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Dietary supplementation with biogenic selenium nanoparticles alleviate oxidative stress-induced intestinal barrier dysfunction


Effects of different dietary SeNPs supplementation on growth performance and Se content of mice

The 10-week supplementation period with different SeNPs-containing diets (0.0-Se, 0.3-Se, and 0.6-Se mg/kg) resulted in marked variations in body weight, food intake and feed conversion rate [FCR = (final weight-initial weight)/feed intake, g/g] of the mice (Fig. 1b–d). Final body weight was higher in mice fed the 0.6-Se diet than in those fed the 0.0-Se diet, but there were no significant differences with the group fed the 0.3-Se diet. The final feed conversion rate of mice in the 0.3-Se and 0.6-Se supplementation groups were significantly lower compared with that of mice in the 0.0-Se group. However, compared with the 0.3-Se group mice, the feed conversion rate of the 0.6-Se group mice was the lowest. This result indicated that the feed conversion efficiency of the 0.6-Se group is the highest. The content of Se in organs, serum, and feces after ten weeks of supplementation was dietary Se concentration-dependent (Fig. 1e–h and Supplementary Fig. 1). Se concentration in liver, kidney, serum, and small intestine of the 0.0-Se group was significantly lower compared with that in the 0.3-Se and 0.6-Se groups, and the Se content in the 0.6-Se group was higher than that in the 0.3-Se group. This implied decreased Se availability in mice in the 0.0-Se and 0.3-Se groups. Se exerts its physiological functions in nutrition, metabolism and immunity largely through selenoproteins. The effects of different SeNPs-containing diets on the expression of selenoprotein genes in the liver and jejunum of mice are shown in Supplementary Fig. 2. Dietary SeNPs supplementation upregulated the expression of most selenoprotein genes in liver (except GPX1, GPX4, SELENOI, SELENOO, and SELENOW) in a dose-dependent manner. Similarly, except for the extremely low expression of the DIO enzyme family and SELENOV in the jejunum, the mRNA expression of most other selenoproteins also increased with the increase of the Se content. Overall, these results supported the notion that the different dietary SeNPs supplementation had an effect on the expression levels of selenoprotein genes in these mice.

Fig. 1: Effects of different dietary SeNPs supplementation on growth performance and Se content of mice.
figure 1

a Schematic diagram of experiment. b Body weight and c food intake was recorded throughout the 10-week period experiment. d Feed conversion rate. eh The Se content in the liver, kidney, serum, and small intestine, respectively, (n = 10). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Effects of different dietary SeNPs supplementation on the intestinal barrier function in mice exposed to diquat

The survival rates of mice exposed to diquat in 0.0-Se, 0.3-Se, and 0.6-Se groups were 66.67%, 100%, and 100%, respectively (Fig. 2a). The detection of biomarkers related to intestinal barrier dysfunction, including FITC-dextran, Diamine oxidase (DAO), D-lactic acid (D-LA), and tight junction proteins (ZO-1, occludin, and claudin1) expression (Fig. 2b–e and Supplementary Fig. 3a–c) revealed that in the normal control group, the activity of DAO and the level of D-LA in the Se-deficient group were significantly higher than those in the dietary SeNPs supplementation group. Conversely, significantly decreased levels of expression of tight junction proteins were detected in mice fed with 0.0-Se diet compared with those in the dietary SeNPs supplementation group. Moreover, compared with the normal control group, the activity of DAO, the levels of D-LA and FITC-dextran in the jejunum of diquat-exposed mice were significantly increased, and the expression of tight junction proteins was significantly decreased. However, dietary biogenic SeNPs supplementation (especially at supernutritional level, 0.6-Se mg/kg) effectively alleviated the intestinal barrier injury caused by diquat. To further assess the jejunum injury, histology was performed on jejunal tissue sections and the villus height and the number of goblet cells in hematoxylin and eosin (H&E) and alcian blue (AB)-periodic acid schiff (PAS) stained tissue sections were determined (Fig. 2f and g). Also, compared with the normal control group, the jejunal villi were disorderly arranged, the villus height was shortened (Supplementary Fig. 3d), and the number of goblet cells was decreased in mice exposed to diquat. However, compared with the diquat-induced oxidative stress model group, dietary 0.6-Se mg/kg supplementation significantly increased the height of the villi and the number of goblet cells (Supplementary Fig. 3e). The mRNA expression levels of mucin 2 (MUC2) and regenerating family member 3gamma (REG3G) in jejunum were detected by qPCR analysis. As shown in Fig. 2h and i, in normal control group, different dietary SeNPs supplements did not affect the expression of MUC2, but compared with the 0.3-Se and 0.6-Se groups, the expression of REG3G in the 0.0-S group was significantly reduced. In the diquat-induced oxidative stress model group, dietary SeNPs supplementation upregulated the expression of MUC2 and REG3G in the jejunum, and had a dose-dependent effect. This indicates that biogenic SeNPs effectively alleviated the diquat-induced intestinal barrier dysfunction.

