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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 187-198

18β-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling


1 Department of Pharmacology, College of Pharmacy, Ningxia Medical University, Yinchuan, China
2 Department of Cardiology, Peking Union Medical College Hospital, Key Lab of Pulmonary Vascular Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
3 Pediatric Intensive Care Unit, General Hospital of Ningxia Medical University, Yinchuan, China
4 Department of Pharmacology, College of Pharmacy; Key Laboratory of Ningxia Ethnomedicine Modernization, Ministry of Education; Ningxia Characteristic Traditional Chinese Medicine Modernization Engineering Technology Research Center, Ningxia Medical University, Yinchuan, China

Date of Submission01-Mar-2022
Date of Decision06-May-2022
Date of Acceptance25-May-2022
Date of Web Publication26-Aug-2022

Correspondence Address:
Dr. Fang Zhao
General Hospital of Ningxia Medical University, 804 Shengli Street, Yinchuan 750004
China
Dr. Ru Zhou
Department of Pharmacology, College of Pharmacy, Ningxia Medical University, 1160 Shengli Street, Yinchuan 750004
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0304-4920.354801

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  Abstract 


Endoplasmic reticulum stress (ERS)-induced inflammation participates in the occurrence of pulmonary arterial hypertension (PAH) by promoting pulmonary vascular remodeling, which involved in the activation of PERK/eIF2α/NF-κB signaling pathway. 18β-Glycyrrhetinic acid (18β-GA) has been found efficacious for attenuating PAH through its anti-remodeling effects in our previous research and it remains unclear whether 18β-GA has an effect on the remodeling caused by ERS-induced inflammation. In this study, we made observations in monocrotaline-induced PAH rats and found improvement of hemodynamic and histopathological parameters, decreases in the right ventricular hypertrophy index, and alleviation of pulmonary vascular remodeling after 18β-GA administration in vivo. Moreover, 18β-GA could significantly inhibit the proliferation and DNA synthesis of human pulmonary arterial smooth muscle cells (HPASMCs) induced by platelet-derived growth factor BB. At the cellular and molecular levels, we found that 18β-GA could significantly reduce the accumulation of misfolded protein in rat lung tissue, inhibit ERS activation, reduce the expression of GRP78, p-PERK, p-eIF2α, and p-NF-κB p65, and increase IκB protein expression. 18β-GA could inhibit the migration of NF-κB into the nucleus, reduce the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatant of HPASMCs, and reduce GRP78, p-PERK, p-eIF2α, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression, increase IκB protein expression in HPASMCs. According to what we observed, this study indicated that 18β-GA could treat PAH, which is related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.

Keywords: 18β-Glycyrrhetinic acid, endoplasmic reticulum stress-induced inflammation, pulmonary arterial hypertension, remodeling


How to cite this article:
Wang JL, Liu H, Jing ZC, Zhao F, Zhou R. 18β-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling. Chin J Physiol 2022;65:187-98

How to cite this URL:
Wang JL, Liu H, Jing ZC, Zhao F, Zhou R. 18β-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling. Chin J Physiol [serial online] 2022 [cited 2022 Oct 6];65:187-98. Available from: https://www.cjphysiology.org/text.asp?2022/65/4/187/354801

#Jia-Ling Wang and Hui Liu contributed equally to this work





  Introduction Top


Pulmonary arterial hypertension (PAH) is a malignant hemodynamic syndrome, which leads to a gradual increase in pulmonary vascular resistance and pulmonary artery pressure, ultimately affects right ventricular (RV) function.[1],[2],[3] For patients with PAH, clinical targeted drugs can reduce pulmonary artery pressure by relaxing the pulmonary vascular, but the singularity of the target limits its application. Chinese medicine contains a large number of active ingredients which can exert pharmacological effects in multiple ways and multiple targets. If we can find effective ingredients that can reverse pulmonary vascular remodeling and explore its mechanism, it will be of practical significance for the study of PAH treatment.

Endoplasmic reticulum stress (ERS) is a defense mechanism of cells. Once the endoplasmic reticulum is stimulated by the outside, unfolded, or misfolded proteins accumulate in the cavity of the endoplasmic reticulum, unfolded protein response (UPR) is activated to maintain the homeostasis of the endoplasmic reticulum.[4],[5],[6] It has been reported that UPR is involved in the up-regulation of inflammation, among them, ERS-induced inflammation mediated by the PERK/eIF2α/NF-κB signaling pathway can promote pulmonary vascular remodeling and participate in the occurrence of PAH.[7] Pulmonary vascular remodeling is one of the key pathophysiological basis of PAH. Inflammatory cells secrete growth factors, such as platelet-derived growth factor (PDGF) could induce abnormal proliferation of pulmonary artery smooth muscle cells and form a positive feedback effect, thereby promoting the formation of pulmonary vascular remodeling.[8],[9] A series of studies have confirmed that ERS inhibitors 4-phenylbutyric acid (4-PBA) and salubrinal could significantly reduce changes in hemodynamic parameters in PAH animal models, improve RV hypertrophy, reduce pulmonary vascular remodeling indicators WT% and WA%, and reduce lung inflammation factors to improve PAH induced by monocrotaline (MCT) or hypoxia.[10],[11] The results of these studies indicate that reducing ERS-induced inflammation and reversing the pulmonary vascular remodeling may become a new approach for the treatment of PAH.

