Progesterone Attenuates SIRT1-Deficiency-Mediated Pre-Eclampsia

Abstract

Pre-eclampsia is a severe hypertensive disorder of pregnancy (HDP), mainly characterized by new-onset hypertension with proteinuria after 20-week gestation. Sirtuin1 (SIRT1), a class III histone deacetylase, is associated with the regulation of various pathophysiological processes, including inflammation, immune response, metabolism, and autophagy. However, the effect of SIRT1 in the pathogenesis of pre-eclampsia remains to be elucidated. In this study, we found that the expression of SIRT1 was relatively lower in the placentas and serum samples of pre-eclampsia patients. Typical pre-eclampsia-like symptoms, such as hypertension, proteinuria, fetal growth restriction, kidney injury, and a narrow placental labyrinth layer, were observed in SIRT1 knockdown (SIRT1^+/-) mice. Of note, these performances could be improved after the intraperitoneal injection of SIRT1 agonist SRT2104. More importantly, we found that the efficacy of progesterone on attenuating symptoms of PE was profoundly better than that of metformin in SIRT1^+/- mice. In addition, our results suggested that progesterone can promote the invasion and inhibit the apoptosis of trophoblasts. These data suggest that SIRT1 plays an important role in pre-eclampsia and that progesterone alleviates pre-eclampsia-like symptoms mediated by SIRT1 deficiency.

Keywords: SIRT1; SRT2104; metformin; pre-eclampsia; pre-eclampsia-like mice; progesterone.

Figure 1

SIRT1 was significantly lower in the placentas and serum samples of pre-eclampsia patients. (A) Western blot analysis of SIRT1 protein levels in placental tissues from normal pregnancy women (NP, n = 7) and women with pre-eclampsia with severe features (sPE, n = 7). (B) Immunohistochemistry for SIRT1 in placental tissues from NP women (n = 7) and sPE women (n = 7). (C) ELISA analysis of the SIRT1 concentration in the serum of PE women (n = 76) and gestational age-matched NP women (n = 95). Error bars, mean ± SD. The data were analyzed by an unpaired Student’s test. ** p < 0.01.

Figure 2

SIRT1^+/− mice demonstrated pre-eclampsia-like symptoms. (A) Schematic diagram of SIRT1^+/− mice construction. (B) Genotype of SIRT1^+/− mice using PCR and agarose gel electrophoresis. (C) Immuofluorescence for SIRT1 in mice placental tissues from SIRT1^flox/flox (n = 3) and SIRT1^+/− (n = 7) mice. (D) Representative appearance of fetuses and placentas from SIRT1^flox/flox and SIRT1^+/− groups. (E–G) The embryo-resorption rate (E), the weight of the placenta (F), and the weight of the live fetus (G) in SIRT1^flox/flox (n = 19) and SIRT1^+/− (n = 31) mice. (H) The gene ratio of fetuses at E18.5 and P28 (E18.5: the 18.5th day of gestation, P28: the 28th day in postnatal age). (I) The systolic blood pressure (SBP) at basic condition, early PG (pregnancy), mid PG, late PG from SIRT1^flox/flox (n = 20), and SIRT1^+/− (n = 33) groups. (J) ΔBP (late PG—basic condition) in SIRT1^flox/flox (n = 20) and SIRT1^+/− (n = 33) groups. (K) The correlation of ΔBP and SIRT1 gene values in fetuses. (L) Masson staining and PAS staining of mice kidney tissues from SIRT1^flox/flox (n = 3) and SIRT1^+/− (n = 7) groups. (M) The concentration of urinary protein in SIRT1^flox/flox (n = 6) and SIRT1^+/− (n = 13) groups at late PG. (N) Masson staining of mice placental tissues in SIRT1^flox/flox (n = 3) and SIRT1^+/− (n = 7) groups. (O) The labyrinth/junctional zone ratio of each group. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student’s test (and nonparametric tests). ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.

Figure 3

SRT2104 significantly improved the PE-like symptoms in SIRT1^+/− mice. (A) Schematic diagram of the treatment of SIRT1^+/− mice from early pregnancy to late pregnancy. (B) Representative appearance of fetuses and placentas from vehicle and SRT2104 groups. (C–G) The embryo-resorption rate (C), the weight of the placenta (D), and the weight of the live fetus (E) in vehicle (n = 8) and SRT2104 (n = 7) groups. (F) The SBP at basic condition, early PG, and mid PG, late PG from vehicle (n = 8) and SRT2104 (n = 9) groups. (G) ΔBP (late PG—basic condition) in vehicle (n = 8) and SRT2104 (n = 9) groups. (H) The concentration of urinary protein in vehicle (n = 4) and SRT2104 (n = 7) group at late PG. (I) Masson staining and PAS staining of mice kidney tissues from vehicle (n = 4) and SRT2104 (n = 4) groups. (J) Masson staining of mice placental tissues in vehicle (n = 4) and SRT2104 (n = 4) groups. (K) The labyrinth/junctional zone ratio of each group. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student’s test (and nonparametric tests). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.

Figure 4

P4 could alleviate hypertension in SIRT1^+/− mice and promote the invasion and inhibit the apoptosis of trophoblasts. (A–E) The embryo-resorption rate (A), the weight of the placenta (B), and the weight of the live fetus (C) in vehicle (n = 8), Met (1/2) (n = 3), Met (n = 5), and P4 (n = 3) groups. (D) ΔBP (late PG—basic condition) in vehicle (n = 8), Met (1/2) (n = 3), Met (n = 5), and P4 (n = 3) groups. (E) The SBP at basic condition, early PG, mid PG, late PG from vehicle (n = 8), Met (1/2) (n = 3), Met (n = 5), and P4 (n = 3) groups. (F,G) The invasion ability of Transwell assay in vehicle, Met (F), and P4 (G) groups of control group. (H) Western blot analysis of SIRT1 protein levels in trophoblasts of SIRT1 KD and control groups. (I) The invasion ability of Transwell assay of vehicle and P4 groups in SIRT1 KD trophoblasts. (J) Statistics of migrating cells. (K) The proliferation function checked by CCK8 at 24 h and 48 h treated with P4 (concentration: 0, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴ mol/L) in SIRT1 KD trophoblasts. (L) The apoptosis assay of vehicle, SRT2104 and P4 groups in SIRT1 KD trophoblasts. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student’s test (and nonparametric tests). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.

Figure 5

Summary diagram. (A)SIRT1 knockdown (SIRT1^+/−) mice showed typical pre-eclampsia-like symptoms, such as hypertension, kidney injury, FGR, and placental pathology. These performances could be improved after intraperitoneal injection of SIRT1 agonist SRT2104 and P4. Metformin could also reduce the elevated blood pressure, but caused FGR. (B) Additionally, P4 could promote the invasion and inhibit the apoptosis of trophoblasts. (PE: pre-eclampsia; FGR: fetal growth restriction; P4: progesterone; Met: metformin.).