Fig. 2: Effects of different dietary SeNPs supplementation on the intestinal barrier in mice exposed to diquat.
figure 2

a Survival rate of mice over a 12-h period during diquat treatment. bd Biomarkers related to intestinal barrier function damage including FITC-dextran (b) (n = 6), DAO (c) (n = 8), D-LA (d) (n = 8). e The expression levels of tight junction proteins (ZO-1, occludin, claudin 1) detected by Western blot analysis (n = 3). f Jejunum morphology was observed by H&E staining (n = 4). g Goblet cells in the jejunum were observed by AB-PAS staining. h mRNA expression levels of MUC2 in the jejunum (n = 6). i mRNA expression levels of REG3G in the jejunum (n = 6). Data are expressed as the fold change versus the 0.3-Se group or 0.3-Se + diquat group (set to 1). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Effects of different dietary SeNPs supplementation on the antioxidant capacity and immune responses

As expected, decreased Se availability weakened the antioxidant capacity of jejunum by decreasing total antioxidant capacity (T-AOC), superoxide dismutase (SOD), GPx and TrxR levels, and increased the level of the lipid peroxidation product, malondialdehyde (MDA). Moreover, diquat exposure significantly aggravated the decline in the antioxidant capacity of the jejunum. Noteworthy, the addition of 0.6-Se mg/kg of SeNPs to the diet can effectively alleviate the decrease in the antioxidant capacity of the mice jejunum caused by diquat (Fig. 3a–e). The levels of biomarkers related to immune responses in the serum and jejunum were also markedly altered by different dietary SeNPs supplementation from diquat-exposed and normal control groups (Fig. 3f–h and Supplementary Fig. 4). The results demonstrated that Se deficiency can lead to an increase in the levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18), and a decrease in the level of secretory immunoglobulin A (sIgA). Moreover, the 0.6-Se group exposed to diquat had significantly lower levels of IL-1β and IL-18 and higher level of sIgA compared with diquat-exposed mice fed either 0.0 or 0.3 mg/kg Se diets. Notably, mice in the 0.3-Se diet were not significantly different from those in the 0.0-Se group. Thus, our results showed that 0.6 mg/kg SeNPs supplementation can improve the antioxidant capacity and relieve intestinal inflammation in diquat-exposed mice.

