Recovering intestinal redox homeostasis to resolve systemic inflammation for preventing remote myocardial injury by oral fullerenes

Significance

Immune disorder is the dominant feature of myocardial injury. Once MI or MI/RI occurs, the intestinal redox homeostasis and epithelial barrier integrity would be disrupted due to the hypoperfusion and unbalanced immune state of the intestine, which could further aggravate the systemic inflammation, in turn accelerating the progression of myocardial injury. Herein, with the aids of quantitative transcriptomics and proteomics, we proposed that OFNS (oral fullerene nanoscavenger) modulated intestinal redox homeostasis and strengthened intestinal barrier integrity to relieve systemic inflammation and immune disorder for remote cardioprotection. We applied our principle to intervene MI in mice and mini-pigs. OFNS present excellent effects on restraining the myocardial injury in MI mice and mini-pigs, the prospect of which in clinic is promising.

Abstract

The unbalanced immune state is the dominant feature of myocardial injury. However, the complicated pathology of cardiovascular diseases and the unique structure of cardiac tissue lead to challenges for effective immunoregulation therapy. Here, we exploited oral fullerene nanoscavenger (OFNS) to maintain intestinal redox homeostasis to resolve systemic inflammation for effectively preventing distal myocardial injury through bidirectional communication along the heart–gut immune axis. Observably, OFNS regulated redox microenvironment to repair cellular injury and reduce inflammation in vitro. Subsequently, OFNS prevented myocardial injury by regulating intestinal redox homeostasis and recovering epithelium barrier integrity in vivo. Based on the profiles of transcriptomics and proteomics, we demonstrated that OFNS balanced intestinal and systemic immune homeostasis for remote cardioprotection. Of note, we applied this principle to intervene myocardial infarction in mice and mini-pigs. These findings highlight that locally addressing intestinal redox to inhibit systemic inflammation could be a potent strategy for resolving remote tissue injury.

Cardiovascular disease is the leading cause of death worldwide (1). Specifically, myocardial infarction (MI) could cause serious myocardial ischemic necrosis due to coronary occlusion (2). The most effective treatment for MI in clinic is reperfusion, e.g., interventional or thrombolytic therapy (3–5). Whereas, these therapies could further induce myocardial ischemia/reperfusion injury (MI/RI) along with more severe myocardial injury. Consistently, the evident pathological feature of myocardial injury under both MI and MI/RI is increased excessive inflammation caused by immune dysfunction (6). However, the unique structure of cardiac tissue induces the restricted retention of intravenous deliveries of immunoregulation agents. Thus, it urgently needs to explore novel therapeutic agents for effective cardiovascular protection. Therefore, effectively regulating the state of myocardial immune homeostasis is urgent for cardioprotection.

Emerging evidences reveal that the intestinal mucosal immune system plays a crucial role in maintaining systemic immune homeostasis. In particular, the intestinal tissue has bidirectional communication with other distal tissues, such as the brain, liver, and heart (7–9). In case of MI or MI/RI, intestinal tissue is more susceptible to bear the brunt because it needs abundant blood supply to absorb nutrients and balance host defense (10–12). In addition, the intestinal redox homeostasis and epithelial barrier integrity would be disrupted, which could further aggravate the systemic inflammation, in turn accelerating the progression of myocardial injury (13–19). Recent researches reveal that regulating intestinal immunity or gut microbiota emerge on treating systemic inflammation-related diseases (20–23). In light of the bidirectional crosstalk between the heart and the intestine, we proposed that targeting intestinal homeostasis to regulate intestinal immunity would be an effective strategy to address systemic inflammation and potentially reverse distal myocardial injury.

Herein, we tailored an oral therapeutic system using fullerene-based reactive oxygen species (ROS) nanoscavenger (OFNS) that locally maintained intestinal redox homeostasis to reduce intestinal and systemic inflammation, thereby preventing against myocardial injury. Conventional attempts have been made to deliver fullerenes through intravenous or local injection (24, 25). However, the injected fullerenes are mostly captured by the reticuloendothelial system, resulting in poor retention in the heart (26–28). In this study, we explored a convenient and desirable oral administration with fullerenes. We demonstrated that OFNS could prominently prevent myocardial injury by sweeping away excessive intestinal ROS and reversing dysfunctional intestinal barrier in MI/RI mice. Further, we explored the quantitative transcriptomic and proteomic profiles to uncover the potential mechanism. It presented that OFNS potentiated complement and coagulation cascades and instigated intestinal immunity, resulting in resolving systemic inflammation and preventing myocardial injury. Subsequently, these results prompted us to investigate the protective role of OFNS in treating ischemic MI both in mice and in mini-pigs. Together, the oral fullerene therapeutic system represents a unique path to prevent the ischemic and reperfusion induced myocardial injury by locally maintaining intestinal redox homeostasis and ameliorating systemic inflammation.

Results

Preparation and Characterization of OFNS.

To be administrated orally, the OFNS were prepared by mixing C₆₀ powder with kinds of amphiphilic supplements including microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), hydroxy propyl cellulose (HPC), and other pharmaceutical excipients (silica gel, copovidone, magnesium stearate) by a mixer-granulator (Fig. 1A). Among them, C₆₀ is the active ingredient. MCC is used as an adsorbent, suspending agent, and bulking agent. CMC and HPC work as the disintegrating agent, which are also used to enhance the stability. Silica gel, copovidone, and magnesium stearate act as glidant, adhesion agent, and lubricating agent, respectively. The mixture was then compressed into tablets. They were further disintegrated into water to form a dispersion liquid for following use. To characterize the size of OFNS dispersion in water, we used environmental scanning electron microscopy. The results showed that the particle size of OFNS dispersion was 1.78 ± 0.35 μm in water (SI Appendix, Fig. S1). The elemental mapping by energy dispersive spectrometer suggested that the most abundant element was C (98.19%), then was O (1.79%) in OFNS (Fig. 1B). In addition, matrix-assisted laser desorption/ionizing time of flight mass spectrometry was applied to determine the type of fullerene, which is pristine C₆₀ in OFNS (SI Appendix, Fig. S2).

