Ginsenoside Rh1 potentiates dexamethasone's anti-inflammatory effects for chronic inflammatory disease by reversing dexamethasone-induced resistance

Abstract

Introduction: Acquired resistance to glucocorticoids constitutes a major clinical challenge, often overlooked in the search for compounds to improve the effect of classic steroids. We sought to unravel how a plant-original compound, ginsenoside Rh1, potentiates dexmethasone (DEX)'s potential anti-inflammation properties.

Methods: Ginsenoside Rh1 combined with DEX was applied in a short-term and long-term treatment protocol for inflammation. Its potential mechanism on anti-inflammation was explored. In addition, the effect of Rh1 on the side-effect induced by DEX was studied. Furthermore, the in vivo anti-inflammatory effects of Rh1 combined with DEX were evaluated in a collagen-induced arthritis (CIA) mice model.

Results: Ginsenoside Rh1 potentiates DEX's anti-inflammatory effects even after prolonged DEX treatment. Rh1 could improve the glucocorticoid receptor (GR)'s transrepression on nuclear factor kappa B (NF-κB) and transactivation on dual specificity protein phosphatase 1 (DUSP1), which is responsible for DEX's anti-inflammatory effects. Parallel Western blot assay and radioligand binding analysis revealed that Rh1 could increase the expression and binding of GR. This is in sharp contrast to DEX alone, showing a direct link among prolonged treatment, decreasing GR and the abolishment of anti-inflammation. Interestingly, Rh1 does not enhance the transactivation of glucocorticoid-responsive elements (GRE) driven genes - gluconeogenic enzyme glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinasee phosphatase (PEPCK) in primary mouse hepatocytes, a mechanism partly held accountable for the metabolic side-effects. Similar results were found in CIA mice.

Conclusion: Rh1 could potentiate DEX's anti-inflammatory effects and does not cause a hyperglycemic side effect. Ginsenoside Rh1 combined with DEX may be a promising candidate treatment option for chronic inflammatory diseases in need of long-term immunosuppression therapies.

Figure 1

Effects of ginsenoside Rh1 combined with DEX on TNF-induced cytokine production. RAW264.7 cells were pretreated with solvent, DEX (1 μM) or DEX combined with Rh1 (10 μM, 1 μM, 0.1 μM ) or Rh1 alone for 2 h (short-term treatment protocol) or 24 h (prolonged treatment protocol), as indicated in the figures. Afterwards, TNF (20 ng /ml) was added for 8 h. (A) Expression of the pro-inflammatory mediators IL-6, IL-17, MMP-1 and TNF-α was monitored by means of quantitative PCR. Gene expression of the housekeeping gene β-actin was used for normalization. (B) Protein expression of IL-6 and IL-17 were also determined by ELISA. Statistical significance was determined by one-way analysis of variance (*P <0.05, **P <0.01 versus TNF group; #P <0.05, ##P <0.01 versus the DEX group). The experiments were replicated three times, and results are representative of at least three independent induction experiments. DEX, dexamethasone; IL, interleukin; MMP-1, matrix matalloproteinase-1; TNF, tumor necrosis factor.

Figure 2

Effects of ginsenoside Rh1 combined with DEX on TNF-induced NF-κB translocation and DUSP1 activation. After pretreatment with solvent, DEX (1 μM), or Rh1 (10 μM) combined with DEX for 2 h or 24 h, (A-B) TNF (20 ng /ml) was added for 30 minutes and localization of p65 was determined by Western blot. (C-D) TNF was added for the indicated time periods (15 and 30 minutes) and expression of phospho-IκBα and total IκBα was determined. The experiment was replicated three times. (E) After pretreatment with solvent, DEX, Rh1 combined with DEX, or Rh1 for 2 h or 24 h, TNF was added for 2 h and DUSP1 was determined by means of quantitative PCR. Gene expression of the housekeeping gene β-actin was used for normalization. Statistical significance was determined by one-way analysis of variance (*P <0.01 versus Control; #P <0.05, ##P <0.01 versus DEX group; ΔP <0.05, ΔΔP <0.01 versus TNF group; &P <0.05, &&P <0.01 versus TNF + DEX group). The experiments were replicated three times, and the results are representative of at least two independent induction experiments. DEX, dexamethasone; DUSP1, dual specificity protein phosphatase 1; TNF, tumor necrosis factor.

Figure 3

Effects of ginsenoside Rh1 combined with DEX on p38 activation. After pretreatment with solvent, DEX (1 μM), or Rh1 (10 μM) combined with DEX for 2 h or 24 h, TNF (20 ng /ml) was added for the indicated times (15 and 30 minutes) and expression of phospho-p38 and total p38 was determined by Western blot. The experiment was replicated three times, and the results are representative of at least two independent induction experiments. DEX, dexamethasone; TNF, tumor necrosis factor.