Fig. 3: Effects of different dietary SeNPs supplementation on the antioxidant capacity and immune response in diquat-exposed mice.
figure 3

ae Oxidative stress response including T-AOC (a), MDA (b), SOD (c), GPx (d), TrxR (e) (n = 8). f Level of IL-1β in the jejunum of mice (n = 8). g Level of IL-18 in the jejunum of mice (n = 8). h Level of sIgA in the jejunum of mice (n = 8). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Dietary SeNPs supplementation effectively alleviated diquat-induced intestinal mitochondrial dysfunction

Se deficiency caused ROS overproduction compared with the 0.3-Se and 0.6-Se groups, and the 0.6-Se group mice exposed to diquat had significantly decreased the levels of ROS in jejunal tissues compared with diquat-exposed mice fed either 0.0 or 0.3 mg/kg Se diets (Fig. 4a and b). As the key organelle for energy metabolism and ROS production, mitochondria are closely related to the redox state of cells. Oxidative stress from mitochondria may be a pathophysiological signal of intestinal barrier dysfunction. Thus, mitochondrial ultrastructure was examined by TEM, and the levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG), ATP, and MMP in jejunal mitochondria were determined. In normal control, compared with the 0.3-Se and 0.6-Se groups, the ultrastructure of mitochondria in 0.0-Se group showed apparent changes, such as the swelling was obvious, the mitochondrial cristae were vague, and the density of matrix was low, less mitochondrial membrane was not intact. In addition, the highest degree of mitochondrial ultrastructural destruction (vacuoles were present, and most mitochondrial membranes were incomplete) was observed in the mitochondria of diquat-exposed mice from the 0.0-Se mg/kg diet group. However, the 0.6-Se group mice were significantly alleviated by the diquat-induced destruction of mitochondria, their mitochondria had a column or mesh shape, the mitochondrial cristae were clear, the density of matrix was normal, and the mitochondrial membrane was intact (Fig. 4c). The destruction of mitochondrial structure also affects mitochondrial function. Compared with the SeNPs supplementation group, the levels of ATP and MMP in the mitochondria of the Se-deficient mice from the normal control decreased significantly. Moreover, compared with the normal control, diquat-exposed further reduced the levels of ATP and MMP, and dietary SeNPs (0.6-Se mg/kg) supplementation effectively alleviated diquat-induced intestinal mitochondrial dysfunction (Fig. 4d and e). The level of 8-OHdG (a biomarker of DNA oxidative damage) in the 0.0-Se group mice was significantly higher than that in the 0.3-Se and 0.6-Se group mice relative to the normal control. However, the 0.6-Se mg/kg SeNPs supplementation was able to significantly protect mitochondrial DNA from diquat-induced oxidative stress damage (Fig. 4f). As shown in Fig. 4g, different dietary SeNP supplements did not affect the mtDNA copy number in the normal control group. In the diquat-exposed group, dietary SeNPs supplementation increased the mtDNA copy number in the jejunum in a dose-dependent manner. Various factors involved in the regulation and repair of mammalian mtDNA replication, including mitochondrial transcription factor A (TFAM), DNA Polymerase gamma (POLG), and DNA Polymerase gamma 2 (POLG2), play an important role in the repair of mitochondrial oxidative stress damage (Fig. 4h–j). Notably, different dietary SeNPs supplementation on the expression of genes related to mitochondrial biogenesis in the normal control group was opposite to that in the diquat-exposed group, specifically, the expression of TFAM, POLG, and POLG2 in the chronic Se-deficient diet group was lower than that in the 0.3-Se and 0.6-Se groups. However, in the diquat-exposed group (acute stress), the expression of genes related to mitochondrial biogenesis in the 0.0-Se group was higher than that in the 0.3-Se and 0.6-Se groups.

Fig. 4: Effects of different dietary SeNPs supplementation on jejunal mitochondrial dysfunction in diquat-exposed mice.
figure 4

a, b ROS production was determined by DHE staining (n = 6). c Mitochondrial ultrastructure was observed by TEM. d Mitochondrial ATP levels in the jejunal mitochondria (n = 8). e MMP was measured by JC-1 staining in the jejunal mitochondria (n = 8). f Levels of 8-OHdG in the jejunal mitochondria (n = 8). g The mtDNA copy number was determined by qPCR analysis (n = 6). h mRNA expression level of TFAM in the jejunum (n = 6). i mRNA expression level of POLG in the jejunum (n = 6). j mRNA expression level of POLG2 in the jejunum (n = 6). Data are expressed as the fold change versus the 0.3-Se group or 0.3-Se + diquat group (set to 1). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Dietary SeNPs supplementation altered the composition and metabolism of gut microbiota in mice

