Apocynin reduced doxycycline-induced acute liver injury in ovariectomized mice

Abstract

To determine the physiological role of estrogen in the development of liver injury, we examined the sensitivities of sham and ovariectomy (ovx) mice against doxycycline (DOXY)-induced acute liver injury. Ovx or sham operation was performed in C57BL/6J wild-type female mice of eight weeks of age. Sham mice and ovx mice were treated with DOXY (240 mg/kg ip) 8 weeks after the operation, 30 min after apocynin (5 mg/kg) or saline administration. Blood and liver samples were obtained at 3 and 6 h after DOXY administration. Liver dysfunction occurred soon after DOXY administration and became more severe in ovx mice than in sham mice. At early phase after DOXY injection, TNF-α and iNOS inductions upregulated almost the same levels in sham and ovx mice. On the other hand, expression levels of IL-6, IL-10, c-fos, cox-2 and HO-1, downstream genes of TNF-α, were significantly increased in ovx mice compared to those in sham mice, correlated with liver dysfunction. In addition, apocynin, a NADPH oxidase (Nox) inhibitor, totally improved DOXY-induced liver injury in both sham and ovx mice, indicating that reactive oxygen species generated through Nox activation by DOXY are responsible for development of acute liver injury.

Keywords: ALF, acute liver failure; ALT, alanine aminotransferase; ARF, acute renal failure; Apocynin; DOXY, doxycycline; Doxycycline-induced liver injury; HO-1, heme oxygenase-1; IL-6, interleukin-6; NADPH oxidase; Nox, NADPH oxidase; Ovariectmized; Ovx, ovariectomy; ROS, reactive oxygen species; SOD, superoxide dismutase; STAT3, signal transducers and activators of transcription-3; TNF-α, tumor necrosis factor-α; cox-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase.

Fig. 1

(A) body weight of sham and ovx mice 8 weeks after operation. The average weights of both group before operation were almost the same (20 ± 0.5 g). (B) Survival plots of sham and ovx mice after DOXY injection. Survival rates of sham and ovx mice treated with saline + DOXY (S + D) or apocynin + DOXY (A + D) are presented. *p < 0.05. All ovx mice (n = 10) died within 48 h after saline and DOXY (240 mg/kg) injection. Survival rate of sham mice (n = 10) at 72 h was better than that of ovx mice. (C) Changes in ALT levels in sham or ovx mice treated with saline + saline (S + S), saline + DOXY (S + D) or apocynin + DOXY (A + D) at 0, 3, 6, 9 and 24 h after DOXY injection. *p < 0.05, **p < 0.01 vs sham S + D mice. ^#p < 0.05, ^##p < 0.01 vs ovx A + D mice. ^Эp < 0.05, ^ЭЭp < 0.01 vs ovx S + S mice. Data are average ± SE. sham S + S (n = 2), sham S + D (n = 13), sham A + D (n = 8), ovx S + S (n = 2), ovx S + D (n = 13), ovx A + D (n = 7).

Fig. 2

Hematoxylin and eosin tissue staining at 3 h after DOXY injection with/without apocynin pretreatment. Sham groups; saline + saline (S + S) (A), apocynin + saline (A + S) (B), saline + DOXY (S + D) (C), apocynin + DOXY (A + D) (D). Ovx groups; saline + saline (S + S) (E), apocynin + saline (A + S) (F), saline + DOXY (S + D) (G), apocynin + DOXY (A + D) (H). Arrows indicate denatured hepatocytes. Normal hepatocyte areas were quantified (I). *p < 0.05, **p < 0.01 vs S + S mice.

Fig. 3

(A) Changes in Nox activity in livers obtained from sham or ovx mice at 3 h after DOXY injection with/without apocynin pretreatment. Sham groups; saline + saline (S + S), saline + DOXY (S + D), apocynin + DOXY (A + D). Ovx groups; saline + saline (S + S), saline + DOXY (S + D), apocynin + DOXY (A + D). Data are average ± SE. *p < 0.05 vs ovx (S + D). ^#p < 0.05 vs sham (A + D). (B) Changes in IL-6 levels in plasma obtained from sham or ovx mice treated with saline (S + S), apocynin + saline (A + S), saline + DOXY (S + D) or apocynin + DOXY (A + D) at 3 and 6 h after DOXY injection. Data are average ± SE. *p < 0.05, **p < 0.01 vs ovx (S + S) or (S + A), ^#p < 0.05 vs sham (S + D).

Fig. 4

Changes in expression levels of TNF-α (A), iNOS (B), HO-1 (C), c-fos (D), IL-6 (E), IL-10 (F) and cox-2 (G) genes in livers obtained from mice treated with saline + saline (S + S), apocynin + saline (A + S), saline + DOXY (S + D) or apocynin + DOXY (A + D) at 3 and 6 h after DOXY injection. Data are average ± SE. *p < 0.05, **p < 0.01 vs S + S mice, ^#p < 0.05, ^##p < 0.01 vs sham mice.