Acid and Neutral Sphingomyelinase Behavior in Radiation-Induced Liver Pyroptosis and in the Protective/Preventive Role of rMnSOD

Abstract

Sphingomyelins (SMs) are a class of relevant bioactive molecules that act as key modulators of different cellular processes, such as growth arrest, exosome formation, and the inflammatory response influenced by many environmental conditions, leading to pyroptosis, a form of programmed cell death due to Caspase-1 involvement. To study liver pyroptosis and hepatic SM metabolism via both lysosomal acid SMase (aSMase) and endoplasmic reticulum/nucleus neutral SMase (nSMase) during the exposure of mice to radiation and to ascertain if this process can be modulated by protective molecules, we used an experimental design (previously used by us) to evaluate the effects of both ionizing radiation and a specific protective molecule (rMnSOD) in the brain in collaboration with the Joint Institute for Nuclear Research, Dubna (Russia). As shown by the Caspase-1 immunostaining of the liver sections, the radiation resulted in the loss of the normal cell structure alongside a progressive and dose-dependent increase of the labelling, treatment, and pretreatment with rMnSOD, which had a significant protective effect on the livers. SM metabolic analyses, performed on aSMase and nSMase gene expression, as well as protein content and activity, proved that rMnSOD was able to significantly reduce radiation-induced damage by playing both a protective role via aSMase and a preventive role via nSMase.

Keywords: SOD; acid sphingomyelinase; liver; neutral sphingomyelinase; radiation.

Figure 1

Mouse liver after irradiation with or without protective or preventive rMnSOD treatment (a) representative liver histology by Caspase-1 immunohistochemical staining. CTR, control mice; rMnSOD, mice treated with human recombinant manganese superoxide dismutase; 0.25 Gy, 0.5 Gy, and 1.0 Gy, mice exposed to increasing radiation doses; 0.25 Gy + rMnSOD, 0.5 Gy + rMnSOD, and 1.0 Gy + rMnSOD, mice exposed to increasing radiation doses and treated with rMnSOD (protective role of rMnSOD); rMnSOD+1.0Gy, mice pretreated with rMnSOD and exposed to 1.0 Gy radiation (preventive role of rMnSOD). Images are representative of 3 similar images from each group of mice (20× magnification). White arrows indicate positive caspase labelling; red arrows indicate cell membrane ruptures. (b) Quantification of Caspase-1 staining was performed using the ImageFocus software. Positive staining is indicated as low (+), medium (++), or high (+++). Only high positive staining was considered and was measured as a percentage of the total area. Data represent the mean + S.D. of three livers for each group. Significance, * p < 0.05 with respect to the CTR, ^§ p < 0.05 with respect to the irradiated samples, ^{^} p < 0.05 with respect to 1.0 Gy + rMnSOD.

Figure 2

Effect of radiation and rMnSOD on SMPD1 and SMPD2 gene expression in the liver. SMPD1 and SMPD2 gene expression evaluated by RTqPCR as reported in the “Materials and Methods” section. Liver from mice treated with increasing doses of radiation with or without rMnSOS. (a) SMPD1 (b) SMPD2. Data are expressed as the mean + SD of three liver samples, each carried out in triplicate. Significance: (a) * p < 0.05 versus the control sample (CTR); (b) ^§ p < 0.05 rMnSOD treated and irradiated samples versus the irradiated samples; (c)^{^} p < 0.05 pretreated and 1.0 Gy irradiated sample versus 1.0 Gy irradiated and rMnSOD treated samples. CTR, control mice; rMnSOD, mice treated with human recombinant manganese superoxide dismutase; 0.25 Gy, 0.5 Gy, and 1.0 Gy, mice exposed to increasing radiation doses; 0.25 Gy + rMnSOD, 0.5 Gy + rMnSOD, and 1.0 Gy + rMnSOD, mice exposed to increasing radiation doses and treated with rMnSOD (protective role of rMnSOD); rMnSOD + 1.0 Gy, mice pretreated with rMnSOD and exposed to 1.0 Gy radiation (preventive role of rMnSOD).

Figure 3

Effect of radiation and rMnSOD on the aSMase and nSMase1 protein level in the liver. The aSMase and nSMase1 level evaluated by Western Blotting, as reported in the “Materials and Methods” section. Liver from the mice treated with increasing doses of radiation with or without rMnSOS. (a) western blotting panel; (b) densitometric analysis performed using the ImageFocus software. Data are expressed as a percentage with respect to the control sample and represent the mean + SD of three liver samples, each carried out in triplicate. Significance: (a) * p < 0.05 versus the control sample (CTR); (b) ^§ p < 0.05 rMnSOD treated and irradiated samples versus irradiated samples; (c) ^{^} p < 0.05 pretreated and 1.0 Gy irradiated samples versus the 1.0 Gy irradiated and rMnSOD treated samples. CTR, control mice; rMnSOD, mice treated with human recombinant manganese superoxide dismutase; 0.25 Gy, 0.5 Gy, and 1.0 Gy, mice exposed to increasing radiation doses; 0.25 Gy + rMnSOD, 0.5 Gy + rMnSOD, and 1.0 Gy + rMnSOD, mice exposed to increasing radiation doses and treated with rMnSOD (protective role of rMnSOD); rMnSOD + 1.0 Gy, mice pretreated with rMnSOD and exposed to 1.0 Gy radiation (preventive role of rMnSOD).

Figure 4

Effect of radiation and rMnSOD on aSMase and nSMase activity in the liver. aSMase and nSMase activity evaluated using an Amplex Red Sphingomyelinase assay kit, as reported in the “Materials and Methods” section. Livers from the mice treated with increasing doses of radiation with or without rMnSOS. (a) aSMase; (b) nSMase. Data are expressed as mU/mg protein/min and represent the mean+SD of three liver samples, each carried out in triplicate. Significance: (a) * p < 0.05 versus the control sample (CTR); (b) ^§ p < 0.05 rMnSOD treated and irradiated samples versus the irradiated samples; (c) ^{^} p < 0.05 pretreated and 1.0 Gy irradiated sample versus the 1.0 Gy irradiated and rMnSOD treated sample. CTR, control mice; rMnSOD, mice treated with human recombinant manganese superoxide dismutase; 0.25 Gy, 0.5 Gy, and 1.0 Gy, mice exposed to increasing radiation doses; 0.25 Gy + rMnSOD, 0.5 Gy + rMnSOD, and 1.0 Gy + rMnSOD, mice exposed to increasing radiation doses and treated with rMnSOD (protective role of rMnSOD); rMnSOD + 1.0 Gy, mice pretreated with rMnSOD and exposed to 1.0 Gy radiation (preventive role of rMnSOD).