Indigo Leaves-Induced Pulmonary Arterial Remodeling without Right Ventricular Hypertrophy in Rats

Abstract

Indigo naturalis (IN), derived from the leaves of the indigo plant, is a traditional Chinese medicine that has historically been used for its anti-inflammatory properties in the treatment of various diseases, including ulcerative colitis (UC). However, long-term use of IN in UC patients is incontrovertibly associated with the onset of pulmonary arterial hypertension (PAH). To investigate the mechanisms by which IN induces PAH, we focused on the raw material of IN, indigo leaves (IL). Only the condition of long-term chronic (6 months) and high-dose (containing 5% IL in the control diet) administration of IL induced medial thickening in the pulmonary arteries without right ventricular hypertrophy in our rat model. IL administration for a month did not induce pulmonary arterial remodeling but increased endothelin-1 (ET-1) expression levels within endothelial cell (EC) layers in the lungs. Gene Expression Omnibus analysis showed that ET-1 is a key regulator of PAH and that the IL component indican and its metabolite IS induced ET-1 mRNA expression via reactive oxygen species-dependent mechanism. We identified the roles of indican and IS in ET-1 expression in ECs, which were linked to pulmonary arterial remodeling in an animal model.

INTRODUCTION

Indigo naturalis (IN), derived from the leaves of indigo plants, is a traditional Chinese medicine historically used for its anti-inflammatory properties in the treatment of various diseases, including ulcerative colitis (UC).^1,2) Its efficacy in UC has been demonstrated in randomized controlled trials.³⁾ The improvement in UC is attributed to the indole compound indigo found in IN, which acts as an agonist of aryl hydrocarbon receptor (AhR) and exerts anti-inflammatory effects.¹⁾ A national survey in Japan conducted from 2017 to 2018 reported that 1.8% of UC patients used IN on their own.⁴⁾ Concurrently, there have been several reported cases in which long-term IN use in UC patients was associated with the onset of pulmonary arterial hypertension (PAH).^4,5) Although warnings regarding its use have been issued in Japan,⁶⁾ the causative components of IN and the pathogenesis of IN-induced PAH remain unclear.

PAH is a progressive, complex, and intractable respiratory disease with a poor prognosis.⁷⁾ It is characterized by an increase in pulmonary arterial pressure due to excessive vasoconstriction or abnormal vascular remodeling, leading to narrowing of the vascular lumen (medial thickening), which results in an increased load on the right ventricle (RV).^8,9) These symptoms progressively worsen, reducing the QOL of the affected patients.

The vacuoles of indigo leaves (IL), which are the raw materials for IN, contain indoxyl-β-D-glucoside (indican), a colorless glycoside with an indole skeleton.¹⁰⁾ In the body, indican is converted into various indole compounds that act as effective agents against UC. Some of these compounds are absorbed and undergo sulfation in the liver to form indoxyl sulfate (IS).¹¹⁾

PAH patients have elevated plasma levels of endothelin-1 (ET-1) and increased expression of ET-1 in the lung tissues.^12,13) The use of endothelin receptor antagonists, such as bosentan, in the treatment of PAH suggests that ET-1 plays a crucial role in the pathogenesis of PAH. ET-1 is a peptide hormone released from the vascular endothelium that causes vasoconstriction and is involved in medial thickening by promoting the proliferation of vascular smooth muscle cells (VSMCs).¹⁴⁾ Although there are reports that IS enhances vascular constriction induced by ET-1 in rat aorta,¹⁵⁾ there are no reports suggesting that IS or its precursors directly influence ET-1 expression.

Therefore, this study focused on the raw material of IN, IL, and evaluated the effects of the components of IL on the pulmonary arteries and the regulation of ET-1 expression by indican and its metabolite IS. These evaluations were conducted using animal models, human umbilical vein endothelial cells (HUVECs), and rat aortic SMCs (RASMCs) to investigate the underlying mechanisms.

MATERIALS AND METHODS

Chemicals

Indican (EI03317) was obtained from Biosynth Chemistry & Biology (Staad, Switzerland). IS potassium salt (I3875) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). An oral spherical activated carbon, AST-120 (Kremezin^®) was purchased from Kureha Chemical (Tokyo, Japan). An AhR inhibitor, CH223191 (16154) was purchased from Cayman Chemical Co. (Ann Arbour, MI, U.S.A.). N-Acetyl-L-cysteine (NAC) (A0905) was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan).