Numerous studies have shown that dietary changes will alter the composition of the gut microbiota. Indeed, in this study, the findings of significant differences in body weight, food intake, and feed conversion rate between the 0.0-Se group and the 0.6-Se group suggest that the dietary SeNPs may have altered the composition of the gut microbiota. As shown in Fig. 5a and b, the ACE and Chao1 indices were used to measure the community richness of gut microbiota. However, supplementation with different concentrations of SeNPs did not significantly alter the overall richness of the gut microbiota. The Shannon and Simpson diversity indices were used to evaluate the diversity of gut microbiota (Fig. 5c and d). Compared to the 0.0-Se group, the Shannon index was increased and the Simpson index was decreased in the 0.6-Se group which indicates that dietary SeNPs supplementation can significantly alter the diversity of the gut microbiota of mice. Beta diversity is shown in Fig. 5e, the samples in the 0.6-Se group have a significantly lower level of dispersion than those in the 0.0-Se group. Additional principal coordinates analysis (PCoA) found that the gut microbiota of mice in different SeNPs supplementation groups showed obvious clustering and the 0.6-Se group was clearly separated from the 0.0-Se group, indicating that SeNPs significantly changed the composition of the gut microbiota (Fig. 5f). Biogenic SeNPs induced modulation of the gut microbiota structure at the phylum level, resulting in an enhanced abundance of Bacteroidetes and a reduced abundance of Verrucomicrobia (Fig. 5g and h). The Firmicutes/Bacteroidetes (F/B) ratio has been suggested as an important index of the health of the gut microbiota. A decreasing trend in the F/B ratio was observed in the 0.6-Se group compared with the 0.0-Se group (P = 0.3385, Fig. 5i). At the genus level, significant increases in the levels of Bacteroides and Clostridium_XlVa and decreases in the level of Desulfovibrio were detected in mice fed with the 0.6-Se diet compared with results for mice fed with the 0.0-Se diet (Fig. 5j and k). Similar alterations in the levels of SCFAs were also observed in the cecal contents of mice fed with different SeNPs supplementation doses. Compared with 0.0-Se group, supranutritional Se increased the content of total SCFAs, butyrate, isobutyrate, valerate, and isovalerate (Fig. 5l). Subsequently, using PICRUSt2 software and the KEGG database, we analyzed the difference and changes in metabolic pathways of functional genes of the gut microbiota. The enriched functional categories in the 0.6-Se group included “Human Diseases” at the KEGG level 1 and “Digestive system” at the KEGG level 2 (Supplementary Figs. 5 and 6).

Fig. 5: Effects of different dietary SeNPs supplementation on gut microbiota and SCFAs levels of mice.
figure 5

ad Alpha diversity index (n ≥ 7). e Beta diversity index (n ≥ 7). f PCoA (n ≥ 7). g The relative abundance composition of fecal microbiota at the phylum level (n ≥ 7). h Differences in fecal microbiota between the phylum level (n ≥ 7). i Ratio of Firmicutes to Bacteroides (n ≥ 7). j The relative abundance composition of fecal microbiota at the genus level (n ≥ 7). k Differences in fecal microbiota between the genus level (n ≥ 7). l SCFAs levels in the cecal contents of mice (n = 5). m Heatmap of Spearman’s correlation between the abundance of gut microbiota and the intestinal barrier dysfunction-related biochemical indexes. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

