Honokiol inhibits inflammation and endoplasmic reticulum stress in a rat model of pregnancy-induced hypertension

Abstract

The levels of inflammatory factors in pregnancy-induced hypertension (PIH) are closely correlated with the severity of the disease. The enhanced endoplasmic reticulum (ER) stress response and upregulated iNOS might contribute to the pathophysiology of preeclampsia. Honokiol has been shown to be a potential anti-inflammatory and anti-ER stress agent. However, the effect of honokiol on the inflammation and ER stress has not been investigated in a gestational hypertensive model. The present study aimed to elucidate the role of honokiol in inflammation, ER stress and endothelial/inducible nitric oxide synthase (eNOS/iNOS) in a PIH rat model. Rats with hypoxia-induced PIH were administered honokiol (0, 200, 600, 2000 μg/kg) daily for one week to different groups by intragastric administration. Systolic blood pressure (SBP) and urinary protein concentration were measured via the tail-cuff method and CBB kit. Proinflammatory cytokines and ER-stress markers, VEGF, NO, iNOS and eNOS in the plasma or placental tissue were analyzed by enzyme-linked immunosorbent assay (ELISA), quantitative real time polymerase chain reaction (RT-qPCR), NO assay kit or western blot. The blood pressure and urinary protein level in PIH rats were significantly decreased after honokiol. Honokiol significantly inhibited the elevated levels of proinflammatory cytokines and ER-stress markers, which were accompanied by the upregulation of eNOS, NO and VEGF mRNA in PIH rats. However, the level of iNOS was reduced by honokiol. Our study suggests a beneficial potential of honokiol in PIH rats through inhibition of proinflammatory cytokines and ER stress. Honokiol could be an intriguing therapeutic approach in ER stress related PIH.

Keywords:

• Pregnancy-induced hypertension
• honokiol
• endoplasmic reticulum stress
• inflammation

Introduction

Pregnancy-induced hypertension (PIH) is considered a major cause of perinatal and maternal mortality, including gestational hypertension (GH) and preeclampsia (PE) [1]. However, the underlying mechanism of PIH remains unclear. GH is related to increased serum levels of proinflammatory cytokines, such as interleukin (IL)-1β, IL-10, and tumour necrosis factor (TNF)-α [2]. A study has found that the expression levels of ER stress markers, including protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2α (eIF2a) and CCAAT/-enhancer-binding protein homologous protein (CHOP), especially including p-PERK, p-eIF2a and p-Ire1, were markedly increased in the placentas of women with PE [3]. And excessive ER stress may contribute to the pathophysiology of PE [4].

Certain macrophages in the placenta synthesize and secrete TNF-α which could maintain normal pregnancy [5]. However, TNF-α beyond the normal level could injure vascular endothelial cells and exacerbate placental impairment. Abnormal secretion of IL-6 can cause excessive accumulation of platelets in blood vessels and form endothelial cell dysfunction. Some evidence has suggested that inflammatory cytokines and nitric oxide (NO) could contribute to the pathological process of PE [4, 6]. A study has found that the levels of inflammatory cytokines in patients with GH are significantly higher than normal pregnancy, while the level of NO in peripheral blood of patients is significantly decreased [6].

The unfolded protein response (UPR) activation in ER resists cell damage induced ER receptors and promotes cell survival. However, cell death would occur if ER stress is chronic or severe [7, 8]. The eIF2ɑ is phosphorylated to halt the initiation of mRNA translation and reduce the protein-folding load on acute ER stress. Chop normally has low expression and is involved in upregulating functional genes in the UPR and directing eIF2ɑ-P dephosphorylation, and restarting global mRNA translation, which is vital for cell survival in case of an acute insult [7]. Prolonged UPR activation can induce cell apoptosis mainly through activation of the PERK-eIF2α-ATF4-CHOP pathway [9]. When gene DDIT3 encoding CHOP was deleted from cells, ER stress caused less protein aggregation in the ER and reduced oxidative stress and apoptosis [10].

NO plays a vital role in the regulation of vascular tone and hemodynamic flow that control the foetal placental-tone by maintaining placental villous vessels in a vasodilated state and attenuating the action of vasoconstrictors [11–13]. Endothelial NO synthase (eNOS) and inducible NOS (iNOS) catalyze the formation of large amounts of NO under the stimulation of shear stress and hypoxia [14]. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor to stimulate eNOS, resulting in NO production and lower BP. It also plays an important role in the maintenance of trophoblast survival in late-gestation [15].

Honokiol is a small-molecule polyphenol isolated from the genus Magnolia, which has been shown to be a potential anti-inflammatory and anti-oxidative agent [8, 16]. However, the effect of honokiol on the inflammation and ER-stress has not been investigated in a PIH rat model. The present study was designed to elucidate the role of honokiol in inflammation, ER stress and eNOS/iNOS in a PIH rat model.

