Plasma-Activated Solutions Mitigates DSS-Induced Colitis via Restoring Redox Homeostasis and Reversing Microbiota Dysbiosis

Abstract

Ulcerative colitis is a chronic disease that increases the risk of developing colorectal cancer. Conventional medications are limited by drug delivery and a weak capacity to modulate the inflammatory microenvironment. Further, gut microbiota dysbiosis caused by mucosal damage and dysregulated redox homeostasis leads to frequent recurrence. Therefore, promoting mucosal healing and restoring redox homeostasis is considered the initial step in treating ulcerative colitis. Plasma-activated solutions (PAS) are liquids rich in various reactive nitrogen species (RNS) and reactive oxygen species (ROS) and are used to treat multiple diseases. However, its effect on ulcerative colitis remains to be examined. Therefore, using a DSS-induced mice colitis model, it is found that PAS has the potential to treat colitis and prevent its recurrence by promoting intestinal mucosal repair, reducing inflammation, improving redox homeostasis, and reversing gut microbiota dysbiosis. Further, an equipment is designed for preparing PAS without using nitrogen; however, after treatment with the Nitro-free PAS, the therapeutic effect of PAS is significantly weakened or even lost, indicating that RNS may be the main mediator by which PAS exerts its therapeutic effects. Overall, this study demonstrates the treatment of ulcerative colitis as a novel application of PAS.

Keywords: microbiota; plasma activated solutions; redox homeostasis; ulcerative colitis.

Figure 1

Experimental setup and preparation of the plasma‐activated solution.

Figure 2

PAS 2 min has therapeutic potential for DSS‐induced colitis. A) Macroscopic images and B) lengths of colons in each group on day 14. On day 14, the colon lengths of mice in the PBS or PAS‐treated groups were shorter than those in the control group. C) Macroscopic images and D) colon lengths of each group on day 21. On day 21, the colon lengths of mice in PBS‐treated, PAS 30 s, and PAS 1 min groups were shorter than those in the control group, whereas the colon lengths of the PAS 2 min, PAS 10 min, and PAS 30 min groups were slightly shorter than those in the control group. E) Daily body weight changes following treatment. The weight of mice in the PAS 2 min, PAS 10 min, and PAS 30 min groups was recovered to that before the DSS intervention; however, the weight of mice in the PBS‐treated, PAS 30 s, and PAS 1 min groups was still lower than that in the control group. F) Disease activity index (DAI) scores following treatment. The DAI scores of mice in the PAS 2 min, PAS 10 min, and PAS 30 min groups were slightly higher than those in the control group, whereas the DAI scores of mice in the PBS‐treated, PAS 30s, and PAS 1 min groups were significantly higher than those in the control. G) Histological scores of colons on day 14. On day14, the histological scores in the PBS‐ and PAS‐treated groups were higher than those in the control group. H) Histological scores of colons on day 21. On day 21, the histological scores in the PBS‐treated, PAS 30 s, and PAS 1 min groups were higher than those in the control group, while those in the PAS 2 min, PAS 10 min, and PAS 30 min groups were lower than those of the above three groups but slightly higher than those in the control; I) H&E‐stained colon sections on day 14. On day 14, H&E staining of both PBS‐treated and PAS‐treated groups still showed obvious colonic injury. J) On day 21, H&E staining of PBS‐treated, PAS 30s, and PAS 1 min groups still showed obvious colonic injury, while that of the PAS 2 min, PAS 10 min, and PAS 30 min groups was almost normal, with only a small amount of inflammatory cell infiltration remaining. Data were presented as Mean ± SD and comparisons were performed with Student's t‐test (B, D, G, H) or two‐way ANOVA with Tukey's test (E, F). Sample size (n) for each statistical analysis was 5. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns: no significance.