A heatmap of Spearman correlation between the altered genera and intestinal barrier dysfunction-related biochemical indexes (the levels of inflammatory cytokines and antioxidant capacity in jejunum) was generated to identify the potential correlation between Se-induced gut microbiota changes and intestinal barrier dysfunction. As shown in Fig. 5m, Allobaculum was negatively correlated with the antioxidant capacity including T-AOC, GPx, and TrxR, while Helicobacter was positively correlated with the activities of T-AOC, GPx, and TrxR. Moreover, Helicobacter and Bacteroides were positively correlated with the level of sIgA, but negatively correlated with Allobaculum. In addition, for SCFAs, Helicobacter was positively correlated with total SCFAs, butyrate, and isobutyrate, Bacteroides was positively correlated with total SCFAs, and Desulfovibrio was negatively correlated with total SCFAs, acetate, and propionate. It’s worth noting that the abundance of Desulfovibrio significantly increased in the 0.0-Se group, and the abundance of Bacteroides was significantly decreased, illustrating that Se deficiency reduces the abundance of SCFAs-producing bacteria and increases the abundance of pathogenic bacteria that cause oxidative stress.

The Nrf2 signaling pathway inhibits diquat-induced NLRP3 inflammasome activation in the jejunum

To begin to understand the possible mechanisms by which dietary SeNPs may influence the responses to diquat exposure in this mice model, we decided to focus our research on the NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. The reasons for this decision are that mitochondrial dysfunction plays a key role in activating the NLRP3 inflammasome, and ROS released by damaged mitochondria can promote its activation and the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) transcription factor is a key player in cytoprotection and activated in stress conditions caused by ROS. Our results indicated that Se deficiency can lead to an increase in the level of the NLRP3 inflammasome and subsequent IL-1β and IL-18 expression, and diquat-exposed further increased its expression. However, the levels of the NLRP3 inflammasome and subsequent IL-1β and IL-18 expression showed decreasing trends along with the increasing levels of dietary Se in the jejunum of mice exposed to diquat (Fig. 6a and b). Nrf2 is a master regulator of the antioxidant response and has been implicated in a range of chronic diseases that are characteristically associated with oxidative stress. As shown in Fig. 6c and d, dietary SeNPs supplementation upregulated the expression of Nrf2 (total Nrf2 and nuclear Nrf2) and downstream antioxidant proteins NADPH dehydrogenase (NQO)-1 and heme oxygenase (HO)-1 in a dose-dependent manner, regardless of whether they were exposed to diquat or not.

Fig. 6: The Nrf2 signaling pathway inhibits the diquat-induced NLRP3 inflammasome activation in the jejunum.
figure 6

a The expressions of the NLRP3 inflammatory proteins were measured by Western blot analysis (n = 3). b Quantitative analysis of the protein expression levels of NLRP3, ASC, pro-caspase 1, cleaved-caspase 1, IL-1β, and IL-18. c Nrf2 activation and expression levels of its downstream proteins were measured by Western blot analysis (n = 3). d Quantitative analysis of the protein expression levels of total Nrf2, nuclear Nrf2, HO-1, and NQO-1. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Effects of FMT on intestinal barrier in mice exposed to diquat

We further investigated whether the effects of dietary SeNP supplementation on the intestinal barrier dysfunction and immune responses are associated with its modulation of the gut microbiota using the FMT method to bypass the influence of Se itself (Fig. 7a). As shown in Fig. 7b, the average body weight of the mice in the 0.0-Se-FMT group was slightly lower than that in the other SeNP supplementation groups, but there was no significant difference between the other groups before exposure to diquat. Mice colonized with the gut microbiota from Se-deficient donors showed more severe symptoms of diquat-induced intestinal barrier dysfunction compared with mice in the diquat-induced oxidative stress model group and 0.6-Se-FMT groups, as indicated by the levels of FITC-dextran and tight junction proteins (ZO-1, occludin, and claudin1) expression, H&E and AB-PAS stained tissue sections, and the mRNA expression levels of MUC2 and REG3G. Compared with the diquat and 0.6-Se-FMT groups, 0.0-Se-FMT significantly increased the gut permeability of diquat-exposed mice, as indicated by the higher levels of serum FITC-dextran and the lower levels of tight junction proteins expression (Fig. 7c–e). Moreover, the FMT from the 0.6-Se group significantly increased the height of villi (Fig. 7f and Supplementary Fig. 7a), the number of goblet cells (Fig. 7g and Supplementary Fig. 7b), and the mRNA expression levels of MUC2 and REG3G compared with the diquat model and 0.0-Se-FMT groups (Fig. 7h–i).