Materials and methods

Reagents

Honokiol (over 99% purity) was purchased from Xi'an Acetar (Xian, China). CBB kit was purchased from Sigma (USA). The tissue was lysed using RIPA lysis buffer (Pierce, MA, USA). The protein content was quantified by a BCA protein assay kit (Beyotime Biotechnology, Haimen, China)). Antibodies against iNOS, VEGF, eNOS, GRP94, eIF2α, phospho-eIF2α, ATF-6 and CHOP were purchased from Abcam (England, UK). The levels of TNF-a, IL-1β and IL-6 were measured by corresponding enzyme linked immunosorbent assay (ELISA) kits (Cloud-Clone Corp., Houston, TX, USA). Nitric oxide (NO) assay kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RNA was extracted from placental tissues using TRIzol (Invitrogen, USA). SuperScript II reverse transcriptase and SYBR-Green Real-Time PCR Master mix were purchased from Thermo Fisher Scientific (MA, USA).

Ethics statement

All animal experiments were performed in accordance with the guidelines approved by Zhejiang Chinese Medical University. All efforts were made to minimize animal suffering.

Animals

Wistar rats were purchased from the Experimental Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China). The rats were housed in groups in a room under controlled environment with temperature (25 ± 1 °C), humidity (65 ± 10%) and 12 h light-12 h dark cycle. Food and water were provided ad libitum and uniform conditions such as temperature and humidity were maintained throughout the experiment. The purchased adult rats (age (12 ± 1) weeks; weight (280 ± 20) g) were used for the present study. Pregnancy was confirmed by taking vaginal smears a day after the animals were bred and the presence of sperm was considered as the day 0 of pregnancy. These rats were normally bred until day 15 of pregnancy. After that, they were assigned to the normoxia with normal blood pressure group (control group, n = 10) and the 10% hypoxia-induced PIH group (PIH group, n = 40). The rats in the PIH group were randomly divided into four subgroups (n = 10 for each subgroup). These four subgroups were respectively administrated honokiol at a dose of 2,006,002,000 μg/kg daily for one week via gavage.

Assessment of systolic blood pressure and urinary protein

Blood pressure was measured via telemetry measured at GD15, GD16, GD17, GD18, GD19. Before each measurement was taken, every rat was preheated for 5 min to 38 °C, three times for averaging. Urine samples were harvested at indicated time points and CBB kits were used to detect urinary protein concentrations.

Western blot analysis

The rats were anesthetized using isoflurane after one week of honokiol treatment and dissected immediately to collect cardiac blood 0.5–1.0 mL in the anticoagulant tube and placental tissue. The rats were sacrificed by euthanasia. Then, the blood was centrifuged to get the supernatant and was stored at 4 °C for subsequent experiments. The placental tissues were separated and placed on dry ice and stored in the refrigerator at −80 °C for further study. Placental tissues were homogenized and lysed in RIPA lysis buffer and then centrifuged at 15,000 rpm for 30 min at 4 °C (Biofuge15R, Heraeus, Germany). The protein concentration was measured by BCA protein assay kit. We separated equal amounts of protein in 10%-polyacrylamide gels and transferred it onto polyvinylidene fluoride membranes. Blots were blocked with 5% nonfat milk powder for 1 h and then incubated overnight with antibodies against iNOS, VEGF, eNOS, GRP94, eIF2α, phospho-eIF2α, ATF-6 and CHOP, each of which was diluted in Tris-buffered saline/5% non-fat milk powder. Subsequently, the samples were incubated with antibody against glyceraldehyde 3-phosphste dehydrogenase (GAPDH) as a loading control. HRP-conjugated secondary antibodies were detected by enhanced chemiluminescence reagents. The expression level of each protein was analyzed using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Western blots were done with 1:1000 antibody dilutions.

ELISA analysis

The levels of TNF-a, IL-1β and IL-6 in the plasma and placental tissues were both measured using enzyme-linked immunosorbent assays according to the manufacturer’s protocol.

Measurement of NO levels

The NO levels were measured using a nitric oxide (NO) assay kit according to the manufacturer’s instructions.

Real-time quantitative polymerase chain reaction (RT-qPCR)

RNA was extracted from placental tissues using TRIzol following the manufacturer’s instructions. The cDNA was synthesized using SuperScript II reverse transcriptase. Quantitative PCR was performed with SYBR-Green Real-Time PCR Master mix. Finally, the quantitative expression data were collected and analyzed by a 7900 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Primers were designed to determine endogenous genes including VEGF and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and GAPDH was used as the endogenous control. The VEGF relative expression levels were calculated using the 2^−ΔCt method. Forward VEGF, (5′–3′) CCT CCT GGC GCT CTG ATA TGT and reverse, (5′–3′) GTG AGT GTG TAG GTG TGC GC primers were used.

