Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways, Causing Liver Injury

doi:10.1016/j.rsci.2023.04.003

Abstract

Abstract:

Ustiloxins are vital cyclopeptide mycotoxins originally isolated from rice false smut balls that form in rice spikelets infected by the fungal pathogen Ustilaginoidea virens. The toxicity of the water extract of rice false smut balls (RBWE) remains to be investigated. Studies have shown that RBWE may be toxic to animals, but toxicological evidence is still lacking. In this study, we found that the IC₅₀ values of RBWE to BNL CL.2 cells at 24 and 48 h were 40.02 and 30.11 μg/mL, respectively, with positive correlations with dose toxicity and time toxicity. After treatment with RBWE, the number of BNL CL.2 cells decreased significantly, and the morphology of BNL CL.2 cells showed atrophy and wall detachment. RBWE induced DNA presynthesis phase arrest of BNL CL.2 cells, increased the proportion of apoptotic cells and inhibited cell proliferation. RBWE up-regulated reactive oxygen species (ROS) levels and lowered mitochondrial membrane potentials. Additionally, Western blot and qRT-PCR results suggested that RBWE exerted the above effects by promoting the Nrf2/HO-1 and caspase-induced apoptosis pathways in vitro and in vivo. The contents of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bile acids in the serum of mice from Institute of Cancer were significantly up-regulated by RBWE. At the same time, RBWE can lead to increases in ROS and malondialdehyde contents, decreases in contents of oxidized glutathione, glutathione and reduced glutathione, as well as decrease in catalase and superoxide dismutase activities in mouse liver tissues, demonstrating that oxidative stress occurred in mice. Moreover, liver damage was further detected by haematoxylin-eosin staining and electron microscopy to verify the damage to the mice caused by RBWE. In general, RBWE may cause hepatotoxicity in vivo and in vitro via the apoptosis pathway, which provides a reference for hepatotoxicity and its mechanism of action.

Key words: water extract, rice false smut ball, ustiloxin, liver injury, Nrf2/HO-1 pathway, apoptosis pathway

Zhang Guomei, Li Han, Liu Shanshan, Zhou Xuming, Lu Mingyang, Tang Liang, Sun Lihua. Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways, Causing Liver Injury[J]. Rice Science, 2023, 30(5): 473-485.

Figures/Tables 8

Fig. 1. Effects of different concentrations of water extract of rice false smut balls (RBWE) on cell proliferation in BNL CL.2 cells. A, Cell viability was measured by Cell Counting Kit-8 (CCK8) assay. Data are Mean ± SE (n = 3). B, Cell morphology changes at 24?h after RBWE treatment. Scale bars, 100 μm. The red arrows indicate vascular degeneration in cells. C, Morphological changes in BNL CL.2 cells were observed under a scanning electron microscope. Scale bars, 10 μm.

Fig. 2. BNL CL.2 cell apoptosis was investigated by cell cycle assay and mitochondrial membrane potential assay. A, Histogram of cell cycle. G2, DNA late synthesis phase; S, DNA synthesis phase; G0/G1, DNA presynthesis phase. B, Histogram of cell apoptosis at 24?h after water extract of rice false smut balls (RBWE) treatment. Data are Mean ± SE (n = 3). **, P < 0.01.

Fig. 3. Effects of water extract of rice false smut balls (RBWE) on reactive oxygen species (ROS) content in BNL CL.2 cells. Data are Mean ± SE (n = 3). *, P < 0.05; **, P < 0.01.

Fig. 4. Water extract of rice false smut balls (RBWE)-induced changes in livers of mice. A, Body weight curve. B, Liver weight. C, Liver weight to body weight ratio. D, Histopathological examination of liver by haematoxylin-eosin staining. Typical sections of the hepatic central venous region in mice. Scale bars, 10 μm. E, Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels. F, Total bile acid (TBA) concentration. *, P ?<? 0.05; **, P? <? 0.01. Values are Mean ± SE (n ?= ?12).

Fig. 5. Water extract of rice false smut balls (RBWE)-induced changes in oxidative stress indices in mouse livers. ROS, Reactive oxygen species; MDA, Malondialdehyde; SOD, Superoxide dismutase; CAT, Catalase; GSH, Glutathione; GSSG, Oxidized glutathione; GSH-Px, Reduced glutathione. *, P ?<? 0.05; **, P? <? 0.01. Values are Mean ± SE (n ?= ?8).

Fig. 6. Water extract of rice false smut balls (RBWE) affected oxidative stress-related proteins and genes. A and B, Nrf2 and Haem oxygenase-1 (HO-1) protein levels in BNL CL.2 cells. C, Nrf2 mRNA expression in BNL CL.2 cells. D, HO-1 mRNA expression in BNL CL.2 cells. E and F, Nrf2 and HO-1 protein levels in mouse livers. G, Nrf2 mRNA expression in mouse livers. H, HO-1 mRNA expression in mouse livers. I-L, Nuclear Nrf2 protein level. I and J, Nuclear Nrf2 protein level in BNL CL.2 cells. K and L, Nuclear Nrf2 protein level in mouse livers. *, P ?<? 0.05; **, P? <? 0.01. Values are Mean ± SE (n ?= ?3).

Fig. 7. Expression of apoptosis- and pathway-related proteins and genes in BNL CL.2 cells. A, Expression levels of apoptotic factors in BNL CL.2 cells assessed by Western blot analysis. B, Content of Bcl-2. C, Content of Bax. D, Bcl-2/Bax relative intensity. E, Content of Caspase 3. F, Content of cleaved Caspase 3. G, Content of poly ADP-ribose polymerase (PARP). H, Content of Caspase 9. I, Content of cleaved Caspase 9. J-L, mRNA expression levels of Bcl-2 (J), Bax (K) and Bcl-2/Bax (L) genes by qRT-PCR. *, P ?<? 0.05; **, P? <? 0.01. Values are Mean ± SE (n ?= ?3).

Table 1. Specific sequences of PCR primers.

Target gene	Marker	Primer sequence (5'-3')
Bcl2	Bcl2-F	TGGAGAGCGTCAACAGGGAG
Bcl2	Bcl2-R	CAGCCAGGAGAAATCAAACAGA
Bax	Bax-F	ACAGATCATGAAGACAGGGGG
Bax	Bax-R	CAAAGTAGAAGAGGGCAACCAC
Nrf2	Nrf2-F	TAGATGACCATGAGTCGCTTGC
Nrf2	Nrf2-R	GCCAAACTTGCTCCATGTCC
HO-1	HO-1-F	GATAGAGCGCAACAAGCAGAA
HO-1	HO-1-R	CAGTGAGGCCCATACCAGAAG
GAPDH	GAPDH-F	AGGTCGGTGTGAACGGATTTG
GAPDH	GAPDH-R	TGTAGACCATGTAGTTGAGGTCA

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