Animal experiments
All the animal experiments were performed in
Sprague‒Dawley (SD) rats (aged 13 –14 weeks) housed at
Ningxia Medical University Laboratory Animal Center (Yinchuan, China).
These rats were housed in a temperature-controlled room (22–24°C) with
a 12-h light/dark cycle and had free access to food and water. Following
a week of acclimatization, they were mated with healthy male SD rats at
a 2:1 ratio. The onset of gestation was identified by the presence of
vaginal sperm plugs on gestational day 1. The most reliable animal model
of PE is the surgically induced reduced uterine
perfusion pressure (RUPP) model. This model has been shown to induce
hypertension, proteinuria, renal dysfunction, an antiangiogenic state, inflammation, oxidative stress, cardiac dysfunction and
intrauterine growth restriction (IUGR), similar to PE in humans. Thus,
on the 14th day of gestation, the rats underwent surgical RUPP under
pentobarbital anesthesia. Briefly, through a midline incision, the
abdominal cavity was opened to expose the lower abdominal aorta. Silver
clips (0.203 mm) are placed around the aorta above the iliac bifurcation
to the RUPP by approximately 40%. Simultaneously, silver clips (0.100
mm) are used to decrease the degree of ovarian collateral circulation of
the bilateral uterine arteries at the ovarian ends of the uterine arch.
The sham group underwent the same procedure as the RUPP group but
without clip placement. Recombinant adeno-associated virus (AAV)
serotype 9 vectors carrying TP53BP2 short hairpin RNA (shRNA)
(AAV-shTP53BP2), recombinant AAV serotype 9 vectors carrying DNMT1 shRNA
(AAV-DNMT1), recombinant AAV serotype 9 vectors carrying G9a shRNA
(AAV-shG9a), or recombinant AAV9 vectors carrying a negative control
(AAV-shNC) were manufactured by GeneChem (Shanghai, China). Ten
microlitres of virus (1.5E+11) was injected into the placenta.
On the 20th day of gestation, the rats were euthanized with
pentobarbital, and the pups were removed and weighed. The placentas were
washed with ice-cold physiological salt solution and preserved at -80°C
for subsequent analysis. The animal experiments were approved by the
Committee on the Ethics of Animal Experiments of Ningxia Medical
University (NO. 2021–250).