Xin-Li formula attenuates heart failure induced by a combination of hyperlipidemia and myocardial infarction in rats via Treg immunomodulation and NLRP3 inflammasome inhibition

Abstract

Background and aim

Heart failure (HF) is a complex clinical syndrome that represents the end result of several pathophysiologic processes. Despite a dramatic evolution in diagnosis and management of HF, most patients eventually become resistant to therapy. Xin-Li Formula (XLF) is a Chinese medicine formula which shows great potential in the treatment of HF according to our previous studies. The present study was designed to investigate the effects of XLF on HF induced by a combination of hyperlipidemia and myocardial infarction (MI) in rats and reveal the underlying mechanism.

Experimental procedure

A rat model of HF induced by hyperlipidemia and MI was established with intragastric administration of XLF and Perindopril. In vitro, CD4⁺ T cells from mouse spleen and LPS/ATP-stimulated THP-1 macrophages were employed.

Results and conclusion

XLF was shown to have markedly protective effects on MI-induced HF with hyperlipidemia in rats, including improvement of left ventricular function, reduction of left ventricular fibrosis and infarct size. Moreover, XLF administration significantly increased the number of Foxp3⁺ Tregs, and inhibited mTOR phosphorylation and NLRP3 signaling pathway. In vitro, we found that XLF had induced Treg activation via the inhibition of mTOR phosphorylation in CD4⁺ T cells. Additionally, XLF inhibited NLRP3 inflammasome activation in LPS/ATP-stimulated THP-1 macrophages. Taken together, this study raises the exciting possibility that Xin-Li Formula may benefit HF patients due to its immunomodulatory and anti-inflammatory effects via Treg activation and NLRP3 inflammasome inhibition.

Graphical abstract

Highlights

• •Xin-Li Formula (XLF) has markedly protective effects on heart failure induced by MI and hyperlipidemia in rat.
• •Treg activation and NLRP3 inflammasome inhibition play important roles in XLF-induced improvement of heart failure.
• •XLF may have potential application in prevention of immune and inflammation associated cardiovascular disease.

List of abbreviations

ACEIs

angiotensin-converting enzyme inhibitors

ARBs

angiotensin receptor blockers

CAD

coronary artery disease

CRP

C-reactive protein

CVDs

cardiovascular diseases

FOXP3

forkhead box protein P3

HF

heart failure

LADCA

left anterior descending coronary artery

LN

lymph node

LPS

lipopolysaccharide

LVEDD

left ventricular end-diastolic diameter

LVEDV

left ventricular end-diastolic volume

LVEF

left ventricular ejection fraction

LVESD

left ventricular end-systolic diameter

LVESV

left ventricular end-systolic volume

LVFS

left ventricular fractional shortening

MI

myocardial infarction

mTOR

mechanistic target of rapamycin

mTORC1

mTOR Complex 1

mTORC2

mTOR Complex 2

NLRP3

Nod-like receptor family pyrin domain-containing 3

NT-proBNP

N-terminal pro-brain natriuretic peptide

PMA

phorbol 12-myristate 13-acetate

PVDF

polyvinylidene fluoride

TCM

Traditional Chinese Medicine

Tregs

regulatory T cells

XLF

Xin-Li Formula

1. Introduction

Heart failure (HF) is the end-stage of various cardiovascular diseases that result from any structural or functional impairment of ventricular filling or ejection of blood. It has become a major public health problem around the world.¹ Epidemiologic data suggest that 1.3% (an estimated 13.7 million people) of the Chinese adult population aged ≥35 years had HF,² and the mortality rate in 5 years is 50%, with high rates of sudden death.³ Growing evidence has revealed that the immune system plays an important role in the pathogenesis of heart failure.⁴ Regulatory T cells (Tregs), a small subset of immune cells, participate in the regulation of immune response by secreting immunoregulatory cytokines, such as IL-10 and TGF-β, and suppressing the function immune effector cells.⁵ Impaired homeostasis of Tregs can trigger abnormal immune responses and lead to chronic diseases including heart failure.⁶ The depletion of Tregs aggravates cardiac inflammation and dysfunction, whereas their expansion or adoptive transfer has protective effects on post-MI heart failure.⁷ A growing body of studies has revealed the role of mTOR signaling pathway in regulating the differentiation and function of Treg cells. Inhibition of mTOR induces lineage marker forkhead box protein P3 (FOXP3) expression in naive T cells and drives the expansion of functional Tregs.⁸ Also, nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays a crucial role in innate immunity and inflammation, triggering the activation of caspase-1 and cleavage of pro-inflammatory cytokines IL-1β and IL-18.⁹ It has been reported that NLRP3 inflammasome activation contribute to development and prognosis of HF involved with cardiac remodeling and fibrosis.¹⁰ Therefore, targeting Tregs and NLRP3 inflammasome may be important strategies to explore anti-inflammatory and immunomodulatory therapy for HF.

