Shaoyao-Gancao Decoction Ameliorates the Inflammation State in Polycystic Ovary Syndrome Rats via Remodeling Gut Microbiota and Suppressing the TLR4/NF-κB Pathway

Abstract

Background: Emerging evidence suggests that gut microbiota plays a vital role in the occurrence of multiple endocrine disorders including polycystic ovary syndrome (PCOS). Shaoyao-Gancao Decoction (SGD), a classical Chinese prescription, has been widely used in the treatment of PCOS for decades. In previous studies, we found that SGD treatment could effectively reduce ovarian inflammation in PCOS rats. However, whether the anti-inflammation effect of SGD involves the regulation of the gut microbiota remains elusive. Methods: Letrozole-induced PCOS rat models were established, and the therapeutic effects of SGD were evaluated. Specifically, body weight, serum hormone concentrations, estrus phase and ovary histopathology were assessed. Then the structure of gut microbiota was determined by 16s rRNA sequencing. Additionally, the serum levels of pro-inflammatory cytokines and LPS were measured by ELISA kits. The key gene and protein expressions of TLR4/NF-κB signaling pathway were detected by quantitative real-time PCR and western blot. Results: SGD could effectively reduce body weight, regulate estrous cycles and ameliorate hyperandrogenism in PCOS rats. In addition, SGD treatment decreased releases of pro-inflammatory cytokines, enhanced the expressions of tight junction (occludin and claudin1), and then prevented a translocation of LPS into bloodstream. SGD could significantly reduce the ratio of Firmicutes to Bacteroidetes, decrease the abundance of LPS-producing pathogens Proteobateria and enrich the abundance of Butyricicoccus, Coprococcus, Akkermansia Blautia and Bacteroides in PCOS rats. Furthermore, SGD blunted the key gene and protein expressions of TLR4/NF-κB signaling pathway both in vivo and in LPS-induced RAW264.7 cells. Conclusion: SGD administration could ameliorate the inflammatory response in PCOS rats by remodeling gut microbiome structure, protecting gut barrier, and suppressing TLR4/NF-κB signaling pathway.

Keywords: TLR4/NF-κB pathway; gut barrier; gut microbiota; polycystic ovary syndrome; shaoyao-gancao decoction.

FIGURE 1

SGD alleviated the symptoms in Letrozole-induced PCOS rats. (A) Animal groups and treatments schedule. (B,C) Effect of SGD on body weight in PCOS rats (n = 10). (D) Representative estrous cycles. (E) Quantitative analysis of estrous cycles (n = 10). (F) HE staining of representative ovaries. “a” indicated the appearances of cystic follicles, “b” indicated the appearances of corpus luteum (magnification, ×10), Scale bar: 1 mm. (G) Quantitative analysis of cystic follicles (n = 5). (H) Quantitative analysis of corpus luteum (n = 5). (I–L) Serum levels of sex hormone indicators including testosterone. (T), 17β-estradiol (E₂), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were determined by using ELISA kits (n = 10). Data were presented as mean ± SEM. ***p < 0.001 vs. normal group, ^# p < 0.05 and ^### p < 0.001 vs. PCOS model group.

FIGURE 2

SGD alleviated the chronic low-grade inflammation in PCOS rats. (A–D) Serum levels of inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-18 were determined by using ELISA kits (n = 10). (E–H) The mRNA levels of inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-18 in rats ovarian tissues were quantitated by real-time PCR (n = 5). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. normal group, ^## p < 0.01 and ^### p < 0.001 vs. PCOS model group.

FIGURE 3

SGD treatment modulated the gut microbiota composition in PCOS rats. Shannon (A) and Simpson (B) diversity index. PCoA (C) and NMDS (D) plot of intestinal microbiota based on OTU level. (E) Column diagram of community composition at phylum level. (F) The relative abundances of Firmicutes, Bacteroides, Verrucomicrobia, and Actinobacteria. (G) Firmicutes/Bacteroides ratio. (H) Column diagram of community composition at genus level. (I) The relative abundances of Turicibacter, Akkermansia, Blautia, Bacteroides, Coprococcus and Butyricicoccus in three groups.

FIGURE 4

SGD treatment reduced the serum LPS level and ameliorated the intestinal barrier function in PCOS rats. (A) Serum levels of LPS were determined by using ELISA kits (n = 10). (B–D) The mRNA levels of tight junction proteins gene including occluding (B), claudin-1 (C), and ZO-1 (D) in rats ileum mucosa were quantitated by real-time PCR (n = 5). Data were presented as mean ± SEM. **p < 0.01 and ***p < 0.001 vs. normal group, ^### p < 0.001 vs. PCOS model group.

FIGURE 5

Heatmap of Spearman’s correlation between the gut microbiota [at phylum level (A) and at genus level (B)] and hormones, cytokines, LPS or tight junction proteins. The value of r represents the degree of correlation (0 > r > 1, positive correlation; −1<r < 0, negative correlation). Significant correlations were marked by *p < 0.05.

FIGURE 6

SGD blunted TLR4/NF-κB signaling pathway in PCOS rats and LPS-stimulated RAW264.7 cells. (A) The mRNA levels of TLR4, PI3K, Akt, and NF-κB p65 in rats ovarian tissues were quantitated by real-time PCR (n = 5). (B) The mRNA levels of TLR4, PI3K, Akt, and NF-κB p65 in RAW264.7 cells were quantitated by real-time PCR (n = 3). (C,D) The protein levels of TLR4, p-PI3K, PI3K, p-Akt, Akt, p-p65, and p65 in rats ovarian tissues were quantitated by Western blotting (n = 3). Data were presented as mean ± SEM. **p < 0.01 vs. normal group, #p < 0.05 and ##p < 0.01 vs. PCOS model group, &&p < 0.01 and &&&p < 0.001 vs. the untreated group, †p < 0.05, ††p < 0.01, and †††p < 0.001 vs. the LPS-stimulated group.