18β-Glycyrrhetinic acid (18β-GA) is an active ingredient extracted from licorice, which has been shown to have a variety of pharmacological effects, including anti-inflammatory, anti-oxidant, and anti-proliferation.[12] At present, a variety of drugs with licorice as the main ingredient, such as compound licorice oral solution, compound licorice tablets, and licorice extract, have been clinically used to treat respiratory diseases such as bronchitis, asthma, and cough. The Chinese medicine prescriptions for the prevention and treatment of the new coronavirus disease (COVID-19) issued by various authoritative organizations involved a total of 100 Chinese medicines, and licorice was used in the second place. After molecular docking with AutoDock Vina 1.1.2, it was found that its main ingredient, 18β-GA, may be one of the most effective ingredients to inhibit the COVID-19.[13],[14]

The previous studies of our group[15],[16] have confirmed that 18β-GA has a good therapeutic effect on PAH rats, this effect is related to reducing the mean pulmonary artery pressure and right ventricular systolic pressure (RVSP) in PAH rats, and reducing RV hypertrophy and histopathological changes, inhibiting oxidative stress and remodeling mediated by RhoA/ROCK signaling pathway, thereby improving PAH. Studies have shown that 18β-GA could inhibit the development of liver cancer by reducing ERS.[17] In addition, 18β-GA has an anti-tumor effect by inhibiting the NF-κB signaling pathway.[18] However, few studies could be found about whether 18β-GA could also alleviate ERS-induced inflammation and inhibit PERK/eIF2α/NF-κB signaling pathway.

According to the control principle in the experimental design, it is often necessary to add a positive control drug group to the study to verify the reliability of the drug treatment effect. Sildenafil has been approved by the United States Food and Drug Administration as a new drug for PAH that inhibits PDE-5 activity, which in turn relaxes vascular smooth muscle, reverses vascular remodeling, and leads to a decrease in pulmonary arterial pressure. Therefore, sildenafil was selected as the positive control drug for 18β-GA in this study.

Here, we hypothesized that 18β-GA could treat PAH through this approach, and designed this experiment to verify our hypothesis.


  Materials and Methods Top


Animals

Male Sprague-Dawley (SD) rats (220–260 g, 7 weeks old) were supplied by the animal experimental center of Ningxia Medical University (SYXK Ningxia 2015-0001). The animal experimental procedures were approved in accordance with the Institutional Animal Care and Use Committee of Ningxia Medical University. All the rats were housed under specific conditions (12 h light/12 h dark cycle, 22°C ± 3°C) and given free access to water and food.

Drugs and reagents

18β-GA with purity greater than 98% was purchased from Yuan Ye Biotechnology (China). MCT was purchased from Sigma-Aldrich (USA). Sildenafil was purchased from Pfizer (USA). Antibodies of GRP78, IκBα, tumor necrosis factor-α (TNF-α), β-actin, and Goat anti-rabbit immunoglobulin G (IgG) were purchased from Proteintech Group (USA). Phospho-PERK antibody and phospho-eIF2 alpha antibody were purchased from Affinity Biosciences (USA). Antibodies of NF-κB p65 and phospho-NF-κB p65 were purchased from Cell Signaling Technology (USA). Antibodies of monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-6 were purchased from Abcam Biotechnology (USA). Recombinant Human PDGF-BB was purchased from ProsPec-Tany TechnoGene Ltd. (Israel). WST-1 Cell Proliferation and Cytotoxicity Assay Kit was purchased from Beyotime Biotechnology (China). A cell proliferation enzyme-linked immunosorbent assay (ELISA), BrdU (colorimetric) kit was purchased from Roche (Germany). Human MCP-1 ELISA Kit, Human TNF-α ELISA Kit, and Human IL-6 ELISA Kit were purchased from Boster Biological Technology (China).

Animal groups and treatments

This study was approved by the experimental animal center of the experimental animal center of Ningxia Medical University. Adult male SD rats weighing 240–300 g were kept under constant condition in 12 h dark/12 h light alternation with standard feed and water. SD rats were randomly divided into 6 groups: control group, model group (MCT 60 mg/kg), sildenafil group (MCT + sildenafil 30 mg/kg/day), and 18β-GA groups (MCT + 18β-GA 100, 50, and 25 mg/kg/day). On day 0, the PAH model was established by a single subcutaneous injection of MCT 60 mg/kg in the neck (control animals subcutaneously injecting saline). Three weeks after MCT injection, rats in each group were treated with different doses of 18β-GA or sildenafil, and treatments in all groups were lasted for 3 weeks.

