TCM, consists of complex formulations that have hitherto been difficult to characterize, thus limiting their widespread clinical use, the disease name of RSA is "slippery fetus" and "multiple abortions", which also belong to the category of "repeated pregnancy and repeated abortion", which refers to abortion or miscarriage occurring for 3 consecutive times or more (31). TCM has a long history in treating menstrual diseases and infertility and has achieved sound effects (32). TCM advocates the principles of pre-cultivation, prevention before pregnancy, survival without death, and prevention of pregnancy and change. The method of tonifying kidney, activating blood circulation and removing blood stasis has been used to treat slippery fetus since ancient times. Recent studies have shown that kidney deficiency and blood stasis are the primary syndrome type of recurrent flow induced by pre-thrombotic and unknown causes. Although Huo Xue drugs are contradictions for pregnancy, they follow the treatment principle of "there was a cause without a disappearance, nor a disappearance, and most of them were lost" in < Suwen · Liuyuan Zhengji Grand Essay >. The doctors should not be prejudiced-out and cautious. The method of Bushen Huoxue can achieve a good curative effect in the treatment of recurrent abortion.
In this study, several network pharmacology-based methods were used to predict potential targets. This approach provides new clues to exploring ethnopharmacology and herbal or even TCM formulas. By analyzing the putative therapeutic target network and biological functions by GO and KEGG function enrichment, the potential pharmacological and molecular mechanisms of BHD in treating RSA were preliminary revealed.
The drug-target network and the RSA network had 26 overlapping genes, were enriched in 19 pathways, and are likely the key pathway involved in RSA treatment. According to the P-value and Ration of KEGG pathway enrichment, we hypothesized that BHD plays a role in tonifying kidney and activating blood stasis by regulating the Neuroactive ligand-receptor interaction. Therefore, we conducted a series of experiments to confirm that BHD participates in this pathway during the treatment of RSA.
Neuroactive steroids are hormones that act as regulators of neurotransmitter receptors to either enhance or suppress neuronal activity. The effect of steroid exhibits marked stereoselectivity, suggesting a ligand-receptor interaction. According to the literature, neuroactive steroid influences the modulation of fetal neurodevelopment (33). Neuroactive ligands affect neuronal function by binding to intracellular receptors, which can bind transcription factors (34). The number of apoptotic cells in the brain and central nervous system of zebrafish embryos was increased in the expression of genes involved in the neuroactive ligand-receptor interaction pathway (35). Based on these, we infer that BHD might serve a crucial role in human reproduction by influencing the pathway of Neuroactive ligand-receptor interaction.
HTR-8/SVneo cell line is considered the closest study model to trophoblast cells derived from early human pregnancy (8 ~ 10 weeks) (36). The HTR-8/SVneo human trophoblast cell line applied in the present study is more similar to primary trophoblasts and normal human physiological conditions. Our previous study showed that the H2O2 treatment of HTR-8/SVneo cells could establish a placental oxidative stress model and simulate the mechanism of recurrent abortion in vitro (37).
A total of 26 genes were screened out by PPI network, among which 7 were enriched in this pathway, 3 of which were selected for experimental verification. Firstly, the safe drug concentration of BHD on cells was screened out by cell morphology and cell activity by CCK-8 assay. The results showed that the cell model of oxidative stress at the 5 mg/mL BHD water extract concentration could approach control cells.
PGF2α receptor (PTGFR), produced by the myometrium and intrauterine tissues of pregnancy, involves five distinct yet integrated physiological events: the rupture of the membranes, cervical ripening and dilatation, contractility of the myometrium, placental separation and uterine involution (38).
PTGFR is an essential uterine activation protein (UAP), promoting the tissues' ability to carry out the process of parturition. Myometrial PTGFR mRNA is elevated at term and preterm birth in humans and rodents (39, 40). Also, infusion of a specific inhibitor, THG113.31, in sheep and mice delays preterm birth and prolongs gestation (41, 42). In rats, myometrial PTGFR mRNA expression rate decreases during pregnancy, and its expression increases significantly again at term (43). We added 5 mg/mL BHD into the cell model of RSA oxidative stress. Western blot results showed that the expression level of PTGFR protein was significantly decreased compared with that of the model group.
Meanwhile, at the RNA level, qRT-PCR results were the same as western blot results. Our results are consistent with previous reports and network pharmacologic results. This suggests that PTGFR plays a central role in both pregnancy maintenance and parturition. PTGFR may act via its receptor to amplify the direct actions of cytokines. Together, these feed-forward mechanisms activate the uterus, trigger uterine contractile stimulants' production, and lead to labor and delivery (44). We suggest that the role of the PTGFR in the human uterus requires further validation prior to pursuing it as a target for RSA treatment.
Angiotensin II type I receptor (AGTR1), a component of the renin-angiotensin system (RAS). AGTR1 encodes the angiotensin II (Ang II) type I receptor belonging to the family of G-protein coupled receptors (45). AGTR1 is a major effector controlling blood pressure in the cardiovascular system and induces diverse signal transduction pathways (46). It regulates the aldosterone secretion and is involved in vascular remodeling, inflammation and endothelial dysfunction (47). We found that AGTR1 gene expression was significantly higher in HTR-8/SVneo oxidative stress cell models than in normal cells, both at protein and RNA levels. Alterations in the maternal renin-angiotensin system (RAS) (48) have been implicated in the pathogenesis of RSA (49, 50). The RAS plays a crucial role in maintaining a normal pregnancy, where plasma levels of renin, angiotensin II, and aldosterone are up-regulated (51). Angiotensin II is the primary signaling molecule of the RAS and its major cardiovascular effects are mediated by angiotensin II receptor type 1 (gene denoted as AGTR1), including vasoconstriction, vascular growth promotion, anti-natriuresis, aldosterone synthesis, and inhibition of renin synthesis and release (52). In RSA, this equilibrium is disrupted, and these proteins' plasma levels decrease toward the normal non-gravid range. The TCM decoction BHD can correct this imbalance. After the addition of a safe concentration of BHD, the expression level of AGTR1 protein in cells was significantly decreased. In addition, the AGTR1 level in RNA was lower compared to the model group. Any imbalance results in a pathological state. These results suggest that BHD altered AGTR1 and RSA's imbalance to reach new homeostasis, which was more suitable for a normal pregnancy.
The oxytocin receptor (OXTR) gene is a protein that works as a receptor for the widely expressed oxytocin, which acts both as a hormone and neurotransmitter.
Studies (53–56) have shown that a decrease in the progesterone level coupled with the activation of OXTR leads to intense inflammation, transforming the uterus from the quiescent state to the activated state and resulting in premature labor or abortion. We found that in the cell model of oxidative stress, the expression level of OXTR was significantly increased compared with the normal cell group, which was consistent with the previous research results and proved that this gene was one of the critical reasons for the development of RSA. OXTR, an important inflammatory factor, plays a vital role in initiating and maintaining delivery (57, 58). Myometrium and decidua from pregnant women who underwent cesarean section or hysterectomy during pregnancy (59), and the results showed that OXTR level was low in early pregnancy, whereas in pregnant women with at least 37-week gestational age or premature labor, OXTR level and the sensitivity to oxytocin were significantly higher. In pregnant women with prolonged pregnancy (> 42-week gestational age), the OXTR level was significantly lower. The mRNA level of OXTR is related to premature labor. Further, progesterone and its receptor directly inhibit the mRNA level of OXTR, thus preventing premature labor (60). Our experimental results showed that BHD had the same effect as progesterone and could significantly reduce the expression of elevated OXTR protein and RNA in the oxidative stress model cells. Mammalian animal studies have shown that the balance between progesterone and its receptor plays a critical role in maintaining normal pregnancy (61); other studies have shown that progesterone maintains uterus stability by inhibiting OXTR (62). These results indicate that OXTR plays a vital role in pregnancy and delivery and TCM can achieve progesterone very well.