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SCH58261, the Antagonist of Adenosine A2A Receptor, Alleviates Cadmium-Induced Preeclampsia Via Sirtiun-1/Hypoxia-Inducible Factor-1Ɑ Pathway in Rats

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SCH58261, the Antagonist of Adenosine A2A Receptor, Alleviates Cadmium-Induced Preeclampsia Via Sirtiun-1/Hypoxia-Inducible Factor-1Ɑ Pathway in Rats European Review for Medical and Pharmacological Sciences 2020; 24: 10941-10953 SCH58261, the antagonist of adenosine A2A receptor, alleviates cadmium-induced preeclampsia via sirtiun-1/hypoxia-inducible factor-1ɑ pathway in rats W.-T. SHEN1,2, Y.-J. HUANG2, Q. ZHANG3, F. LIN2, X. WANG2, D.-Y. YE4, Y.-P. HUANG1,2 1The First Clinical Medical College, Wenzhou Medical University, Wenzhou, Zhejiang, China 2Departments of Obstetrics and Gynecology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China 3Departments of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China 4Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology Abstract. – OBJECTIVE: To identify the role of Introduction adenosine A2A receptor (A2AR) in cadmium-in- duced preeclampsia (PE) rats and the potential molecular mechanism. Preeclampsia (PE) is a syndrome limited to PATIENTS AND METHODS: The expression of pregnancy that presents with the hallmark clin- A2AR in placentae obtained from PE women and ical manifestations of new-onset hypertension normal pregnant (NP) women were measured. and proteinuria after the 20th gestational week1, The pregnant rats were randomly divided into which could increase considerably the risk of four groups, including NP rats, PE rats, SCH+NP foetal growth restriction (FGR)2. This disease rats, and SCH+PE rats. The 0.125 mg/kg/d CdCl2 was used to establish a PE rat model in PE and is implicated in 2% to 8% of pregnancies and is SCH+PE rats. SCH58261 was used as the specif- considered as a common trigger for maternal and ic antagonist of A2AR with a concentration of 0.2 perinatal morbidity and mortality worldwide3. mg/kg in SCH+NP and SCH+PE rats. The condi- Placenta, a multifunctional organ, plays a central tions of mother, foetus, and placenta were test- conciliatory role during pregnancy, whose proper ed. The placental expression of A2AR, sirtuin-1 a a structure and function are integral for a success- (sirt1), and Hypoxia-Inducible Factor-1 (HIF-1 ) ful and smooth pregnancy4. Increasing evidence was measured by Western blot (WB) and immu- nohistochemistry (IHC) staining. supports the view that the PE is primarily a pla- 5,6 RESULTS: A2AR and HIF-1a increased, and cental dysfunctional disease . sirt1 decreased in placenta in both PE women Cadmium, a nonbiological heavy metal, exists and cadmium-induced PE rats. After treatment ubiquitous in the environment, especially in the with SCH58261, the sirt1 increased and HIF-1a plant roots, such as rice, tobacco, etc. The pop- decreased in cadmium-induced PE rats along ulation intakes cadmium via diet and smoking. with the amelioration of maternal outcomes, foe- tal and placental growth. Pregnant women are more susceptible to it due to CONCLUSIONS: This paper firstly revealed the increased intestine absorbency and respirato- that placental A2AR mediated cadmium-induced ry rate7. Placenta is recognized as the major organ PE, and A2AR suppression could attenuate pla- target of cadmium. Besides, epidemiologic study cental impairment by acting on the expression has suggested that gestational cadmium exposure of sirt1 and sirt1-mediated regulation of HIF-1a. contributes to the development of PE although the mechanism remains mysterious. Key Words: The available data have shown that cadmium Preeclampsia, Adenosine A2A receptor, Sirtuin-1, exposure during pregnancy links with the ab- Hypoxia-inducible factor-1a. normal increase of adenosine8,9. The proposed Corresponding Author: Yinping Huang, MD; e-mail: [email protected] 10941 W.-T. Shen, Y.-J. Huang, Q. Zhang, F. Lin, X. Wang, D.-Y. Ye, Y.-P. Huang mechanism of PE has included adenosine-de- Rats and Treatments pendent biological effects, adenosine receptors, Adult female SD rats (220±10 g) purchased and the associated adenosine signalling path- from the Animal Centre of Chongqing Medical way10-13. Adenosine usually exhibits its effects University were maintained with a standard chow by the attachment of specific receptors, such as diet and standard 12:12 h light-dark cycle at a 14 A1R, A2AR, A2BR, and A3R , whose activation controlled temperature of 25°C. Female rats were has been distinguished as danger signals for mated with male rats overnight. The day on which placental maldevelopment11,15,16. Among them, sperm was identified from the vaginal smear was A2AR is one type of high-affinity adenosine defined as gestational day 0 (GD0). The pregnant receptors, playing as an alarm sensor to tissue animals were randomly assigned to the following damage. As expected, the elevation of placental four groups containing five rats each, the nor- 17 A2AR occurs in PE patients , and is exaggerat- mal pregnancy (NP) rats, normal pregnancy with ed when complicated by FGR17,18. Besides, this SCH58261 (SCH+NP) rats, preeclampsia (PE) receptor is strongly positively correlated with rats, and preeclampsia with SCH58261 (SCH+PE) the expression of HIF-1a in human placenta rats. From GD9 to GD18, SCH groups (SCH+NP throughout pregnancy with or without PE, and and SCH+PE rats) were intraperitoneally injected proved to be able to act with HIF-1a to sustain with SCH58261 (Beyotime, Shanghai, China) at a tissue damage16,19. concentration of 0.2 mg/kg/d at 7:30, and the rest Although the enhancing effect of cadmium in of the rats were treated with saline at an equiva- trophoblast HIF-1a expression in the development lent volume at the same time. From GD9 to GD14, of PE has also been well recognized in previous rats in the CdCl2-treated groups (PE and SCH+PE 20-23 studies , the role of A2AR in cadmium-related rats) were intraperitoneally injected with CdCl2 PE remains unknown. Thus, the purpose of the (0.125 mg/kg/d, obtained from Sinopharm Chem- present work is to identify the effect of cadmi- ical Reagent Co., Ltd., Mainland, China) at 8:00; um on the expression of A2AR in placenta and rats in the other groups were treated with saline at address the role of receptor in cadmium-induced an equivalent volume at the same time. The dos- preeclamptic rats. age of SCH58261 used was determined based on the literature25. All procedures used in this study complied with the Institutional Animal Care and Patients and Methods Use Committee (IACUC). Patients Measurement of Systolic Blood Pressure According to ACOG, PE was diagnosed by the (SBP) new onset of hypertension (systolic blood pres- All female rats were trained to adapt to blood sure ≥140 mmHg or diastolic blood pressure ≥90 pressure measurement before the start of the ex- mmHg), and proteinuria (≥300 mg/24 h or ran- periment. SBP was measured between 8:00 and dom urine protein ≥0 (+)) after the 20th gesta- 12:00 every three days from GD3 to GD18 with tional week24. The blood pressure and urine pro- a noninvasive pressure monitor (BP-98A, Softron tein levels of all recruited volunteers recovered Co., Ltd., Beijing, China). The average value from to normal levels within six weeks after delivery. three consecutive SBP readings (that differed by The inclusive criteria of participants in this pa- no more than six mmHg) was viewed as the SBP per were from singleton pregnancies at term, the value for that day. age of the pregnancy women was between 26 and 28. The exclusive criteria is that those pregnant Urine Protein Quantitation women suffering from cardiovascular disease, On GD3 and GD18, the rats were fasted in met- kidney disease, or other complications of preg- abolic cages with water for 24 h. The urine was nancies were excluded. Normotensive pregnant collected, and protein was quantified by the bicin- women were randomly employed as controls. choninic acid assay (BCA) method purchased from Normotensive pregnant women (n=5) and preg- Thermo Fisher Scientific, Waltham, MA, USA). nant patients with PE (n=5) were included in the study. All protocols applied in our research were Sample Collection approved by the Clinical Research Committee of Rat pups were delivered on GD19 by cesarean The First Affiliated Hospital of Wenzhou Medi- section after anesthetization with an intraperito- cal University. neal injection of 1% pentobarbital. One kidney 10942 SCH58261 alleviates cadmium-induced PE via sirtiun-1/HIF-1a and three placentae were randomly chosen from ary antibody (the secondary antibodies were from each maternal rat and processed with 4% para- Sangon, Shanghai, China) for 60 min. Finally, the formaldehyde for histological evaluation. The bands were developed with an enhanced chemi- remaining placentae were kept at -80°C. Fetal luminescence (ECL) solution (Pierce, Rockford, weight, fetal length, placental diameter, and pla- IL, USA) in a Bio-Rad ChemiDoc XRS system cental weight were measured. Placental efficiency (USA), and the relative bands densities were eval- was defined by fetal weight/placental weight26. uated with AlphaEaseFC software. The FGR rate was defined as the proportion of foetuses born below the 10th weight percentile for RT-qPCR the gestational age27. Total RNA was extracted from placental tissues with RNAios Plus solution following the man- Morphological Assay and ufacturer’s instructions. The RNA was reverse Immunohistochemistry (IHC) Staining transcribed into cDNA with a PrimeScript RT Re- Paraffin slices (4-μm thick) of the kidney and agent Kit and then amplified with TB Green® Pre- placentae were processed by HE staining for mi- mix Ex Taq™ II. The comparative cycle threshold crostructural observation. The 4-μm thick placen- method was employed for the analysis of relative tal slices were stained with anti-A2AR, anti-sirt1, gene expression against b-actin. All reagent kits and anti-HIF-1a, respectively, in a wet darkroom used in the experiments described above were at 4°C overnight, followed by an anti-rabbit/ provided by TaKaRa (Dalian, China). The follow- mouse auto-immunostaining (ZSGB-BIO, Bei- ing primers were used in our study: sirt1 forward jing, China).
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