Currently, adrenaline b-receptor blockers, angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) are first-line medication in treating HF. However, unsatisfactory side-effects have been found during long-term treatment, and mortality and morbidity associated with heart failure remains high. Hence, there are increasing calls for further exploration of pathophysiological mechanisms and effective treatment strategies for heart failure. Traditional Chinese Medicine (TCM) is becoming increasingly popular in Western countries for its supposed tonic effect and potential curative properties. TCM syndrome differentiation theory regards “Deficiency of Yin and Yang (陰陽兩虛 yīn yáng liǎng xū)” and “phlegm-blood stasis (痰瘀 tán yū)” as the crucial causes for heart failure. Therefore, “dispelling dampness and phlegm (祛濕化痰 qù shī huà tán), tonifying Qi (益氣 yì qì) and activating blood (活血 huò xuě)” has been a recommended therapeutic strategy to cope with heart failure. Xin-Li Formula (心力方 xīn lì fāng) is a Chinese medicine formula created by Academician Chen Ke-ji who is a well-known traditional Chinese medicine practitioner. The primary ingredients in the formula are Plantaginis Herba (車前草 chē qián cǎo, the whole plant of Plantago asiatica L.), Curcumae Rhizoma (莪術 é zhú, the dry rhizomes of Curcuma phaeocaulis Val.), Ginseng Radix et Rhizoma Rubra (紅參 hòng shēn, the dry roots and rhizomes of Panax ginseng C. A. Mey after steaming), Astragali Radix (黃芪 huáng qí, the dry roots of Astragalus membranaceus (Fisch.) Bge.), Astragali Radix Praeparata Cum Melle (炙黃芪 zhì huáng qí, the dry roots of Astragalus membranaceus (Fisch.) Bge. after processing) and Corni Fructus (山茱萸 shān zhū yú, the dry fruits of Cornus officinalis Sieb. et Zucc) in line with the standards of Chinese Pharmacopoeia 151 WS1-160(Z-150)-99(Z). Xin-Li Formula (XLF) was formulated according to TCM syndrome differentiation for HF known as “Deficiency of Yin and Yang” and “phlegm-blood stasis”. Based on the TCM compatibility theory “Jun-Chen-Zuo-Shi (君臣佐使 Jūn Chén-Zuǒ-Shǐ”, Plantaginis Herba acts as the sovereign (Jun) with the function of dispelling dampness and phlegm, Curcumae Rhizoma and Ginseng Radix et Rhizoma Rubra serve as the minister (Chen) with the effect of activating blood, removing stasis and tonifying Qi, Astragali Radix, Astragali Radix Praeparata Cum Melle and Corni Fructus serve as the assistant (Zuo) and the courier (Shi) with the function of tonifying Qi.

Our previous clinical study showed a significant decrease in serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels of HF patients after oral administration of XLF for 4 weeks, without obvious adverse effects (data have not been published). Modern pharmacological studies have demonstrated the protective effects of Plantago asiatica L against cardiomyocyte injury¹¹ and hyperlipidemia.¹² Plantamajoside and verbascoside are the main bioactive compounds of Plantago asiatica L. A previous study revealed the anti-inflammatory effect of plantamajoside via suppressing NF-κB and MAPK activation.¹³ Additionally, verbascoside has been shown to inhibit the progression of atherosclerosis in rat models.¹⁴ Panax ginseng C. A. Mey and Astragalus membranaceus (Fisch.) Bge., as well as their active compounds (such as ginsenoside and astragaloside) are often used for treating HF with the advantages of being multitarget and affordable.15, 16, 17 In addition, the pharmacodynamics material basis for activating blood and removing stasis of Curcuma phaeocaulis Val. has been identified by spectrum-effect relationship analysis and network pharmacology.¹⁸ These results indicated that XLF is a clinically effective herb formula for treating HF. Although XLF shows great potential in treating HF, the underlying mechanism of XLF has yet to be elucidated.

2. Materials and methods

2.1. Materials

Curcumae Rhizoma (No. 210501591), Ginseng Radix et Rhizoma Rubra (No. 200401), Astragali Radix (No. 210500571), Astragali Radix Praeparata Cum Melle (No. 210600421) and Corni Fructus (No. 210502031) were purchased from Kangmei Pharmaceutical Co. Ltd (Guangdong, China). Plantaginis Herba (No. 210102) was purchased from Zisun Pharmaceutical Co. Ltd (Guangdong, China). Identification and authentication of the herb materials were carried by Prof. Yong Li from Guangdong Provincial Key Laboratory of Advanced Drug Delivery Systems, Guangdong Pharmaceutical University. Perindopril was purchased from Servier (Tianjin) Pharmaceutical Co. Ltd (Tianjin, China). Sodium deoxycholate and propylthiouracil were purchased from Dalian Meilun Biological Technology Co. Ltd (Dalian, China). Cholesterol and 1–2 propanediol were purchased from Shanghai Macklin Biochemical Co.Ltd (Shanghai, China). CD4 (L3T4) MicroBeads (130-117-043) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). RPMI-1640 medium, fetal bovine serum, penicillin and streptomycin were purchasedfrom Gibco (Invitrogen, Carlsbad, CA, USA). PMA/Ionomycin Mixture was purchased from MultiSciences (Lianke) Biotech (Hangzhou, China). IL-2 was purchased from Peprotech (Rocky Hill, NJ, USA). Anti-CD4 (FITC, 11-0040-82), anti-CD25 (PE, 12-0390-82), anti-CD25 (PE-Cyanine 7, 25-0251-82), anti-Foxp3 (APC, 17-5773-82) and Foxp3/transcription factor staining buffer set (00-5523-00) were purchased from eBioscience (San Diego, CA, USA). IL-1β and IL-18 ELISA Kit were purchased from R&D Systems (Minneapolis, MN, USA). N-terminal pro-brain natriuretic peptide (NT-proBNP) and C-reactive protein (CRP) ELISA Kits were purchased from Cusabio (Wuhan, China). FastPure Cell/Tissue Total RNA Isolation Kit was purchased from Vazyme Biotech Co. Ltd (Nanjing, China). DAB chromogenic reagent kit was purchased from Maxim Biological technology development co., LTD (Fuzhou, China). MCC950 was purchased from MedChem Express (Monmouth Junction, NJ, USA). Lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA) and ATP were purchased from Sigma (St Louis, MO, USA). Foxp3 antibody (ab215206) was purchased from Abcam (Cambridge, MA, USA). Antibodies against NLRP3 (NBP2-12446), caspase-1(NBP100-56565) and IL-1β (NB600-633) were purchased from Novus (Centennial, CO, USA). Primary antibodies against mTOR (2972), phosphor-mTOR (2971), GAPDH, and secondary antibody HRP-anti-Mouse IgG (7076), HRP-anti-rabbit IgG (7074) were purchased from Cell Signaling Technology (Boston, MASS, USA).