Fig. 7: Effects of FMT on the intestinal barrier in mice exposed to diquat.
figure 7

a Schematic diagram of the FMT experiment. b Body weight during FMT. c FITC-dextran levels in the serum of mice. (n = 6). d, e The expression levels of tight junction proteins (ZO-1, occludin, claudin1) were measured by Western blot analysis (n = 3). f Jejunum morphology was observed by H&E staining (n = 4). g Goblet cells in the jejunum were observed by AB-PAS staining. h mRNA expression level of MUC2 in the jejunum (n = 6). i mRNA expression level of REG3G in the jejunum (n = 6). Data are expressed as the fold change versus the 0.3-Se group or the 0.3-Se + diquat group (set to 1). Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Effects of FMT on antioxidant capacity and immune responses in mice exposed to diquat

As shown in Fig. 8a–e, there was a significant decrease in T-AOC and SOD levels and an increase in the MDA level in diquat-exposed mice, which were strongly inhibited by 0.6-Se-FMT. However, 0.0-Se-FMT led to a significant decrease in the level of T-AOC and an increase in the level of MDA compared with the diquat-induced model group. In the diquat-induced model group, the 0.0-Se-FMT significantly reduced their activity, while 0.6-Se-FMT pretreatment increased their activity. In addition, increased levels of IL-1β and IL-18 and decreased levels of sIgA were observed in mice exposed to diquat compared with the normal control group. However, pretreatment with 0.6-Se-FMT significantly inhibited the increase of the IL-1β level and decrease of sIgA level compared to the diquat-induced model group. Remarkably, 0.0-Se-FMT aggravated the increase of pro-inflammatory cytokines and the decrease of sIgA induced by diquat (Fig. 8f–h).

Fig. 8: Effects of FMT on the antioxidant capacity and immune response in mice exposed to diquat.
figure 8

ae Oxidative stress response markers including T-AOC (a), MDA (b), SOD (c), GPx (d), TrxR (e) (n = 6). f Level of IL-1β in the jejunum of mice (n = 6). g Level of IL-18 in the jejunum of mice (n = 6). h Level of sIgA in the jejunum of mice (n = 6). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Effects of FMT on gut microbiota in mice exposed to diquat

16S rDNA sequencing was used to analyze the changes caused by FMT in the gut microbiota at Se concentrations from 0.0-Se and 0.6-Se. Compared with the diquat-induced model group, the ACE index of the 0.0-Se-FMT group decreased significantly and the Simpson index of the 0.6-Se-FMT group decreased significantly (Fig. 9a and b). Further PCoA analysis revealed that the gut microbiota of mice in different groups had obvious clustering, indicating that FMT and/or diquat significantly changed the composition of the gut microbiota (Fig. 9c). At the phylum level, 0.0-Se-FMT significantly increased the F/B ratio compared with the diquat model and 0.6-Se-FMT groups, which means that the gut microbiota of this group is disordered (Fig. 9d and e). At the genus level, compared with the normal control group, diquat exposure led to significant increases in the levels of Desulfovibrio and decreases in the level of Candidatus_Saccharimonas and Rikenella. In addition, 0.0-Se-FMT further reduced the abundance of Candidatus_Saccharimonas, and 0.6-Se-FMT decreased the abundance of Desulfovibrio and increased the abundance of Candidatus_Saccharimonas and Rikenella compared with the diquat exposure model group (Fig. 9f–h).

