Soyasaponin I alleviates hypertensive intracerebral hemorrhage by inhibiting the renin–angiotensin–aldosterone system

ABSTRACT

Background

Hypertensive intracerebral hemorrhage (HICH) is a life-threatening disease and lacks effective treatments. Previous studies have confirmed that metabolic profiles altered after ischemic stroke, but how brain metabolism changes after HICH was unclear. This study aimed to explore the metabolic profiles after HICH and the therapeutic effects of soyasaponin I on HICH.

Methods

HICH model was established first. Hematoxylin and eosin staining was used to estimate the pathological changes after HICH. Western blot and Evans blue extravasation assay were applied to determine the integrity of the blood–brain barrier (BBB). Enzyme-linked immunosorbent assay was used to detect the activation of the renin–angiotensin–aldosterone system (RAAS). Next, liquid chromatography–mass spectrometry-untargeted metabolomics was utilized to analyze the metabolic profiles of brain tissues after HICH. Finally, soyasaponin I was administered to HICH rats, and the severity of HICH and activation of the RAAS were further assessed.

Results

We successfully constructed HICH model. HICH significantly impaired BBB integrity and activated RAAS. HICH increased PE(14:0/24:1(15Z)), arachidonoyl serinol, PS(18:0/22:6(4Z, 7Z, 10Z, 13Z, 16Z, and 19Z)), PS(20:1(11Z)/20:5(5Z, 8Z, 11Z, 14Z, and 17Z)), glucose 1-phosphate, etc., in the brain, whereas decreased creatine, tripamide, D-N-(carboxyacetyl)alanine, N-acetylaspartate, N-acetylaspartylglutamic acid, and so on in the hemorrhagic hemisphere. Cerebral soyasaponin I was found to be downregulated after HICH and supplementation of soyasaponin I inactivated the RAAS and alleviated HICH.

Conclusion

The metabolic profiles of the brains changed after HICH. Soyasaponin I alleviated HICH via inhibiting the RAAS and may serve as an effective drug for the treatment of HICH in the future.

KEYWORDS:

• Soyasaponin I
• hypertensive intracerebral hemorrhage
• blood–brain barrier
• renin–angiotensin–aldosterone system
• metabolomics

Introduction

Intracerebral hemorrhage (ICH) is a devastating disorder caused by non-traumatic rupture of blood vessels in the brain parenchyma, including primary ICH and secondary ICH (1). ICH accounts for about 28% of all stroke subtypes and is one of the leading causes of human death and disability worldwide (2). Hypertension, the main cause of ICH, leads to hyalinization and fibrosis of brain microvessels and alternation of cerebrovascular hemodynamics. These pathological changes dampen cerebrovascular wall elasticity, ultimately resulting in hypertensive ICH (HICH) under fluctuating blood pressure (3). Currently, with the change of people’s lifestyle, the incidence of HICH is increasing year by year, and due to the lack of effective treatment, HICH has an extremely high mortality and disability rate, imposing onerous burdens on families and society (4). Therefore, it is urgent to clarify the pathological mechanisms of HICH and find effective therapeutic approaches.

The renin–angiotensin–aldosterone system (RAAS) is an important humoral regulatory system composed of a series of peptide hormones and corresponding enzymes, whose main function is to regulate and maintain the balance of blood pressure and water–electrolyte metabolism in the body (5). Aberrant activation of RAAS is a key mechanism in the pathogenesis of hypertension, and RAAS inhibitors, such as angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) have been widely used to treat hypertension in the clinic, greatly improving the prognosis of HICH patients (6). Thus, inactivation of RAAS is an effective strategy for the treatment of HICH. The brain is the most metabolically active organ in the body and is functionally regulated by various metabolites (7–10). After stroke onset, the brain and peripheral organs undergo stress responses and alterations in energy metabolic pathways. Differentially expressed metabolites, such as glucose and lipids, have been used in stroke diagnosis and prognosis (11). With the development of metabolomics techniques, an increasing number of metabolites have been found to participate in the pathogenesis of stroke and showed great potential in stroke diagnosis and prognosis prediction (12). However, most studies only focused on the effects of peripheral circulating metabolites on brain function after stroke, especially ischemic stroke. Yet, it remains unclear how the metabolic profiles of brain tissues change after HICH and whether these altered metabolites interact with the RAAS system.

In the present study, we analyzed brain tissue metabolite profiles of normal and HICH rats using untargeted metabolomics, and soyasaponin I was found to be significantly reduced after HICH. We also explored the therapeutic potential of soyasaponin I against HICH, hoping to provide new approaches for the treatment of HICH.