2.2. Preparation of Xin-Li formula

XLF is composed of six Chinese medicinal herbs as shown in Appendix Table A.1. Decoction formulation XLF was prepared under standardized process. In brief, Plantaginis Herba, Curcumae Rhizoma, Ginseng Radix et Rhizoma Rubra, Astragali Radix, Astragali Radix Praeparata Cum Melle and Corni Fructus were mixed, immersed in 800 mL water for 30 min, then decoct for 30 min in the first time, collect the liquid to a clean measuring cup, decoct for 30 min with 600 mL water for the second time. Herbal decoction was evaporated to obtain condensed extract at a high concentration (2 g/mL) and a low concentration (1 g/mL).

2.3. Chromatographic analysis of Xin-Li formula

0.1 g of the XLF extract lyophilized powder was transferred into an Erlenmeyer flask and 5 mL of 70% methanol was added. After ultrasonic extraction at 40 kHz, 30 °C for 20 min, the extract was filtered through a 0.22 μm nylon membrane filter for UPLC analysis.

Chromatographic analysis was performed on a UPLC-Q-Exactive Orbitrap MS (Thermo Fisher Scientific, USA). The sample was separated on a Waters Acquity UPLC C18 column (2.1 mm × 100 mm × 1.7 μm). The mobile phase was consisted of acetonitrile (eluent A) and 0.1% formic acid in water (eluent B). A linear gradient elution was used according to the following profile: 0–2.5 min, 95% of B; 2.5–3 min, 95%–85% of B; 3–10 min, 85%–70% of B; 10–24 min, 70%–50% of B; 24–41 min, 50%–5% of B; 41–41.1 min, 5%–95% of B; 41.1–45 min, 95% of B. The flow rate was 0.3 mL/min, and the column temperature was maintained at 35 °C. 2 μL of sample solution was injected in each run.

The mass spectrometer equipped with an H-ESI source that operated in positive and negative ion mode. Using the Full MS/dd-MS2 as scanning mode, the resolution of Full MS and dd-MS2 were 70,000 and 17,500 respectively. The scanning range was from 100 to 1500 m/z. The sheath gas pressure was 40 arb, and nebulizer pressure was 10 arb. The spray voltage was set to 3.5 kV (+) and 3.0 kV (−). The temperature of capillary and auxiliary heating temperature was 325 °C and 350 °C. S-lens RF level was 50. Collision energy were 20, 40, 60 eV and 10, 25, 40 eV. (Considering the large difference in the collision energy required for each chemical component, two collision energies are used).

2.4. Animal model and grouping

Male Sprague-Dawley rats (specific-pathogen-free, weighing 250–280 g) were obtained from the Experimental Animal Center of Guangdong Province. All rats were housed under standard conditions with 12 h light/dark cycles and constant room temperature of 22 ± 2 °C and 60 ± 5%.

Before conducting myocardial infarction-induced heart failure model, rats were administrated Lipid emulsion (lard 100 g, cholesterol 40 g, propylthiouracil 4 g, Sodium deoxycholate 8 g, 1–2 propanediol 80 mL, Tween 80 100 mL, ddH₂O120mL) via oral gavage at a rate of 1 mL/100 g/day for 2 weeks. Myocardial infarction (MI) was established by ligating left anterior descending coronary artery (LADCA). Briefly, SD rats were anesthetized with 1.5%Pentobarbital sodium (75 mg/kg intraperitoneally) and LADCA of rat hearts were ligated at 2 mm below the edge of the left atrium. The area supplied by the LADCA turning bright red to pale and electrocardiogram change of elevated ST segment suggest that LADCA was successfully ligated. One week after MI, echocardiography was performed to measure left ventricular ejection fraction (LVEF). The rats with LVEF <45% were considered as heart failure rats and used for further experiments.

The heart failure rats (LVEF <45%) were randomized into Model group, Xin-Li Formula high-dose (XLF-H) group, Xin-Li Formula low-dose (XLF-L) group and Perindopril group (n = 10). Rats in Model group were treated with vehicle, while rats in XLF-H group and XLF-L group were respectively administrated with high-dose (20 g/kg/day, 1 mL/100 g with 2 g/mL XLF decoction) and low-dose (10 g/kg/day, 1 mL/100 g with 1 g/mL XLF decoction) oral gavage for 4 weeks. Perindopril (0.8 mg/kg/day) was administrated as positive control. Rats that underwent the same surgery but without ligating LADCA were used as sham group.

2.5. Cell culture and treatment

Male C57BL/6 mice (specific-pathogen-free, weighing 23–25 g) obtained from the Experimental Animal Center of Guangdong Province were used for CD4⁺ cells isolation. Total splenocytes were isolated from the spleen and red blood cell lysis was performed using buffer containing ammonium chloride. CD4⁺ cells were sorted out using mouse CD4 (L3T4) MicroBeads following the standard manufacturer protocol. The purity of the sorted T cells was routinely checked by flow cytometry and determined to be >90%. Then, CD4⁺ T cells were cultured in complete RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, and stimulated with PMA/Ionomycin Mixture (250X) and IL-2 (10 ng/mL) for 3 days. CD4⁺ T cells were treated with indicated concentrations of XLF or 60 μM 3BDO (mTOR activator) with XLF, or 100 nM rapamycin (mTOR inhibitor) for 72 h.