Figure 3

Nitrogen in PAS may be the main component contributing to its therapeutic effect. A) H&E‐stained colon sections. H&E staining showed obvious colonic inflammation and injury after the Nitro‐free PAS treatment. B) Macroscopic images of colons and F) the lengths of colon in each group. After treatment with PAS for two weeks, the colons in the PAS 2 min group were longer than those in the PBS‐treated group. However, after treatment with Nitro‐free PAS, the therapeutic effect disappeared and the colons in Nitro‐free PAS were shorter than those in the PAS 2 min group. C) Daily body weight changes following treatment. After treatment with Nitro‐free PAS, the recovery of weight in the PAS 2 min group disappeared. The weight of mice in the Nitro‐free PAS group was significantly reduced compared to that in the control group; D) Concentrations of nitrate and nitrite in PAS 2 min and Nitro‐Free PAS measured using ion chromatography. Nitrite concentrations in Nitro‐free PAS were significantly lower than those in the PAS 2 min group while nitrite could not even be detected in the Nitro‐Free PAS. E) DAI scores following treatment. The DAI scores of the Nitro‐Free PAS group were significantly higher than those in the control and PAS 2 min groups. G) Histological scores of colons. On day 21, H&E staining of Nitro‐Free PAS groups still showed obvious colonic injury, whereas H&E staining of the PAS 2 min group almost returned to normal. H) Representative graph of nitrate and nitrite concentrations measured using ion chromatography. I) The concentration of nitrite under different treatment durations. The overall trend was that the concentration of nitrite increased gradually with the extension of treatment time. The concentration of nitrite in the PAS 30 min group was significantly higher than that in the other groups. No statistically significant difference was found between the PAS 2 min and 10 min groups, but the levels in both were higher than those in the shorter PAS treatment. J–L) The concentration of nitrite under different storage conditions. The concentration of nitrite in PAS was stable at room temperature within 24 h. M) The concentration of nitrate under different treatment durations. The concentration of nitrite increased gradually with the extension of treatment time. No statistically significant difference was found between PAS 2 and 10 min, but the concentrations in both were higher than those in the shorter PAS treatments. N–P) The concentration of nitrate under different storage conditions. At room temperature, the concentration of nitrate remained stable within 24 h. Data were presented as Mean ± SD and comparisons were performed with Student's t‐test (D, F, G, I‐P) or two‐way ANOVA with Tukey's test (C, E). Sample size (n) for each statistical analysis was 5. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns: no significance.

Figure 4

The PAS 2 min treatment alleviated inflammation, regulated redox homeostasis, and promoted mucosal repairing. A) Daily body weight changes following treatment. After PAS 2 min treatment for two weeks, the weight of the mice returned to normal and was significantly higher than that of the PBS‐treated animals. Further, there was no statistically significant difference in weight between the control and control+PAS groups. B) Disease activity index (DAI) scores following treatment. The DAI scores of mice returned to normal levels after 2 weeks of PAS 2 min treatment, whereas the scores of the PBS group remained at a high level. C) Macroscopic images of the colons and D) colon lengths. The colon lengths of mice in the PBS‐treated groups were still shorter than those in the control group, whereas those in the PAS 2 min group were only slightly shorter than those in the control group. E) Histological scores of colons. Histological scores of colons in the PBS‐treated group were higher than those in the control group, whereas those in the PAS 2 min group were lower than those in the PBS‐treated group but slightly higher than those in the control. F–H) The concentrations of TNF‐α, IL‐6, and IL‐1β in colon tissue. The increased concentrations of TNF‐α, IL‐6, and IL‐1β in colon tissue induced by DSS could be reversed by the PAS 2 min treatment for 2 weeks. I) MDA concentrations in colon tissue. The concentration of MDA was upregulated in the DSS+PBS group compared with that in the PAS 2 min and control groups. J, K) Concentrations of SOD/CAT in the colon tissue. The concentrations of SOD/CAT were decreased in the DSS+PBS group compared with those in the PAS 2 min and control group. N) Representative fluorescent images of TUNEL staining in the colonic sections. Scale bars represent 100 µm. O) Representative transmission electron microscopy (TEM) images for the microstructure of colonic epithelia. P) Expression of ZO‐1 determined using confocal immunofluorescence. Q) Expression of occludin determined using confocal immunofluorescence. Data were presented as Mean ± SD and comparisons were performed with Student's t‐test (D‐K) or two‐way ANOVA with Tukey's test (A, B). Sample size (n) for each statistical analysis was 5. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns: no significance.

Figure 5

PAS microbiota transplantation relieves colitis better than the PAS‐derived sterile filtrate. A) Daily body weight changes following treatment. The weight of mice in the PAS‐FMT group was significantly higher than the that in the PAS‐SFF, CON‐FMT, and CON‐SFF groups. B) Disease activity index (DAI) scores following treatment. DAI scores in the PAS‐FMT group were significantly lower than those in the PAS‐SFF, CON‐FMT, and CON‐SFF groups. C) Macroscopic images of colons. The mice in the PAS‐FMT group had the least severe colon damage and inflammation. D) Colon lengths. The colons of mice in the PAS‐FMT group were significantly longer than those in the PAS‐SFF, CON‐FMT, and CON‐SFF groups. E) Histological scores of colons. The histological scores of colons in the PAS‐FMT group were the lowest among the four groups. F–H) Concentrations of pro‐inflammatory cytokines. I) MDA concentrations in colon tissue. The concentration of MDA in the PAS‐FMT group was lower than that in the other three groups. J) Concentrations of CAT in colon tissue. The concentrations of CAT in the PAS‐FMT and PAS‐SFF groups were lower than those in the other two groups, but the difference between the PAS‐FMT and PAS‐SFF groups was not statistically significant. K) SOD concentrations in colon tissue. The concentration of SOD in the PAS‐FMT group was lower than that in the other three groups. L) H&E‐stained colon sections. Colon injury was the mildest in mice of the PAS‐FMT group among the four groups. M) Alcian blue‐periodic acid‐Schiff (AB‐PAS)‐stained colon sections. N) Representative fluorescence images of TUNEL staining in the colonic sections. Scale bars represent 100 µm; O) Representative transmission electron microscopy (TEM) images for the microstructure of colonic epithelia. Data were presented as Mean ± SD and comparisons were performed with Student's t‐test (D–K) or two‐way ANOVA with Tukey's test (A, B). Sample size (n) for each statistical analysis was 5. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns: no significance.

