Forty-eight adult male C57BL/6 mice were randomly divided into three groups (n = 16 each), including control group, model group (bilateral
femoral fracture and removal of 35% of the total blood volume), and AGM group (
trauma/
hemorrhage and AGM 200 mg/kg). Eight mice in each group were sacrificed at 3 hours and 24 hours, respectively, after modeling, and blood samples and tissue homogenate of spleen and liver were collected. The contents of
tumor necrosis factor-α (TNF-α),
interleukins (IL-6, IL-1β) in serum and liver tissue were determined with
enzyme linked
immunosorbent assay (ELISA). Serum
aspartate transaminase (AST),
alanine aminotransferase (ALT) and lactic
dehydrogenase (LDH) were determined with automatic biochemistry analyzer. Spleen proliferation response stimulated with
concanavalin A (ConA) was evaluated with methyl thiazolyl tetrazolium colourimetry (MTT). γ-
interferon (IFN-γ) and
IL-2 releases were determined with ELISA.
RESULTS: Compared with control group, 3 hours after
trauma/
hemorrhage, the levels of serum TNF-α,
IL-6, and IL-1β in model group were significantly elevated [TNF-α (ng/L): 145.38±31.50 vs. 23.06±11.14,
IL-6 (ng/L): 496.94±50.76 vs. 47.13±17.47, IL-1β (ng/L): 321.31±43.02 vs. 29.25±16.24,all P < 0.01]. It was found that AGM treatment could alleviate the increase in serum pro-inflammatory mediators induced by
trauma/
hemorrhage, such as TNF-α (ng/L: 111.56±25.47 vs. 145.38±31.50),
IL-6 (ng/L: 412.56±44.33 vs. 496.94±50.76), IL-1β (ng/L: 273.38±45.25 vs. 321.31±43.02, P < 0.05 or P < 0.01). Twenty-four hours after
trauma/
hemorrhage, serum pro-inflammatory mediators were recovered to the levels in control group. There was no significant difference in TNF-α and
IL-6 levels at 3 hours after
trauma/
hemorrhage among groups. Compared with control group, the expressions of liver TNF-α and
IL-6 in model group were increased at 24 hours following
trauma [TNF-α (ng/mg): 32.93±4.90 vs. 26.58±2.33,IL-6 (ng/mg): 11.20±1.66 vs. 8.38±0.89,both P < 0.01]. However, AGM inhibited the level of TNF-α (ng/mg: 28.92±3.16 vs. 32.93±4.90) and
IL-6 (ng/mg: 9.03±1.28 vs. 11.20±1.66) in the liver as induced by
trauma/
hemorrhage (P < 0.05 and P < 0.01). At 24 hours after modeling, model group and AGM group had distinctly higher serum AST, ALT, LDH levels than those of control group [AST (U/L): 405.9±31.2, 245.7±22.1 vs. 128.2±15.9; ALT (U/L): 92.1±6.3, 51.6±5.0 vs. 30.1±3.2; LDH (U/L): 606.7±36.3, 478.7±25.3 vs. 384.0±16.6, all P < 0.01]. Nevertheless,the increase in serum AST, ALT and LDH was alleviated in AGM group (all P < 0.01). Meantime,
trauma/
hemorrhage produced a noticeable depression of proliferation of splenic cells and IFN-γ and
IL-2 release stimulated with ConA compared with control group [proliferation rate: (40.97±4.13)% vs. (89.99±7.76)%, IFN-γ (ng/L): 91.6±12.3 vs. 353.2±21.5,IL-2 (ng/L): 53.4±6.4 vs. 91.0±12.2,all P < 0.01]. In contrast, AGM notably restored the capacity of proliferation response of splenic cells [proliferation rate: (74.86±5.75)% vs. (40.97±4.13)%,P < 0.01],enhanced the release of IFN-γ and
IL-2 stimulated with ConA [IFN-γ (ng/L): 327.8±23.6 vs. 91.6±12.3,
IL-2 (ng/L): 74.8±10.4 vs. 53.4±6.4, both P < 0.01].
CONCLUSIONS: