Moringa oleifera leaf supplementation relieves oxidative stress and regulates intestinal flora to ameliorate polycystic ovary syndrome in letrozole-induced rats

Abstract

This study investigated the effects of supplementation Moringa oleifera leaf (MOL) on relieving oxidative stress, anti-inflammation, changed the relative abundance of multiple intestinal flora and blood biochemical indices during letrozole-induced polycystic ovary syndrome (PCOS). Previous studies have shown that MOL has anti-inflammatory, anti-oxidation, insulin-sensitizing effects. However, whether MOL has beneficial effects on PCOS remains to be elucidated. In the current study, 10-week-old female Sprague-Dawley rats received letrozole to induce PCOS-like rats, and subsequently were treated with a MOL diet. Then, the body weight and estrus cycles were measured regularly in this period. Finally, the ovarian morphology, blood biochemical indices, anti-oxidative, intestinal flora, and anti-inflammation were observed at the end of the experiment. We found that MOL supplementation markedly decreased the body weight, significantly upregulated the expression of Sirt1, FoxO1, PGC-1α, IGF1, and substantially modulated the sex hormone level and improved insulin resistance, which may be associated with the relieves oxidative stress. Moreover, the supplementation of MOL changed the relative abundance of multiple intestinal flora, the relative abundance of Fusobacterium, Prevotella were decreased, and Blautia and Parabacteroides were increased. These results indicate that MOL is potentially a supplementary medication for the management of PCOS.

Keywords: Moringa oleifera leaf; inflammation; intestinal flora; oxidative stress; polycystic ovary syndrome.

FIGURE 1

Enrichment analysis for KEGG enrichment analysis of differential genes.

FIGURE 2

Targets site prediction of the effect of MOL on polycystic ovary.

FIGURE 3

MOL‐PCOS target PPI network.

FIGURE 4

GO biological function enrichment analysis of MOL‐PCOS target.

FIGURE 5

KEGG enrichment analysis bubble chart.

FIGURE 6

Component‐target‐pathway diagram of MOL in the treatment of PCOS.

FIGURE 7

Molecular docking results.

FIGURE 8

Schematic diagram of docking results of some core compounds: (a) 3D model of PIK3CA (PDBID:4OVU) crystal structure docking. (b) 3D model of HRAS (PDBID:4XVR) crystal structure docking. (c) 3D model of TLR4 (PDBID:6Z62) crystal structure docking. (d) 3D model of Sirt1 (PDBID:4I5I) crystal structure docking. (e) 3D model of FoxO1 (PDBID:4LG0) crystal structure docking. (f) 3D model of PGC‐1α (PDBID:7E2E) crystal structure docking.

FIGURE 9

LET‐induced vaginal smear, ovarian morphology, and reproductive hormone level alterations in conventional and MOL‐treated rats. (a) Schematic representation of the above experimental design. (b) Estrous cycle examination, (c) body weight before treatment, (d) body weight after 7 weeks treatment, (e) H&E staining of representative ovaries. Scale bar = 200 μm. (f) Serum T level, (g) serum LH levels, (h) serum FSH levels, (i) serum E2 levels, (j) ratio of LH/FSH. Results of vaginal smear. (D) Diestrus, (P) Proestrus, (E) Estrus, (M) Metestrus. (*p < .05, ** p < .01 vs. control; #p < .05, ##p < .01 vs. L group.)

FIGURE 10

Results of pancreas tissues morphology and the glucose metabolism changes, (a) H&E staining of representative pancreas, (b) serum FBG levels, (c) serum FINS levels, (d) the level of HOMA‐IR. (*p < .05, ** p < .01 vs. control; #p < .05, ##p < .01 vs. L group.)

FIGURE 11

Effect of MOL treatment on the oxidative stress expression in the ovary of PCOS‐like rats. (a, c, d) IHC analysis of Sirt1/FOX01/IGF1 protein expression on the ovaries of PCOS‐like rats after different treatments. (b) Western bolt analysis of the effects of MOL treatment on the protein levels of Sirt1, PGC‐1α, and FOX01 expression in the ovarian from PCOS‐like rats (*p < .05, **p < .01 vs. control; #p < .05, ##p < .01 vs L group.)

FIGURE 12

Effect of MOL treatment on the gut barrier function and permeability expression in the colon of PCOS‐like rats. (a) H&E staining of representative colon, (b, c) Effect of MOL treatment on the occludin, claudin‐1 expression levels in the colon tissues from PCOS‐like rats. (*p < .05, ** p < .01 vs. control; #p < .05, ##p < .01 vs., L group.)

FIGURE 13

Taxonomic profile of the gut microbiomes of the samples collected from the four groups. (a) The OUT numbers of Phylum, Class, Order, Family, Genus, (b–e) the microbial diversity and richness of the microbiota in the feces. (f) Beta diversity of the microbiota in the feces. (g–i) Relative abundance of gut microbial species at the phylum levels in the feces of rats. (j, k) Linear discriminant analysis (LDA) effect size (LEfSe) results on gut microbiota (*p < .05, ** p < .01 vs. control; #p < .05, ##p < .01 vs. L group).

FIGURE 14

Effect of MOL treatment on the inflammation expression in the ovary, colon, and intestinal flora of PCOS‐like rats. (a) IHC analysis of TLR4 protein expression on the colon of PCOS‐like rats after different treatment. (b) Effect of MOL treatment on the TLR4 expression levels in the ovaries tissues from PCOS‐like rats. (c–h) Effect of MOL treatment on relative abundance of intestinal flora species at the genus levels related to inflammation in the feces of rats. (*p < .05, **p < .01 vs. control; #p < .05, ##p < .01 vs. L group.)

FIGURE 15

Correlation analyses between relative abundance (%) of microbiota and other related indicators. (a) Relative abundance of the phylum levels and biochemical indices. (b) Relative abundance of the Top 20 genus levels and biochemical indices. (c) Relative abundance of the differential genus levels and biochemical indices. Relative abundance of gut microbial species at the genus levels in the feces of rats.