efficacy of low molecular heparin on preeclampsia by

Research ArticleEfficacy of Low Molecular Heparin on Preeclampsia by InhibitingApoptosis of Trophoblasts via the p38MAPK Signaling Pathway

Dandan Quan,1 Li Li,2 and Manzhen Zuo 1

1Department of Obstetrics and Gynecology, The People’s Hospital of China Three Gorges University, The First Hospital of Yichang,Hubei 443000, China2Department of Obstetrics and Gynecology, The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing 211100, China

Correspondence should be addressed to Manzhen Zuo; [email protected]

Received 30 April 2021; Revised 5 July 2021; Accepted 17 July 2021; Published 3 August 2021

Academic Editor: Tao Huang

Copyright © 2021 Dandan Quan et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. To explore the efficacy of low molecular heparin on preeclampsia by inhibiting apoptosis of trophoblasts via thep38MAPK signaling pathway. Methods. A preeclampsia rat model was established, and the effects of low molecular heparin onpreeclampsia via the p38MAPK signaling pathway were analyzed based on intervention of the rats with different combinationsof low molecular heparin and p38MAPK signaling pathway activator. Furthermore, a hypoxia/reoxygenation model oftrophoblasts in vitro was established to explore the effects of low molecular heparin on trophoblasts via the p38MAPK signalingpathway. Results. After treatment with low molecular heparin, pregnant rats in the heparin group showed significantly decreasedblood pressure, 24 h proteinuria, and p38MAPK protein levels in placenta tissues and decreased apoptosis rate of placenta tissuecells (all P < 0:05) and showed more fetal rats and lowered weight of them (both P < 0:05) but showed no significant change inthe weight of placenta (all P > 0:05). Pregnant rats treated with low molecular heparin and p38MAPK activator showedsignificantly higher blood pressure, 24 h proteinuria, and p38MAPK protein levels in placenta tissues and apoptosis rate ofplacenta tissue cells than those of pregnant rats in the heparin group (all P < 0:05) and also showed less fetal rats and lighterfetal rats than those in the heparin group (both P < 0:05) but showed no difference with them in the weight of placenta(P > 0:05). Further analysis revealed that low molecular heparin could protect the survival and migration of trophoblasts underhypoxia/reoxygenation conditions and reduce apoptosis of them (all P < 0:05). Conclusion. Low molecular heparin can alleviatepreeclampsia by inhibiting the p38MAPK signaling pathway and can inhibit apoptosis of trophoblasts and promoteproliferation and migration of them.

1. Introduction

Preeclampsia is a leading cause of premature delivery ofinfants and maternal and infant mortality. Its clinical mani-festations include hypertension and placenta, and its inci-dence is about 2%-5% [1]. At present, some scholars believethat preeclampsia is correlated with maternal childbearingage and placenta, and it is generally believed that preeclamp-sia is caused by reperfusion injury after placental ischemia,oxidative stress, and imbalance of angiogenesis/antiangio-genic factors, and it is prone to cause lifelong complicationsof cardiovascular and kidney diseases in women, increasingthe risk of stroke in women by 6 times [2, 3].

Low molecular heparin is often used to prevent pre-eclampsia, which can lower the occurrence of preeclampsiaand reduce fetal death [4, 5]. However, as many researchesonly focus on the analysis of the efficacy of lowmolecular hep-arin, the mechanism of it in preventing preeclampsia has notbeen fully studied. Trophoblasts are the main cellular formof placenta, with an important role in the development andprogression of preeclampsia [6]. Some studies have reportedthat low molecular heparin can protect trophoblasts andmaintain their functions [7, 8]. Interestingly, the p38MAPKsignaling pathway has effects similar to those of lowmolecularheparin [9, 10], which suggests that low molecular heparinmay affect preeclampsia like the p38MAPK signaling pathway.

HindawiComputational and Mathematical Methods in MedicineVolume 2021, Article ID 3337514, 7 pageshttps://doi.org/10.1155/2021/3337514

https://orcid.org/0000-0001-5272-4025

https://creativecommons.org/licenses/by/4.0/

https://creativecommons.org/licenses/by/4.0/

https://doi.org/10.1155/2021/3337514

It has been reported that activating the MAPK signaling path-way is an action mechanism of low molecular heparin in pro-moting the migration of trophoblasts [11].