Figure 6

PAS 2 min treatment can restore intestinal bacterial homeostasis. A) Operational taxonomic unit (OTU)‐based group rank Abundance curve. The Rank Abundance Curve indicated that the richness and diversity of intestinal bacteria in the PBS intervention group were significantly lower than those in the control and PAS treatment groups. B) Shannon index and C) Chao1 index between the three groups. The Shannon and Chao1 indices were higher in the PAS intervention group than in the PBS intervention group. D) Weighted Unifrac distance principal coordinate analysis (PCoA) analysis based on OTUs. The bacterial composition of the control and PAS groups was similar, whereas that of the PBS group was significantly different. E) Phylogenetic relationships at the genus level based on OTUs. The Lactobacillus genus was increased after the PAS 2 min treatment. F,G) Bar chart of the relative abundance at the family and genus level of the top ten groups. H) Species notes and abundance information heat maps for each sample at the genus level. I) SIMPER (Similarity Percentage) analysis at the genus level. J) Difference between DSS+PBS and DSS+PAS at the genus level. PAS 2 min treatment increased the abundance of Lachnoclostridium. K) Analysis of differences in microbial taxa shown by Linear discriminant analysis Effect Size (LEfSe). At the species level, a significant enrichment in Helicobacter typhlonius was observed in the PBS treatment group, whereas enrichment in Lactobacillus reuteri could be seen at the species level in the PAS‐treated group; L) FAPROTAX functional prediction enrichment analysis based on OTUs. The significant enrichment in diarrhea pathways in the PBS group was reversed after PAS intervention.

Figure 7

PAS 2 min pretreatment can partly prevent DSS‐induced mucosal damage. Daily body weight changes following treatment. After 2 weeks of PAS 2 min pre‐treatment, the weight loss of mice was not as obvious as that in the DSS group. B) Disease activity index (DAI) scores following treatment. The DAI scores in the PAS 2 min pre‐treatment group were lower than those in the DSS group. C) Macroscopic images of colons. PAS 2 min pre‐treatment can partly prevent DSS‐induced colon injury and inflammation. D) The lengths of colons in each group. The lengths of colons in the PAS 2 min pre‐treatment group were longer than those in the DSS group. E) Histological scores of colons. The histological scores of colons in the PAS 2 min pre‐treatment group were lower than those in the DSS group. F) The concentrations of TNF‐α in colon tissue. The concentration of TNF‐α was higher in the DSS group than that in the PAS 2 min pre‐treatment group. G) The concentrations of IL‐6 in colon tissue. There were no statistically significant differences between the PAS 2 min pre‐treatment and DSS groups, and both showed upregulation compared with that in the control group. H) The concentrations of IL‐1β in colon tissue. The concentration of IL‐1β was higher in the DSS group than in the PAS 2 min pre‐treatment group. I) The concentrations of MDA in colon tissue. There was no statistically significant difference between the PAS 2 min pre‐treatment and DSS groups, and both were upregulated than those in the control group; J) The concentrations of SOD and K) CAT in colon tissue. The concentrations of SOD/CAT were downregulated in both the DSS and PAS 2 min pre‐treatment groupd compared with those in the control group. L) H&E stained and M) Alcian blue‐periodic acid‐Schiff (AB‐PAS)‐stained colon sections. N) Representative fluorescence images of TUNEL staining in the colonic sections. Scale bars represent 100 µm. O) Representative transmission electron microscopic images (TEM) for colonic epithelial microstructure. Data were presented as Mean ± SD and comparisons were performed with Student's t‐test (D–K) or two‐way ANOVA with Tukey's test (A, B). Sample size (n) for each statistical analysis was 5. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns: no significance.