Hydrogen sulfide against preeclampsia exposure-induced oxidative mitochondrial damage in HTR-8/SVneo cells

Extravillous trophoblast invasion disorder caused by oxidative stress is involved in the pathogenesis of preeclampsia (PE). In order to identify whether hydrogen sulfide (H2S) can prevent oxidative stress injury in extravillous trophoblasts. HTR-8/SVneo cells were detected by H2S inhibiting H2O2 induced oxidative mitochondrial damage. Reactive oxygen species (ROS) were detected, as well as malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD). JC-1 detected the potential of the mitochondrial membrane in this experiment. Then to detect the expression level of the apoptosis-inducing protein B-cell lymphoma-2 (Bcl-2) associated X protein (Bax), caspase 3, p53, p-p53, the apoptosis-inhibiting protein Bcl-2, PRAP, and the mitochondria fission protein Drp1, p-Drp1. CCK-8 assay, it was demonstrated that cell proliferation in the NaHS group was significantly higher than that in the Mod group, indicating that H2S may induce cell proliferation. Transwell assay elucidated that cell invasion in the NaHS group was recovered compared to the Mod group. ROS concentration no matter in cells or mitochondria was decreased by NaHS, which we could get from the comparison between the Mod group, PAG group, and NaHS group. The concentration of MDA was significantly lower in the NaHS group, and the concentration of SOD was extremely high in the NaHS group. Utilized JC-1 to detect mitochondrial membrane potential and found that cells from the NaHS group had a stable potential while cells from the Mod group and PAG group partly lost their potential, which could demonstrate that NaHS could maintain mitochondrial membrane potential. The western blot results revealed that p-Drp1 had a significant decline in the NaHS group, which means mitochondria fission was decreased in the NaHS group. The expression level of Bax and caspase 3 was significantly lower than in the Mod group and PAG group, and the expression level of Bcl-and PRAP was significantly higher in the NaHS group. That could prove that NaHS protect HTR-8/SVneo cell by inhibiting cell apoptosis. These promising results show that H2S elicits its effects on cell apoptosis by decreasing ROS concentration, maintaining mitochondrial membrane stability, and promoting apoptosis-inhibiting protein expression in cells.


Introduction
There is no known pathophysiology for preeclampsia (PE), which is a pregnancy-related multisystem disorder. In developing countries, it accounts for 15% of all maternal deaths, neonatal and fetal deaths, and preterm births (1,2). The condition is characterized by uncontrolled hypertension during pregnancy accompanied by proteinuria, with neurological symptoms such as seizures at the end of pregnancy (3,4). Despite the fact that the pathophysiology of PE remains unclear, previous studies have indicated that it is closely associated with oxidative stress in trophoblast cells. This led to altered physiologic transformation of spiral arteries, leading to placental hypoperfusion and hypoxia, and ultimately placental insufficiency. During normal placental implantation, extravillous trophoblast migrates into the spiral arteries of the maternal uterus during placental implantation, forming vascular sinuses at the fetal-maternal interface to supply nutrients to the fetus (5). While during PE, trophoblast failed to remodel the spiral artery, leading to placental ischemia and other syndromes of PE.
Oxidative stress may occur as a result of hypoxia and reoxygenation of the placenta caused by a poor spiral artery invasion (6,7). One of the key features of PE is oxidative stress in the trophoblast, which is partially caused by mitochondrial dysfunction (8). Mitochondrial outer membrane permeabilization (MOMP) is associated with mitochondrial function. When MOMP is destroyed, reactive oxygen species (ROS) increase and cell apoptosis are induced by mitochondrial pathways (9). Pores formed by Bax, a protein from the Bcl-2 family, contribute to the loss of MOMP and then release factors such as cytochrome c to activate caspase, which finally leads to cell apoptosis. Thus, mitochondria could be a trigger for curing PE.
H 2 S has been proved by many scientists that it may participate in a lot of pathophysiologic processes, which are quite important in the human body, such as apoptosis, oxidative stress, inflammation, and angiogenesis (10). As a physiologic vascular regulator, H 2 S inhibits proliferation and regulates apoptosis and autophagy of vascular cells, thus affecting vascular diseases in various ways (11). In the meantime, it is well known that H 2 S regulates long-term potentiation and calcium levels in neuronal cells, so disturbances of H 2 S levels in cells have been implicated in neurodegenerative diseases such as Alzheimer's, Parkinson's, strokes, and traumatic brain injuries, which attract a lot of attention (12). H 2 S has also been proven to promote cancer cell death, inhibit cancer angiogenesis, and metastasis (13). The mitochondrial enzyme superoxide dismutase (SOD) and ROS are regulated by H 2 S, which protects cardiomyocytes and inhibits apoptosis (14,15). That is to say, H 2 S exerts its protective effect on cells through the mitochondria pathway. The L-cysteine/H 2 S pathway has been linked to the development of PE in previous studies (16), and H 2 S donors may have prevented PE (17)(18)(19). As we mentioned above, we postulate that H 2 S might be able to protect the placenta in PE. In line with our hypothesis, a study has demonstrated that AP39, a novel mitochondriatargeted hydrogen sulfide donor, could restore mitochondrial health through increasing active mitochondrial content, and decreasing superoxide production (20). We suppose to identify the protection of H 2 S on the trophoblast of PE and the mechanism behind that.

