Chlorogenic acid attenuates high mobility group box 1 (HMGB1) and enhances host defense mechanisms in murine sepsis

Abstract

Sepsis is a complex, multifactorial, rapidly progressive disease characterized by an overwhelming activation of the immune system and the countervailing antiinflammatory response. In the current study in murine peritoneal macrophages, chlorogenic acid suppressed endotoxin-induced high mobility group box 1 (HMGB1) release in a concentration-dependent manner. Administration of chlorogenic acid also attenuated systemic HMGB1 accumulation in vivo and prevented mortality induced by endotoxemia and polymicrobial sepsis. The mechanisms of action of chlorogenic acid included attenuation of the increase in toll-like receptor (TLR)-4 expression and suppression of sepsis-induced signaling pathways, such as c-Jun NH₂-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB, which are critical for cytokine release. The protection conferred by chlorogenic acid was achieved through modulation of cytokine and chemokine release, suppression of immune cell apoptosis and augmentation of bacterial elimination. Chlorogenic acid warrants further evaluation as a potential therapeutic agent for the treatment of sepsis and other potentially fatal systemic inflammatory disorders.

Figure 1

The effect of chlorogenic acid on in vitro cytokine and chemokine release. Cytokine and chemokine release from macrophages were determined after 16 h of LPS stimulation in the presence or absence of various concentrations of chlorogenic acid (CA). The effects of CA on basal and LPS-induced release of HMGB1 (A), MIP-2 (B), MCP-1 (C), TNF-α (D), IL-1β (E), IL-6 (F) and IL-12 (G) were determined. Data are presented as mean ± SEM; n = 8. Statistically significant differences from the control group (**P < 0.01). Statistically significant differences from the LPS-treated control group (⁺P < 0.05 and ⁺⁺P < 0.01).

Figure 2

Chlorogenic acid protected against sepsis-induced mortality. Chlorogenic acid was administered at various doses after the induction of sepsis via an intraperitoneal injection of LPS or CLP. Mice were monitored for survival for up to 10 d. (A) LPS-injected mice were given chlorogenic acid or ethyl pyruvate. (B) CLP mice were given various doses of chlorogenic acid or ethyl pyruvate. The results of treatment groups were compared with the vehicle-treated group. n = 10–30 per group.

Figure 3

Chlorogenic acid protected against CLP-induced organ injury. Assessment of serum enzyme activities and histological analysis were performed to evaluate the protective effects of chlorogenic acid on multiorgan dysfunction induced by sepsis. (A) Serum levels of LDH, BUN, creatinine, ALT and AST were determined at various time points after CLP. Histological analysis of heart (B), kidney (C), liver (D) and lung tissue (E) collected 24 h after CLP. Significant histopathological changes (such as inflammatory cell infiltration, congestion, necrosis and degeneration) were observed in the CLP group (200×, scale bar 50 μm), and these pathological changes were ameliorated by the administration of chlorogenic acid (20 mg/kg). The serum levels of soluble ICAM-1 were measured 6, 12, 18 and 24 h after CLP (F), and the changes in the level of ICAM-1 in heart, kidney, liver and lung tissues were determined 18 h after CLP (G). Treatment with chlorogenic acid (20 mg/kg) suppressed the increase in ICAM-1 levels induced by CLP. Data are presented as mean ± SEM; n = 8–10. Statistically significant differences compared with the sham group (*P < 0.05 and **P < 0.01). Statistically significant differences compared with the vehicle-treated CLP group (⁺P < 0.05 and ⁺⁺P < 0.01).

Figure 4

The effects of chlorogenic acid on CLP-induced cytokine and chemokine release. The levels of HMGB1 (A), MIP-2 (B), MCP-1 (C), TNF-α (D), (E) IL-1β (E), IL-6 (F), IFN-γ (G), IL-2 (H), IL-12 (I) and IL-10 (J) were determined after CLP, with or without treatment with chlorogenic acid (20 mg/kg). Data are presented as mean ± SEM; n = 8–10. Statistically significant differences compared with the sham group (*P < 0.05 and **P < 0.01). Statistically significant differences compared with the vehicle-treated CLP group (⁺P < 0.05 and ⁺⁺P < 0.01).

Figure 5

The effects of chlorogenic acid on TLR4 and signaling pathways. The influences of chlorogenic acid on TLR4 and MAPK activation were evaluated to understand the mechanisms underlying chlorogenic acid–mediated survival. TLR4 mRNA expression was quantified in heart (A), kidney (B), liver (C) and lung tissues (D) at various time points after CLP. (E) The protein expression of TLR4 was increased 6 h after CLP in the liver, and this increase was attenuated by chlorogenic acid treatment (20 mg/kg). The phosphorylation of JNK (F), p38 (G) and ERK (H) were assessed 6 h after CLP-induced sepsis. (I) NF-κB activation was determined in nuclear fraction of liver samples collected 6 h after CLP. (J) The decrease in the level of cytosolic IκB-α expression after CLP was significantly attenuated by the administration of chlorogenic acid (20 mg/kg). Data are presented as mean ± SEM; n = 6–8. Statistically significant differences compared with the sham group (*P < 0.05 and **P < 0.01). Statistically significant differences compared with the vehicle-treated CLP group (⁺P < 0.05 and ⁺⁺P < 0.01).

Figure 6

Lymphocyte apoptosis determined by annexin V staining. Lymphocytes were identified by characteristic forward and side scatter properties. The percentage of lymphocytes undergoing apoptosis was quantified by annexin V in the following experimental groups: sham, vehicle-treated CLP and chlorogenic acid (20 mg/kg)-treated CLP groups 24 h after CLP-induced sepsis. Data are presented as mean ± SEM; n = 5–7. Statistically significant differences compared with the sham group (**P < 0.01). Statistically significant differences compared with the vehicle-treated CLP group (⁺⁺P < 0.01).

Figure 7

Chlorogenic acid enhanced in vivo bacterial clearance. CFUs were counted in peritoneal fluid (A), blood (B), lung (C) and spleen (D) 24 h after CLP to examine whether the administration of chlorogenic acid (20 mg/kg) enhanced the clearance of CLP-induced bacteria. After serial dilutions with PBS, peritoneal fluid, blood and tissue homogenates were cultured overnight on blood-agar base plates maintained at 37°C. Data are presented as mean ± SEM; n = 8. Statistically significant differences compared with the sham group (**P < 0.01). Statistically significant differences compared with the vehicle-treated CLP group (⁺P < 0.05 and ⁺⁺P < 0.01).