Figure 1.
Effects of YHB (0.5, 1, 2 and 4 mg/kg) on organ injury in mice 12 h after LPS (20 mg/kg) injection.
(A) Representative M-mode echocardiograms in control (upper), LPS (middle) and 1 mg/kg YHB+LPS (lower) groups. (B, C, D, E, F) Changes in ejection fraction (EF), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), stroke volume (SV) and cardiac output (CO). (G) Serum alanine aminotransferase (ALT) activity. (H) Blood urea nitrogen (BUN) level. (I) Lung wet-to-dry weight (W/D) ratio. n = 8–10. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with LPS group.
Figure 2.
Effects of YHB (1, 2 or 4 mg/kg) on cardiac and plasma TNF-α and NO levels in LPS-challenged mice.
(A and B) Cardiac and plasma TNF-α levels were examined at 1 h after 20 mg/kg LPS challenge (n = 10). (C and D) Cardiac and plasma NO levels were determined at 12 h after 20 mg/kg LPS injection (n = 10). *P<0.05, **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with LPS group.
Figure 3.
Cardiac and lung α2- AR levels and cardiac localization of α2- AR subtypes in YHB or/and LPS-challenged mice.
(A and B) Levels of α2A, α2B and α2C-AR protein in the heart and lung (n = 8). LPS (20 mg/kg) or normal saline was injected intraperitoneally 1 h after intragastrical treatment with YHB (1 mg/kg) or water, the α2A, α2B and α2C-AR proteins were determined using Western blotting at 4 h after LPS injection. *P<0.05, **P<0.01 compared with control group; #P<0.05 compared with LPS group. (C) Representative confocal images of normal mouse cardiac sections. The sections were stained with antibodies against cardiac troponin I (blue), α2-AR subtypes (green) and synaptophysin (SYP, a mark for presynaptic terminals, red) or CD34 (a marker for endothelial cells, red). Insets, high-power magnification of the area indicated by arrows. Scale bar = 20 µm.
Figure 4.
Effects of YHB or/and reserpine (RSP) on cardiac and plasma NE levels, TNF-α and NO production, myocardial inducible nitric oxide synthase (iNOS) expression and left ventricular EF in LPS-challenged mice.
(A and B) Cardiac and plasma NE levels at 0.5 and 2 h after LPS or normal saline injection (n = 10). Mice were injected intraperitoneally with LPS (20 mg/kg) or normal saline at 1 h after intragastrical treatment with YHB (1 mg/kg) or water. In separate experiments, mice first received subcutaneous injection of RSP (4.5 mg/kg) or normal saline once a day for 2 consecutive days, then exposed to YHB (1 mg/kg) or/and LPS (20 mg/kg) on the 4th day after last RSP administration. LPS or normal saline was injected intraperitoneally at 1 h after YHB treatment. NE concentrations of the heart (C) and plasma (D) was detected at 0.5 h after LPS injection (n = 8). (E) Left ventricular EF 12 h after LPS injection (n = 8). (F and G) TNF-α production of the heart and plasma at 2 h after LPS challenge (n = 10). (H and I) Cardiac and plasma NO levels at 12 h after LPS injection (n = 8). (J) Cardiac iNOS expression was detected by Western blot assay at 6 h after LPS challenge (n = 7). *P<0.05, **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with LPS group; ΔP<0.05, ΔΔP<0.01 compared with YHB+LPS group.
Figure 5.
Effects of YHB or/and reserpine (RSP) on the cardiomyocyte apoptosis in LPS-challenged mice.
(A and B) Representative confocal images of cardiac troponin I, DAPI and TUNEL-stained cardiac sections are shown from LPS and YHB+LPS groups, respectively. (C) Apoptotic index (AI) of cardiomyocytes at 12 h after LPS injection (n = 10). (D) Cardiac caspase 3/7 activity at 2 h after LPS injection (n = 10). **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with LPS group.
Figure 6.
Effects of prazosin (PRA), atenolol (ATE) and ICI 118551 (ICI) on the cardioprotective action of YHB in LPS-challenged mice.
PRA (α1-AR antagonist, 2 mg/kg), ATE (β1-AR antagonist, 10 mg/kg), ICI (β2-AR antagonist, 10 mg/kg) or vehicle were delivered intraperitoneally and followed by intragastrical administration of YHB (1 mg/kg) or water. LPS (20 mg/kg) or normal saline was injected intraperitoneally 1 h after treatment with YHB or water. (A, B and C) The left ventricle EF was examined at 12 h after LPS injection (n = 8). (D, E, F) Cardiac caspase-3/7 activity (n = 8) and (G) cardiomyocyte apoptotic index (AI, n = 10) were detected at 2 h and 12 h after LPS injection, respectively. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with LPS group; ΔP<0.05, ΔΔP<0.01 compared with YHB+LPS group.
Figure 7.
Proposed mechanisms involved in improvement of LPS-induced cardiac dysfunction by YHB.
Besides α2 -AR in infiltrated macrophages, YHB blocked cardiac presynaptic α2A-AR and in turn increases cardiac NE release during endotoxemia. Elevated cardiac NE inhibits cardiac TNF-α and iNOS expression, attenuates cardiomyocyte apoptosis via stimulating α1-AR and β2-AR, directly activates β1-AR and thereby improves LPS-induced decreased EF.