Hemodynamic indexes

Treatment for 3 weeks later, each group of rats received external jugular vein catheterization after anesthesia (10% urethane). During intubation, the location of the catheter was judged by the waveform and pressure value displayed by the BL-420S biological signal acquisition and analysis system (TaiMeng, China), and the measured RVSP and mean pulmonary artery pressure (mPAP) were recorded.

Right ventricular hypertrophy index

After the hemodynamic measurement, the heart was taken out quickly. First, the left and right atria were removed, and then the RV, left ventricular and septum (LV + S) of rats were separated and weighed separately. The ratio of (LV + S)/RV was calculated and considered as right ventricular hypertrophy index (RVHI).

Pulmonary vascular pathological sections

Lung tissues in all rat groups were cut into 4 μm-thick sections and subjected to hematoxylin and eosin (H and E) staining and Sirius Red Staining. Twenty small pulmonary vessels with diameters of 50–300 μm were randomly selected from each group and analyzed at a magnification of × 400. Pulmonary vascular remodeling and collagen deposition were characterized by microscopic evaluation. Two indices reflecting pulmonary arterial remodeling were calculated as follows: (1) ratio of pulmonary arterial wall thickness (WT%) =100% × (external diameter - internal diameter)/external diameter; (2) ratio of pulmonary arterial wall area (WA%) =100% × (transection area of the walls - lumen area)/transection area of the walls.

Thioflavin T staining assay

The rat lung tissue was embedded with optimum cutting temperature compound, and 5 μm sections were cut using a cryostat, and then stored at −20°C. The fresh frozen rat lung tissue sections at −20°C were taken out, placed at room temperature for 5 min, and then transferred to 4% formaldehyde fixative solution for fixation for 20 min. The sections were rinsed with tap water for 3 min, and then placed in a freshly prepared 500 mol Thioflavin T (ThT) staining solution for 3 min. Finally, the sections were rinsed with tap water for 3 min and then covered with a cover glass, and the fluorescence intensity was observed using a fluorescence microscope.

Western blot

The total protein was extracted from lung tissue and human pulmonary arterial smooth muscle cells (HPASMCs) in each animal group. The equivalent amounts of protein lysates in each group were separated by 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with blocking PBST (phosphate-buffered saline [PBS] involving 0.1% Tween-20) buffer at room temperature for 2 h and then incubated overnight at 4°C with primary antibodies to GRP78, p-PERK, p-eIF2α, IκB, p-NF-κB p65, TNF-α, IL-6, MCP-1, or β-actin. Subsequently, the membranes were washed and incubated with goat anti-rabbit IgG antibody for 2 h at room temperature. The grayscale values of each band on the blots were analyzed with Quantitative One (Bio-Rad Company, CA, USA).

Cell culture

HPASMCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 supplemented with 10% fetal bovine serum in an incubator (at 37°C with 5% CO2). Cells were used between passages 3 and 8 in this study.

Measurement of cell viability and proliferation

To determine the effect of 18β-GA on HPASMCs viability, the effect of different doses of 18β-GA (0-90 μM) on 48 proliferations was investigated using the WST-1 cell proliferation assay according to the manufacturer's protocol. HPASMCs were grown to 60%–70% confluence and then serum-starved for 24 h prior to use in the experiments. Cells were treated with PDGF-BB (20 ng/ml) for 24 h, then treated with 18β-GA for an additional 48 h and incubated with WST-1 for the last 2 h. Cell proliferation was determined by measuring the absorbance at 450 nm.

Measurement of cell DNA synthesis

Cell proliferation was assessed using a BrdU ELISA kit. Briefly, HPASMCs were seeded at a density of 96 into 5 × 104 cells in 96-well cell culture plates. According to the manufacturer's protocol, BrdU was incorporated into proliferating cells. The absorbance was measured at 370 nm using a microplate reader.

Immunofluorescence

Cells were washed with PBS buffer, fixed in 4% paraformaldehyde for 30 min at room temperature, washed for three times with PBS buffer, and broke the membrane with 0.5% TritonX-100 for 30 min, washed for three times with PBS buffer, added 5% bovine serum albumin (BSA) + 0.1% TritonX-100 and blocked at room temperature for 30 min. Next, cells were washed again with PBS buffer for three times, followed by the addition of NF-κB p65 antibody at 4°C overnight. Three times of washing with PBS buffer, and the addition of secondary antibody DAPI (4, 6-diamino-2-phenylindole)-labeled rabbit-anti-rat IgG for 30 min of incubation at 37°C in the dark. After 30 min of blocking and antibody incubation, cells were washed again with PBS buffer and observed under a fluorescence microscope (Olympus, Japan).

Enzyme-linked immunosorbent assay

Initially, we extracted collect the pulmonary artery smooth muscle cell culture supernatant, then the contents of TNF-α, MCP-1, and IL-6 were assessed by ELISA kit according to the manufacturers' instructions. Finally, we read the absorbance on a microplate reader at a wavelength of 450 nm immediately and generated a standard curve which was used to determine the unknown sample concentration.