Chemicals
NaHS was used as an H 2 S donor When NaHS reacts with water, it provides a solution of H 2 S with a concentration of approximately 33% of its original concentration (21). NaHS was purchased from Aladdin (China). Our previous experimental results showed that NaHS within 400 µM promoted the proliferation of HTR-8/Svneo cells. Referring to other kinds of literature and our result (22,23), we chose the concentration of 200 µM for our experiments. PAG (DL-Propargylglycine), an endogenous hydrogen sulfide generating enzyme inhibitor was purchased from Sigma (USA). Dulbecco's modified Eagle's medium (DMEM), the cell culture medium, was purchased from BasalMedia (China), and fetal bovine serum (FBS), penicillin, and streptomycin were purchased from GIBCO-BRL (USA). CCK-8 (Cell Counting Kit-8) was purchased from Beyotime (China). Rabbit polyclonal antibodies to GAPDH, Bax, Bcl-2, p53, p-p53, pro-PRAP, and COX IV were purchased from Proteintech (USA). Mice antibodies to p-p53 and Drp1 were purchased from Bioss (USA), and antibodies to procaspase 3, and p-Drp1 were purchased from Cell Signaling Technology (USA).
Cell culture HTR-8/Svneo cell lines (HTR-8) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd., DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 g/mL streptomycin, at 37 • C, 5% CO 2 , and saturated humidity were used to culture HTR-8 cells. A 0.25% concentration of trypsin was used to digest logarithmic phase cells for the experiment. A suspension of individual cells was prepared and seeded into an appropriate plate. A suitable condition for dividing cells was achieved by dividing them into four groups: the Control group, the Mod group, the PAG group and the NaHS group. After detection, we chose 0.

CCK-8 assay
We evaluated cell viability by Cell counting kit 8 assay. Each group got 100 µL of one of the medicine solutions we mentioned above, and the Control group got 100 µL of serum-free DMEM medium. A 100 µL of CCK-8 was added to each culture plate hole after 24 h of cultivation and incubated at 37 • C for 3 h. In order to determine the relative survival rate, we selected the wavelength of 450 nm and measured the absorbance of each well on the plate reader (Tecan, Germany, Infinite M Nano).