This study has analyzed the effects of low molecular hep-arin on trophoblasts to explore its therapeutic mechanism.

2. Materials and Methods

2.1. Research Objects. Sixty specific pathogen-free (SPF) Wis-tar rats were purchased from the Animal Center of SouthernMedical University, with animal license of SCXK (Yue) 2016-0041, including 30 female rats between 8 and 10 weeks old,with weight of 200-220 g and 30 male rats between 9 and 11weeks old, with weight of 300-330 g. The rats were raised in30 cages with one male rat and one female rat in each cageat room temperature of 20-25°C under relative humidity of40%-70% and a light/dark cycle of 12 hours, and they wereallowed to freely drink water. Then, the vaginal suppositoryof each female rat was analyzed on the next day. All animalexperiments were carried out under the approval by the Ani-mal Care and Use Committee of our hospital and the guidingprinciples of the Council for International Organization ofMedical Sciences (CIOMS).

2.2. Establishment of Preeclampsia Model. On the 13th dayafter the formation of vaginal suppository in female rats,the blood pressure of each female rat was measured andrecorded. Then, 30 female rats were selected by the randomnumber table method, and each selected female rat was sub-cutaneously injected with 200mg/(kg·d) L-nitroargininemethyl ester (Sigma Company, the United States) for 4 con-secutive days. If the blood pressure of a selected rat increasedby more than 30mmHg, the modeling of the rat was consid-ered successful. The used animal sphygmomanometer waspurchased from Shanghai Yuyan Scientific Instruments Co.,Ltd.

2.3. Grouping and Treatment of Rats. Twenty-four of the 30female rats were successfully modeled, and they wereassigned to a model group, a lowmolecular heparin interven-tion group (hereinafter referred to as the heparin group), anda low molecular heparin combined with p38MAPK activatorintervention group (hereinafter referred to as the combina-tion group), 8 rats in each group. The rest 10 female rats weretaken as a control group. Female rats in the heparin groupwere injected intravenously with 40μL/(kg d) low molecularheparin at the 15th day after vaginal suppository was formed,and rats in the combination group were injected intrave-nously with 0.5mg/kg p38MAPK activator through tail veinon the basis of treatment to those in the heparin group. Ratsin the control group and the model group were injected sub-cutaneously with the same volume of normal saline as that oflow molecular heparin. On the 21st day after pregnancy ofeach rat, the pregnant rat was anesthetized with 3% pento-barbital sodium by intraperitoneal injection to take the fetalrats and placenta out, and the number of fetal rats and weightof them and apoptosis rate of placenta tissue cells wererecorded. The low molecular heparin was purchased fromGlaxoSmithKline (Tianjing, China), and the p38MAPK acti-

vator (Dehydrocorydaline chloride) was from the MCECompany in China. In addition, pentobarbital sodium waspurchased from Shanghai Kefeng Chemical Reagent Co.,Ltd., and electronic scales from Beijing Jinda Sunshine Tech-nology Co., Ltd.

2.4. Blood Pressure and Proteinuria Detection. The bloodpressure and 24h proteinuria of pregnant rats were recordedbefore modeling, after 15 days of pregnancy, and 21 days ofpregnancy, respectively, and the 24 h proteinuria was ana-lyzed using an automatic biochemistry analyzer (SYSMEXBX-4000 full-automatic biochemical analyzer) from SYS-MEX Medical Electronics (Shanghai) Co., Ltd.