Statistical analysis

For the multiple comparisons, analysis of variance (ANOVA) was used. Post-hoc analysis was performed with t test. p < 0.05 was considered statistically significant. The statistical analyses were performed based on GraphPad Software (San Diego, CA, USA).

Results and discussion

Hypoxia induced PIH like syndrome

The PIH rat model established by hypoxia exhibited significant and characteristic hypertension and proteinuria compared to the control pregnant group. This indicated that the PIH model was successful.

Blood pressure

There were no significant differences between the blood pressure in the PIH group and the control group before hypoxia exposure. After hypoxia treatment of pregnant rats on GD 16, SBP was significantly increased from GD16 to GD21 compared with that of the normoxia group. After honokiol treatment, SBP was significantly decreased in PIH rats (Figure 1(A)).

Figure 1. Honokiol attenuated SBP (A) and proteinuria (B) in PIH rats. Note: (A) SBP was detected at indicated time points. (B) Urinary protein concentrations were measured at indicated time points. Data are expressed as (mean ± SD) [n = 10]; error bars represent standard deviation (±SD). ***p < 0.001 compared with the control group. ^##p < 0.01 and ^###p < 0.001 compared with the PIH group.

Proteinuria

The urinary protein levels of each group were similar before hypoxia exposure. After hypoxia treatment of pregnant rats on GD 16, this level was significantly elevated on GD16 to GD21, relative to that of the normoxia-treated pregnant group. After honokiol treatment, urinary protein content was significantly decreased in PIH rats (Figure 1(B)).

Honokiol decreased the levels of TNF-a, IL-1β and IL-6 in the plasma and placenta

Inflammation markers, IL-1β, IL-6 and TNF-α levels, were determined in the plasma and placenta to assess the effect of honokiol on inflammation. The levels of proinflammatory factors, including TNF-α, IL-1β and IL-6, in the plasma and placenta in PIH rats were significantly decreased (Figure 2).

Figure 2. Honokiol decreased the levels of TNF-α, IL-1β and IL-6 in the plasma (A) and the placenta (B) of PIH rats. Note: ELISA results. ***p < 0.001 compared with the control group. Data are expressed as (mean ± SD) [n = 10]; error bars represent standard deviation (±SD). ^##p < 0.01 and ^###p < 0.001 compared with the PIH group.

Effect of honokiol treatment on placental ER stress marker proteins

The levels of GRP94, eIF2α, p-eIF2α, ATF-6 and CHOP in the placental tissue were detected by western blot. The levels of GRP94, ATF-6, CHOP and p-eIF2α were significantly lower in the honokiol group. There was no significant difference in the eIF2α levels between PIH rats and control rats (Figure 3).

Figure 3. Honokiol attenuated placental ER-stress in PIH rats. Western blot of protein levels of GRP94, eIF2α, p-eIF2α, ATF-6 and CHOP in rat placentas and relative expression. Honokiol doses: 200 μg/kg (L), 600 μg/kg (M) and 2000 μg/kg (H). Note: Relative expression was calculated by dividing the value obtained in the experimental group by the value in the control group. Data are expressed as (mean ± SD) [n = 10]; error bars represent standard deviation (±SD). ***p < 0.001 compared with the control group. ^##p < 0.01 and ^###p < 0.001 compared with PIH group.

Effect of honokiol treatment on iNOS, eNOS and VEGF levels in the placenta, and the NO levels in the plasma in PIH rats

The western blot analyses indicated that the eNOS and VEGF levels increased (Figure 4(A,C,E)), whereas the iNOS levels were decreased following honokiol treatment (Figure 4B). Honokiol treatment was associated with significant upregulation in the expression of VEGF mRNA (Figure 4(F)). The NO levels in the plasma were increased in the rats that received honokiol treatment (Figure 4(D)).

Figure 4. Honokiol altered the levels of iNOS, eNOS, VEGF and NO in PIH rats. (A) Western blot of protein levels of iNOS, eNOS and VEGF in rat placentas and relative expression (B, C, E). (F) Expression of VEGF mRNA detected by RT-qPCR. (D) NO levels measured using a NO assay kit. Honokiol doses: 200 μg/kg (L), 600 μg/kg (M) and 2000 μg/kg (H). Note: Data are expressed as (mean ± SD) [n = 10]; error bars represent standard deviation (±SD). ***p < 0.001 compared with the control group. ^##p < 0.01 and ^###p < 0.001 compared with PIH group.

