Suppressive effects of electrolyzed reduced water on alloxan-induced apoptosis and type 1 diabetes mellitus

Abstract

Electrolyzed reduced water, which is capable of scavenging reactive oxygen species, is attracting recent attention because it has shown improved efficacy against several types of diseases including diabetes mellitus. Alloxan produces reactive oxygen species and causes type 1 diabetes mellitus in experimental animals by irreversible oxidative damage to insulin-producing β-cells. Here, we showed that electrolyzed reduced water prevented alloxan-induced DNA fragmentation and the production of cells in sub-G1 phase in HIT-T15 pancreatic β-cells. Blood glucose levels in alloxan-induced type 1 diabetes model mice were also significantly suppressed by feeding the mice with electrolyzed reduced water. These results suggest that electrolyzed reduced water can prevent apoptosis of pancreatic β-cells and the development of symptoms in type 1 diabetes model mice by alleviating the alloxan-derived generation of reactive oxygen species.

Fig. 1

Anti-apoptotic effects of ERW on alloxan (ALX)-treated HIT-T15 cells assessed by TUNEL assay (a), and sub-G1 analysis (b). (a) Anti-apoptotic effects of ERW on ALX-treated HIT-Ti 5 cells. HIT-T15 cells were treated with 1 mM ALX for 4 h after cultivation in ultrapure water (UPW) containing 2 mM NaOH-based medium or ERW-based medium for 24 h. The pHs of UPW containing 2 mM NaOH and ERW were neutralized by 12 mM HEPES in media. Apoptotic cells were then detected by the TUNEL procedure and analyzed by flow cytometry. UPW(−) means UPW containing 2 mM NaOH-based medium without ALX treatment; UPW(+) means ultrapure water containing 2 mM NaOH with 1 mM ALX treatment; ERW(+) means ERW-based medium with ALX treatment. Values are means ± S.E.M. from three independent experiments. Plus symbols represent significant differences observed between UPW(−) and UPW(+); p < 0.001. Asterisks represent significant differences observed between UPW(+) and ERW(+); *** p < 0.001. (b) Effects of ERW on the cell population in the sub-G1 phase. Cell cycle analysis was carried out using PI staining and flow cytometry, after HIT-T15 cells were treated in the same conditions as in Fig. 1a. Sub-diploidy apoptotic cells (sub-G1) are expressed as a percentage on the left side of each profile. The graph shown is a typical result of three independent experiments. Sample designations are the same as in Fig. 1a

Fig. 2

Schematic representation of the in vivo study protocol. Six-week-old CD-1 mice were treated with UPW or ERW for up to 9 weeks. Blood samples were taken at the indicated time points and used for the blood glucose assay (B.G.A. indicated by upward arrow, ↑). Alloxan (ALX) was administered intraperitoneally (I.P.), as indicated by a downward arrow at the indicated time points (↓). Further detail is given in the Materials and methods

Fig. 3

The effects of ERW on blood glucose levels of normal and ALX-induced diabetic CD-1 mice. CD-1 mice were treated as described in the study protocol (Fig. 2). Blood glucose levels at the 0, 4 and 8th week time points are presented. Each value denotes the means ± S. E. M., calculated from the data of survived mice. Plus symbols represent significant differences observed between UPW-administrated mice without ALX treatment (UPW(−)ALX) and UPW-administrated mice with ALX treatment (UPW(+)ALX) (⁺⁺⁺p < 0.001) or ERW-administrated mice with ALX treatment (ERW(+)ALX) (⁺p < 0.05). Asterisks represent significant differences observed between UPW(+)ALX and ERW(+)ALX (*** p < 0.001)

Fig. 4

The effect of ERW on serum insulin levels of ALX-administered CD-1 mice. At the end of the study shown in Fig. 2, blood samples collected via orbital sinus puncture was centrifuged and sera were collected. The serum insulin concentration was determined using an insulin ELISA kit. Values are means ± S.E.M., calculated from the data of survived mice. Asterisks represent significant differences observed between UPW or ERW fed mice followed by ALX administration; *** p < 0.001. Sample designations are the same as in Fig. 3

Fig. 5

The effects of ERW on body weight (a), food (b) and water (c) consumptions of ALX-induced type 1 diabetes model mice. (a) Body weight was measured at the time of experimental initiation (designated as 0 weeks), and the 4th and 8th week. Asterisks represent significant differences observed between UPW(−)ALX and ERW(+)ALX; ** p < 0.01. Values are means ± S.E.M., calculated from the data of survived mice. The time course of food (b) and water (c) consumptions in normal and ALX-administrated mice was measured at the indicated time points. In the case of food and water consumption measurements, three mice were placed in a cage and one group is composed of three cages, and food and water were measured as a total weight per cage. Thus the values are expressed as means ± S.E.M. (n = 3). * p < 0.05 and *** p < 0.001, compared with UPW(+)ALX. Sample designations are the same as in Fig. 3