Statistical analysis

All data are presented as the mean ± standard error of the mean. For the comparison of multiple groups, data were analyzed by one-way analysis of variance followed by the Student–Newman–Keuls test. P < 0.05 was considered statistically significant. SPSS software (version 26.0; SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.


  Results Top


18β-Glycyrrhetinic acid decreased hemodynamic indexes in monocrotaline-induced pulmonary arterial hypertension rats

We assessed mPAP and RVSP as two main hemodynamic indexes in animal models. Rats in MCT group had higher mPAP and RVSP versus the control group [Figure 1]A, [Figure 1]B, P < 0.05]; after 18β-GA (50, 100 mg/kg) administration, mPAP and RVSP were significantly decreased compared with the MCT group.
Figure 1: Effect of 18β-GA treatment on MCT-induced pulmonary arterial hypertension. (A) Effect of 18β-GA on mPAP. (B) Effect of 18β-GA on RVSP. (C) Effect of 18β-GA on RVHI. (D) Effect of 18β-glycyrrhetinic acid on MCT-induced pulmonary vascular remodeling. Representative photomicrographs of pulmonary small arteries by H and E staining (magnification × 200). (E) Left: Effect of 18β-glycyrrhetinic acid on the ratio of the vascular walls thickness (WT%), Right: Effect of 18β-glycyrrhetinic acid on the ratio of the vascular wall area (WA%). (a) Control group; (b) MCT group; (c) 18β-GA 25 mg/kg group; (d) 18β-GA 50 mg/kg group; (e) 18β-GA 100 mg/kg group; (f) Sildenafil group. Data are expressed as mean ± SEM (n = 10). ##P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus MCT group. 18β-GA: 18β-glycyrrhetinic acid, MCT: monocrotaline, mPAP: Mean pulmonary arterial pressure, RVSP: Right ventricular systolic pressure, RVHI: Right ventricular hypertrophy index, WT%: The ratio of the vascular walls thickness, WA%: The ratio of the vascular wall area.

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18β-Glycyrrhetinic acid attenuated right heart failure in monocrotaline-induced pulmonary arterial hypertension rats

Similar results were observed in the ratio of RV to LV ± S. As shown in [Figure 1]C, the ratio of RV/LV ± S in the MCT group was much higher than that in the control group (P < 0.05 and P < 0.01). Nevertheless, after 6 weeks of treatment, the RV/LV ± S ratio in the 18β-GA group was significantly lower than in the MCT group [[Figure 1]C; P < 0.01], indicating that 18β-GA attenuated MCT-induced RV hypertrophy.

18β-Glycyrrhetinic acid attenuated pulmonary artery remodeling in monocrotaline-induced pulmonary arterial hypertension rats

The results of H.E staining showed that the thickness of the pulmonary artery wall in the MCT group increased and the lumen area was narrowed. After 18β-GA (50, 100 mg/kg) and sildenafil (30 mg/kg) treatment, the pathological changes of pulmonary blood vessels in rats have been significantly improved [Figure 1]D. Furthermore, the results of H.E staining showed a significant increase in WA% and WT% in the lung tissue of MCT-exposed rats compared with the control group, which were treated as the indicators for evaluating vascular remodeling. The MCT-induced elevations in the WT% and WA% were markedly inhibited by 18β-GA treatment [P < 0.05 and P < 0.01, [Figure 1]E]. Sirius scarlet staining results showed that there was significant collagen deposition around the pulmonary blood vessels in the MCT group, and the arrangement of collagen fibers was disordered, while the collagen deposition in the 18β-GA and sildenafil treatment groups was significantly improved [Figure 2].
Figure 2: Effect of 18β-glycyrrhetinic acid on the expression level of collagen in pulmonary artery of rats. Representative photomicrographs of pulmonary small arteries by Picro Sirius Red staining (magnification × 200): (a) Control group; (b) MCT group; (c) 18β-GA 25 mg/kg group; (d) 18β-GA 50 mg/kg group; (e) 18β-GA 100 mg/kg group; (f) Sildenafil 30 mg/kg group. MCT: Monocrotaline, 18β-GA: 18β-glycyrrhetinic acid.

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18β-Glycyrrhetinic acid decreased accumulation of misfolded proteins

ThT is a small molecule that shows enhanced fluorescence when it binds to protein aggregates. The enhanced ThT-fluorescence is directly related to the activation of the UPR. In this experiment, ThT staining was used to detect misfolded protein aggregates. It was found that the accumulation of misfolded protein in the lung tissue of the MCT group increased, which proved the occurrence of ERS. Treatment with 18β-GA could reduce the accumulation of misfolded protein and improved ERS [P < 0.01, [Figure 3]].
Figure 3: Effect of 18β-glycyrrhetinic acid on ERS. (a) Representative photomicrographs of the lung tissue by ThT staining (magnification × 200). (b) Effect of 18β-GA on fluorescence intensity. Data are expressed as mean ± SEM (n = 6). ##P < 0.01 versus control group, **P < 0.01 versus MCT group. MCT: Monocrotaline, 18β-GA: 18β-glycyrrhetinic acid, SEM: Standard error of the mean, ThT: Thioflavin T, ERS: Endoplasmic reticulum stress.