THP-1 cells were cultured in RPMI-1640 medium supplemented with PMA (160 nM) for 24 h to transform into adherent macrophages. The THP-1 derived macrophage were pretreated with indicated concentrations of XLF or vehicle for 24 h, and then stimulated by LPS (1 μg/mL) for 4 h. Medium was removed and replaced with serum free medium containing 10 μM MCC950 (NLRP3 inhibitor) for 1 h, and then stimulated by 10 mM ATP (NLRP3 activator) for 30 min.

2.6. Echocardiography

The echocardiography was performed one week after MI for grouping and 4 weeks after intragastric administration by using Visual Sonics Vevo 2100 system (Visualsonics Inc., Toronto, Canada). Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were measured, while left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated in parasternal short-axis view at the level of the papillary muscle by M-mode tracing method.

2.7. Histomorphology

Rat hearts were isolated, and cross section at the level of the papillary muscle were stored in 4% paraformal dehyde for more than 24 h and then embedded in paraffin wax. Tissue blocks were sectioned to 3.5 μm in thickness and stained with Masson's trichrome staining to assess left ventricular fibrosis and infarct size according to standard protocols. Infarct size was calculated as a percentage of total infarct circumference divided by total left ventricular circumferences. Peri-infarct interstitial fibrosis (% area) of rat heart was quantified in the LV border zone. Collagen (blue) staining threshold was initially adjusted so that all fibrotic areas were captured, and tissue fibrosis was then determined from 4 to 6 high-power fields per section, expressed as a percentage of the blue area to the total tissue area.

2.8. Immunohistochemistry

The sections of heart tissues were dewaxed and antigen retrieved in citrate buffer (pH = 6.0). After blocked with 3% H₂O₂ and 5% bovine serum albumin, sections were incubated with primary anti-Foxp3 antibody at 4 °C overnight, then incubated with secondary antibody HRP-anti-Mouse IgG at room temperature for 30 min. A DAB chromogenic reagent kit was used for coloration. The stained sections were observed under microscope and the densities of Foxp3 were measured by using Image J.

2.9. Flow cytometry

Draining lymph node (LN) and spleen cells were harvested and stained with anti-CD4-FITC and anti-CD25-PE antibodies in the dark for 30 min. The CD4⁺ T cells from mouse spleen were collected and stained with anti- CD25-PE-Cyanine 7 antibodies in the dark for 30 min. After being treated with Foxp3/transcription factor fixation/permeabilization at room temperature for 30 min, the cells were further stained with anti-Foxp3-APC antibody for 30 min. The stained cells were washed and finally analyzed using a flow cytometer (FACS Calibur, BD Biosciences).

2.10. Western blotting

The protein extractions were obtained from cells and frozen cardiac tissues and the protein concentrations detected by BCA Protein Assay Kit and subjected to Western blot analysis. The protein was then separated by 12% SDS-PAGE gels electrophoresis and electro-transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% nonfat milk for 1.5 h at room temperature and then incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibody at room temperature for 1.5 h with gentle rotation. The blots were detected by enhanced chemiluminescence (ECL) and quantification of band intensity was performed using BioRad Image Lab Software.

Primary antibodies used were:NLRP3 (1:3500 dilution), caspase-1 (1:2000 dilution), phosphor-mTOR (1:1000 dilution), mTOR (1:1000 dilution), Foxp3 (1:1000 dilution), IL-1β (1:1000 dilution) and GAPDH (1:10000 dilution).

2.11. Quantitative real-time PCR analysis

Total RNA was extracted from rat heart tissues by FastPure Cell/Tissue Total RNA Isolation Kit. The reverse transcription step involved use of oligo(dT) primers, and then underwent quantitative real-time PCR with the primer pair sequences for Foxp3, forward, 5′-TCACACGCATGTTCGCCTACTTC-3′, and reverse, 5′-CTCACTCTCCACTCGCACAAAGC-3′; and GAPDH, forward, 5′-GACATGCCGCCTGGAGAAAC-3′ and reverse, 5′-AGCCCAGGATGCCCTTTAGT-3′, as a housekeeping gene. Relative gene expression was normalized to GAPDH levels. Quantitative PCR (qPCR) reactions involved use of ChamQ Universal SYBR qPCR Master Mix and CFX Manager 2.1 system (Bio-rad). The fold change in RNA level was calculated by the 2^−ΔΔCt method.

2.12. Statistical analysis

Data are presented as the mean ± S.D. Statistical comparison among normally distributed variables in multiple groups was performed using one-way ANOVA followed by LSD test (Equal Variance assumed) or Dunnett's T3 test (Equal Variance not assumed). SPSS statistical software version 19.0 was used for all data analysis. A value of P < 0.05 was considered statistically significant.

3. Results

3.1. Chemical profile of XLF by UPLC-Q-Exactive Orbitrap MS

A total of 73 components from all six composed herbal medicines were characterized by UPLC-Q-Exactive Orbitrap MS analysis, including 8 components from Plantaginis Herba, 4 components from Curcumae Rhizoma, 22 components from Ginseng Radix et Rhizoma Rubra, 23 components from Astragali Radixand Astragali Radix Praeparata Cum Melle, 4 components from Corni Fructus, and 13 unknown components (Fig. 1 & Appendix Table A.2).

Fig. 1.

Base peak intensity (BPI) chromatograms of XLF in the positive (A) and negative ion mode (B).

3.2. XLF improves left ventricular function in HF rats induced by hyperlipidemia and MI

The echocardiography was performed 4 weeks after intragastric administration of XLF. As shown in Fig. 2, rats in the model group showed a significant reduction in EF and FS value, while an elevation in LVESV, LVEDV, LVESD and LVEDD compared with the sham group (P < 0.01). The EF and FS values significantly increased, while LVESV and LVESD significantly decreased in the XLF-H, XLF-L and Perindopril groups compared with the model group (P < 0.01). Additionally, compared with the model rats, a significant reduction of LVEDV and LVEDD was found in Perindopril group. LVEDV and LVEDD also decreased in XLF-H, XLF-L groups but without a statistical difference. These results suggest that similar to perindopril, XLF improves left ventricular function in HF rats induced by hyperlipidemia and MI, and high doses of XLF are more effective than low doses.