Methods

Animals and HICH model

This study was approved by the ethics review committee of The Affiliated Changsha Hospital of Xiangya School of Medicine, Central South University (KX-2020101). Male adult Sprague–Dawley (SD) rats were purchased from Hunan SJA Laboratory Animal Co., Ltd. The SD rats were acclimatized and housed in a specific pathogen-free animal house for 1 week before experiments. Rats were randomly divided into sham group, HICH group, and soyasaponin I treatment group. After anesthesia, hair removal, and skin disinfection, the rats were fixed on a brain stereotaxic apparatus (#ZS-FD/S, Zhongshi Science & Technology, China) and the fontanel was fully exposed. Five microliters of collagenase IV (#9001-12-1, Sigma, USA) was injected into the right caudate nucleus (0.2 mm anterior to fontanel, 3 mm to the right of the sagittal line, and 5.5 mm in depth) using a microinjector with an injection rate of 0.2 μL/min. After the injection, the syringe was left for 10 min to prevent collagenase reflux, and the needle hole was finally sealed with bone wax. Rats in the sham group received 5 μL of 0.9% saline injection, and the rest of the operations were the same as that of the HICH group. For the soyasaponin I treatment group, 10 mg/kg soyasaponin I was administered once a day by gavage, while the sham and HICH groups were given equal amounts of distilled water. Seven days later, rats were sacrificed, and brain tissues and peripheral blood were collected for further detection.

Hematoxylin and eosin staining

Paraffin sections of the brain tissue were prepared using a microtome. Sections were first subjected to xylene dewaxing and gradient ethanol rehydration. Following that, hematoxylin and eosin (HE) (#AWI0020a, Abiowell, China) were successively applied to stain the sections for 10 and 5 min. Next, sections were dehydrated by gradient alcohol and vitrified by xylene. Finally, after sealing with neutral gum, the sections were observed and pictured under a microscope.

Western blot

Total proteins of brain tissues were extracted using RIPA (radio-immunoprecipitation) lysis buffer (#AWB0136, Abiowell, China). After detecting concentrations by BCA (Bicinchoninic Acid Assay) assay, proteins were separated using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and electrotransferred on PVDF membranes (#1620177, Bio-Rad, USA). After blocking with 5% skim milk (#AWB0004, Abiowell, China), the blots were incubated with primary antibodies against Occludin (#27260-1-AP, Proteintech, USA), Collagen IV (#ab6586, Abcam, UK), or β-actin (#66009-1-Ig, Proteintech, USA) at 4°C overnight. The next day, the blots were washed with PBS (Phosphate-Buffered Saline) and incubated with horseradish peroxidase–conjugated second antibodies (#SA00001-1 and #SA00001-2, Proteintech, USA). Finally, the blots were treated with ECL (enhanced chemiluminescence) chemiluminescent solution (#AWB0005a, Abiowell, China), and the protein expressions were quantified after imaging.

Evans blue extravasation assay

Blood–brain barrier (BBB) integrity was determined using the Evans blue extravasation assay. Briefly, 3 mL/kg of Evans blue dye (#314-13-6, Sigma, USA) was injected into rats through the tail vein, and 2 hours later, rats were perfused with 0.9% saline after anesthesia. After perfusion, rats were decapitated and the brain tissues were collected. After weighing, brain tissues were homogenized with formamide (#F810079, Macklin, China) and then underwent centrifugation. Following that, the supernatant was extracted, and the absorbance value at 630 nm was obtained using a microplate reader. Finally, the amount of extravasated Evans blue was calculated by referring to the standard curve.

Detections of renin, angiotensin, aldosterone, and prostaglandin E2

Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to detect serum concentrations of renin (#KT-60668, Kamiya Biomed, Japan), angiotensin (#KT-33516, Kamiya Biomed, Japan), aldosterone (#KT-59377, Kamiya Biomed, Japan), and cerebral concentration of prostaglandin E2 (PGE2) (#ab287802, Abcam, UK) according to the manufacturers’ instructions.