2.5. Detection of p38MAPK Signaling Pathway-RelatedProteins. A Western blot assay was carried out to detect thep38MAPK level, and the protein of tissues and cells wasextracted using the freeze-thaw method. The concentrationof the protein was determined using the bicinchoninic acid(BCA) method and adjusted to 4μg/μL. The protein was sep-arated through 12% polyacrylamide gel electrophoresis. Theinitial voltage was 90V, and then, the voltage was increasedto 120V to move the sample to an appropriate position ofthe separation gel. After electrophoresis, the protein wastransferred to a membrane under 100V constant voltagefor 100min and blocked at 37°C for 60min. Subsequently,the membrane was blocked with 5% skim milk powder forfuture immune response. The membrane was incubated withprimary antibody (1 : 1000) at 4°C for one night and thenwashed with warm phosphate buffer saline (PBS) three times,5min each time. After washing, the membrane was incubatedwith secondary antibody (1: 1000) at room temperature for1 h. After incubation, the protein was developed and immo-bilized with electrochemiluminescence (ECL) agent. Thescanned protein band was analyzed using the Quantity Onesoftware, and the relative protein expression level wasrecorded as the gray value of the band/gray value reference.BCA protein kit, ECL kit, and trypsin with item numbers of23250, 35055, and 90058 were all purchased from ThermoScientific™, and rabbit anti-p38MAPK, bcl-2, bax monoclo-nal antibodies, and goat anti-rabbit IgG secondary antibodieswith item numbers of ab170099, ab185002, ab32503, andab6721 were all purchased from the Abcam Company inthe United States.

2.6. Cell Apoptosis Experiment by the Tunel Method. The apo-ptosis rates of placenta tissue cells and chorionic villus cellswere measured using the Tunel method as follows: The num-ber of apoptotic cells and the total number of cells under fiveselected fields under an optical microscope were counted,and the apoptosis rate was calculated.

2.7. Cell Sources. Human chorionic villus cells (HTR-8/Svneo) with number of CRL-3271 purchased from theAmerican Type Culture Collection (ATCC) were culturedin RPMI-1640 medium with 5% fetal bovine serum in 95%air and 5% carbon dioxide at 37°C.

2.8. Cell Grouping and Intervention. The cells were assignedinto a blank group, a hypoxia group, a heparin group, and a

2 Computational and Mathematical Methods in Medicine

combination group. Except for cells in the blank group, cellsin other groups were all treated with hypoxia/reoxygenation(hypoxia for 2 hours and reoxygenation for 4 hours). Cellsin the heparin group in medium were added with 5% hepa-rin, and cells in the combination group were added with 5%heparin and 5% p38MAPK activator. Afterwards, the apo-ptosis and migration of the cells were detected.

2.9. Cell Proliferation Assay by Cell Counting Kit-8 (CCK8). Atotal of 1 ∗ 103 cells were routinely seeded into a 96-wellplate, and 10μL CCK8 solution was added into the cells at24 h, 48 h, 72 h, and 96h after culturing, respectively. Theoptical density of each well at 450 nm was measured using amicroplate reader (Bio Rad, Hercules, California, the UnitedStates) after each addition of CCK8 solution.

2.10. Determination of Migration and Invasion of Cells. Atotal of 5 × 104 cells were transferred to the upper compart-ment, and the lower compartment was added with L-15medium with 10% FBS. Twenty-four hours later, the cellsunder the microporous membrane were immobilized with75% methanol and stained with crystal violet. Subsequently,

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Figure 1: Efficacy of different interventions on preeclampsia in pregnant rats. (a) Blood pressure; (b) 24 h proteinuria; (c) the number of fetalrats; (d) the weight of fetal rats; (e) the weight of placenta. a indicates that in comparison with the control group, P < 0:05; b indicates that incomparison with the model group, P < 0:05, and c indicates that in comparison with the heparin group, P < 0:05.

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Figure 2: Effects of different interventions on the p38MAPKprotein level in placenta tissues of pregnant rats. a indicates that incomparison with the control group, P < 0:05; b indicates that incomparison with the model group, P < 0:05, and c indicates thatin comparison with the heparin group, P < 0:05.

3Computational and Mathematical Methods in Medicine

the number of cells penetrating the membrane in five selectedfields under an optical microscope was calculated, and threeparallel experiments were carried out. The Transwell insertand related reagents were purchased from Corning (NewYork, the United States).

2.11. Statistical Analysis. In this study, the data were statisti-cally analyzed and visualized into figures using GraphpadPrism 8, and measurement data were expressed as themean ± standard deviation. Comparison among multiplegroups was conducted using the one-way ANOVA, and backtesting was carried out using LSD. In addition, Pearson’sanalysis was used for correlation analysis. P < 0:05 indicateda significant difference.

