Experimental strategy and mechanism. (A) Nano-bubble disk. (B) Hydrogen emission from nano-bubbles. (C) Molecular hydrogen blood–brain barrier penetration. (D) The anti-inflammatory effect of hydrogen. (E) The antioxidant effect of hydrogen. (F) The regulation of intestinal flora by hydrogen. (G) Behavioral tests. (H) Biochemical index detection. (I) Fluorescence observation of zebrafish larvae.

Device component schematic and safety verification. (A) Schematic diagram of the capture device. (a) The change in hydrogen concentration in the unit over a 12-h period. (B) SEM image of nano-bubble disk. (C) Minimum distance between individuals. (D) Mean distance. (E) The Trace heat maps of unstart group, start group, 5 min after the shutdown (n = 10 per group). The data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significance was defined as follows versus unstart group: NS - not significant.

(A) Diagram of T-maze device. (B) Time trend diagram of zebrafish finding deep water area in different groups. (C) Time it takes the zebrafish to find deep water at 0 h. (D) Time it takes the zebrafish to find deep water at 3 h. (E) Time it takes the zebrafish to find deep water at 24 h (n = 5 per group). The data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significance was defined as follows versus AD group: *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant.

(A) Schematic diagram of NTT device. (B) Track chart and heat map of each group during capture time. (C) The average speed of each group. (D) The total distance traveled. (E) The number of entries the top. NTT (n = 7 per group). The data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significance was defined as follows versus AD group: *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant.

(A) Schematic diagram of the shoal installation. (B) Track heat map of each group during capture time. (C) Minimum distance between individuals. (D) Average speed (n = 10 per group). The data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significance was defined as follows versus AD group *p < 0.05; **p < 0.01.

According to different zebrafish groups, the brain neutral particle fluorescence intensity, whole brain pathological section and whole brain tissue inflammatory factor levels. (A) Fluorescent aggregation of neutrophils in the brain of zebrafish (n = 3 per group). (B) Total fluorescence intensity. (C) Brain H&E staining, (D) TNF-α. (E) IL-6. (F) IL-1β. (G) IL-10 (n = 12 per group). The data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significance was defined as follows versus AD group *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Detection of brain AD factor and liver sEHs in Zebrafish. (A) Inspection operation flow. (B) Aβ1-42. (C) sEH (n = 12 per group). The data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significance was defined as follows versus AD group: *p < 0.05, ****p < 0.0001.

The whole brain oxidative stress factor and whole body ROS fluorescence intensity were analyzed according to different groups of zebrafish. (A) ROS juvenile fish dye fluorescence. (B) MDA. (C) CAT. (D) GSH. (E) SOD (n = 12 per group). The data are expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significance was defined as follows versus AD group *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Effects of HRW on intestinal microorganisms of AD zebrafish. (A) Simpson index was used to characterize the diversity and uniformity of species distribution in zebrafish intestinal communities. (B) Shannon index was used to determine intestinal community diversity of zebrafish. (C) Comparison of intestinal flora β diversity between control group, AD group, and HRW group. (D) Venn diagram of species abundance. (E) Genus level intestinal microbial abundance. (F) Heat maps of the top 10 genera with average abundance were obtained by UPGMA clustering.

The mechanism of hydrogen-based therapy for AD: (A) Antioxidant effect. (B) Anti-inflammatory effect. (C) Regulation of hepatic sEH level. (D) Regulation of intestinal flora homeostasis.