at 50μg/ml and the cells were then performed with the immunofluorenscence procedure to see the distribution of them in cells.2) RAW264.7 cells were incubated with Rgl or Re at 10,30,50 or 70μg/ml and then stimulated with Alexa Fluor 594-LPS at 10μg/ml. After removing the supernatant, cells were performed with the immunofluorenscence procedure.3) RAW-BlueTM cells were cultured with Rgl or Re at 10,30,50 or 70μg/ml for 1h prior to or after LPS stimulation. After 20 h, supernatants were collected for SEAP.4) RAW264.7 cells were cultured with Rg1 or Re at 50μg/ml for 1 h prior to LPS stimulation, after 20 h, supernatants were collected for pro-inflammatory cytokines and mediators determination. Results 1) Rg1 distributed both extracellularly and intracellularly but Re only located extracellularly.2) Pretreatment of mouse macrophages with Rgl and Re prior to LPS, could strongly down-regulate LPS-mediated signaling, including LPS binding to TLR4, LPS-induced NF-κB activation and expression of multiple pro-inflammatory mediators. However, no inhibition was found in post-treatment.3. Protective effect of ginsenosides Rgl and Re on LPS-induced sepsis in animalsObjective To find whether Rgl and Re could protect animals from LPS-induced sepsis. Methods 1) SD rats were randomly divided into 5 groups with 6 in each. The rats were intravenously administrated saline,1 mg/kg Rg1,1 mg/kg Re or 1 mg/kg TAK-242.15 minutes later, rats were challenged with LPS. Body temperature was measured and blood samples for white blood cell (WBC) countsand serum pro-inflammatory mediators determination were collected at indicated time points.2) Rgl and Re were tested in separated experiments for their preventive effect on LPS-induced lethality. BALB/c mice were randomly divided into 7 groups with 10 mice in each. The mice were first subcutaneously injected with Rgl/Re (0,2.5,5,10 or 20 mg/kg) or TAK-242 (5 mg/kg) 3 times with 30 min intervals.15 minutes later, the mice except for the control were intraperitoneally challenged with LPS. Observe the behavior and survival rate of mice; 3) BALB/c mice were divided into 5 groups with 10 mice in each. The mice except for the control were intraperitoneally injected with LPS.15 minutes later, mice were subcutaneously injected with Rgl (10 mg/kg) or Re (20 mg/kg) or TAK-242 (5 mg/kg) or saline (control) for 3 times with 30 min intervals. Survival was recorded within 60 h.4) 45 BALB/c mice were randomly divided into 5 groups with 9 each. The mice in the first 4 groups were subcutaneously injected with 10 mg/kg Rgl,20 mg/kg Re,5 mg/kg TAK-242 for 3 times with 30 min intervals or left untreated, and then challenged with 20 mg/kg LPS 15 min later. The mice in the fifth group of mice were injected with LPS first and then subcutaneously administered with 10 mg/kg Rgl for 3 times with 30 min intervals. Three mice in each group were euthanized at 4,8, or 12h post LPS administration for blood collection. White blood cell counts and serum pro-inflammatory mediators production were analysed. Results Pretreatment with Rgl and Re effectively attenuated LPS-induced fever, increasement of white blood cell population and high production of various pro-inflammatory cytokines (interleukin-1β, tumor necrosis factor-α and interleukin-6) and other mediators (inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide and prostaglandin E2) in rats; 2) In the mouse endotoxin shock model, pre-treatment with Rg1 and Re for three times significantly attenuated LPS-induced decline in the population of WBC expecially neutrophils and excessive expression of multiple pro-inflammatory factors (interleukin-1β, tumor necrosis factor-α and interleukin-6, chemokine KC, macrophage inflammatory protein-2, prostaglandin E2 and nitric oxide), and effectively increased the survival rate of mice. Administration with Rgl post infection still effectively inhibited LPS-induced inflammatory responses, and protected 90% mice from death, while no protection was found in Re post-treatment.Ginsenoside Rgl in combination with polymyxin B attenuates an acute sepsis induced by Escherichia coli in rabbitsObjective To evaluate the protective effect of Rgl combined with a low dose of polymyxin B against an acute sepsis induced by Escherichia coli (E.coli). Methods Rabbits were randomly divided into 4 groups with 5 rabbits in each. One day prior to the experiment, basal body temperature was recorded. All the rabbits were subcutaneously inoculated with E. coli suspension at a dose of 1×109 CFU/kg. Immediately after the inoculation, animals were intravenously (left ear vein) injected with saline, Rgl (12 mg/kg), PMB (1 mg/kg) or Rgl (12 mg/kg) plus PMB (1 mg/kg), respectively. Body temperature was measured every hour within 12 hours, at 24 and 48 hours post inoculation. Blood samples were harvested from the right ear vein in pyrogen-free vacuum tubes with or without EDTA for white blood cell counts and analysis of bacteriology, endotoxin and cytokines. Results The combination treatment effectively reduced the bacterial burden and LPS in blood and attenuated LPS-induced fever, changes in the population of white blood cell counts including neutrophils and lymphocytes and high production of serum pro-inflammatory mediators (interleukin-1β, tumor necrosis factor-α and interleukin-6). PMB in a low dose alone only displayed short-term effect on inflammatory responses, no significant inhibitory effect was observed after 8 hours post infection. Single Rgl treatment had little therapeutic effect against E.Coli infection.Taken together, these results indicated that Rgl and Re could act as adjuvant through TLR4 signaling pathway; Rg1 and Re competed with LPS to inhibit LPS binding to TLR4; Rgl effectively prevented endotoxin-induced sepsis in rats and mice; Combined administration with Rgl and a safe dose of PMB could be a promising treatment against E.Coli infection. Thus, further studies are needed to evaluate the protective effect of ginsenoside Rgl as a potential anti-microbial agent against gram-negative bacteria. |
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