Renoprotective and neuroprotective effects of enteric hydrogen generation from Si-based agent

Abstract

We have developed Si-based agent which can generate a large amount of hydrogen. Si-based agent continues generating hydrogen for more than 24 h by the reaction with water under conditions similar to those in bowels, i.e., pH8.3 and 36 °C, and generates ~400 mL hydrogen. To investigate beneficial effects for diseases associated with oxidative stress, Si-based agent is administered to remnant kidney rats and Parkinson's disease mice. Rats are fed with control or Si-based agent-containing diet for 8 weeks. Si-based agent is found to greatly suppress the development of renal failure and the parameters of oxidative stress. Treatment with Si-based agent in a mouse model of hemi-Parkinson's disease induced by 6-hydroxydopamine attenuated degeneration of dopaminergic neurons and prevented impairment of motor balance and coordination. These findings indicate that the Si-based agent shows renoprotective and neuroprotective effects presumably via suppression of oxidative stress by generation of hydrogen.

Figure 1

Hydrogen volume generated by the reaction of Si-based agent with water at 36 °C. (a) pH of the solution is adjusted at 8.3 using NaHCO₃. (b,c) pHs of the solutions are adjusted at 8.5 and 9.0, respectively, using NaHCO₃ plus Na₂CO₃. Si-based agent is composed of Si fine particles covered with silicon oxide which includes high amounts of suboxide species (cf. supplementary Fig. 3), i.e., Si atoms bound to one, two, and three oxygen atoms, each. Si fine particles aggregate to the sizes larger than 0.1 μm.

Figure 2

The effects of Si-based agent on renal function and the impacts of oxidative stress. (a) The effects of Si-based agent on serum creatinine. (b) The effects of Si-based agent on urinary protein. (c) 8-hydroxydeoxyguanosine (8-OHdG) staining after for measuring the impact of oxidative stress. The dark brown dots (arrow) correspond to representative 8-OHdG-positive nuclei in the control group (i) or the Si-based agent (0.1%–1.0%) groups (ii-iv). Scale bar: 100 µm. We counted the number of 8-OHdG-positive cells per 100 cells in ten random areas of each rats (v). In addition, we measured the urinary 8-OHdG level with ELISA kit (vi). Data were expressed as mean +/− SD. **p < 0.01, *p < 0.05 vs. the control group, respectively.

Figure 3

Representative morphological changes in the control group (a,e) or the Si-based agent (0.1wt.%: b,f, 0.5wt.%: c,g, 1.0wt.%: d,h) groups, respectively. Scale bar: 100 µm. The dilations of glomeruli and the increase of mesangial matrix (arrowhead) are examined. The diameters of glomeruli were assessed using a color image analyzer and compared among each group (i). Mesangial matrix levels were assessed according to previously described method scored from 0 to 4 with α-SMA staining: 0, no mesangial matrix expansion; 1, minor; 2, weak; 3, moderate; and 4. The scores were averaged and compared among each group (j). (**p < 0.01, *p < 0.05).

Figure 4

The anti-apoptotic and anti-inflammatory effects of Si-based agent on renal function. (a) Interstitial phenotypic changes are assessed by immunohistochemical staining of α-SMA (arrow) in the control group (i) or the Si-based agent (0.1wt.%-1.0wt.%) groups (ii-iv). The positive area was quantitatively assessed using a color image analyzer (v). (b) TUNEL method was performed for measuring the levels of apoptosis in each group as describe above (i-iv). We counted the number of TUNEL-positive cells (arrow) per 100 cells in ten random areas of each rats (v). Scale bar: 100 µm. (c-f) The normalized messenger RNA (mRNA) level of caspase-3 (c), C-C motif chemokine ligand 2 (CCL2) (d), interleukin-6 (IL-6) (e), and a tissue inhibitor of metalloproteinases (TIMP-1, f) were measured. The levels are normalized to β-actin levels for each sample. The y-axis values represent the number of copies relative the number of copies in the same samples. The levels are normalized to β-actin levels for each sample. The y-axis values represent the number of copies relative the number of copies in the same samples. Data were expressed as mean +/− SD. **p < 0.01, *p < 0.05 vs. the control group, respectively.

Figure 5

Neuroprotective effects of Si-based agent in 6-OHDA-induced hemiparkinsonian mice. (a) The histogram of the number of an unaffected side turn (right hand turn) in apomorphine-induced rotation test. The control group (white bar, first from left), the Si-based agent group (black bar, second from left), the 6-OHDA group (white bar, third from left) and the 6-OHDA + Si-based agent group (black bar, fourth from left). Data were expressed as means ± SEM of six (Control group), six (Si-based agent group), seven (6-OHDA group) or eight animals (6-OHDA + Si-based agent group). **P < 0.01 vs. the 6-OHDA group (Student’s t-test). (b) The micrographs of tyrosine hydroxylase staining for substantia nigra pars compacta (left side) and striatum (right side). White arrow indicated lesion site. Opposite side was contralateral control. Upper images: the 6-OHDA group. Lower images: 6-OHDA and silicon groups. Scale bar: 200 µm (left) and 1 mm (right). (c) The histogram of the time that mice stayed on a rotating rod in rotarod test. (d) The histogram of the spontaneous motor activity measured by Supermex system. (e–g) The histograms of the following parameters evaluated by Open field test: total travel distance (e), the average speed (f) and Active or inactive time (g). Control group (white bar, first from left), silicon group (black bar, second from left), 6-OHDA group (white bar, third from left) and 6-OHDA + silicon group (black bar, fourth from left). All data were expressed as means ± SEM of six (Control group), six (Si-based agent group), seven (6-OHDA group) or eight animals (6-OHDA + Si-based agent group). *P < 0.05 or **P < 0.01 vs. 6-OHDA group (Student’s t-test).