Animal Experiments

IL (Persicaria tinctoria) were obtained from Bon-Arm. Co., Ltd. (Tokushima, Japan). All experimental procedures were performed in accordance with the guidelines of the Animal Research Committee of Tokushima University Graduate School and the protocol was approved by the Institutional Review Board of Tokushima University Graduate School (Permit Number: T2020-80). Wistar male rats (4-weeks-old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The animals were raised under controlled environmental conditions with 12 h light/dark cycles in a temperature-controlled room at 25 °C. The rats were randomly divided into the following groups: normal diet (control), IL, and IL plus AST-120 groups. The control group was fed a normal diet (CLEA Rodent Diet CE-2; Nippon CLEA, Tokyo, Japan), whereas the IL group was fed a normal diet containing 5% IL powder. The IL plus AST-120 group was fed with the diet used for IL group plus 5% AST-120. Water and food were provided ad libitum. After 1 or 6 months of feeding, blood samples were collected from the inferior vena cava under anesthesia with chloral hydrate (300 mg/kg), allowed to rest at room temperature, and centrifuged at 10000 × g for 15 min to collect serum samples, which were frozen at −80 °C for later analysis. After euthanasia using a large dose of chloral hydrate, organs (lungs and hearts) were harvested after refluxing with normal saline for further investigation.

Morphological Analysis

The heart and lungs were perfused with 0.9% normal saline. The lungs were fixed in 10% formalin at 4 °C for 72 h and embedded in paraffin. Paraffin-embedded lung sections were subjected to hematoxylin–eosin (H&E) staining and photographed under a microscope (BZ-9000; Keyence, Osaka, Japan). Morphological analysis of the pulmonary arteries (inner diameter: <100 µm) was performed at 200× magnification. The medial wall thickness of the pulmonary arteries was calculated using the following formula: medial wall thickness (µm) = (outer diameter − inner diameter)/2 or the ratio of medial wall thickness to outer diameter. For quantitative analysis, 20–35 randomly selected vessels from each sample were analyzed, and the mean value was calculated for each sample. To assess right ventricular hypertrophy, the free walls of the RV and left ventricle plus the septum (LV + S) were dissected and weighed, and the Fulton index was calculated using the formula: RV/(LV + S). Measurements were performed using ImageJ 1.52a software (National Institute of Health, Bethesda, MD, U.S.A.).

Immunofluorescence Staining

Lung tissue samples were fixed in 10% formalin for 16 h, followed by three overnight incubations in 30% sucrose and were embedded in optimal cutting temperature compound (4583; Sakura Finetek, Japan Co., Ltd., Tokyo, Japan). Transverse sections (8 µm thickness) were cut with a cryostat, placed on glass slides for immunohistochemistry, and stored at −80 °C. The sections were fixed with acetone and permeabilized with 0.1% Triton X-100. After blocking the samples with 10% goat serum, anti-ET-1 antibody (1 : 500, PA3-067; Invitrogen, CA, U.S.A.), anti-CD31 antibody (1 : 200, 550274, or 550300; BD Biosciences, Franklin Lakes, NJ, U.S.A.), and Hoechst 33342 solution (1 : 2000; Dojindo, Kumamoto, Japan) were used for immunohistochemistry. Each primary antibody detection was performed using Alexa Fluor 488 goat anti-mouse (1 : 2000, ab150113; Abcam, Cambridgeshire, U.K.), Alexa Fluor 568 goat anti-rabbit (1 : 2000, ab175471; Abcam), Alexa Fluor 488 goat anti-rat (1 : 2000, ab150157; Abcam), Alexa Fluor 568 goat anti-rabbit (1 : 2000, A11036; Invitrogen), or Alexa Fluor 568 goat anti-mouse secondary antibodies (1 : 2000, ab175473; Abcam). Tissue imaging was performed using a confocal laser microscope (A1R; Nikon, Tokyo, Japan). Although we used two anti-CD31 antibodies (550274 and 550300; BD Biosciences) for EC layer detection, we confirmed that the staining pattern was almost the same when these two antibodies were used, as shown in Supplementary Fig. 1. The mean intensity of ET-1 staining within the EC layers (CD31 positive area) was measured using ImageJ 1.52a software (National Institute of Health).

HPLC Analysis

The HPLC system consisted of an LC-10AD intelligent HPLC Pump (Shimadzu, Kyoto, Japan), an RF-10A fluorescence detector (Shimadzu), and a CTO-10AC column oven (Shimadzu). A 20-µL sample was injected into the column. Separation was carried out on a TSKgel ODS-80Ts column (C18, 4.6 × 15 cm, 5 µm; Tosoh, Tokyo, Japan) at 40 °C. The mobile phase consisted of 0.2% 2,2,2-trifluoroacetic acid (TFA) in water and acetonitrile (95 : 5, v/v) and was delivered at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were 290 and 400 nm, respectively. Data obtained from the measuring instruments were converted to a digital format using a PicoLog ADC-20 data logger (Pico Technology Ltd., England, U.K.). The digital data were imported into Microsoft Excel for further analysis and evaluation.