Liquid chromatography–mass spectrometry untargeted metabolomics

Samples were first dissolved and extracted using methanol. Equal amounts were taken from all samples and then mixed as quality control (QC) samples. Liquid chromatography–mass spectrometry (LC–MS) analysis was performed on a high-performance liquid chromatograph (#AB ExionLC, AB Sciex, USA) and a high-resolution mass spectrometer (#QE, ThermoFisher, USA). The chromatographic column was ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm, Waters, USA) with the column temperature set at 40°C. The mobile phase A was water (containing 0.1% formic acid), the mobile phase B was acetonitrile (containing 0.1% formic acid), and the flow rate was set at 0.35 mL/min with an injection volume of 5 μL. The gradient elution program was as follows: 0 min A:B = 95:5; 1.5 min A:B = 95:5; 3 min A:B = 70:30; 7 min A:B = 40:60; 9 min A:B = 10:90; and 11 min A:B = 10:90. The MS signal acquisition was performed in positive and negative ion scan mode, and the MS parameters were set as follows: spray voltage, 3500 V; capillary temperature, 320°C; probe heater temperature, 350°C; sheath gas flow rate, 40 Arb; auxillary gas flow rate, 10 Arb; S-lens RF level, 50; mass range, 100–1000 m/z. Finally, metabolomic analysis of LC–MS data was commissioned from Shanghai Lu-Ming Biotech Co., Ltd.

High-performance liquid chromatography analysis

The contents of adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in the brain were determined using high-performance liquid chromatography (HPLC). The HPLC analysis was performed on a Triple TOF 5600 System (AB Sciex, USA). The chromatographic column was an HSS T3 column (100 × 2.1 mm, 1.7 μm, Waters, USA) with the column temperature set at 40°C. The mobile phase A was water (containing 0.1% formic acid) and the mobile phase B was acetonitrile (containing 0.1% formic acid), and the gradient elution program was 0.01 min, 99% A; 1.5 min, 99% A; 13 min, 1% A; 16.5 min, 1% A; 16.6 min, and 99% A; 20 min, stop.

Statistical analysis

GraphPad Prism 7 software was applied to analyze the data. Student’s t-test was used to determine the statistical significances between two groups. The data were shown as mean ± standard deviation, and p < .05 was considered to be statistically significant.

Results

The RAAS was activated after HICH

First, HICH model was established and the pathological changes were evaluated using HE staining. As shown in Figure 1A, clumps of red blood cells were observed within the hematoma of the right caudate nucleus after HICH, and normal tissue surrounding the hematoma is distorted by compression. BBB is a physical barrier that separates the central nervous system from the periphery (13). To evaluate the integrity of BBB, we detected BBB-related protein expressions and performed Evans blue extravasation assay. HICH significantly reduced protein levels of Occludin and Collagen IV in the caudate nucleus (Figure 1B-C), whereas increased Evans blue leakage in the hemorrhagic hemisphere (Figure 1D). Furthermore, activation of the RAAS was also estimated. The results showed that serum levels of renin, angiotensin, and aldosterone were markedly enhanced in rats after HICH (Figure 1E). These results indicated that HICH model was successfully constructed and the RAAS was activated after HICH.

Figure 1. The RAAS was activated after HICH. (a) HE staining of the right caudate nucleus. (b) WB analysis of Occludin and collagen IV in brain tissues. (c) Quantification of WB band intensities of Occludin and collagen IV in brain tissues. (d) Analysis of Evans blue extravasation in rats. (e) Serum contents of renin, angiotensin, and aldosterone were detected by ELISA. *p < .05 versus sham group.

HICH altered metabolic profiles of the brain

To assess how the metabolic profiles of brain tissues alter after HICH, we collected brain tissues and conducted LC–MS-untargeted metabolomics. First, base peak chromatograms (BPCs) of positive and negative ion scan mode are shown in Figure 2A. Then, principal component analysis (PCA) was used to determine the stability of the MS system. As shown in Figure 2B, QC samples were closely clustered together, indicating that our experiment was stable and reproducible. To clarify the differences between sham and HICH groups, partial least squares discriminant analysis (PLS-DA) and orthogonal PLS-DA (OPLS-DA) were implemented. Samples from sham and HICH groups were clearly separated (Figure 3A-B), suggesting a pronounced difference in metabolic profiles between the sham and HICH groups. Volcano plot exhibited 427 differentially expressed metabolites between sham and HICH groups (Figure 3C), and top 50 metabolites in terms of variable important in projection values are visualized by heatmap (Figure 3D). These data demonstrated that HICH altered metabolic profiles of the brain.

Figure 2. Evaluation of the stability of the LC–MS-untargeted metabolomics. (a) BPC of positive and negative ion scan modes. (b) PCA of samples from QC, sham, and HICH groups.

Figure 3. HICH changed metabolic profiles of the brain. (a) PLS-DA and (b) OPLS-DA of samples from QC, sham, and HICH groups. (c) Volcano plot of differentially expressed metabolites between sham and HICH groups. (d) Heatmap of top 50 differentially expressed metabolites between sham and HICH group. A represents sham group, and B and C represent HICH group.