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Effects of 18β-glycyrrhetinic acid on the expression of GRP 78, p-PERK, p-eIF2α, IκB, and p-NF-κB p65 in rat lung tissue

In order to explore the correlation between the therapeutic effects of 18β-GA on PAH rats and the PERK/eIF2α/NF-κB signaling pathway, we used Western blot to determine GRP 78, p-PERK, p-eIF2α, IκB, and p-NF-κB p65 protein expression in rat lung tissue. The results showed that the expression levels of GRP78, p-PERK, p-eIF2α, and p-NF-κB p65 in the lung tissue of the MCT group were significantly higher than those in the normal control group, and the expression level of IκB was significantly decreased [P < 0.01, [Figure 4]]. Treatment with 18β-GA (100 mg/kg) can reverse the expression changes of GRP78, p-PERK, p-eIF2α, IκB, and p-NF-κB p65 in the lung tissue of PAH rats [P < 0.05, P < 0.01, [Figure 4]]. The results preliminarily suggested that the therapeutic effect of 18β-GA on PAH rats may be related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.
Figure 4: Effects of 18β-glycyrrhetinic acid on the expression of GRP 78, p-PERK, p-eIF 2α, IκB, and p-NF-κB p65 in the lung tissue. (a) Representative Western blot band of GRP 78 activation in the lung tissues. (b) Representative Western blot band of p-PERK activation in the lung tissues. (c) Representative Western blot band of p-eIF 2α activation in the lung tissues. (d) Representative Western blot band of IκB activation in the lung tissues. (e) Representative Western blot band of p-NF-κB p65 activation in the lung tissues. Data are expressed as mean ± SEM (n = 6). ##P < 0.01 vs. control group, *P < 0.05, **P < 0.01 versus MCT group. MCT: monocrotaline, 18β-GA: 18β-glycyrrhetinic acid, SEM: Standard error of the mean.

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Effects of 18β-glycyrrhetinic acid on the viability of human pulmonary arterial smooth muscle cells

In order to explore the effect of 18β-GA on the activity of HPASMCs, we used 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM or 90 μM 18β-GA to intervene cells for 48 h. The WST-1 method was used to detect the viability of HPASMCs and it was found that compared with the control group, 18β-GA at a concentration below 80 μM would not have a significant effect on the viability of HPASMCs [P > 0.05, [Figure 5]]. The above results indicated that 18β-GA at a concentration below 80 μM did not cause cytotoxicity to HPASMCs.
Figure 5: Effect of 18β-glycyrrhetinic acid on the viability of HPASMCs at 48 h. Data are expressed as the mean OD ± standard deviation, n = 6. *P < 0.05, **P < 0.01 versus control group. 18β-GA: 18β-glycyrrhetinic acid, HPASMCs: Human pulmonary artery smooth muscle cells; OD: Optical density.

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18β-Glycyrrhetinic acid inhibited the proliferation and DNA synthesis of human pulmonary arterial smooth muscle cells

In order to explore the effect of 18β-GA on the proliferation of HPASMCs, PDGF-BB was used to construct a proliferation model of HPASMCs, and then 18β-GA was given for intervention. The results of WST-1 showed that HPASMCs proliferated significantly after being stimulated with PDGF-BB for 24 h [P < 0.01, [Figure 6]a]. After the intervention of 18β-GA at the concentration of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, and 80 μM for 48 h, the results showed that the intervention of 18β-GA at a concentration of 50 μM and above could significantly inhibit the proliferation of HPASMCs induced by PDGF-BB [P < 0.01, P < 0.05, [Figure 6]a]. After analyzing the results of BrdU detection, it was found that the use of PDGF-BB to stimulate HPASMCs for 24 h could significantly increase the DNA synthesis of HPASMCs [P < 0.01, [Figure 6]b], while the treatment of HPASMCs with different concentrations of 18β-GA for 48 h, DNA synthesis of HPASMCs decreased; the result was consistent with the WST-1 method, reflecting the inhibitory effect of 18β-GA on the proliferation of HPASMCs induced by PDGF-BB [P < 0.01, P < 0.05, [Figure 6]b]. According to the results of WST-1 and BrdU, the concentration at 70 μM was selected for the next experiment.
Figure 6: Effects of 18β-glycyrrhetinic acid on PDGF-BB induced HPASMCs proliferation and DNA synthesis at 48 h. Cell proliferation was examined using (a) the WST-1, and (b) bromodeoxyuridine incorporation was determined using an enzyme-linked immunosorbent-based assay. Data are expressed as the mean OD ± standard deviation. ##P < 0.01 versus the control group; *P < 0.05, **P < 0.01 versus cells exposed to PDGF-BB alone; n = 6. PDGF: Platelet-derived growth factor, HPASMCs: Human pulmonary artery smooth muscle cells, OD: Optical density.