Measurement of Serum IS

Serum IS levells were determined using HPLC, based on previously reported techniques.¹⁶⁾ Indican and IS were weighed and dissolved in methanol to obtain standard solutions. Sixty microliters of serum were deproteinized with 180 µL of methanol, and the supernatant was obtained after centrifugation at 15000 rpm for 15 min. The supernatant was filtered through a 0.22-µm membrane filter and analyzed using HPLC. Serum concentrations were measured from the obtained peak areas and calibration curves were generated using standard IS solutions.

Indican/IS Binding Assay to AST-120

The binding affinity between indican or IS and AST-120 was measured using HPLC. IS and indican were used as the measurement samples. Each sample was dissolved in 0.05 M phosphate buffer (pH 7.4) to a concentration of 10 mg/dL. AST120 (500 mg) was added to 50 mL of sample solution and the mixture was shaken at 37 °C for 3 h. After filtering the solution through a 0.45-µm membrane filter, IS or indican remaining in the solution was analyzed using HPLC.

Gene Expression Omnibus (GEO) Database Analysis

We used Genevestigator software (https://genevestigator.com/, version: 9.5.0, Release Date: 2022-10-06) to analyze the data retrieved from the GEO database (https://www.ncbi.nlm.nih.gov/geo/), that is, datasets GSE117261 and GSE16624. Changes in gene expression in each dataset were calculated using by “Differential Expression” analysis and the false discovery rate (FDR), p-value, and log₂FC for each gene were obtained. Genes were defined as significantly upregulated based on FDR < 0.1, p < 0.05, and log₂FC > 0.6. Official human gene symbols were converted to official rat gene symbols using GeneCardsSuite (https://www.genecards.org) website.

Cell Culture

HUVECs (C2517A) were purchased from Lonza (Basel, Switzerland) and cultured in EBM™-2 endothelial cell basal medium (CC-3156; Lonza, Basel, Switzerland) supplemented with EGM™-2eEndothelial cell growth medium-2 singleQuots™ supplements and growth factors (CC-4176; Lonza). RASMCs were purchased from Lonza and cultured in Dulbecco’s modified Eagle’s medium/F12 (048-29785; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (S1810-500; BioWest, Nuaillé, France), 30 µg/mL gentamicin sulfate (071-06453; FUJIFILM Wako), and 15 ng/mL amphotericin B (017-27971; FUJIFILM Wako). All the cells were cultured in 5% CO₂ and 95% room air (21% O₂) at 37 °C.

Quantitative Real-Time PCR

The cells were seeded in 6-well plates at a concentration of 0.6 × 10⁵ cells/well three days before stimulation. The medium was replaced 28 h before the stimulation, and the AhR inhibitor, CH223191 (10 µM), or NAC (10 µM) was added 1 h before the stimulation of indican or IS for 12 h. Total RNA was extracted from cells or lung tissues according to the modified acid guanidinium-phenol-chloroform method using RNAiso Plus (9109; TaKaRa Bio, Kusatsu, Shiga, Japan) according to the manufacturer’s instructions. Total RNA (1000 ng) was used for cDNA synthesis using ReverTra Ace qPCR RT Master Mix (FSQ-301; TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. The PCR mixture contained cDNA (equivalent to 33 ng of total RNA), forward and reverse primers, and THUNDERBIRD SYBER qPCR Mix (QPS-201; TOYOBO). Quantitative PCR was performed in an Applied Biosystems 7500 (Applied Biosystems, Foster City, CA, U.S.A.) under the following conditions: 1 min at 95 °C and 45 cycles of 10 s at 95 °C and 40 s at 60 °C. β-Actin gene expression was used to normalize sample amplification. The primers used in this study are listed in Tables 1 and 2.

Table 1. Human Oligonucleotide Primers Used in Quantitative Real-Time PCR

Human	Forward	Reverse
ET-1	5′- CAGGGCTGAAGACATTATGGAGA-3′	5′-CATGGTCTCCGACCTGGTTT-3′
ETB	5′-GCTTGCTTCATCCCGTTCAGA-3′	5′-CTTCCCGTCTCTGCTTTAGGTG-3′
CYP1A1	5′-TCGGCCACGGAGTTTCTTC-3′	5′-GGTCAGCATGTGCCCAATCA-3′
β-Actin	5′-GCGGGAAATCGTGCGTGACATTA-3′	5′-ATGGAGTTGAAGGTAGTTTCGTG-3′