HICH decreased soyasaponin I level in the brain

Next, Pearson correlation analysis was used to estimate the association between differential metabolites in brain tissues. As visualized in Figure 4A, red represented positive correlation and blue represented negative correlation. Darker colors and larger circles indicated stronger correlations. Interestingly, we detected soyasaponin I in the brains of sham and HICH rats, and cerebral soyasaponin I levels were strikingly reduced after HICH (Figure 4B). Besides, HICH also resulted in downregulation of RAAS-related metabolites including ADP, AMP, and PGE2 in the brains (Figure 4B). These results implied that cerebral soyasaponin I expression was decreased after HICH.

Figure 4. Soyasaponin I level in the brain was decreased after HICH. (a) Pearson correlation analysis of differentially expressed metabolites between sham and HICH groups. (b) The expressions of soyasaponin I, ADP, AMP, and PGE2 in the brains. *p < .05 versus sham group.

HICH altered metabolic pathways of the brain

To clarify the metabolic pathways involved in differential metabolites, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed. As shown in Figure 5A and B, alanine, aspartate, and glutamate metabolism, regulation of lipolysis in adipocytes, purine metabolism, protein digestion and absorption, pyrimidine metabolism, ABC transporters, aminoacyl-tRNA biosynthesis, central carbon metabolism in cancer, choline metabolism in cancer, and thermogenesis were top 10 metabolic pathways enriched by the KEGG analysis. These data showed that HICH changed metabolic pathways of the brain.

Figure 5. HICH changed metabolic pathways of the brain. (a) Bubble plot of the KEGG pathway of differentially expressed metabolites between sham and HICH groups. (b) KEGG enrichment analysis of differentially expressed metabolites between sham and HICH groups.

Soyasaponin I inactivated the RAAS and alleviated HICH

Since previous results proved that cerebral soyasaponin I levels were strikingly reduced after HICH, we next explored whether soyasaponin I has therapeutic effects on HICH. ELISA showed that serum concentrations of renin, angiotensin, and aldosterone were profoundly decreased in HICH rats upon soyasaponin I treatment (Figure 6A). HE staining exhibited a significant reduction in hemorrhage in the caudate nucleus after soyasaponin I administration (Figure 6B). Meanwhile, soyasaponin I remarkably decreased Evans blue leakage (Figure 6C), whereas increased ADP (Figure 6D), AMP (Figure 6E), and PGE2 (Figure 6F) expressions in the hemorrhagic hemisphere. These data indicated that soyasaponin I inactivated the RAAS and alleviated HICH.

Figure 6. Soyasaponin I inhibited RAAS activation and mitigated HICH. (a) Serum contents of renin, angiotensin, and aldosterone were detected by ELISA. (b) HE staining of the right caudate nucleus. (c) Analysis of Evans blue extravasation in the brains of rats. The expressions of (d) ADP, (e) AMP, and (f) PGE2 in the brains. *p < .05 versus sham group.

Discussion

The pathological mechanism of neurological impairment caused by HICH mainly includes the occupying effect of the hematoma, as well as cerebral edema, intracranial hypertension, altered local cerebral blood flow, and abnormalities of the coagulation and fibrinolytic system induced by hematoma breakdown products and vasoactive substances released from damaged brain tissues (14). Hematoma compression alone is not sufficient to cause severe damage to the brain tissues, whereas impaired cerebral circulation and metabolism caused by secondary cerebral edema are thought to be critical factors triggering brain damage after HICH (15,16). Overactivated RAAS is an important contributor to the development of hypertension and HICH. Renin, an aspartic protease synthesized and secreted by paraglomerular cells, initiates RAAS activation by converting angiotensinogen into angiotensin I. The catalytic hydrolysis of angiotensin I by ACE generates angiotensin II, which then acts on the adrenal cortex and leads to aldosterone release (17). Overactivation of RAAS can cause vasoconstriction and water and sodium retention, increase blood pressure, and myocardial contractility, thereby increasing the effective circulating blood volume and exacerbating hypertension (18). It has been clearly demonstrated clinically that the application of RAAS inhibitors such as ACEIs and ARBs significantly improved the prognosis of HICH (6). Therefore, inhibition of RAAS activation is an effective way to mitigate HICH.

