Asiaticoside ameliorates uterine injury induced by zearalenone in mice by reversing endometrial barrier disruption, oxidative stress and apoptosis

Abstract

Zearalenone (ZEA) is a mycotoxin produced by Fusarium fungi that has been shown to have adverse effects on human and animal health, particularly on the fertility of females. As a saponin derived from the medicinal plant Centella asiatica, asiaticoside (AS) has multiple bioactivities. This study aimed to investigate the protective effects of AS on ZEA-induced uterine injury and the underlying mechanism. In the present study, we demonstrated that AS could rescue ZEA-induced uterine histopathological damage and modulate the secretion of sex hormones, including progesterone (P4), luteinizing hormone (LH), and estradiol (E2), in ZEA-treated mice. Moreover, AS alleviated ZEA-induced damage to endometrial barrier function by upregulating the expression of tight junction proteins (ZO-1, occludin, and claudin-3). Further mechanistic investigations indicated that ZEA reduces the antioxidant capacity of uterine tissues, whereas AS improves the antioxidant capacity through activating the Nrf2 signaling pathway. Most notably, the protective effect of AS was blocked in Nrf2 gene knockout (Nrf2^-/-) mice. Moreover, the p38/ERK MAPK pathway has been implicated in regulating ZEA toxicity and the beneficial effect of AS. Additionally, an Nrf2 inhibitor (ML385) weaken the suppressive effect of AS on the oxidative stress and MAPK pathway. AS also inhibits ZEA-induced apoptosis in uterine tissues via the PI3K/Akt signaling pathway. However, when the PI3K small molecule inhibitor LY294002 was co-administered, the ability of AS to suppress the expression of apoptosis-related proteins and inhibit ZEA-induced apoptosis decreased. Collectively, these findings reveal the involvement of multiple pathways and targets in the protective effect of AS against ZEA-induced uterine injury, providing a new perspective for the application of AS and the development of a ZEA antidote.

Keywords: Apoptosis; Asiaticoside; Nrf2; Uterine injury; Zearalenone.

Fig. 1

The effects of AS on ZEA-induced uterine injury. (A) The chemical structure of AS. (B) Scheme of the drug delivery procedure. (C) Uterine morphology observation. Body weight (D), uterine weight (E) and uterine index analysis (F). (G) Histopathologic section of the uterus (the scale bar represents 50 μm). (H) Histopathological scores of uterine sections. (I) The average endometrial thickness in each group. (n = 8, the values are presented as the means ± SEMs). Compared with the Ctrl group: ^##p < 0.01; compared with the ZEA group, ^*p < 0.05, ^**p < 0.01

Fig. 2

Effects of AS on the serum levels of P4, LH and E2. (A) Serum P4 content. (B) Serum LH content. (C) Serum E2 content. (n = 8, the values are presented as the means ± SEMs). Compared with the Ctrl group: ^##p < 0.01; compared with the ZEA group, ^**p < 0.01

Fig. 3

AS upregulated the protein expression of TJ proteins in ZEA-treated mice. (A) Representative immunoblots of ZO-1, Occludin and Claudin-3. (B) Relative protein expression of ZO-1, Occludin and Claudin-3. (n = 6–8, the values are presented as the means ± SEMs). Compared with the Ctrl group: ^##p < 0.01; compared with the ZEA group, ^**p < 0.01

Fig. 4

AS inhibits ZEA-induced oxidative stress by activating the Nrf2 pathway. Mouse uterus (A) SOD activity; (B) CAT activity; (C) MDA content. (D) Representative immunoblots of Nrf2, HO-1, NQO1 and nuclear Nrf2. (E) Densitometric quantification of Nrf2, HO-1, NQO1 and nuclear Nrf2 expression levels. (F) Scheme of the drug delivery procedure. (G) Representative immunoblots of Nrf2, HO-1, NQO1 and nuclear Nrf2. (H) Relative protein expression of Nrf2, HO-1, NQO1 and nuclear Nrf2. (I-K) Uterine SOD, CAT and MDA levels in the mice. (n = 6–8, the values are presented as the means ± SEMs). Compared with the Ctrl group: ^##p < 0.01; ^**p < 0.01; ns: not statistically significant

Fig. 5

Nrf2 knockout abolishes the effect of AS on attenuating ZEA-induced uterine injury. (A) Experimental design. (B) Uterine morphology observation. Body weight (C), uterine weight (D) and uterine index analysis (E). (F) Histopathologic section of the uterus (the scale bar represents 50 μm). (G) Histopathological scores of uterine sections. (H) The average endometrial thickness in each group. (I, J) The protein expression of Nrf2, HO-1 and NQO1 in the mouse uterus. (K‒M) Uterine SOD, CAT and MDA levels in the mice. (n = 6–8, the values are presented as the means ± SEMs). ^*p < 0.05, ^**p < 0.01, ns: no statistical significance

Fig. 6

AS inhibits ZEA-induced apoptosis by activating the PI3K/AKT signaling pathway. (A) Experimental design. (B, C) The protein ratios of p-PI3K/PI3K, p-AKT/AKT, Bcl-2/Bax and C-Casp3/Pro-Casp3 in the mouse uterus. (D, E) IHC staining of Ki67 in the uterine tissues of the mice. (F, G) Apoptosis levels were detected by TUNEL staining. (n = 6–8, the values are presented as the means ± SEMs). ^**p < 0.01, ns: not statistically significant

Fig. 7

AS suppresses the MAPK signaling pathway by promoting the Nrf2 pathway in ZEA-exposed mice. (A, B) The protein ratios of p-ERK/ERK, p-JNK/JNK and p-p38/p38 in the mouse uterus. (C, D) Western blot analysis of the expression of Nrf2, Nuclear Nrf2, p-ERK/ERK, p-JNK/JNK and p-p38/p38. (n = 6–8, the values are presented as the means ± SEMs). Compared with the Ctrl group: ^##p < 0.01; ^*p < 0.05, ^**p < 0.01

Fig. 8

A proposed signaling pathway involved in AS against ZEA-induced uterine injury in mice