Table 2. Rat Oligonucleotide Primers Used in Quantitative Real-Time PCR

Rat	Forward	Reverse
ET-1	5′-GTTTTGCATTGAGTTCCATTTGC-3′	5′-GAAAGCCACAAACAGCAGAGAGA-3′
ETA	5′-ATCGGGATCCCCTTGATTAC-3′	5′-CCAGTCCTTCACGTCTTGGT-3′
ETB	5′-CGATTGTATCATGCCTCGT-3′	5′-GGGACCATTTCTCATGCACT-3′
Cyp1a1	5′-ATGAGTTTGGGGAGGTTACTGGT-3′	5′-ACTTCTTATTCAAGTCCTTGAAGGCA-3′
Nt5e	5′-GGCTATCTGAAGGTTGAGT-3′	5′-CCGAGTTCCTGTGTAGAATA-3′
Rgs1	5′-AACTCCTTGCCAACCAGATG-3′	5′-TGTGGGAGTTGGTGTTTTGA-3′
Thy1	5′-AGCTATTGGCACCATGAACC-3′	5′-GCTGATCACCCTCTGTCCTC-3′
Aspn	5′-TGTCCAACAGTGCCAAAGATG-3′	5′-CCAACAACGCAGCGAAAC-3′
Cpa3	5′-TGCACCAGAGTCCGAGAAAG-3′	5′-TCGGTGGCAGTTTGATTGTG-3′
Cpe	5′-CTCCTGGTCATCGAGCTGTCT-3′	5′-TCGTGTGCTGTGGATCAGGTT-3′
Frzb	5′-ATTTGGCTCGCTGTATTGAC-3′	5′-GTGCACATTTCACTGTCCTG-3′
Gem	5′-TAGTGCGGTGTCGAGAAGTG -3′	5′-GACAGGTCATGGCAGGATTT-3′
Ltbp2	5′-GCTACACTTGTGACTGCTT-3′	5′-GCGATAGGAACCCTCTGT-3′
β-Actin	5′-GGAGATTACTGCCCTGGCTCCTA-3′	5′-GACTCATCGTACTCCTGCTTGCTG-3′

3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide (MTT) Assay

RASMCs were seeded in 24-well plates at a concentration of 1 × 10⁴ cells/well two days before the stimulation. One day before the stimulation, the medium was changed to 1% FBS-containing medium and the cells were stimulated with indican or IS (25, 100, and 250 µM). After stimulation for 24 h, MTT reagent (349–01824; Dojindo) dissolved in phosphate buffered saline (PBS) was added (0.5 mg/mL final), and the cells were incubated for 3 h. After incubation, the medium was removed and dimethyl sulfoxide was added to dissolve the formazan crystals. To evaluate cell proliferation, absorbance was measured at 538 nm.

Measurement of Intracellular Reactive Oxygen Species (ROS) Level

Intracellular ROS were detected using dichlorofluorescein diacetate (DCFH-DA, D6883; Sigma-Aldrich). HUVECs were incubated with DCFH-DA (10 µM) for 30 min at 37 °C and then washed and stimulated with indican (100 µM), IS (100 µM), or H₂O₂ (400 µM) for 30 min. NAC (10 µM) was added 1 h before the stimulation. ROS production was quantified at excitation/emission wavelength of 488/532 nm using a microplate reader (Infinite F200; Tecan Austria GmbH, Austria).

Statistical Analysis

Data are presented as mean ± standard error of the mean (S.E.M.). Unpaired t-test was used for comparison between two groups. For multiple group comparisons, Dunnett’s test or one-way ANOVA followed by Tukey’s test was used to assess statistical significance. p-value < 0.05 was defined as indicating statistical significance. Detailed statistical analysis methods are described along with each method in this section and the figure legends.

RESULTS

Only the Condition of Long-Term Chronic and High-Dose Administration of IL Induced Medial Thickening in Pulmonary Arteries without Right Ventricular Hypertrophy in Rats

Significant medial thickening in the pulmonary arteries was induced in rats fed a diet containing 5% IL for 6 months compared with the normal diet, and AST-120 administration completely suppressed this effect (Figs. 1A–C). IL and AST-120 administration did not affect body weight during the experimental period (Fig. 1D). The food intake did not differ between the IL and control groups (data not shown). Medial thickening of the pulmonary arteries is a major morphological change in PAH patients, but IL administration did not affect right ventricular weight (Fig. 1E). Pulmonary arterial remodeling was not observed in rats fed a diet containing 5% IL for a month, which was shorter than 6 months (Fig. 2). These results suggested that long-term chronic and high-dose administration of IL induces medial thickening of the pulmonary arteries as an initial step in PAH in rats.