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Effects of 18β-glycyrrhetinic acid on nuclear translocation of NF-κB p65 in human pulmonary arterial smooth muscle cells

The analysis of immunofluorescence pictures revealed that NF-κB p65 was mainly expressed in the cytoplasm of HPASMCs in the control group, with holes in the nucleus, indicating that there was almost no expression of NF-κB p65. In the PDGF-BB group, we found strong red fluorescence in the nuclei of HPASMCs, indicating that NF-κB p65 had undergone nuclear translocation at this time. After intervention with 18β-GA, the overlap of blue and red fluorescence in HPASMCs was smaller than that of PDGF-BB group, and the red fluorescence intensity in the nucleus was also weakened [Figure 7]. This result showed that 18β-GA could effectively block the nuclear metastasis of NF-κB p65 in HPASMCs.
Figure 7: Effects of 18β-glycyrrhetinic acid on NF-κB nuclear translocation in human pulmonary arterial smooth muscle cells (magnification × 400). Cells were analyzed by fluorescence microscope, and individual and merged stainings are shown. 18β-GA: 18β-glycyrrhetinic acid, PDGF-BB: Platelet-derived growth factor-BB.

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Effects of 18β-glycyrrhetinic acid on the contents of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 in human pulmonary arterial smooth muscle cells culture supernatant

The results of ELISA showed that the contents of TNF-α, IL-6, and MCP-1 in the HPASMCs culture supernatant of the PDGF-BB group were significantly higher than those in the Control group, and 18β-GA was administered. It could significantly down-regulate the content of TNF-α, IL-6, and MCP-1 in PASMCs induced by PDGF-BB [P < 0.01, [Figure 8]].
Figure 8: Effect of 18β-glycyrrhetinic acid on the content of (a) TNF-α, (b) IL-6, and (c) MCP-1 in the cell supernatant. Data are expressed as the mean ± standard deviation, n = 6. ##P < 0.01 versus control group, **P < 0.01 versus PDGF-BB group. PDGF-BB: Platelet-derived growth factor-BB, 18β-GA: 18β-glycyrrhetinic acid, TNF-α: Tumor necrosis factor-α, IL-6: interleukin, MCP-1: Monocyte chemoattractant protein-1.

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Effects of 18β-glycyrrhetinic acid on the expression of GRP78, p-PERK, p-eIF2α, IκB, p-NF-κB p65, tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 in human pulmonary arterial smooth muscle cells

In order to further explore the inhibitory effect of 18β-GA on the PERK/eIF2α/NF-κB pathway on the basis of animal experiments, we used western blot to detect GRP78, p-PERK, p-eIF2α, IκB, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression, the results showed that compared with the control group, the expression of GRP78, p-PERK, p-eIF2α, IκB, p-NF-κB p65, TNF-α, IL-6, and MCP-1 in PDGF-BB group HPASMCs was significantly up-regulated, and IκB protein expression was down-regulated, and the intervention of 18β-GA could reverse the PDGF-BB-induced GRP78, p-PERK, p-eIF2α, IκB, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression changes [P < 0.05, P < 0.01, [Figure 9]].
Figure 9: Effects of 18β-glycyrrhetinic acid on the expression of GRP 78, p-PERK, p-eIF 2α, IκB, p-NF-κB p65, TNF-α, IL-6, and MCP-1 in the HPASMCs. (a) Representative Western blot band of GRP 78 activation in the HPASMCs. (b) Representative Western blot band of p-PERK activation in the HPASMCs. (c) Representative Western blot band of p-eIF 2α activation in the HPASMCs. (d) Representative Western blot band of IκB activation in the HPASMCs. (e) Representative Western blot band of p-NF-κB p65 activation in the HPASMCs. (f) Representative Western blot band of TNF-α activation in the HPASMCs. (g) Representative Western blot band of IL-6 activation in the HPASMCs. (h) Representative Western blot band of MCP-1 activation in the HPASMCs. Data are expressed as mean ± SEM (n = 3). ##P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus PDGF-BB group. PDGF-BB: Platelet-derived growth factor-BB, 18β-GA: 18β-glycyrrhetinic acid, TNF-α: Tumor necrosis factor-α, IL-6: interleukin-6, MCP-1: Monocyte chemoattractant protein-1, HPASMCs: Human pulmonary arterial smooth muscle cells, SEM: Standard error of the mean.

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  Discussion Top


Our study demonstrates that 18β-GA has a good therapeutic effect on PAH by inhibiting pulmonary vascular remodeling. The results are complementary to our previous research.[15],[16] Considering the potential regulatory effect of 18β-GA on ERS-induced inflammation, the present experiments aim to evaluate whether 18β-GA improves PAH and how it operates more comprehensively. The results demonstrate for the first time that 18β-GA can mitigate pulmonary vascular remodeling and relieves PAH by regulating PERK/eIF2α/NF-κB signaling pathway and its downstream factors.