Transwell assay
A concentration of 1 × 10 5 cells/well was seeded into the filters in 200 µL of FBS-free medium for 24 h, followed by a different treatment. In the lower chambers, 500 µL of medium containing 30% FBS was used. Using a tipped swab, cells on the top side were removed 24 h after treatment Crystal violet (E607309, Sangon Biotech, China) was used to stain cell migration to the lower side. For each group of testing, at least three independent wells were included, and five to six photographs were taken for each group, and the results were analyzed using Image J (Nation Institutes of Health, Bethesda, MD, USA).

Assay of reactive oxygen species generation
A membrane-permeable fluorescent probe, 2 , 7dichlorofluorescin diacetate (DCFH-DA), and a fluorescence microscope (ECLIPSE NI, NIKON, Japan) were used to detect intracellular ROS. The fluorescence is detected at 525 nm after excitation at 488 nm. The concentration of mitochondrial ROS was detected utilizing MitoSOX Red Mitochondrial Superoxide Indicator (40778ES50, Yeasen, China) and fluorescence microscope (ECLIPSE NI, NIKON, Japan). The fluorescence is detected at 580 nm after excitation at 510 nm.

Detection of mitochondrial membrane potential
An JC-1 array of fluorescent dyes were used to detect mitochondrial membrane potential ( m ) (10009172, Cayman, Japan). As mitochondrial membrane potential declines, the red fluorescence of the JC-1 assay changes to green. A total of 300 µL of JC-1 staining solution was added to each well after HTR-8 cells were treated with different medicine solutions for 24 h. A 20 min incubation period was performed in the incubator on the plate. Fluorescence microscopy was used to photograph the plate (ECLIPSE Ts2, NIKON, JapanJC-1 monomers were detected at 485 and 535 nm, and JC-1 aggregates were detected at 535 and 595 nm.

Analyses of statistics
The mean and standard deviation are expressed as mean ± SEM. For multiple comparisons, a one-way ANOVA was used followed by a post hoc analysis adjusted with a leastsignificant-difference correction (SPSS Inc., Chicago, IL, USA). The significance level was considered if * P < 0.05, * * P < 0.01, * * * P < 0.001.

H 2 O 2 -induced cell apoptosis
We utilized DAPI and flow cytometry to confirm cell apoptosis. After attaching to AT regions of DNA, DAPI stains nuclear and chromosomes, which emit blue fluorescence. Thus, it can indicate morphological changes in the nuclear. Nuclear sequestration is a distinctive feature of apoptosis, and DAPI stains the nucleus and shows its morphology. While apoptosis occurs, Phosphatidylserine (PS) transfers from the cell's inner membrane to the outer membrane. Thus, we could use Annexin V to combine PS in the outer membrane of cells to indicate apoptotic cells. PAG is one of the cystathionine γ-lyase (CSE) inhibitors, which can inhibit the endogenous synthesis of H 2 S. As shown in Figure 2C, after being treated with H 2 O 2 , we can easily find the change in nuclear morphology from round shapes to irregular shapes. Treated with H 2 O 2 and PAG even increased the sequestration, indicating that H 2 S might be involved in the HTR-8 cell apoptosis process. Meanwhile, the result of the NaHS (0.2 mM) group showed that NaHS can partly relieve the influence of H 2 O 2 . Flow cytometry results gave further proof of the above results. Figures 2A,B presented that the proportion

NaHS promoted cell proliferation and invasion after H 2 O 2 treatment
In order to demonstrate the effect of NaHS, we analyzed cell proliferation ability by CCK-8 assay and cell invasion ability by invasion assay. CCK-8 assay represented that the survival rate of the NaHS group was significantly higher than the Mod group and the PAG group, indicating that H 2 S could induce HTR-8 cells proliferation at 0.2 mM after H 2 O 2 treatment, as shown in Figure 3A. The invasion assay showed that there were more HTR-8 cells transferring to the lower chambers in the NaHS group (Figures 3B,C), by which we could demonstrate that H 2 S had the ability to accelerate HTR-8 cell invasion. While in the Mod group, cells were treated with 0.1 mM H 2 O 2 for 24 h, thus causing the attenuation of cell invasion. Above all, we could find that H 2 S was sufficient to protect HTR-8 cells from oxidative stress, because of its ability to induce cell proliferation and cell invasion.

NaHS reduced the concentration of reactive oxygen species
A ROS is a molecule that contains oxygen and reacts chemically. Excessive ROS can induce apoptosis through both the different pathways and thus do harm to the human body (26). We detected the concentration of intracellular and mitochondrial ROS utilizing DCFH-DA (for cell) and MitoSOX Red (for mitochondria). DCFH-DA is a cell-permeable probe that could be oxidized by ROS and emits green fluorescence, and MitoSOX Red could penetrate into cells and selectively targets mitochondria to visualize the process of ROS production in the mitochondria. In Figure 4A, red fluorescence intensity is significantly higher after H 2 O 2 treatment, especially in H 2 O 2 plus PAG group. And the intensity suppressed in the NaHS group demonstrated that H 2 S could inhibit the production of mitochondrial ROS. It is also obvious that NaHS decreased ROS levels in cells. In line with a previous study, H 2 S exerts its antioxidant effect by decreasing ROS.
SOD and catalase (CAT) have an important role in scavenging the levels of ROS. Their activity and concentration are highly related to the oxidative condition in cells. Malondialdehyde (MDA) is cytotoxic and commonly used as a lipid peroxidation indicator. After detecting the concentration of SOD, CAT, and MDA (in Figure 4C), we could find a higher level of MDA and lower levels of SOD and CAT in the H 2 O 2 group and the PAG group, which is consistent with the previous results of ROS. After being treated with NaHS, the concentration of SOD and CAT became significantly higher which indicated that H 2 S could protect HTR-8 cells through cleaving ROS.

NaHS restored mitochondrial function
Perturbation of mitochondrial function is associated with loss of the mitochondrial transmembrane potential and the release of apoptogenic factors. Mitochondrial membrane potential ( m) is a sensitive indicator of mitochondrial damage (27). JC-1 is a commonly used fluorescent probe, which is used for the detection of m . When m is high, JC-1 emits red fluorescence. Unlike in a high m condition, it is not possible for JC-1 to gather together at lower min the mitochondrial matrix and produce green fluorescence. As shown in Figure 4B, H 2 O 2 treatment for 24 h could extremely increase green fluorescence, and H 2 O 2 plus PAG undoubtedly made the green fluorescence lighter, which means H 2 O 2 has a harmful effect on mitochondrial membrane potential. NaHS reduced the effects of H 2 O 2 on m which is proved by the increased red fluorescence in the NaHS group.
Mitochondria are complex organelles, and their cleavage and fusion are precisely regulated by genes, but this balance can be disrupted when apoptosis occurs. In the pro-apoptotic Western blot was used to detect the expression of p-Drp1 and Drp1 within and outside the mitochondria. As shown in and PAG can promote mitochondrial lysis, implying that this lysis may be related to H 2 S concentration. And NaHS have a protective effect on mitochondria by inhibiting its lysis, from which we purposed that it might be related to cell apoptosis.