Fig. 9: Effects of FMT on gut microbiota in mice exposed to diquat.
figure 9

a, b Alpha diversity index (n = 6). c PCoA (n = 6). d The relative abundance composition of gut microbiota at the phylum level (n = 6). e Ratio of Firmicutes to Bacteroides (n = 6). fh Differences in gut microbiota between the genus level (n = 6). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

FMT from 0.6-Se activated the Nrf2 signaling pathway to inhibit diquat-induced intestinal mitochondrial dysfunction

As shown in Fig. 10a and b, treatment with diquat obviously induced the overproduction of ROS in the jejunum, which was clearly abolished by FMT from the 0.6-Se pretreatment. Representative images of mitochondrial ultrastructure from each group are shown in Fig. 10c, which reveal that the 0.6-Se-FMT can protect the mitochondrial morphology from diquat-induced oxidative stress damage. In addition, compared with the normal control group, the ATP content and MMP level were significantly decreased in the jejunal mitochondria from mice of the diquat exposure model group. However, the 0.6-Se-FMT effectively alleviated diquat-induced intestinal mitochondrial dysfunction (Fig. 10d and e). In addition, the level of 8-OHdG in the 0.6-Se-FMT group was significantly lower than that in the diquat exposure model and 0.0-Se-FMT groups (Fig. 10f). The mtDNA copy number can be used as an important feature of mitochondrial function. Therefore, the relative level of mtDNA copy number was measured by qPCR analysis. As shown in Fig. 10g, pretreatment with the 0.6-Se-FMT significantly alleviated the reduction in the mtDNA copy number in mice exposed to diquat. The mtDNA copy number is correlated to the expression of mitochondrial biogenesis genes. Notably, the expression of TFAM, POLG, and POLG2 in the diquat model exposure group was higher than that in the normal control group and pretreatment with the 0.6-Se-FMT significantly down-regulated the mRNA levels of TFAM, POLG, and POLG2 in the jejunum when compared with the group treated with diquat alone (Fig. 10h). We also examined whether NLRP3 and Nrf2 are involved in the antioxidative effect of FMT from the 0.6-Se. Western blot analysis showed that diquat exposure can lead to an increase in the level of the NLRP3 inflammasome and subsequent IL-1β and IL-18 expression, and FMT from 0.0-Se further increased their expression. However, pretreatment with the 0.6-Se-FMT significantly inhibited the activation of the NLRP3 inflammasome compared to the diquat exposure model group and 0.0-Se-FMT group (Fig. 10i and Supplementary Fig. 8a). Furthermore, the expression of Nrf2 (total Nrf2 and nuclear Nrf2), NQO-1, and HO-1 in the jejunum from mice in the diquat exposure model group decreased significantly compared with the normal control group, and the FMT from 0.0-Se further decreased their expression. However, the 0.6-Se-FMT effectively improved the expression level of Nrf2 and downstream antioxidant proteins compared with the diquat exposure model and 0.0-Se-FMT groups, indicating that FMT from the 0.6-Se activated the Nrf2 signaling pathway to inhibit the diquat-induced intestinal mitochondrial dysfunction (Fig. 10j and Supplementary Fig. 8b).

Fig. 10: FMT from the 0.6-Se group activated the Nrf2 signaling pathway to inhibit the diquat-induced intestinal mitochondrial dysfunction.
figure 10

a, b ROS production was determined by DHE staining (n = 7). c Mitochondrial ultrastructure was observed by TEM. d Mitochondrial ATP level in the jejunal mitochondria (n = 6). e MMP was measured by JC-1 staining in the jejunal mitochondria (n = 6). f Level of 8-OHdG in the jejunal mitochondria (n = 6). g The mtDNA copy number was analyzed by qPCR (n = 6). h The mRNA expression levels of TFAM, POLG, and POLG2 in the jejunum (n = 6). i The expression levels of NLRP3 inflammatory protein were measured by Western blot analysis (n = 3). j Nrf2 activation and expression levels of its downstream proteins measured by Western blot analysis (n = 3). Data are expressed as the fold change versus the control group (set to 1). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.



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