Fig. 2.

Left ventricular function. Left ventricular function was measured by echocardiography. Representative M-mode echocardiograms are shown in (A). (B–G) LVEF, LVFS, LVESV, LVESD, LVEDV, LVEDD are shown as mean ± S.D (n = 6). ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group. ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

3.3. XLF reduces left ventricular fibrosis, infarct size and cardiac index in HF rats induced by hyperlipidemia and MI

Masson's trichrome staining was used to assess left ventricular fibrosis and infarct size. As shown in Fig. 3A–D, there was a significant larger infarct in the model group compared with the sham rats. Cardiac peri-infarct zone interstitial fibrosis in the model group was also markedly increased compared with the sham rats. XLF and perindopril treatment reduced left ventricular fibrosis and infarct size. Additionally, cardiac index for each rat was calculated using the following formula: heart weight/bodyweight × 100%. As shown in Fig. 3E, rats in the model group developed a significant increase in cardiac index compared with the sham rats (P < 0.01). Cardiac index in the XLF-H and Perindopril groups significantly declined compared with the model group, while there were no significant differences between the XLF-L group and the model group (P > 0.05). These results suggest that similar to perindopril, XLF reduces the left ventricular fibrosis, infarct size and cardiac index in HF rats induced by hyperlipidemia and MI, and high doses of XLF produce better effects than low doses of XLF.

Fig. 3.

Left ventricular fibrosis, infarct size and cardiac index. Left ventricular fibrosis and infarct size were assessed by Masson staining. Representative images are shown in (A & B). Scale bar = 50 μm in (A). (C–D) The percentages of infarct size and quantitation of peri-infarct interstitial fibrosis (% area) are shown as mean ± S.D (n = 5). (E) Cardiac index for each rat was calculated using the following formula: heart weight/bodyweight × 100%. Data are shown as mean ± S.D. (n = 5–6). ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

3.4. XLF increases the number of CD25+Foxp3+ Tregs in lymph nodes and spleen cells

Draining lymph node and spleen cells were isolated after echocardiography measurement, and CD4⁺CD25⁺Foxp3⁺ Tregs were detected using flow cytometric analysis. As shown in Fig. 4, rats in the model group showed a significant reduction of CD4⁺CD25⁺Foxp3⁺ Tregs numbers in draining lymph nodes and spleens compared with the sham group (P < 0.01). The numbers of CD4⁺CD25⁺Foxp3⁺ Tregs in draining lymph nodes and spleens significantly increased in the XLF-H groups (P < 0.01), while there was mild increase in the Perindopril group (P > 0.05) compared with the model group. These results showed that XLF increases the number of CD25+Foxp3⁺ Tregs in lymph nodes and spleen cells from HF rats induced by hyperlipidemia and MI, and XLF had better effects on Treg activation than perindopril.

Fig. 4.

CD4⁺CD25⁺Foxp3⁺ Tregs frequencies in lymph node and spleen cells. Draining lymph node and spleen cells were isolated and CD4⁺CD25⁺Foxp3⁺ Tregs were detected using flow cytometric analysis. Representative images are shown in (A). (B–C) The percentages of CD4⁺CD25⁺Foxp3⁺ Tregs in LN and spleen are shown as mean ± S.D (n = 6). ∗P < 0.05 versus Sham group, ##P < 0.01 versus Model group.

3.5. XLF inhibits mTOR phosphorylation and activates Foxp3 expression in HF rats induced by hyperlipidemia and MI

To examine the involvement of mTOR/Treg axis in the effect of XLF on HF induced by hyperlipidemia and MI, the protein expression levels of mTOR and phosphor-mTOR were analyzed by Western blotting, and Foxp3 expression was analyzed by qPCR, Western blotting and immunohistochemistry. As shown in Fig. 5A&B, MI resulted in significant activation of mTOR phosphorylation compared with the sham rats; XLF and perindopril treatment reversed the activation of mTOR phosphorylation without altering the protein expression of total mTOR. Quantitative assessment of Foxp3 protein expression by Western blotting and Foxp3 distribution by immunohistochemistry both showed there was a significant up-regulation of Foxp3 expression in the model group, and further up-regulation was found in the XLF groups (Fig. 5A, C&D, E). Similar results were also found for mRNA level with further up-regulation of Foxp3 in both the XLF and the Perindopril groups (Fig. 5F). These results indicated that both the inhibition of mTOR phosphorylation and the activation of Foxp3 expression, which play important roles in Treg immunomodulation, were involved in the effect of XLF on preventing MI-induced HF with hyperlipidemia.

Fig. 5.

The expressions of mTOR, p-mTOR and Foxp3 in rat hearts. (A)The protein expressions of mTOR, p-mTOR and Foxp3 in heart tissues were determined by Western blot analysis. p-mTOR/mTOR ratio and Foxp3/GAPDH ratio were quantified as shown in (B) and (C). The distribution of Foxp3 in heart tissues were also assessed by immunohistochemistry (scale bar = 50 μm). Representative images are shown in (D) and quantitative assessment of Foxp3 positive areas are shown in (E). (F) The expressions of Foxp3 in mRNA level were analyzed by qPCR. Measurements were performed in three different samples and data are shown as mean ± S.D. ∗P < 0.05 versus Sham group, ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

3.6. XLF inhibits NLRP3 signaling pathway in HF rats induced by hyperlipidemia and MI

To examine the involvement of NLRP3 signaling pathway in the effect of XLF on HF induced by hyperlipidemia and MI, the protein expression levels of NLRP3, pro-caspase-1, caspase-1, pro-IL-1β and IL-1β were analyzed by Western blotting. The levels of IL-1β in the serum were also detected using ELISA kits. As shown in Fig. 6A–F, MI resulted in significant up-regulation of NLRP3, pro-caspase-1, caspase-1, pro-IL-1β and IL-1β, each of which was reversed by XLF and perindopril treatment. In addition, the serum IL-1β levels of model rats increased compared with the sham rats, whereas those of rats in the XLF and Perindopril groups declined compared with the model group (Fig. 6G). These results indicated that the inhibition of NLRP3 signaling pathway was involved in the effect of XLF on HF rats induced by hyperlipidemia and MI.