PAH is a complex clinical syndrome that starts from the pulmonary circulation, which leads to a gradual increase in pulmonary vascular resistance and pulmonary artery pressure, and ultimately affects RV function.[19] Many studies on clinical patients and animal models of PAH have shown that the mechanism of PAH is complex, and pulmonary vascular remodeling is closely related to the formation and development of PAH, which is also a difficulty in the clinical treatment of PAH.[20] The pulmonary artery media is mainly composed of pulmonary artery smooth muscle cells, and its abnormal proliferation is an important link in the occurrence of pulmonary vascular remodeling.[21] Therefore, how to inhibit the proliferation of pulmonary artery smooth muscle cells and reverse pulmonary vascular remodeling is the key to the treatment of PAH.

The successful construction of PAH animal model is the key to proving the occurrence and development of PAH and drug screening. MCT-induced PAH animal model is widely used in the research of PAH prevention and drug screening. Its preparation method is a single injection of MCT subcutaneously in the neck, which has the advantages of simple operation, low price, and high success rate.[22] Therefore, this method on adult male SD rats was used to construct a PAH model to verify the therapeutic effect of 18β-GA on PAH and explore the mechanism. Human PAH is characterized by abnormally elevated mPAP, RVSP, and RV hypertrophy. Therefore, hemodynamic parameters and RV hypertrophy index can directly reflect the success of the PAH rat model.[23] The results of the right heart catheterization method and organ weighing method in this experiment showed that mPAP, RVSP, and RVHI in the MCT group increased significantly, and 18β-GA treatment inhibited the increase, indicating that 18β-GA has a great therapeutic effect on PAH rats. Thickening of the vascular wall of pulmonary arteries is one of the pathological features of human PAH.[24] In previous studies,WT%, WA%, and collagen deposition were often used to assess the degree of pulmonary vascular remodeling.[25],[26] In this experiment, the H and E stained sections and Sirius scarlet stained sections of rat lung tissues were observed, and it was found that the WT% and WA% of rats in the MCT group were significantly increased, there were obvious collagen deposition around the pulmonary blood vessels, and the arrangement of collagen fibers was disordered, 18β-GA treatment could significantly improve this change, once again confirming that 18β-GA can treat PAH rats by inhibiting pulmonary vascular remodeling.

PDGF can stimulate the division and proliferation of a variety of cells, and it has a variety of forms of dimer structure. Among them, PDGF-BB has a rough proliferation effect on vascular smooth muscle cells and has been widely used in cell models of PAH mechanism research.[27],[28] Therefore, this experiment used this method to construct a model of HPASMCs proliferation, and explored the mechanism of 18β-GA's inhibitory effect on the proliferation of HPASMCs. WST-1 is a reagent widely used in the detection of cytotoxicity and cell proliferation, which is simpler and more sensitive than MTT. The results of WST-1 showed that stimulation with 20 ng/ml PDGF-BB for 24 h could significantly induce the proliferation of HPASMCs, suggesting that the HPASMCs proliferation model was successfully constructed. The WST-1 method was used to detect the effect of 18β-GA on the activity and proliferation of HPASMCs. The results showed that the intervention of 18β-GA at a concentration below 80 μM for 48 h would not have a significant effect on the activity of HPASMCs, and it could be significantly inhibited the proliferation of HPASMCs induced by PDGF-BB starting from a concentration of 50 μM. The results of the BrdU indicated that 18β-GA could significantly inhibit the DNA synthesis of HPASMCs in the PDGF-BB group, which is consistent with the WST-1 results, further confirming that 18β-GA has an inhibitory effect on the proliferation of HPASMCs induced by PDGF-BB.

Studies have shown that ERS participates in pulmonary vascular remodeling by mediating inflammation, which is an important mechanism for the occurrence and development of PAH.[29] A series of studies have found that in different PAH animal models, ERS inhibitors 4-PBA and TUDCA could reduce PAH animal hemodynamic parameters, pulmonary vascular remodeling indicators WT%, WA%, and improve RV hypertrophy.[30] UPR has the effect of maintaining the homeostasis of the endoplasmic reticulum. PERK is a transmembrane protein receptor that senses ERS to initiate UPR signal transmission. In resting cells, it binds to GRP78 and remains in an inactive state. Once ERS occurs, GRP78 will dissociate from the PERK receptor PERK and triggers UPR. Studies have reported that UPR is involved in the up-regulation process of inflammation. Among them, the activation of PERK has been shown to induce inflammation by mediating the downstream NF-κB signaling pathway, which plays an important role in cell proliferation.[11] PERK is a type I transmembrane protein kinase upstream of eIF2α. When ERS occurs, PERK first autophosphorylates and then specifically phosphorylates downstream eIF2α, leading to downstream IκB transcriptional inhibition, and NF-κB is released and quickly transferred to the nucleus, participating in inflammation. Transcriptional regulation, initiated pulmonary vascular inflammation cascade can promote abnormal pulmonary vascular remodeling, leading to PAH.[31] Studies have confirmed that salubrinal, an inhibitor of ERS, could improve the MCT-induced PAH model and reduce the recruitment of pulmonary inflammatory factors.[11] Studies by Koyama et al. found that the expression of PERK and its downstream molecules in the lung tissue of PAH mice induced by hypoxia increased after treatment with the ERS inhibitor 4-PBA, the expression of inflammatory factors was significantly reduced, and the expression of PERK and its downstream molecules induced by hypoxia was inhibited, pulmonary artery proliferation was improved.[30] In summary, the inflammatory response induced by ERS promotes pulmonary vascular remodeling and participates in the occurrence and development of PAH.