Fig. 1. Effects of IL Administration for 6 Months on Pulmonary Arterial Remodeling in Rats

(A) Morphological changes in pulmonary arteries with H&E-stained tissue sections. Scale bar: 10 µm. (B) Medial thickness (µm) of the pulmonary arteries. Data were analyzed using one-way ANOVA followed by the Tukey’s test; ** p < 0.01. Mean ± S.E.M., n = 9, 9, 5. (C) Ratio of the medial thickness to the outer diameter of the pulmonary arteries. Data were analyzed using one-way ANOVA followed by Tukey’s test; * p < 0.05. Mean ± S.E.M., n = 9, 9, 5. (D) BW changes in the three groups during the experimental period. Data were analyzed using one-way ANOVA followed by Tukey’s test. Mean ± S.E.M., n = 9, 9, 5. (E) Ratio of the right ventricular weight to the left ventricle plus septum weight (RV/(LV + S)). Data were analyzed using one-way ANOVA followed by Tukey’s test. Mean ± S.E.M., n = 9, 9, 5.

Fig. 2. Effects of IL Administration for a Month on Pulmonary Arterial Remodeling in Rats

(A) Morphological changes in pulmonary arteries with H&E-stained tissue sections. IL administration for a month did not induce medial thickening of the pulmonary arteries in our rat model. Scale bar: 10 µm. (B) Medial thickness (µm) of the pulmonary arteries. Data were analyzed using unpaired t-test. Mean ± S.E.M., n = 3. (C) Ratio of the medial thickness to the outer diameter of the pulmonary arteries. Data were analyzed using unpaired t-test. Mean ± S.E.M., n = 3.

Serum IS Levels Were Increased in IL-Administered Rats, and This Effect Was Suppressed by AST-120

Serum IS levels were higher in IL-administered rats than in control rats, and AST-120 administration decreased this effect (Figs. 3A, B). Serum indican levels were below the detection limit of our HPLC method (data not shown); however, we succeeded in detecting indican in IL (Supplementary Fig. 2). A previous report showed that oral administration of indican increased serum indican levels but suggested that indican rapidly transformed into IS in rats.¹¹⁾ IL contain various IS precursors (indole analogs), with indican as the major component of them.¹⁰⁾ They can be absorbed from the surface of the intestinal epithelium into the circulating blood and converted into the final product, IS, in the liver.¹¹⁾ In our in vitro experiments, indican and IS dissolved in phosphate buffer were eliminated after 3 h of incubation with AST-120 (Fig. 3C). AST-120 is activated charcoal and is not absorbed into the circulating blood; therefore, these results and the reports strengthened the idea that the IL-derived IS precursor, indican, was absorbed into the rat body in the IL without AST-120 group, suggesting that it could bind to AST-120 and be trapped by AST-120 on the surface of the intestinal epithelium in the IL with AST-120 group.

Fig. 3. Serum IS Levels Were Increased in IL-Administered Rats and Were Suppressed by AST-120

(A) Typical HPLC chromatogram of rat serum samples collected at the endpoint of our six-month long-term chronic administration model. The black arrow indicates the peak of IS. (B) Serum IS concentration (µM) calculated from the peak area of IS. A concentration below 2 µM was defined as not detected (N.D.). Data were analyzed using one-way ANOVA followed by Tukey’s test; ** p < 0.01. Mean ± S.E.M., n = 9, 9, 5. (C) The peak of indican or IS in the HPLC chromatogram was abolished in the samples incubated with AST-120 for 3 h.

SMC Proliferation Was Induced by IS but Not Indican

Next, we examined the direct effects of indican or IS on SMC proliferation, which leads to medial thickening. Indican did not induce SMC proliferation, whereas IS induced it through AhR-independent mechanisms (Figs. 4A, B). The mRNA expression of the target gene of AhR, Cyp1a1 was induced by indican, and this induction was suppressed by the AhR inhibitor, CH223191 (Fig. 4C). Although IS, a major AhR agonist, also induced Cyp1a1 mRNA expression, it was not statistically significant among multiple comparisons (Fig. 4C). These results indicated that only IS directly induced SMC proliferation.

Fig. 4. Effects of Indican and IS on SMC Proliferation

(A, B) MTT assay revealed that IS induced SMC proliferation but indican did not. The data for Figs. 4A and B were acquired at the same time, and both the control and CH223191 alone groups were common between the two graphs. Data were analyzed using Dunnett’s test for dose-dependent comparisons or one-way ANOVA followed by Tukey’s test for the comparisons between the two groups with and without CH223191 at each dose; ** p < 0.01. Mean ± S.E.M., n = 18, 12. (C) Cyp1a1 mRNA expression in RASMCs. Data were analyzed using one-way ANOVA followed by Tukey’s test; ** p < 0.01. Mean ± S.E.M., n = 10.

