Transcutaneous Auricular Vagus Nerve Stimulation Alleviates Preeclampsia-Induced Placental Trophoblast Apoptosis by Inhibiting Mitochondrial Unfolded Protein Response

Research Background

Preeclampsia is a severe obstetric complication affecting 2%-8% of pregnancies worldwide. This disease not only impacts the health of pregnant women but also seriously threatens the life safety of fetuses. Currently, the pathogenesis of preeclampsia is not fully elucidated, and there is a lack of effective preventive measures. In recent years, research has found that transcutaneous auricular vagus nerve stimulation (TAVNS), as a new non-pharmacological treatment, has potential therapeutic effects on preeclampsia. However, its specific mechanism remains unclear.

Research Source

This study was jointly completed by seven researchers including Zhao Jing, Yang Yanan, Qin Jiayi, et al. from the School of Acupuncture-Moxibustion and Tuina, School of Public Health, and School of Medical Technology at Shaanxi University of Chinese Medicine, and published in the journal “Neurosci. Bull.” (DOI: 10.1007/s12264-024-01244-9).

Research Process

Animal and Cell Models

This study used Sprague-Dawley rats and the HTR-8/SVneo placental trophoblast cell line. Rat experiments were conducted under the guidance of the Animal Ethics Committee of Shaanxi University of Chinese Medicine. In the in vivo study, researchers established a reduced uterine perfusion pressure (RUPP) model to simulate placental ischemia and hypoxia environment for evaluating the effects of TAVNS. In cell experiments, cobalt chloride (CoCl2) treatment was used to simulate a hypoxic environment to study the effects of Ach on hypoxia-induced trophoblast apoptosis.

TAVNS Treatment

An electroacupuncture device was used to stimulate the auricular concha of both ears in rats for 30 minutes daily for 7 consecutive days. The study groups included Normal Pregnancy (NP) group, RUPP group, and RUPP+TAVNS group. Blood pressure measurements, ELISA, immunofluorescence, and Western Blot were used to assess the effects of TAVNS on blood pressure, placental tissue morphology, and inflammatory factors.

Main Research Findings

Blood Pressure and Inflammatory Factors

The results showed that systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly increased in the RUPP group, while TAVNS significantly reduced RUPP-induced SBP and DBP. Additionally, levels of inflammatory factors TNF-α and IL-6 in placental tissue were significantly elevated in the RUPP group, while TAVNS significantly lowered their levels.

Placental Structure and Mitochondrial Morphology

Microscopic observation revealed damage to the spongy trophoblast layer and labyrinth layer in the placenta of RUPP rats, while TAVNS treatment significantly restored the normal placental structure. Moreover, RUPP rats showed enlarged mitochondrial area, swelling, and cristae disorder in placental trophoblasts, which were significantly alleviated by TAVNS.

Acetylcholine (Ach) and Its Receptors

Mass spectrometry analysis showed that TAVNS could increase Ach concentration in placental tissue. Immunofluorescence and Western Blot experiments also indicated that TAVNS significantly increased the expression of M3 acetylcholine receptor (M3AChR) in placental tissue.

ROS, mtROS, and Uprmt

RUPP led to a significant increase in placental ROS levels, while TAVNS significantly reduced ROS levels. Western Blot experiments showed that the expression of HSP70 and LONP1 was significantly upregulated in the RUPP group, while TAVNS significantly downregulated their expression. Furthermore, the expression of pro-apoptotic factors cleaved caspase-3 and NF-κB-p65, as well as cytochrome C, was increased in placental tissue of the RUPP group, while TAVNS significantly reduced the expression of these factors.

M3AChR Gene Knockdown Experiment

Using a mouse retrovirus (LV)-shRNA system to knockdown M3AChR, the results showed that knocking down M3AChR significantly increased the expression of HSP70, LONP1, cleaved caspase-3, and NF-κB-p65, reversing the protective effect of TAVNS, further confirming the important role of M3AChR in the protective effect of TAVNS.

Hypoxia-Induced Cell Model Experiments

In the in vitro cell model, Ach significantly improved CoCl2-induced decrease in cell viability, cell apoptosis, and excessive generation of mtROS in a concentration-dependent manner. TEM observation results showed that Ach could significantly alleviate CoCl2-induced mitochondrial swelling and cristae disorder. Additionally, flow cytometry results indicated that Ach could significantly increase the number of JC-1 red aggregates, suggesting its protective effect on mitochondrial function. Western Blot experiments also showed that Ach could significantly downregulate the expression of HSP70 and LONP1, and inhibit the release of cytochrome C.

Research Conclusions

This study revealed for the first time that TAVNS alleviates preeclampsia by promoting Ach release, inhibiting mtROS generation, and subsequently downregulating Uprmt, thereby reducing placental trophoblast apoptosis and improving placental function. The research suggests that Uprmt may be a new molecular target for protecting placental trophoblasts, and the mitochondrial protective effect of Ach plays a key role in alleviating preeclampsia-related damage.

Research Significance and Highlights

This study not only broadens the understanding of the pathophysiological mechanism of preeclampsia but also provides new insights for the treatment of preeclampsia. As a non-pharmacological treatment, TAVNS has the advantages of high safety and fewer side effects, showing important clinical application potential. The study also discovered the inhibitory effect of Ach on mitochondrial unfolded protein response and apoptosis pathways, with M3AChR playing a key role in its protective effect, providing new molecular targets for future targeted drug development.