Spironolactone in Combination with α-glycosyl Isoquercitrin Prevents Steatosis-related Early Hepatocarcinogenesis in Rats through the Observed NADPH Oxidase Modulation

Abstract

Administration of the diuretic, spironolactone (SR), can inhibit chronic liver diseases. We determined the effects of SR alone or in combination with the antioxidant α-glycosyl isoquercitrin (AGIQ) on hyperlipidemia- and steatosis-related precancerous lesions in high-fat diet (HFD)-fed rats subjected to a two-stage hepatocarcinogenesis model. Rats were fed with control basal diet or HFD, which was administered with SR alone or in combination with an antioxidant AGIQ in drinking water. An HFD increased body weight, intra-abdominal fat (adipose) tissue weight, and plasma lipids, which were reduced by coadministration of SR and AGIQ. SR and AGIQ coadministration also reduced hepatic steatosis and preneoplastic glutathione S-transferase placental form-positive foci, in association with decrease in NADPH oxidase (NOX) subunit p22phox-positive cells and an increase in active-caspase-3-positive cells in the foci. Hepatic gene expression analysis revealed that the coadministration of SR and AGIQ altered mRNA levels of lipogenic enzymes ( Scd1 and Fasn), antioxidant-related enzymes ( Catalase), NOX component ( P67phox), and anti-inflammatory transcriptional factor ( Pparg). Our results indicated that SR in combination with AGIQ had the potential of suppressing hyperlipidemia- and steatosis-related early hepatocarcinogenesis through the reduced expression of NOX subunits.

Immunohistochemical expression of autophagosome markers LC3 and p62 in preneoplastic liver foci in high fat diet-fed rats

“…Effects of the HFD on GST-P-positive preneoplastic liver foci were variable in our studies, probably reflecting variations due to the study condition and diet lot. Both the area and number of foci were significantly increased in the HDF group compared with the CTL group in our previous study (Murayama et al, 2018) or were both weakly increased without a significant difference in another study (Nakamura et al, 2018). The present study revealed only a minor effect on the number of GST-P-positive preneoplastic liver foci.…”

“…Analysis of mRNA levels of the genes listed in Supplemental Table 2 in liver tissues (n = 6 animals per group) was performed with real-time reverse transcription-polymerase chain reaction (RT-PCR) as previously reported (Murayama et al, 2018). The relative differences in gene expression were calculated using the threshold cycle (CT) values that were first normalized to those of the hypoxanthine phosphoribosyl transferase 1 (Hprt1) gene, the endogenous control in the same sample, and then relative to a control CT value using the 2−ΔΔCT method (Livak and Schmittgen, 2001).…”

“…Recently, our group (Yoshida et al, 2017) developed a steatosis-related early hepatocarcinogenesis model in HFD-fed rats based on a medium-term liver assay for 8 weeks (Ito et al, 2003). The HFD-induced hyperlipidemia and steatosis and preneoplastic lesions in the liver, and the effects on the liver were related to altered lipid metabolism and oxidative stress (Murayama et al, 2018;Nakamura et al, 2018).…”

“…In this study, to elucidate the roles of autophagy in NAFLD/NASH-related hepatocarcinogenesis, we determined the expression of the autophagosome markers LC3 and p62 in preneoplastic liver foci, together with gene expression analysis for several autophagy-related genes, including Atg5 and Atg7, and the effects of an autophagy inducer or repressor were assessed in a steatosis-related hepatocarcinogenesis model (Yoshida et al, 2017;Murayama et al, 2018;Nakamura et al, 2018).…”

The Role of Oxidative Stress in NAFLD–NASH–HCC Transition—Focus on NADPH Oxidases

“…Its expression seems to affect cancer progression and metastasis, being implicated in the modulation of KRAS–RAF oncogenic signaling [ 54 ]. Yoshida et al observed that the NOX subunit p22phox is expressed in preneoplastic hepatic foci during HFD-induced hepatocarcinogenesis, and the mineralocorticoid spironolactone in combination with β-glycosyl isoquercitrin is able to prevent these steatosis-induced precancerous lesions by reducing p67phox expression and the number of p22phox-positive cells [ 73 ].…”