Discussion
There are many complications associated with pregnancy, but one of the most feared is preeclampsia. PE usually begins as hypertension and proteinuria in the third trimester and can quickly progress to more serious conditions, such as death for both mother and child. It is still unclear what causes PE, but clinical and pathological studies suggest the placenta plays a pivotal role in their pathogenesis (5). Also, endogenous H 2 S pathway could contribute to the development of PE both in gene level and protein level (30). Based on previous studies, abnormal placental development and angiogenesis are key features of PE, which progresses and eventually leads to ischemia and hypoxia of the placenta. The excessive apoptosis of trophoblast cells is one of the factors that subsequently lead to poor placental development and angiogenesis. In this study, we verified the inhibitory effect of H 2 S on apoptosis of trophoblast cells and further investigated its mechanism. Finally, we revealed the protective effect of H 2 S on the placenta in PE and its mechanism.
HTR-8/Svneo cell line is an immortalized extravillous trophoblast cell line derived from normal human early pregnancy placenta. It is a common cell line used to study the function of early pregnancy trophoblast in vitro (31). In this experiment, we confirmed that H 2 S could promote the proliferation and invasion of HTR-8 cells after H 2 O 2 treatment which is consistent with our previous study (23), and its antioxidant effect reduced the production of ROS, maintained the function of mitochondria, and suppress the synthesis of apoptosis-related proteins. To confirm the role of H 2 S in promoting cell proliferation and invasion, CCK-8 assay and invasion assay were utilized to detect both the proliferation and invasion abilities of cells, and the results showed that the survival rate of cells and the migration of cells were significantly increased after the addition of NaHS compared with the Mod group. This verified the protective effect of H 2 S on HTR-8 cells under excessive oxidative stress.
H 2 S inhibits cell apoptosis at low concentrations. There are three ways in which H 2 S inhibits apoptosis: First, it regulates the MAPK pathway through ATP-sensitive potassium channels and thus inhibits apoptosis; Second, H 2 S is oxidized in the cytoplasm and can hypersulfide caspase 9 and other proteins, thus disabling them and the subsequent apoptotic process will be inhibited; Third, H 2 S hypersulfates NF-κB and directly induced it's combining with anti-apoptosis gene promoter. H 2 S exerts its effect on mitochondria through regulating mitochondrial fission and fusion, which is achieved by regulating the Drp1 protein, a major factor controlling mitochondrial fission, which can form helical oligomers and thus contribute to mitochondrial division (32). Our study confirmed that low concentrations of H 2 S can reduce the intracellular p-Drp1/Drp1 ratio, thus reducing mitochondrial cleavage and further reducing the accumulation of ROS and thus inhibiting apoptosis. We also demonstrated that H 2 S could restore mitochondrial membrane potential, reduce mitochondrial cleavage, and decrease ROS levels in mitochondria, which lead to the inference that the inhibitory effect of H 2 S on apoptosis in HTR-8 cells is partly dependent on the mitochondrial pathway.
p53 is an intracellular antitumor factor that is used in the field of tumor therapy because of its ability to inhibit cell growth and promote apoptosis. When cells are stimulated by an internal or external environment (e.g., DNA damage or hypoxia), the p53 expression level will be up-regulated, thus causing apoptosis. p53 expression promotes the up-regulation of the expression of the pro-apoptotic signal Bax (Bcl-2 associated × protein) and the down-regulation of the expression of the anti-apoptotic signal Bcl-2 in the Bcl-2 gene family. The Bax protein forms pores in the mitochondrial membrane, resulting in destroying mitochondrial permeability and subsequent loss of mitochondrial membrane potential and release of cytochrome c (33). Caspase 9, a protein of the aspartate family (caspase family) that is essential in the mitochondrial apoptotic pathway, is upstream of the entire cascade reaction and can have an effect by activating downstream acting proteins, and is an initiator protein. When activated, the initiator protein can activate downstream caspase 3, thus activating DNA degradation by deoxyribonucleases, triggering apoptosis. Our study confirmed that H 2 S can inhibit apoptosis in the mitochondrial pathway by down-regulating the expression of Bax and caspase 3 proteins and up-regulating the expression of Bcl-2, PRAP proteins.
However, many details still need to be added in the future. We verified at the cellular level that H 2 S inhibited apoptosis of HTR-8 cells after H 2 O 2 treatment via the mitochondrial pathway, and confirmed that its antioxidant effect was sufficient to promote the proliferation and invasion of HTR-8 cells after H 2 O 2 treatment. PE is a disease which is quite difficult to research, because of its unique characteristics, we here provide a potential way to cure or prevent the happening of PE. And we also replenish the function of H 2 S which is a core-compound in our laboratory. Even though, these results need to be confirmed at the animal level in order to have a better clinical meaning.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.