Fig. 6.

The protein expressions of NLRP3/caspase-1/IL-1β signaling pathway in rat hearts. (A) The protein expressions of NLRP3 and caspase-1 in heart tissues were determined by Western blot analysis. NLRP3/GAPDH ratio, pro-caspase-1/GAPDH ratio, caspase-1/GAPDH ratio, pro-IL-1β/GAPDH ratio and IL-1β/GAPDH ratio were quantified as shown in (B–F). Measurements were performed in three different samples and data are shown as mean ± S.D. (G) Serums were collected to evaluate the level of IL-1β. Data are shown as mean ± S.D (n = 6). ∗P < 0.05 versus Sham group, ∗∗P < 0.01 versus Sham group, #P < 0.05 versus Model group, ##P < 0.01 versus Model group.

3.7. XLF increases the number of CD25+Foxp3⁺ Tregs and inhibits mTOR phosphorylation in CD4⁺ T cells

Cell viability was monitored by CCK-8 assay. As shown in Fig. 7A, XLF has no obvious cytotoxicity with concentrations lower than 1 mg/mL in CD4⁺ T cells. To determine the effects of XLF on Treg regulation in vitro, CD4⁺ T cells from mouse spleen were treated with indicated concentrations of XLF and CD25⁺Foxp3⁺ Tregs were detected using flow cytometric analysis. As shown in Fig. 7B&C, XLF significantly increases the number of CD25⁺Foxp3⁺ Tregs at a dose of 100 μg/mL. Moreover, to confirm the role of mTOR in activation effect of XLF on Treg regulation, CD4⁺ T cells were treated with 100 μg/mL XLF, or 60 μM3BDO (mTOR activator) with XLF, or 100 nM rapamycin (mTOR inhibitor). Then, CD25⁺Foxp3⁺ Tregs were detected using flow cytometric analysis and the protein expression levels of mTOR and phosphor-mTOR were analyzed by Western blotting. The results showed that XLF-induced Treg up-regulation was abolished by mTOR activator 3BDO, and the inhibition of mTOR with rapamycin significantly increased the number of CD25⁺Foxp3⁺ Tregs (Fig. 7B&C). Additionally, XLF and rapamycin significantly inhibited mTOR phosphorylation, and XLF-induced inhibition of mTOR phosphorylation was abolished by mTOR activator 3BDO (Fig. 7D&E). These results indicated that the Treg activation via inhibition of mTOR phosphorylation was involved in XLF-induced immunomodulation in the CD4⁺ T cells.

Fig. 7.

CD25⁺Foxp3⁺ Tregs frequencies and mTOR phosphorylation in CD4⁺ T cells. (A) CD4⁺ T cells were treated with indicated concentrations of XLF for 72 h, and cell viability was analyzed by CCK-8 kit. (B) CD25⁺Foxp3⁺ Tregs were detected using flow cytometric analysis. (C) The percentages of CD25⁺Foxp3⁺ Tregs in CD4⁺ T cells are shown as mean ± S.D. (D) The protein expressions of mTOR and p-mTOR in CD4⁺ T cells were determined by Western blot analysis. p-mTOR/mTOR ratio was quantified as shown in (E). Data were presented as mean ± SD (n = 3). ∗∗P < 0.01 versus untreated control, ##P < 0.01 versus XLF treatment.

3.8. XLF inhibits NLRP3 inflammasome activation in LPS/ATP-stimulated THP-1 macrophages

Cell viability was monitored by CCK-8 assay. As shown in Fig. 8A, XLF has no obvious cytotoxicity with concentrations lower than 1 mg/mL in THP-1 macrophages. To further determine the inhibitory effect of XLF on NLRP3 inflammasome activation in vitro, LPS/ATP-stimulated THP-1 macrophages were treated with indicated concentrations of XLF with or without MCC950 (NLRP3 inhibitor). The IL-1β and IL-18 levels in the supernatant were detected with ELISA Kits, and the NLRP3 protein expression level was analyzed by Western blotting. As shown in Fig. 8B&C, LPS/ATP stimulation increased the level of IL-1β and IL-18, which were reversed by either XLF (100 μg/mL) or MCC950 administration. Co-treatment with XLF and MCC950 had superior inhibitory effects. Similar results were also found in the protein expression level for NLRP3. As shown in Fig. 8D&E, the protein expression levels of NLRP3 were increased in the LPS/ATP stimulated-macrophages, while both XLF and MCC950 administration reversed the LPS/ATP-induced NLRP3 inflammasome activation. These results indicated that the inhibition of NLRP3 inflammasome activation was involved in the protective effect of XLF on LPS-induced inflammation.

Fig. 8.

IL-1β and IL-18 levels and NLRP3 expression in LPS/ATP-induced THP-1 macrophages. (A) THP-1 macrophages were treated with indicated concentrations of XLF for 24 h, and cell viability was analyzed by CCK-8. (B–C) The supernatants of THP-1 macrophages were collected to evaluate the level of IL-1β and IL-18 after indicated treatment. (D) The protein expression levels of NLRP3 in THP-1 macrophages were analyzed by Western blotting. NLRP3/GAPDH ratio was quantified as shown in (E). Data were presented as mean ± SD (n = 3). ∗∗P < 0.01 versus untreated control, ^P < 0.05 versus LPS/ATP, ^^ P < 0.01 versus LPS/ATP, #P < 0.05 versus XLF treatment.

4. Discussion

A myocardial infarction (MI)-induced HF rat model which has been widely accepted by researchers in the field is characterized by left ventricular myocardial dysfunction including reduced EF and FS, elevated LVESD and LVEDD. Phlegm-blood stasis syndrome, which is characterized by combination of phlegm turbidity and blood stasis, is the core pathogenesis dynamically throughout the occurrence, development, and prognosis of HF. Phlegm turbidity, one of the primary syndromes of HF, is characterized as hyperlipidemia often caused by obesity and a high-fat diet. Blood stasis, another primary syndrome of HF, is characterized by poor blood flow and blocked arteries, and is common in patients with thrombotic and vascular diseases. In the present study, a rat model of HF induced by hyperlipidemia and MI was introduced by ligating LADCA and oral administration of lipid emulsion to simulate the TCM syndrome of “phlegm-blood stasis” in HF. The results showed significant reductions in EF and FS values of HF rats induced by hyperlipidemia and MI compared with the sham rats and with the MI-induced HF rats (data shown in Appendix Fig. A.1). More severe fibrosis and larger infarcts were also found in HF rats induced by hyperlipidemia and MI compared with sham rats and the MI-induced HF rats (data shown in Appendix Fig. A.2). Moreover, the levels of TC, TG, LDL-C, NT-proBNP and C-reactive protein (CRP) in the serum of HF rats induced by hyperlipidemia and MI were significantly higher than those of sham rats and the MI-induced HF rats (data shown in Appendix Fig. A.3). These findings suggest that lipid metabolism disorder caused by hyperlipidemia promoted MI-induced myocardial injury leading to more severe LV dysfunction and fibrosis. The rat model of HF induced by hyperlipidemia and MI, which is different from the known HF model, was successfully established in current study, and well simulated the characteristics of TCM syndrome in HF.

In recent years, Traditional Chinese Medicine (TCM) has become increasingly popular worldwide due to its advantages in promoting the functional recovery of various organs, reducing adverse reactions, and improving the quality of life of patients. As such, several Chinese herbal medicines have been successfully applied to HF prevention and treatment.^19,20 Xin-Li Formula (XLF), a Chinese medicine formula created by a well-known Academician Chen Ke-ji, was formulated according to TCM syndrome differentiation of HF known as “Deficiency of Yin and Yang” and “phlegm-blood stasis”. Our present study has shown that XLF has protective effects on HF rats induced by hyperlipidemia and MI, including the improvement of left ventricular function, reduction of left ventricular fibrosis, infarct size and cardiac index. Along with positive results from our previous clinical study that XLF lowers the NT-proBNP levels of HF patients, we may consider XLF a beneficial therapy for HF patients, especially those with diagnosis of “phlegm-blood stasis” in TCM.

It is generally accepted that inappropriately sustained inflammation with the loss of normal immunosuppression is involved in the pathogenesis of heart failure. Tregs, which can be identified by the lineage marker forkhead box protein P3 (FOXP3), contribute to suppressing excessive immune activation and maintain immune homeostasis.²¹ Emerging evidence indicates that Tregs play critical roles in immune suppression that impart beneficial effects in HF. Moreover, clinical studies has demonstrated reduced peripheral blood Treg-cells in patients with chronic heart failure,²² and low levels of circulating Tregs can be used to predict the development of acute cardiovascular events in humans.²³ Previous murine studies have revealed that Tregs are activated after myocardial infarction, followed by limiting inflammation and modulating macrophage phenotype, leading to facilitate cardiac wound healing and remodeling.^24,25 In other mouse models of ischemic HF, the expanded Tregs have been found to be dysfunctional which resulted in promoting immune activation and left ventricular remodeling.^26,27 By using flow cytometry, Western blot, qPCR and immunohistochemistry, we found that the numbers of naturally occurring CD4⁺CD25⁺Foxp3⁺ Tregs in lymph node and spleen cells from post-MI HF rats declined significantly, while the protein and mRNA expressions of Foxp3 in the heart tissue were up-regulated. Our results are consistent with the findings of previous reports that Tregs play crucial roles in the pathogenesis of MI-induced HF. Given that, the restoration of normal Treg function may be a potential solution for therapeutic immunomodulation in HF.

Recent studies have confirmed that Tregs reconstituted after ablation exhibit restoration of immunosuppressive capacity and reversed LV remodeling and dysfunction.²⁸ The IL-2/anti-IL-2 complex (IL-2C), a mediator of Treg expansion, has been reported to attenuate adverse remodeling after MI by directly inhibiting the infiltration of inflammatory macrophages and facilitating the polarization of anti-inflammatory M2 macrophages.^29,30 The expansion of Tregs by adoptive transfer or a CD28 super agonistic antibody (JJ316) led to an increased Foxp3⁺ Tregs numbers in the infarcted heart, and improved cardiac function in post-MI rats.³¹ In our current study, the intervention of XLF significantly increased the numbers of CD4⁺CD25⁺Foxp3⁺ Tregs in draining lymph nodes and spleen from post-MI HF rats, and it had better effects than perindopril, which is used for the conventional therapy of HF. Moreover, up-regulations of Foxp3 protein expression as well as mRNA expression in the heart were found in XLF- and Perindopril-treated groups compared with HF rats. We further confirmed the effects of XLF on Treg up-regulation in vitro by using CD4⁺ T cells from mouse spleens. All of these results indicated that Treg activation plays an important role in the therapeutic effects of XLF on myocardial infarction-induced heart failure.

Although Treg-targeted therapies might provide a promising approach to the prevention and treatment of HF, the underlying mechanisms are still to be elucidated. mTOR (mechanistic target of rapamycin), a conserved serine/threonine kinase, is firstly identified as a direct target of rapamycin in 1994.³² mTOR integrates signals that coordinate cell growth with environmental conditions and is important in cell and organismal physiology.^33,34 mTOR Complex 1 (mTORC1) and 2 (mTORC2) are the two catalytic subunits of mTOR. T cells rely on mTOR activity for their homeostasis and functional fitness.³⁵ The inhibition of mTOR signaling increases in Treg generation, while mTORC1 plays a critical role in the development of Tregs’ suppressive activity.³⁶ Rapamycin, the first selective inhibitor of mTOR, has been revealed to decrease the production of effector T cells and increased the generation of Tregs.³⁷ In previous clinical studies, rapamycin has been shown to promote the expansion of functional CD4⁺CD25⁺Foxp3⁺ Tregs in type 1 diabetic patients and kidney transplant recipients.^38,39 In addition, azithromycin (a bacteriostatic macrolide with mTOR inhibitory activity) and rapamycin have both been reported to benefit the expansion of Foxp3⁺ Tregs in vitro.⁴⁰ In the present study, we found that MI resulted in a significant activation of mTOR phosphorylation compared with sham rats. XLF and perindopril treatment reversed the activation of mTOR phosphorylation without altering the protein expression of total mTOR. In vitro, we found that XLF-induced Treg up-regulation was abolished by mTOR activator 3BDO, and mTOR inhibition with rapamycin significantly increased the number of CD25⁺Foxp3⁺ Tregs. Additionally, XLF and rapamycin significantly inhibited mTOR phosphorylation without affecting total mTOR expression in CD4⁺ T cells. These results provide further evidence that Treg immunomodulation-based therapies by targeting mTOR may be a viable approach to XLF in preventing myocardial infarction-induced heart failure. Despite these extensive advances, it remains a major challenge for us to translate the understanding of mTOR in immune cells into treatment for human diseases.

In addition to Treg immunomodulation, the central role of inflammation in the development and progression of heart failure has been well established.⁴¹ Elevated levels of plasma pro-inflammatory biomarkers have been observed in HF patients.⁴² NLRP3, which are classified into the nod-like receptor (NLR) family, recruit the oligomerization of a common adaptor protein ASC to activate pro-inflammatory caspase-1, and have been implicated in the pathogenesis of various inflammatory diseases.⁴³ Caspase-1 cleaves pro–IL-1β and pro–IL-18 into their mature and active forms followed by releasing the pro-inflammatory factor IL-1β and IL-18. A growing body of evidence highlights the relevance of NLRP3 inflammasome in the pathogenesis of HF.⁴⁴ The pharmacological blockade of NLRP3 pathways with drugs currently employed in the clinical practice, as well as phytochemicals, exert beneficial effects in both patients with HF^45,46 and animal models of MI.^47,48 All of these studies indicate that the inhibition of NLRP3 pathway could be a promising therapeutic approach for the prevention and management of HF. Consistent with previous reports, our present study revealed an increase in serum IL-1β levels in post-MI HF rats compared with sham rats, while XLF and Perindopril administration reduced the level of IL-1β. In addition, the protein expressions of NLRP3, pro-caspase-1, caspase-1, pro-IL-1β and IL-1β in HF rat hearts were significantly up-regulated which were also reversed by XLF and perindopril treatment. To further explore the inhibitory effects of XLF on NLRP3 inflammasome activation, LPS/ATP-stimulated THP-1 macrophages were employed in the present study. Similar to the NLRP3 inhibitor MCC950, we found that XLF reduced the levels of IL-1β and IL-18 in the supernatant of LPS/ATP-stimulated THP-1 macrophages by inhibiting the protein expression of NLRP3 inflammasome. The results of our study raise the exciting possibility that we might be able to benefit from Xin-Li Formula due to its anti-inflammatory effects on HF by inhibiting NLRP3 pathway.

5. Conclusions

Our findings highlight the crucial roles of mTOR-induced Treg dysfunction and NLRP3 inflammasome-induced inflammation in the pathogenesis of heart failure induced by a combination of hyperlipidemia and MI. More importantly, our findings demonstrate the protective effects of Xin-Li Formula on HF due to its immunomodulatory and anti-inflammatory effects via Treg activation and NLRP3 inflammasome inhibition. Nonetheless, the effects of XLF on functional suppressive activity and adoptive transfer of Tregs in rats subjected to myocardial infarction with hyperlipidemia remain unknown. In addition, the upstream mechanism of NLRP3 and its relationship with Tregs are in need of further exploration.

Ethics statement

The animal experiment was approved by the Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese medicine. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.

Funding

The present work was supported by Guangzhou Basic and Applied Basic Research Foundation (Grant No. 2023A03J0742, 202201020348), National Natural Science Foundation of China (Grant No. 81874432 and 82074369), Science and Technology Planning Project of Guangdong Province (Grant No. 2018B030322012), and the Specific Fund of State Key Laboratory of Dampness Syndrome of Chinese Medicine (Grant No. SZ2021ZZ12, SZ2021ZZ26, SZ2021ZZ46).

CRediT authorship contribution statement

Taohua Lan: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft. Qiaohuang Zeng: Formal analysis, Investigation. Ying Zhu: Investigation. Guangjuan Zheng: Writing – review & editing. Keji Chen: Supervision. Wei Jiang: Writing – review & editing, Supervision, Funding acquisition. Weihui Lu: Writing – review & editing, Funding acquisition.

Declaration of competing interest

None.

Appendix A. Supplementary data

The following are the Supplementary data to this article.