Ginsenoside Rhl Combined With Dexamethasone Attenuates Inflammation Without Provoking Resistance

BackGRound：Glucocorticoids (GCs) are widely used in treating inflammatoryand immune related diseases. However, although the first impressions were positive, itsoon became clear that the frequent and prolonged administration of GCs influence adiverse set of key biological functions and cause severe, sometimes irreversible,side-effects. The physiological and pharmacologic effects of GC are mainly mediated byglucocorticoid receptors (GRs). It is reported that the quantity and binding capacity of GRsare important in determinanting the curative effects of GCs. Even if the level of GCs arehigh in vivo, the effects of GCs are still poor for few quantities and binding of GR, whichcould binding with ligand are very small. In our prior studies, the GRs expression andbinding capacity in peripheral blood mononuclear cells (PBMC) from systemic lupuserythematosus patients with GRs resistance are lower than those in GCs sensitive patiens.For good curative effects, we often treat the patients with high-dose GCs, or combinedwith immunosuppressive agents, whih give rise to various of side effects. Moreover,high-dose GCs used in a long time is account for further reduced expresson and bindingcapacity of GRs, which will make the subsequent therapy more difficult. As a result, a newmedicine which can regulate GCs effectively is still the hot ports.In recent years, Chinese herbs exert an increasingly effect in clinical treatment. Inour previous research, we found Ginsenosides (GSS) could induce a homologousupregulation of GRs and enhance the effects of GCs. In the subsequent clinical studies, it isrevealed that GSS could enhance the effects of GCs in treating systemic lupuserythematosus patients, and reduce the dose of GCs without reduced the clinical effects.The GSS includes about40Ginsenosides,such as Rb1、Rb2、Rb3、Rc、Rd、Re、Ro、Rf、Rg1、Rg2、Rg3、Rh1、20(S)-Rh2、20(R)-Rh2、F1、F2,ect.Because of its complicatedcompositions,GSS can not be used in clinical treatment widely,and it is difficult to clarifyits possible mechanism in clinic.OBJECTIVE:1.Pick out one or several Ginsenoside(s),which can upregulate thelevel of the expression and binding capacity of GRs. Then find out the most effectivecomponent on GRs from them.2.Study the selected-Ginsenoside’s effects on inflammationmodels on GCs resistance in vivo and in vitro, and exlpore its possible mechanism.METHODS:1.We first pick out the Ginsenoside(s) which can upregulate the GRsprotein levels by Western-Blot and quantity RT-PCR methods. Radioligand binding analysis is used for GR binding capacity.2.Observe the anti-inflammatory effects of Dex combined with the selected-Ginsenoside in vitro and vivo and explore its possible mechanism.（1）We compared the therapeutic potential of Dex combined with the selected-Ginsenoside&Dex alone treatment protocols in two different experimental settings. FirstRAW264.7cells were pretreated with solvent, Dex (1μM) or the selected-Ginsenoside (10μM) combined with Dex (1μM) for2h (short-term treatment protocol) or24h (prolongedtreatment protocol).Then we use quantity RT-PCR method to test the pro-inflammatorymediators IL-6, IL-17, MMP-1and TNFα.（2）We tested whether the enhancement of the inhibition of Dex combined with theselected-Ginsenoside on pro-infalmmatroy cytokines is in a NF-κB dependent way. Theshort-term and the long-time treatment protocol was performed as above described.First,weuse Western-Blot method to test whether Dex combined with the selected-Ginsenosideinhibit the translocation of P65more efficiently compared with Dex alone in both theshort-term and the long-time treatment protocols.And in order to investigate the molecularbasis of the altered subcellular localization of P65, we detect phospho-IκBα and total IκBαprotein still by Western-Blot method.（3）Because the selected Ginsenoside can upregulate GRs in quantity and bindingcapacity, we tset whether this upregulation would result in enhancement of GRE promotoractivity by dual-luciferase reporter gene assay. We use RAW264.7cells,which werepretreated with solvent, Dex (1μM) or Dex (1μM) combined with the selected-Ginsenoside (10μM) for12h. Meanwhile we use primary hepatocytes which were treatedwith solvent, Dex alone or Dex combined with the selected-Ginsenoside for12h toobserve whether the upregulation of GRs would cause a hyperglycemic side effect.（4）We also use bovine type II collagen-induced arthritis (CIA) mouse model ofDBA-1mouse to anti-inflammation properties in vivo.The arthritis (CIA) mice wererandomly divided into3groups according to the scores of inflammation as follows: normalsaline (NS) group,Dex (1.5mg/kg·d) group, Dex（1.5mg/kg·d）+Rh1(25mg/kg·d)group.They were given treatment one time every day,in continuous10days.We observedtheir inflammatory symptoms and scored them everyday.The arthritis mice weresacrificed after10days treatment.Then we detect glycemic levels,and tested the level ofGR、G-6-P、PEPCK by quantity RT-PCR method.RESULTS:1. Ginsenoside Rb1、Rb3、Rh1can upregulate the level of GR in quantity and binding capacity. Ginsenoside Rh1is the most effective in upregulating GR amongthem in the future study.2. In vitro study, Dex and Dex combined with Rh1both can repress the mRNAexpression of IL-6, IL-17, MMP-1and TNFα efficiently, which induced by TNF-αin theshort-term treatment protocol (P<0.01). However, Dex combined with Rh1could represspro-inflammatory cytokine mRNA expression efficiently even in the long-time treatmentprotocol, while Dex couldn’t. The parallel western blot experiment and radioligand bindinganalysis confirmed that, in the prolonged treatment protocol, the effect of Dex on GRexpression and binding capacity became more pronounced than in the short-term treatmentprotocol, thereby explaining the abolishment of its anti-inflammatory effect. The sameresults were also found in P65pathway.3. For up-regulating GRs expression and binding of Rh1, GRE promotor activity wasdetermined. Interstingly, the results of the dual-luciferase reporter gene assay showedthat compared to Dex alone,Dex combined with Rh1enhance the Dex-induced GREpromotor activity slightly in RAW264.7cells, but could not improve the expression ofPEPCK or G6P like Dex alone.4. In vivo investigations in the arthritis (CIA) mouse model of DBA-1mouse showedthat Dex combined with Rh1could inhibit the inflammatory symptoms of arthritis moreefficiently, compared to Dex alone(P<0.01). However it did not affect blood glucose leveland downregulated the express of GRs, while Dex group had a significant increase inblood glucose levels and lower GRs expression(P<0.01).CONCLUSIONS: Ginsenoside Rh1could ameliorate GCs resistance in vivo and invitro without inducing side effects, such as hyperglycemia.