Pro-inflammatory cytokines, ER-related markers, VEGF mRNA and iNOS/eNOS, are positively correlated with urine proteins and systolic blood pressure, indicating that these indexes could reflect the severity of the disease to a certain degree. In our study, proinflammatory cytokines including IL-1β, TNF-α and IL-6 were decreased following honokiol treatment in PIH rats. The inhibition of NF-κB pathway is a pivotal process in the anti-inflammatory activity of Honokiol. A study has shown that honokiol inhibited inflammation in human umbilical cord derived mesenchymal stem cells via blocking NF-κB pathway [17]. It might indicate that honokiol is probably involved in the regulation of the NF-κB pathway.

Inflammatory factors in pregnant women are closely correlated with the occurrence of hypertensive disorder complicating pregnancy. For example, high maternal TNF-α could suppress normal extravillous trophoblast cell proliferation, migration, and integration, which was associated with PE [18]. Our results might imply that honokiol exerts a protective effect partly via inhibiting inflammation.

A previous study showed that excessive ER stress and upregulated iNOS are probably associated with increased apoptosis in placenta of PE patients [4]. In our study, ER stress and iNOS levels were increased in PIH rats. The results might imply that honokiol inhibited apoptosis in the placental tissues of PIH rats. Although honokiol induced cell apoptosis by triggering ER stress in cancer, it also exerted potential effects through downregulation of certain gene expression levels in ER-mediated apoptotic pathways [19]. It might be attributed to pro- and anti-survival effects of UPR-pathway activation on cells [7]. PERK and ATF6 signals could initiate a pro-survival pathway. However, excessive and prolonged ER-stress mediated growth inhibition by activation of apoptotic signalling molecule CHOP [20]. eIF2α is involved in the PERK signalling pathway and is an upstream molecule of CHOP [21]. Glucose regulated protein 94 (GRP94) is a downstream target of ATF6 and could reflect the level of ER stress to some extent [22]. In our study, increased p-eIF2α, ATF-6 and CHOP implied that excessive ER-stress might activate the pro-apoptosis pathway induced by CHOP. Moreover, honokiol significantly reduced p-eIF2α, ATF-6 and CHOP levels.

Studies have found that honokiol exerts neuroprotective effect by downregulating GRP-78 and CHOP in the hippocampus region, and downregulating p-eIF2 and CHOP to prevent against ER stress-related cell apoptosis and pathologic changes in testicular torsion/detorsion-induced testicular injury [8, 23]. They suggested that honokiol inhibition on ER-stress could produce potent protective effects.

NO could maintain normal peripheral resistance and blood pressure in the face of an increased cardiac output and plasma volume during pregnancy [24]. A previous study reported an upregulation in eNOS/NO signalling in healthy pregnant women [25]. Thus, eNOS/NO signalling performs a vital role during normal pregnancy. Our study revealed that honokiol significantly increased NO levels in the PIH rats. However, the levels of iNOS were decreased as opposed to eNOS. A study has suggested that iNOS levels were decreased by honokiol in rats subjected to acute kidney injury induced by cecal ligation and puncture [26]. Furthermore, eNOS knockout caused elevation of blood pressure and decreased the production of NO. VEGF induced vasodilation and increased blood flow in diverse vascular beds, whose effects were mediated partly through stimulating endothelial cells to produce NO [27]. In addition, VEGF might increase NO release by augmenting endothelial calcium signalling [28]. Thus, the upregulation of NO levels by honokiol may be partly mediated via increasing VEGF levels. Further, reduction of VEGF might cause hypoperfusion and subsequent hypoxia of the foetus in hypertensive pregnancy [24, 29]. Moreover, the upregulation of VEGF through small activating RNAs enhanced migration and tube formation ability in human trophoblast cells [18]. Honokiol significantly increased the expression of VEGF mRNA in our study. It might imply that honokiol possesses potent pro-angiogenic property.

Hypertension, proteinuria and diabetes gestational syndromes may be regarded as the epiphenomenon of a failed response to the vasodilatory and pro-angiogenic challenge that pregnancy imposes to the mother [30]. Our results might imply that honokiol could enhance the adaptive ability of patients with PIH to the changes of vasodilatory and pro-angiogenic factors.

Conclusions

This study demonstrated that administration of honokiol by gavage to PIH rats can decrease inflammation, ER stress and upregulate eNOs and VEGF mRNA levels. Honokiol treatment was associated with decreased hypoxia-induced hypertension and urinary protein levels. We speculate that honokiol plays a vital role in regulating vasodilatory and pro-angiogenic factors in patients with PIH. The obtained results suggest that honokiol exerts a protective effect on PIH rats through decreasing inflammation, ER-stress, upregulating eNOs and VEGF mRNA levels. The study suggests that honokiol deserves further investigation as a potential drug for the pathological conditions in patients with PIH.

Disclosure statement

No potential conflict of interest was reported by the authors.