GEO Analysis Revealed That ET-1 Is a Key Regulator of PAH in Both Patients and a Rat Model

To determine the types of key PAH-related regulators that were altered in the lungs of IL-administered rats, we first analyzed GEO datasets from PAH patient samples or rat lung samples in a rat PAH model. In APAH (associated pulmonary arterial hypertension) and IPAH (idiopathic pulmonary arterial hypertension) patients, 163 and 131 genes, respectively, were upregulated in the lung tissue (#GSE117261).¹⁷⁾ In the lungs of the rat PAH model (#GSE16624), 444 genes were upregualted.¹⁸⁾ The ten genes indicated in Table 3 were common among these genes (Fig. 5A). Thus, our GEO analysis indicated that these 10 genes were key regulators of PAH in both patients and rats. We checked the mRNA expression levels of these 10 genes in the lungs of control and IL-administered rats and found that only ET-1 (Edn1) mRNA expression levels normalized to CD31 mRNA expression were induced according to the unpaired t-test (Fig. 5B). These results suggested that EC-derived ET-1 mRNA expression is a key regulator of PAH and that its expression, mainly in ECs, was induced in the lungs by IL administration for 6 months in our rat model.

Table 3. The 10 Genes Which Were Common among the Up-Regulated Genes in APAH Patients (GSE117261), in IPAH Patients (GSE117261) and in the PAH Rat Model (GSE16624)

Gene symbol (rat)	Description
Edn1	Endothelin 1
Nt5e	5′ Nucleotidase, ecto
Rgs1	Regulator of G-protein signaling 1
Thy1	Thy-1 cell surface antigen
Aspn	Asporin
Cpa3	Carboxypeptidase A3
Cpe	Carboxypeptidase E
Frzb	Frizzled-related protein
Gem	GTP binding protein overexpressed in skeletal muscle
Ltbp2	Latent transforming growth factor beta binding protein 2

Fig. 5. GEO Analysis Revealed That ET-1 Is a Key Regulator of PAH

(A) According to GEO analysis, the 10 genes listed in Table 3 were common among the 163 upregulated genes in APAH patients (GSE117261), 131 up-regulated genes in IPAH patients (GSE117261), and 444 upregulated genes in the PAH rat model (GSE16624). Genes were defined as significantly upregulated based on a false discovery rate (FDR) < 0.1, p < 0.05, and log₂FC > 0.6. (B) Effects of IL administration for 6 months on mRNA expression levels of the extracted 10 genes via GEO analysis in rat lungs. Each mRNA expression level was normalized to CD31 mRNA expression level. Data were analyzed using unpaired t-test; * p < 0.05. Mean ± S.E.M., n = 9.

EC-Derived ET-1 Expression Was Elevated in the Lungs of IL-Administered Rats

As shown in Fig. 5, ET-1 receptor antagonists are widely used clinically for PAH therapy and EC-derived ET-1 is a crucial regulator of PAH development and progression. Immunofluorescence analysis showed that ET-1 protein expression level within CD31 positive area (EC layer) in the pulmonary arteries was significantly induced by IL administration for a month (Figs. 6A, B), even though medial thickening was not observed (Fig. 2). After IL administration for 6 months, significant medial thickening was observed, and ET-1 mRNA expression levels normalized to CD31 (ET-1/CD31) mRNA expression were significantly induced compared with the control group, according to the unpaired t-test for two-group comparisons (Fig. 5B). According to one-way ANOVA followed by Tukey’s test for multiple group comparisons, we observed that IL administration for 6 months tended to induce ET-1/CD31 mRNA expression (p = 0.0726) (Fig. 6C). Although we usually normalize mRNA expression levels by β-actin mRNA level as described in Materials and Methods, to investigate EC-derived mRNA expression levels in lung tissues, we normalized ET-1 mRNA level by CD31 mRNA level. As shown in Supplementary Fig. 3, there was no significance in CD31 mRNA expression level in lung samples among the three groups in our rat model. Our immunofluorescence analysis showed a similar result, but this was not statistically significant after 6 months of IL administration (Figs. 6D, E). The mRNA expression levels of two ET-1 receptors, ET_A and ET_B, were not altered by IL administration for 6 months (Figs. 6F, G). This result was consistent with the fact that neither indican nor IS affected the mRNA expression levels of these receptors in SMCs (Supplementary Fig. 4). Cyp1a1 mRNA expression in the lungs was induced by IL administration (Fig. 6H), suggesting that IL-derived indole analogs, such as indican and IS, were absorbed into the circulating blood and affected the pulmonary arteries. AST-120, however, could not suppress this effect, suggesting that AST-120 could not trap some IL-derived AhR activators (except indican or IS) which did not affect medial thickening. These results suggested that EC-derived ET-1 induction by IL-derived components in the early phase plays a key role in medial thickening.