3. Results

3.1. Efficacy of Different Interventions on Preeclampsia inPregnant Rats. Compared with pregnant rats in the controlgroup, pregnant rats in the model group showed significantlyhigher blood pressure and 24 h proteinuria levels (both P <0:05) and showed significantly less fetal rats, lighter fetal rats,and placenta (all P < 0:05), suggesting that the modeling wassuccessful. In addition, after treatment with low molecularheparin, pregnant rats in the heparin group showed signifi-cantly decreased blood pressure and 24h proteinuria levels

(both P < 0:05) and showed an increase in the number offetal rats and weight of them but showed no significantchange in the weight of placenta (P > 0:05). Rats in the com-bination group showed significantly higher blood pressureand 24 h proteinuria, less fetal rats, and lighter fetal rats than

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Figure 3: Effects of different interventions on apoptosis of placenta tissue cells of pregnant rats. (a) Apoptosis rate of placenta tissue cells; (b)the bax protein level in placenta tissues; (c) the bcl-2 protein level in placenta tissues; (d) the correlation between p38MAPK protein level andapoptosis rate in placenta tissue cells; (e) the correlation between p38MAPK and bcl-2 protein level in placenta tissues; (f) the correlationbetween p38MAPK and bax protein level in placenta tissues. a indicates that in comparison with the control group, P < 0:05; b indicatesthat in comparison with the model group, P < 0:05, and c indicates that in comparison with the heparin group, P < 0:05.

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Figure 4: Effects of different interventions on the p38MAPKprotein level in chorionic villus cells. a indicates that incomparison with the control group, P < 0:05; b indicates that incomparison with the hypoxia group, P < 0:05, and c indicates thatin comparison with the heparin group, P < 0:05.


those in the heparin group (all P < 0:05) but showed no dif-ference with them in the weight of placenta (P > 0:05)(Figure 1).

3.2. Effects of Different Interventions on the p38MAPK ProteinLevel in Placenta Tissues of Pregnant Rats. The p38MAPKprotein level in placenta tissues was seen in the model group,the combination group, the heparin group, and the controlgroup from high to low (all P < 0:05) (Figure 2).

3.3. Effects of Different Interventions on Apoptosis of PlacentaTissue Cells of Pregnant Rats. The apoptosis of placenta tissuecells and the level of apoptosis-related protein (bax) wereboth seen in the model group, the combination group, theheparin group, and the control group from high to low (allP < 0:05), and the level of apoptosis-related protein (bcl-2)was seen in them from low to high (all P < 0:05). Correlationanalysis showed that the level of p38MAPK protein was sig-nificantly linearly correlated with the apoptosis rate, bax,and bcl-2 levels of placenta tissue cells (all P < 0:05)(Figure 3).

3.4. Effects of Different Interventions on the p38MAPK ProteinLevel in Chorionic Villus Cells. The level of p38MAPK proteinin chorionic villus cells was seen in the blank group, the hep-arin group, the combination group, and the hypoxia groupfrom low to high (all P < 0:05) (Figure 4).

3.5. Effects of Different Interventions on Apoptosis ofChorionic Villus Cells. The apoptosis rate of chorionic villuscells and the level of apoptosis-related protein (bax) wereboth seen in the hypoxia group, the combination group, theheparin group, and the control group from high to low (allP < 0:05), and the level of apoptosis-related protein (bcl-2)was seen in them from low to high (all P < 0:05) (Figure 5).

3.6. Effects of Different Interventions on Proliferation andMigration of Chorionic Villus Cells. The proliferation andmigration abilities of chorionic villus cells were seen in thehypoxia group, the combination group, the heparin group,

and the control group from low to high (all P < 0:05)(Figure 6).

4. Discussion

Preeclampsia is the major cause of perinatal morbidity andmortality for pregnant and lying-in women all over theworld. Excessive death of placental cells is a crucial clinicalmanifestation of women with preeclampsia, which leads tomaternal placental dysfunction and induces thrombosis,abortion, etc. [12, 13]. Low molecular heparin can improvethe placental function by increasing blood flow to theimplantation site and reduce the thrombosis [14]. Studiesshow that low molecular heparin can promote the angiogen-esis and suppress apoptosis of trophoblasts [15, 16]. How-ever, the mechanism of this action of low molecularheparin remains unclear. This study found that low molecu-lar heparin could suppress the apoptosis of trophoblasts andcould promote proliferation and migration of them by inhi-biting the p38MAPK signaling pathway.