Metabolomics is a technique that systematically investigates the metabolic profiles of biological samples, such as cells, tissues, and body fluids, allowing qualitative and quantitative analysis of alterations in metabolic intermediates and end products of organisms after stimulations (19). Compared with transcriptomics and proteomics, metabolomics focuses on the changes of metabolites due to genetic modifications or external environmental modifications and is the most reflective of the phenotype and overall functions of organisms (20). Metabolomics has been widely used in the screening of diagnostic and prognostic markers for ischemic stroke. For example, Marklund et al. found that lower linoleic acid levels were associated with higher incidence of cardiovascular disease and ischemic stroke (21). Sun et al. identified that serum tetradecanedioate and hexadecanedioate levels were positively associated with the incidence of ischemic stroke and cardiogenic stroke independently of other risk factors (22). Kimberly et al. revealed the role of branched-chain amino acid levels in stroke diagnosis, as they found that serum levels of valine, leucine, and isoleucine were lower in patients with cardiogenic stroke than in patients with transient ischemic attack and were associated with poor neurological outcomes (23).

However, there are very few metabolomic studies focusing on ICH, which may be due to the ease of diagnosis of ICH by cranial CT. Zhang et al. found that blood glutarylcarnitine, 3-hydroxylbutyrylcarnitine, hydroxystearoylcarnitine, myristoylcarnitine, and so on levels were effective in distinguishing healthy individuals, patients with ICH, and patients with cerebral infarction (24). In the present study, LC-MS untargeted metabolomics was used to determine the metabolic profiles in the brains of HICH rats. We found that HICH significantly increased cerebral PE(14:0/24:1(15Z)), arachidonoyl serinol, PS(18:0/22:6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)), PS(20:1(11Z)/20:5(5Z, 8Z, 11Z, 14Z, 17Z)), glucose 1-phosphate, etc., whereas reduced the contents of creatine, tripamide, D-N-(carboxyacetyl)alanine, N-acetylaspartate, N-acetylaspartylglutamic acid, and so on. Meanwhile, we found that these altered metabolites involved alanine, aspartate, and glutamate metabolism, regulation of lipolysis in adipocytes, purine metabolism, protein digestion and absorption, pyrimidine metabolism and other metabolic processes. Our data contributed to a better understanding of cerebral metabolic changes after HICH.

Soyasaponin, a bioactive compound widely present in the legumes, belongs to the triterpene saponins and is classified into two groups (A and B) (25). Soyasaponin B is the most abundant component of soyasaponin and is considered to serve as the main substance for the biological functions of soyasaponin (26). As the main constituent of soyasaponin B, soyasaponin I has shown therapeutic effects in a variety of diseases. Soyasaponin I inhibited the progression of breast cancer (27), melanoma (28), ovarian cancer (29), and colon cancer (30). Soyasaponin I also exerted anti-inflammatory effects by inhibiting the NF-κB pathway (31,32), and it promoted recovery of cognitive functions in memory-deficient rats (33). However, the role of soyasaponin I in HICH is still unclear. In the present study, brain soyasaponin I levels were found to be significantly reduced after HICH, indicating that soyasaponin I might be involved in the pathological process of HICH. To determine the effect of soyasaponin I on HICH, HICH rats were supplemented with exogenous soyasaponin I, and the severity of HICH was evaluated. Administration of soyasaponin I resulted in restoration of HICH-induced pathological changes, increased BBB integrity, and significantly reduced RAAS activation. Consistent with our data, previous studies have confirmed that soyasaponin I was a natural renin inhibitor (34–36). Theoretical approaches such as Docking Simulation and Molecular Dynamics Simulation have confirmed that soyasaponin I inhibited renin at the structural level. Soyasaponin I could bind to the active site of renin and a region near the active site, showing inhibition effect on renin via competing with the substrate (36). Our study clearly confirmed that soyasaponin I showed inhibitory effects on RAAS in vivo. Considering the crucial role of soyasaponin I in aggravating HICH, our data indicated that soyasaponin I might alleviate brain injury after HICH by inhibiting RAAS overactivation.

It is worth noting that some limitations existed in this research. Neurological damage of HICH rats needs to be further evaluated, and the molecular mechanism of soyasaponin I alleviating HICH by inhibiting RAAS needs to be further confirmed by rescue experiments. In addition, our study lacks the support of in vitro experimental data. In the future, we intend to further confirm the therapeutic effects of soyasaponin I on HICH, including additional blood pressure assays and neurological damage assays in rats, additional in vitro experiments, and in-depth investigation of molecular mechanisms. Furthermore, we intend to conduct clinical studies on the effect of soyasaponin I on HICH, aiming to provide new methods and new ideas for the treatment of HICH.

Conclusions

In conclusion, our study identified the alterations of metabolic profiles within the brain after HICH, and at the same time, we revealed a potential role of the RAAS inhibitor soyasaponin I in improving HICH. This study suggested that soyasaponin I may serve as an effective drug for the treatment of HICH in the future.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [82160153], Scientific research plan of Hunan Provincial Health Commission [202204042966], and Changsha Natural Science Foundation [kq2202007].