A Study On The Slective Glucocorticoid-like Effects Of Ginsenosides Rg1

BackgroundGlucocorticoid (GC), a steroid hormone secreted by the adrenal gland, regulates many important physiological processes in the body, including growth, metabolism, and immunological reactivity. GC is also one of the most commonly used medicines in treating chronic asthma, rheumatoid arthritis, etc. However, their use is limited by the unavoidable side effects, such as osteoporosis, diabetes, redistribution of fat and sodium retention, etc. The physiological and pharmacologic effects of GC are mainly mediated by glucocorticoid receptor (GR), a ligand dependent transcription factor. Binding of GC to GR activates GR. Activated GR translocates to nucleus as a dimer, and then regulates gene transcription directly or indirectly. The transrepression is generally considered the key mechanism for the anti-inflammatory activity of GC. In contrast, several side effects are thought to be predominantly mediated via transactivation. Thus, ligands that preferentially induce the transrepression and not transactivation function of GR and could be as effective as standard GC but with fewer undesirable effects would be more valuable for the clinical use of GC.It has been demonstrated that many Chinese herbs, herbs'monomers and herbs compounds may exert anti-inflammatory effects. Many herbs' extraction also has a similar structure to steroid hormones. Ginsenosides (GSS) which have a steroid-like structure and possess anti-inflammatory, anti-stress and modulating immunology effects are the main activity components of ginseng. In our previous clinical studies, GSS may enhance the effectiveness of prednisolone and attenuate its side effects, such as Cushing's syndrome, Shingles and Bacterial infection, etc. Furthermore, GSS combined with dexamethasone (Dex) could prevent and cure the injury of liver and kidney after transcatheter arterial chemoembolization (TACE) effectively. Previous studies have reported that Ginsenosides Rgl (G-Rgl) could transactivate the activity of GRE-luciferase reporter gene as a GR functional ligand. Therefore, we supposed that G-Rgl may exert GC-like anti-inflammatory effect but with little side effects.ObjectiveTo observe the anti-inflammatory effects in vitro and in vivo and the effects on glucose and bone metabolism of G-Rgl; to investigate the molecular mechanisms of G-Rg1 effects; to study whether G-Rgl may repress NF-κB through GR then participate anti-inflammation and induce little GC-like side effects meanwhile.Methods(1) Anti-inflammatory effect and side effects of G-Rgl in vitro. Eight hours after stimulation of Raw264.7 cells with lipopolysaccharide (LPS), supernatant was collected. Tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) were determined with enzyme-linked immunosorbent assay (ELISA). The effect of G-Rgl on mouse primary osteoblasts proliferation was examined by MTT method.(2) Anti-inflammatory effect and side effects of G-Rg1 in vivo. The anti-inflammatory effect of G-Rg1 in vivo was examined in two mouse models, zymosan-induced inflamed paw model and bovine type II collagen-induced arthritis (CIA) mouse model. Glucose level was determined 6 h after administration of G-Rgl. Body, spleen and thymus weight and bone metabolism were determined following a four-week mouse administration.(3) The impacts of G-Rgl on the nuclear translocation and phosphorylation of GR, NF-κB and MAPK pathway were investigated by western blot to study the mechanisms of G-Rg1 actions.Results(1) Anti-inflammatory effect and side effects of G-Rg1 in vitroG-Rgl may significantly inhibit the secretion of TNF-a and IL-6 in a concentration-dependent manner. Its anti-inflammatory effect was less than Dex at the same concentration. Dex exhibited a proliferation inhibition effect on mouse primary osteoblasts cells in a concentration-dependent manner, but G-Rg1 had little inhibition effect on mouse primary osteoblasts cells at indicated concentrations (10-9-10-5 M).(2) Anti-inflammatory effect and side effects of G-Rg1 in vivoBoth G-Rg1 and Dex could exert anti-inflammatory effects in zymosan-induced inflamed paw model (P<0.05 or P<0.01, vs vehicle group), but no significant difference was found between the two groups (P>0.05). In CIA mouse model, after 2-week treatment with G-Rgl or Dex, the symptom score was significantly less than that of vehicle group (P<0.01) and there was no significant difference between G-Rg1 and Dex groups (P>0.05). Six hours after intraperitoneal injection of Dex, mouse blood glucose level significantly increased compared with vehicle; however, there was no significant difference between G-Rgl and vehicle groups (P>0.05). after 4 weeks treatment of Dex, mouse body weight notablely decreased (P<0.01), but G-Rg1 treatment did not decrease the body weight compared with vehicle (P>0.05). The spleen weight in Dex treatment group was light than that of G-Rg1 and vehicle groups; no notable difference was detected between the G-Rgl and vehicle groups(P>0.05). The thymus weight of Dex treated mice was also much light than that treated with G-Rgl or vehicle (P<0.01); G-Rg1 may not affect the thymus weight (P>0.05). To eliminate the affect of body weight changes on spleen and thymus weight, we also calculated the spleen and thymus index, consistent with spleen and thymus weight changes, the spleen and thymus index of Dex treated mice were also lower than of G-Rgl and vehicle treated mice (P<0.01); there was no significant difference of the thymus index between G-Rgl and vehicle groups (P>0.05), but significant difference was found of spleen index between the two groups (P<0.05). There was no significant difference of mouse bone trabeculae, cortex and total density between the three groups (P>0.05); however, the cortex thickness of Dex group was thinner than that of G-Rgl and vehicle groups (P<0.05), and the bone area was also less that another two groups (P<0.01; P<0.05). The bone mineral content was decreased by four-week injection of Dex (P<0.01), but G-Rg1 didn't decrease that compared with vehicle. There was also a significant difference between Dex and G-Rgl groups (P<0.05).(3) Mechanisms of G-Rgl effectsOne hour after the treatment, both G-Rgl and Dex could induce nuclear translocation of GR. G-Rgl could not induce the phosphorylation of GR Ser-211 site, but Dex may induce the phosphorylation of GR Ser-211 site significantly. LPS stimulation could increase the level of p65 protein in nucleus, but G-Rgl pretreatment may attenuate it. IκB was degraded by LPS and was completely degraded after 30 min of LPS treatment. After 60 min of LPS treatment, new synthetical IκB can be detected again. Two hours pretreatment of G-Rgl could markedly increase the stability of IκB, attenuate its degradation. Fifteen minutes after LPS stimulation, phosphorylation of JNK, p38, ERK was increased significantly; pretreatment with G-Rgl may inhibit LPS induced-phosphorylation of ERK, JNK and p38, indicating that inhibiting the phosphorylation of MAPK pathway, then inhibiting inflammatory cytokines secretion is one of the mechanisms of anti-inflammatory effects of G-Rgl. Conclusion(1) G-Rgl may exert anti-inflammatory effect in vitro and in vivo;(2) G-Rgl may not induce GC-like side effects on the glucose metabolism, body and lymphoid organs weight and bone metabolism;(3) G-Rgl may exert anti-inflammatory effect through inhibiting MAPK phosphorylation and NF-κB activation; the mechanism that G-Rgl may not induce GC-like side effects maybe related with non-phosphorylation of GR at Ser 211 site.(4) G-Rgl is a potential anti-inflammatory medicine with better therapy index (anti-inflammatory effect/side effects).