Xiaochaihutang Improves the Cortical Astrocyte Edema in Thioacetamide-Induced Rat Acute Hepatic Encephalopathy by Activating NRF2 Pathway

Abstract

Oxidative stress induced by high ammonia, which leads to astrocyte edema, is the key to acute hepatic encephalopathy (AHE). Nuclear factor erythroid 2-related factor 2 (NRF2) has been implicated in oxidative stress, but the mechanism of NRF2 against ammonia-induced astrocytes edema has not been fully studied. We confirmed that the NRF2 pathway is related to brain edema caused by AHE and found that Xiaochaihutang (XCHT) could effectively activate the NRF2 pathway to treat AHE. The model of AHE was established with thioacetamide (TAA) in rats. Rat behaviors were observed, brain water content, blood ammonia levels, glutamine synthetase (GS), malondialdehyde (MDA), and total superoxide dismutase (T-SOD) were determined after XCHT treatment. Furthermore, the expression of NRF2 pathway proteins and mRNA, glial fibrillary acidic protein (GFAP) and aquaporins 4 (AQP4) were examined. In order to determine whether XCHT has a direct effect on cerebral edema caused by high ammonia, we examined the effect of XCHT compound serum on cortical astrocytes in the presence of ammonia, through microscopic observation and immunofluorescence (IF). Results showed that AHE induced by TAA changed the behavior of the rats, and increased brain water content, blood ammonia levels, GS and MDA content meanwhile decreasing T-SOD, but these symptoms were improved by treatment with XCHT. XCHT protected brain edema by activating the NRF2 pathway and increasing the expression of downstream proteins and genes. Astrocytes treated with 5 mM ammonia also showed an increase in the AQP4 protein expression but a decrease in XCHT compound serum and ammonia-induced cell edema groups. This study demonstrates that the NRF2 pathway is involved in the brain edema in AHE, and XCHT may represent a useful prescription for the treatment of AHE.

Keywords: NRF2 pathway; Xiaochaihutang; acute hepatic encephalopathy; astrocyte edema; brain edema.

Figure 1

Acute hepatic encephalopathy (AHE) was induces by repeated injection of 300 mg/kg/day thioacetamide (TAA) for 3 days (1 mL/kg in saline, i.p.). From the 4th day, the animals in treatment groups were given the XCHT granules (1.0 g/kg, 2.0 g/kg and 4.0 g/kg, 1mL/100 g/day, i.g.) for 2 weeks. (A, B) Measurement of the rat behaviors [(A) Animal behavior scoring. (B) Open field test. Rats in each group were evaluated for total distance traveled]. (C) Brain water content ([(wet weight – dry weight)/wet weight] × 100%). Data were expressed as mean ± SD (n = 5-7). ^#p < 0.05, ^##p< 0.01 vs. the vehicle group. *p < 0.05, **p < 0.01 vs. the TAA group.

Figure 2

Effect of XCHT on ammonia level in TAA induced AHE rats. (A) Ammonia plasma levels in all groups of rats. (B) GS levels in cerebral cortex of rats. Data were expressed as mean ± SD (n = 5-7). ^#p < 0.05, ^##p < 0.01 vs. the vehicle group. *p < 0.05, **p < 0.01 vs. the TAA group.

Figure 3

Effects of different doses of XCHT on oxidative stress level [MDA (A) and T-SOD (B)] in cerebral cortex of TAA induced AHE rats. Data were expressed as mean ± SD (n = 5-7). ^##p < 0.01 vs. the vehicle group. *p < 0.05, **p < 0.01 vs. the TAA group.

Figure 4

Effect of XCHT on structure of the astrocytes in cerebral cortex of AHE rats. GFAP expression in cerebral cortex of rat brain (IHC, 200×). The decrease of GFAP immunostaining in cortex of TAA group rats compared to the vehicle group, which indicated that the structure of astrocytes was destroyed and brain edema occurred. In contrast, the expression of GFAP in astrocytes increased in the treatment groups with different doses.

Figure 5

Effect of XCHT on TAA-induced AHE in rats by activating cortical astrocytes NRF2 pathway. (A) The mRNA expression of NRF2, KEAP-1, HO-1, NQO-1, GCLC, GCLM, GFAP and AQP4. (B) The protein expression of Nucl-NRF2. (C, D) The protein expression of Cyto-NRF2, KEAP-1, HO-1, NQO-1, GCLC, GCLM, GFAP and AQP4. Data were expressed as mean ± SD (n = 5-7). ^#p < 0.05, *p < 0.01 vs. the TAA group.

Figure 6

Effect of XCHT compound serum on ammonia-induced astrocyte swelling. (A) Purity of primary astrocyte culture. Immunolabeling of primary rat astrocyte cultures with the markers GFAP (Red) revealed pure primary astrocyte culture. Nucleus were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Blue). (Staining, 100×) (B) Morphological overview of astrocyte culture after treatment. (C) Immunolabeling of primary rat astrocyte cultures with the markers AQP4 (Green), Nucleus were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Blue).