Fig. 6. Effects of IL Administration on EC-Derived ET-1 Expression Levels in Rat Lungs

(A) ET-1/CD31 double immunostaining of IL-administered (for a month) rat lung tissue sections. Scale bar: 10 µm. (B) Mean intensity of ET-1 staining within CD31 positive area (EC layer). Data were analyzed using unpaired t-test; * p < 0.05. Mean ± S.E.M., n = 3. (C) ET-1 mRNA expression level normalized to CD31 mRNA expression in IL-administered (for 6 months) rat lungs. Data were analyzed using one-way ANOVA followed by Tukey’s test. Mean ± S.E.M., n = 9, 9, 5. Only the comparison between the control and IL group is included in Fig. 5B. (D) ET-1/CD31 double immunostaining of IL-administered (for 6 months) rat lung tissue sections. Scale bar: 10 µm. (E) Mean intensity of ET-1 staining within CD31 positive area (EC layer). Data were analyzed using one-way ANOVA followed by Tukey’s test. Mean ± S.E.M., n = 9, 9, 5. (F, G, H) mRNA expression changes in the ET_A receptor (F), ET_B receptor (G), and Cyp1a1 (H) in IL-administered (for 6 months) rat lungs. Data were analyzed using one-way ANOVA followed by Tukey’s test; * p < 0.05, ** p < 0.01. Mean ± S.E.M., n = 9, 9, 5.

Both Indican and IS Induced ET-1 Expression via ROS Production in ECs

Finally, we investigated how IL-derived indican or IS contributed to ET-1 induction in ECs. Both indican and IS induced ET-1 mRNA expression in HUVECs, but CH223191 did not suppress this effect (Fig. 7A). While the ET_A receptor was hardly expressed in ECs, ET_B receptor mRNA expression was induced by indican and IS, and CH223191 did not suppress this effect (Fig. 7B). CYP1A1 mRNA expression was induced by indican and its effect was clearly suppressed by CH223191 (Fig. 7C). IS induced CYP1A1 mRNA expression and CH223191 suppressed this effect (Fig. 7C). These results suggested that indican and IS induced ET-1 mRNA expression via an AhR-independent pathway. Although some reports have shown that hypoxia-inducible factor-1α (HIF-1α) regulates ET-1 expression,¹⁹⁾ we found that indican and IS did not induce HIF-1α protein expression (Supplementary Fig. 5). This result is consistent with another report showing that IS does not induce HIF-1α expression but suppresses hypoxia-induced HIF-1α elevation.²⁰⁾ Next, we focused on the effects of ROS production. Indican, IS, and hydrogen peroxide (positive control) induced ROS production, but a ROS inhibitor, NAC, completely suppressed those effects in HUVECs (Fig. 7D). NAC suppressed indican- and IS-induced ET-1 mRNA expression (Fig. 7E). These results suggested that both indican and IS induced ET-1 expression via ROS production in ECs. To the best of our knowledge, this is the first study to show that indican induces ET-1 expression via ROS production in ECs and contributes to pulmonary arterial remodeling.

Fig. 7. Mechanisms of Indican- and IS-Induced ET-1 mRNA Expression in HUVECs

(A) Effects of AhR activation on indican- or IS-induced ET-1 mRNA expression. Data were analyzed using one-way ANOVA followed by Tukey’s test; * p < 0.05, ** p < 0.01. Mean ± S.E.M., n = 15, 10. (B) Effects of indican, IS, and CH223191 on ET_B receptor mRNA expression. Data were analyzed using one-way ANOVA followed by Tukey’s test; * p < 0.05, ** p < 0.01. Mean ± S.E.M., n = 15, 10. (C) Effects of indican, IS, and CH223191 on CYP1A1 mRNA expression. Data were analyzed using one-way ANOVA followed by Tukey’s test; ** p < 0.01. Mean ± S.E.M., n = 15, 10. (D) Effects of indican and IS on ROS production. Data were analyzed using one-way ANOVA followed by Tukey’s test; ** p < 0.01. Mean ± S.E.M., n = 8. (E) Effects of NAC on indican- or IS-induced ET-1 mRNA expression. Data were analyzed using one-way ANOVA followed by Tukey’s test; * p < 0.05, ** p < 0.01. Mean ± S.E.M., n = 25, 10.

