Abstract
Impaired decidualization was recognized as one of the crucial pathomechanisms accounting for unexplained recurrent spontaneous abortion (URSA). Currently, the exact molecular mechanism and targeted clinical decision are still in exploration. Bushen Huoxue decoction (BSHXD) has previously been proved effective in treating URSA, but its mechanism remains to be elucidated. This study aimed to explore the regulation mechanism of BSHXD in decidualization from its intervention in autophagy so as to rationalize its potential as a novel therapeutic regime for URSA. Decidua tissues were collected from patients with URSA and healthy pregnant women who underwent legal terminations for non-medical reasons at the first trimester. Besides, cell line T-hESCs was utilized to establish induced decidualization model, and were randomly divided into ESC group, DSC group, 3-MA group, AMPK siRNA group, scrambled siRNA group and AMPK siRNA + BSHXD group. Transmission electron microscopy, Monodansylcadaverine (MDC) assay, qRT-PCR, immunohistochemistry, immunofluorescence and Western blotting were used to evaluate the level of decidualization, autophagy and activation of AMPK signaling pathway in decidua tissues and cell experiments. Experiments on decidua tissues showed that decidualization was impaired in URSA with inhibited autophagy. Besides, pAMPK T172 and pULK1 S556 were decreased, and pmTOR S2448 and pULK1 S757 were increased. Cell experiments showed that the level of autophagy increased during induced decidualization, but when autophagy was inhibited, decidualization was impaired. In addition, AMPK/mTOR/ULK1 affected decidualization by mediating autophagy, and BSHXD improved decidualization through this mechanism. In conclusion, this study clarified that the inhibition of autophagy mediated by AMPK/mTOR/ULK1 was associated with impaired decidualization, and the intervention of BSHXD on this pathological process may be a vital mechanism for its treatment of URSA. This study laid the foundation for further research and application of BSHXD.
Highlights
Recurrent spontaneous abortion (RSA) is defined as three or more consecutive abortions prior to the 20th week of gestation
Results showed that the mRNA levels of IGFBP1 and PRL in the unexplained recurrent spontaneous abortion (URSA) group were significantly lower than the control group (p < 0.05 and p < 0.01, respectively) (Figure 3A), indicating impaired decidualization in URSA
The mRNA levels of PRL and IGFBP1 were significantly increased in AMPK small interfering RNA (siRNA) + Bushen Huoxue decoction (BSHXD) group when compared with AMPK siRNA group (p < 0.01) (Figure 6J). These results suggested that BSHXD can regulate the phosphorylation of AMPK/mTOR/ULK1 to affect the level of autophagy in decidua stromal cells (DSCs) and improve decidualization, which was conducive to the treatment of URSA
Summary
Recurrent spontaneous abortion (RSA) is defined as three or more consecutive abortions prior to the 20th week of gestation. 50% of RSA cases remain elusive, which is defined as unexplained recurrent spontaneous abortion (URSA). Decidualization is defined as the significant morphological, biochemical and functional changes of endometrial stromal cells (ESCs) to decidua stromal cells (DSCs) so as to better adapt to embryo implantation (Ng et al, 2020; Ochoa-Bernal and Fazleabas, 2020). This process is regulated by complex molecular signals, involving huge energy changes and intracellular remodeling, both of which are essential signals for the initiation of autophagy (Popli et al, 2021). Current studies have confirmed that inhibition of autophagy is associated with impaired decidualization and pregnancy complication (Kim and Lee, 2014; Nakashima et al, 2019), but the regulation mechanism is still perplexing
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