Figure 4

Effects of ginsenoside Rh1 combined with DEX on GR. (A-B) After pretreatment with solvent, DEX (1 μM) or Rh1 (10 μM) combined with DEX for 2 h or 24 h, TNF (20 ng/ml) was added for 8 h. (A) Western blot analysis was performed in parallel on total extracts with an anti-GR antibody. The detection of β-actin was used as a loading control. (B) Specific binding of GR was also determined in parallel on RAW 264.7 cells. RAW264.7 cells (1 × 10⁶) were incubated 3 h with 10⁻⁶ M [³H] DEX, with or without excess unlabeled DEX. Specific binding was determined. *P <0.05, **P <0.01 versus control, #P <0.01 versus DEX group. (C-D) RAW264.7 cells were treated with solvent, DEX alone or Rh1 combined with DEX for 24 h. (C) Transcription was then stopped by addition of actinomycin D (0.25, 0.5, 1 ng/ml) in the presence of Rh1. GR mRNA levels were assessed by means of quantitative PCR. β-actin was used for normalization. The left panel is the quantitative result and the right panel is RNA electrophoretic figures. *P <0. 01 versus control; #P <0.01 versus DEX group; ΔP <0.01 versus Rh1 + DEX group. (D) Protein synthesis was stopped by addition of cycloheximide (5, 2, 1 μg/ml) in the presence of Rh1. GR levels were determined by Western blot. The detection of β-actin was used as a loading control. The experiment was replicated three times and results are representative of at least two independent induction experiments. DEX, dexamethasone; GR, glucocorticoid receptor; TNF, tumor necrosis factor.

Figure 5

Effect of Rh1 combined with DEX on GRE activity and some gluconeogenesis-related genes. RAW264.7 cells were treated with solvent, DEX alone or Rh1 combined with DEX for 24 h. (A) Cell lysates were assayed for luciferase activities. Promoter activities are expressed as relative induction factor, that is, the ratio of expression levels recorded either at induced and non-induced conditions, with the latter taken to be 1. Assays were performed in triplicate, and results are representative of at least two independent induction experiments. (B) Primary hepatocytes were treated with solvent, DEX alone or Rh1 combined with DEX for 24 h. RNA was isolated and reverse transcribed. The resulting cDNA was subjected to PCR analysis with primers to detect the household gene GAPDH (loading control) or the gene coding for DUSP1, PEPCK and G6P in the same sample. *P <0.01 versus control, #P <0.01 versus DEX group. DEX, dexamethasone; DUSP1, dual specificity protein phosphatase 1; G6P, glucose-6-phosphatase; GRE, glucocorticoid receptor elements; PEPCK, phosphoenolpyruvate carboxykinasee phosphatase; TNF, tumor necrosis factor.

Figure 6

Anti-inflammatory potential of ginsenoside Rh1 combined DEX in CIA. (A) After onset of arthritis, CIA mice were randomized in a PBS, DEX (1 mg/kg) treatment or Rh1 (10 mg/kg) combined with DEX (1 mg/kg) treatment protocol. Disease severity was monitored daily. Dots and bars represent mean ± SEM. *P <0.05, **P <0.01 compared with PBS, # P <0.05 compared with DEX. (B) Histopathology of paw joints 10 days post-arthritis onset. HE staining of the metacarpophalangeal joint: a, b, c and d are representative images for blank (normal mice), PBS-, DEX- and Rh1 combined with DEX-treated mice, respectively. The different parameters assessed are 1) influx of inflammatory cells in joint capsule, 2) cartilage destruction, 3) joint cavity narrowing, 4) influx of inflammatory cells in marrow cavities, and 5) and periosteal thickening. (C) Real time PCR was performed in parallel on liver tissues extracts with GR primers. Gene expression of the housekeeping gene GAPDH was used for normalization. *P <0.05, **P <0.01 compared with blank, ##P <0.01 compared with DEX. (D) The blood glucose concentration was determined 6 h after treatment (mice were fasted 18 h before the blood samples were taken). The normal mice were as blank. Values are expressed as means ± SD. *P <0.01 versus blank??; #P <0.01 versus DEX group. (E) Liver samples were taken from the CIA mice after 10 days of treatment. PCR analysis with primers detects the household gene β-actin (loading control) or the gene coding for PEPCK and G6P in the same sample. *P <0.05, **P <0.01 compared with blank, #P <0.05, ##P <0.01 compared with DEX. CIA, collagen-induced arthritis; DEX, dexamethasone; G6P, glucose-6-phosphatase; GR, glucocorticoid receptor; PBS, phosphate-buffered saline; PEPCK, phosphoenolpyruvate carboxykinasee phosphatase.