ThT is a benzothiazole dye that shows enhanced fluorescence when combined with a protein-rich in β-sheet structure, which can ERS induced by different conditions and compounds.[32],[33] In this experiment, ThT staining was used to detect misfolded protein aggregates, and to evaluate the activation of ERS in rat lung tissue. It was found that the accumulation of misfolded protein in the lung tissue of rats in the MCT group increased, which proved the occurrence of ERS, and the administration of 18β-GA reduced the accumulation of misfolded proteins. Elevated GRP78 expression is an important sign of ERS.[34] Western blot results showed that the expression of GRP78 protein in the lung tissue of the MCT group was significantly increased, confirming the occurrence of ERS again, and 18β-GA could inhibit the expression of GRP78, p-PERK, and p-eIF2α, and p-NF-κB p65, increase IκB protein expression in the lung tissue of PAH rats. The above results preliminarily prove that the therapeutic effect of 18β-GA on PAH rats may be related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.

NF-κB target genes regulate a broad family of inflammatory cytokines, such as TNF-α, ICAM-1, IL-6, and MCP-1. Previous studies on SU/Hx-induced PAH have found that the levels of inflammatory factors such as TNF-α and IL-6 in the lung are elevated.[35],[36] Studies have confirmed that salubrinal, an ERS inhibitor, could reduce the expression of inflammatory factors in the lung, and treatment with ERS inhibitor 4-PBA in PAH mice could significantly reduce MCP-1, IL-6, and TNF-α expression and inhibited the proliferation of pulmonary artery smooth muscle stimulated by hypoxia.[37] Therefore, inhibiting the proliferation of pulmonary artery smooth muscle cells by inhibiting the PERK/eIF2α/NF-κB signaling pathway is an effective way to improve pulmonary vascular remodeling.

In the study of NF-κB activation, immunofluorescence detection of p65 nuclear translocation is the most common method.[38] In this experiment, the immunofluorescence results found that compared with the control group, the red fluorescence in the nucleus of HPASMCs in the PDGF-BB group was stronger, indicating that NF-κB had undergone nuclear transfer. After the intervention of 18β-GA, the red fluorescence intensity in the nucleus was weakened, indicating that 18β-GA could effectively block NF-κB nuclear transfer. ELISA results showed that the intervention of 18β-GA could significantly reduce the increase of TNF-α, IL-6, and MCP-1 in the culture supernatant of HPASMCs induced by PDGF-BB. Western blot showed that compared with the control group, GRP78, p-PERK, p-eIF2α, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression levels in the PDGF-BB group increased significantly, and IκB expression levels decreased; 18β-GA intervention could reverse the above changes in protein expression, which indicated that the inhibitory effect of 18β-GA on the proliferation of PASMCs induced by PDGF-BB was related to the inhibition of the PERK/eIF2α/NF-κB signaling pathway.


  Conclusion Top


In summary, this project established an MCT-induced PAH rat model and confirmed that 18β-GA could improve hemodynamic parameters, RV hypertrophy, and histopathological changes in PAH rats, and reduced ERS-induced inflammation, reversed pulmonary vascular remodeling, and its effect in treating PAH may be related to the inhibition of PERK/eIF2α/NF-κB signaling pathway. Furthermore, by establishing a PDGF-BB-induced HPASMCs proliferation model, it was further confirmed that 18β-GA had a significant inhibitory effect on PDGF-BB-induced HPASMCs proliferation and DNA synthesis, which could prevent NF-κB from entering the nucleus and reduce the content of inflammatory factors TNF-α, IL-6, MCP-1 in HPASMCs, improved ERS-induced inflammation and inhibited the activation of PERK/eIF2α/NF-κB signaling pathway, thereby inhibiting the proliferation of HPASMCs. However, this study only uses western blot technology to explore the mechanism of 18β-GA in the treatment of PAH and has not been verified by other methods, which has certain shortcomings. Therefore, in follow-up experiments, we will explore the specific targets of 18β-GA in the treatment of PAH through siRNA and other technologies. The experimental results will provide experimental data and theoretical basis for the study of PAH treatment mechanisms.

Acknowledgment

I sincerely thank Ningxia Medical University and the General Hospital of Ningxia Medical University for their scientific research platforms.

Financial support and sponsorship

This project was supported by Ningxia Hui Autonomous Region Key Research and Development Project (Grant No. 2019BFG02027); 2017 Ningxia Hui Autonomous Region Science and Technology innovation leader training project (Grant No. KJT2017005); Ningxia Hui Autonomous Region Key Research and Development Project Foreign Science and Technology Cooperation Special (Grant No. 2018BFH03019).

Conflicts of interest

There are no conflicts of interest.



 
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