In this study, we constructed preeclampsia rat modelsthrough L-nitroarginine methyl ester [17], finding that bloodpressure and 24h proteinuria levels of the rats significantlyincreased and the number of fetal rats and weight of themand the weight of placenta in them decreased significantly.In addition, we also found that the apoptosis rate of placentatissue cells of the rats increased significantly, while afterintervention of low molecular heparin, those manifestationswere improved. The results were consistent with previousresearch results. Low molecular heparin can positively acton preeclampsia [18, 19]. The p38MAPK signaling pathwayhas been reported to be related to the growth and migrationof trophoblasts. For example, a study by Zhou et al. [20]has revealed that transforming growth factor-β1 couldinhibit the apoptosis of trophoblasts induced by mercuricchlorides by suppressing the p38MAPK signaling pathway,and one other study by Ebegboni et al. [21] also reported thatflavonoids promoted the self-renewal and invasion of spher-ical stem cells of trophoblasts by inhibiting p38MAPK

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Figure 5: Effects of different interventions on apoptosis of chorionic villus cells. (a) Apoptosis rate of chorionic villus cells; (b) bax proteinlevel in chorionic villus cells; (c) bcl-2 protein level in chorionic villus cells. a indicates that in comparison with the control group, P < 0:05; b indicates that in comparison with the hypoxia group, P < 0:05, and c indicates that in comparison with the heparin group, P < 0:05.


signaling pathway. In addition, one study by Che et al. [22]also found that knockout of heparanase gene inhibits theinvasion of human trophoblasts by activating the p38MAPKsignaling pathway. Heparin has also been reported to inhibitexcessive circulation and remodeling of porcine pulmonaryartery via the p38MAPK signaling pathway, thus alleviatingthe pulmonary hypertension [23]. Low molecular heparin,as a derivative of heparin, may also play its role by regulatingthe p38MAPK signaling pathway, but no research on it hasbeen reported so far. In this study, we found that low molec-ular heparin could inhibit the p38MAPK signaling pathway.According to the design idea of rescue experiment, we acti-vated the p38MAPK signaling pathway under the interven-tion with low molecular heparin, finding that the efficacy oflow molecular heparin was strongly inhibited and the apo-ptosis of placental tissue cells was also significantly lowered.Therefore, we can conclude that lowmolecular heparin couldalleviate preeclampsia by inhibiting the p38MAPK signalingpathway and could lower the apoptosis of placenta tissuecells. In order to further verify the effects of low molecularheparin on trophoblasts via the p38MAPK signaling path-way, we used an in vitro hypoxia-reoxygenation model tosimulate the living environment of trophoblasts in pre-eclampsia patients, finding that low molecular heparin couldprotect trophoblasts via the p38MAPK signaling pathway.

There are some deficiencies in this study. For example,the effects of low molecular heparin on inhibiting thep38MAPK signaling pathway have been verified only in rats,but the onset of preeclampsia is complicated, such as signifi-cant differences in the condition and prognosis betweenpatients with early-onset preeclampsia and those with late-onset preeclampsia. In addition, one study by Bolnick et al.[7] has uncovered that low molecular heparin induces extra-cellular differentiation of trophoblasts through epidermalgrowth factor-like growth factor signals and reduces apopto-sis of them during oxidative stress, which indicates that thereare more mechanisms for the influence of lowmolecular hep-arin on trophoblasts, so a large amount of research is neededfor exploration of the mechanisms.

To sum up, low molecular heparin can alleviate pre-eclampsia by inhibiting the p38MAPK signaling pathwayand can inhibit apoptosis of trophoblasts and promote prolif-eration and migration of them.

Data Availability

All the raw data could be accessed by contacting the corre-sponding author if any qualified researcher need.

Conflicts of Interest

The authors declare no conflict of interest.

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efficacy of low molecular heparin on preeclampsia by

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