DISCUSSION

In this study, we focused on the raw material of IN, IL, to investigate the pathogenesis of IN-induced PAH. However, only long-term chronic (6 months) and high-dose (containing 5% IL in the control diet) administration of IL induced medial thickening in the pulmonary arteries without right ventricular hypertrophy in our rat model. Furthermore, despite using a higher dose for IL administration in our rat model than the dose (containing 1% IL in the control diet) used in a study by Tsuji et al.,²¹⁾ we did not observe pulmonary arterial remodeling after IL administration for a month. We hypothesized that the components of IL have a weak ability to induce PAH and that other components of IN may play key roles in PAH development. For example, Masaki et al. reported that indirubin, which is also present in IN, induces PAH via AhR activation.²²⁾ However, we found that high-dose and chronic administration of IL induced medial thickening in the pulmonary arteries after EC-derived ET-1 induction. EC-derived ET-1 induction causes SMC migration and proliferation which links to medial thickening.²³⁾ But it should take time for such morphological change formation, we supposed that ET-1 induction in early phase was very important.

ET-1 is a key regulator of PAH, and our bioinformatic analysis identified it as a key regulator of PAH in both patients and rats. Therefore, IL administration-induced ET-1 expression in ECs, followed by medial thickening of the pulmonary arteries, was a reasonable cause of pulmonary arterial remodeling. We also elucidated that a major IL component indican and its final metabolite IS induced ET-1 expression via AhR independent and HIF-1α independent pathways but via ROS production in ECs. Several reports have shown that increased ROS levels contribute to ET-1 induction,²⁴⁾ which is consistent with our results. Furthermore, some papers have already shown that IS induces ROS production through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation.^25,26) Unexpectedly, CH223191 increased mRNA expression levels of ET-1 and ET_B receptors in ECs. We used 10 µM of CH223191 in our in vitro studies. This dose is often used in the most of papers^27,28) and not too high. As shown in Fig. 7C, CH223191 certainly showed its effect in our in vitro studies, but we could not conclude whether CH223191 induced the mRNA expressions of ET-1 and ET_B receptors through AhR-dependent or -independent manner. The roles of IL components, especially indican, in living systems remain unclear. To the best of our knowledge, this is the first study to elucidate the mechanisms by which indican contributes to ET-1 induction, which is linked to pulmonary arterial remodeling. The mRNA expression levels of the two receptors of ET-1, ET_A and ET_B, in the lungs were not changed by IL components or their metabolites in our rat model.

Although the serum indican levels in IL-administered rats were below the detection limit of our HPLC method, we successfully detected the indican present in IL. As indican is soluble in water, we hypothesized that it would be absorbed from the surface of the intestinal epithelium into the circulating blood in IL-administered rats and affect the pulmonary arteries. A previous report showed that oral administration of indican increased serum indican levels and that indican was transformed into IS in rats.¹¹⁾ We found that indican and IS were trapped by AST-120 and that AST-120 administration suppressed serum IS levels and medial thickening in the pulmonary arteries induced by IL administration in our rat model. Thus, our results showed that indican was absorbed into the circulating blood and reached the pulmonary arteries.

In conclusion, we elucidated the roles of a major IL component, indican, and its final metabolite, IS, in ET-1 expression in ECs and in pulmonary arterial remodeling. In addition, our in vitro study using a specific AhR-inhibitor^29,30) showed that IS, but not indican, had the direct ability to induce SMC proliferation through AhR-independent mechanisms, although several papers have shown that AhR activation contributes to SMC proliferation.^31,32) However, these effects were mild in our experiments, and it is expected that other IN components may play key roles in PAH development. Hiraide et al. reported that Qing-Dai (IN) induces PAH accompanied by right ventricular hypertrophy via an AhR-dependent pathway in a mouse model.³³⁾ Thus, we elucidated the unique ability of indican and IS to induce pulmonary arterial remodeling.

Acknowledgments

We appreciate the excellent technical support of the Support Center for Advanced Medical Sciences, Tokushima University Graduate School of Biomedical Sciences.

Funding

This work was partially supported by JSPS KAKENHI Grant Number: 22K20711 (to KT) and was also supported by JST SPRING, Grant Number: JPMJSP2113 (to HT).

Author Contributions

HT, MI, and YH performed bioinformatics analyses. HT, MI, YU, YH, MM, and AK performed experiments and analyzed data. M I-S, YH, and KT performed the histological analyses. LM, MF, YH, and YI contributed to the interpretation of the data and edited the manuscript. HT, MI, and KT interpreted the data and wrote the manuscript. KT planned and generated the study design and obtained funding.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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