Fig. 1.

Preparation and characterization of OFNS in vitro. (A) Schematic diagram of OFNS synthesis. C₆₀ is the active ingredient; MCC is the bulking agent; PVP/VA is the adhesion agent; magnesium stearate acts as the lubricating agent; Silica gel is the glidant; HPC/CMC is the disintegrating agent. They were mixed and compressed into tablets and then disintegrated into OFNS. (B) Elemental mapping of OFNS by TEM. (Scale bar, 500 nm.) (C) The ESR spectra of •OH captured by DMPO after pretreated with H₂O₂ (100 mM) and treated with supplements (100 μg/mL) or OFNS (100 μg/mL). (D) The relative •OH scavenging activity of supplements or OFNS by the ESR spectra. (E) The relative intensity of DCF after treated with OFNS or traditional antioxidants (VC, NAC, and MT). “a.u.” is the abbreviation of “arbitrary units”. (F) The relative cell viability of IEC-6 cells after incubation with OFNS (0, 20, 40, 60, 80, and 100 μg/mL) for 24 h. (G) Relative cell viability of IEC-6 cells pretreated with H₂O₂ for 1 h and treated with different concentrations of OFNS (0, 10, 20, 40, 80, and 100 μg/mL) for 3 h. (H) The intracellular ROS levels of IEC-6 cells pretreated with H₂O₂ for 1 h and treated with/without OFNS for 3 h by FCM. (I) The protein expression of ZO-1 in IEC-6 cells pretreated with H₂O₂ and treated with/without OFNS. (J) Relative cell viability of HUVECs pretreated with H₂O₂ for 1 h and treated with different concentrations of OFNS (0, 20, 40, 80, and 100 μg/mL) for 3 h. (K) The intracellular ROS levels of HUVECs pretreated with H₂O₂ for 1 h and treated with/without OFNS (80 μg/mL) by FCM. (L) The intracellular ROS levels of HUVECs pretreated with H₂O₂ for 1 h and treated with/without OFNS (80 μg/mL) by confocal. The yellow arrow represents OFNS. (Scale bar, 20 μm.) (M) Schematic diagram of the effect of OFNS on RAW264.7 cells treated with LPS. (N) The intracellular ROS levels of RAW264.7 cells treated with LPS for 6 h and treated with/without OFNS (100 μg/mL) for 3 h by FCM. (O) The content of IL-6 and TNF-α in RAW264.7 cells’ supernatant by ELISA. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Considering that hydroxyl radical (•OH) is the most oxidizing free radical among ROS (29–31), we performed electron spin-resonance (ESR) spectrum to test the •OH scavenging capacity of OFNS. The results revealed that OFNS exhibited notable •OH clearance rate (49.63 ± 1.67%) due to the existence of fullerene, and the supplement was scarcely with any scavenging capacity of •OH (Fig. 1 C and D). To compare the antioxidative property of OFNS with the traditional anti-antioxidants, we used a commonly used ROS-sensitive probe, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) to detect the contents of ROS. It showed that OFNS owned prominent antioxidative ability than vitamin C (VC), N-Acetyl-L-cysteine (NAC), and melatonin (MT) at the same concentration of 500 μg/mL (Fig. 1E). All of those suggested that OFNS had a superior antioxidative capability.

We further investigated the regulation effects on cellular redox homeostasis in three kinds of cell models, including epithelial, endothelial, and inflammatory cells. For rat small intestinal crypt epithelial cells (IEC-6 cells), we first measured the cell internalization of OFNS using a cellular transmission electron microscope (TEM). It revealed that most of OFNS did not enter the cells, which mainly distributed in extracellular environment (SI Appendix, Fig. S3). Then, we detected the cytotoxicity of OFNS. Our data showed that the relative cell viability of IEC-6 cells was not observably reduced after incubation with OFNS up to 100 μg/mL (Fig. 1F). After that, we established an oxidative injury model induced by H₂O₂ in IEC-6 cells and found that OFNS could significantly increase the cell viability with a concentration-dependent manner (Fig. 1G). In addition, the intensity of 2′,7′-dichlorofluorescein (DCF) by flow cytometry (FCM) was reduced after OFNS treatment (Fig. 1H), suggesting that OFNS could regulate the intracelluar redox homeostasis via quenching extracellular excessive ROS. Given the important role of the cellular junctions in cell viability, we further tested the effect of OFNS on tight junction (e.g., ZO-1). It found that the protein expression of ZO-1 in H₂O₂-treated IEC-6 cells was obviously down-regulated due to a high ROS environment measured by western blotting (WB). Whereas, OFNS could significantly potentiate the cellular junction by up-regulating the protein expression of ZO-1 (Fig. 1I).

In addition, we found that OFNS could also regulate cellular redox homeostasis and decrease cell apoptosis in human umbilical vein endothelial cells (HUVECs). Specifically, it observed that OFNS did not cause severe cytotoxicity toward HUVECs up to 100 μg/mL (SI Appendix, Fig. S4) and obviously ameliorate the cellular viability and diminish the excessive ROS of HUVECs in the H₂O₂ oxidative injury model (Fig. 1 J–L). Then, we used the classical apoptotic-necrosis Annexin V-FITC/PI costaining system to evaluate the effect of OFNS on cell apoptosis in H₂O₂ oxidative injury model. The viable cells (Annexin V-FITC⁻/PI⁻) and early apoptotic cells (Annexin V-FITC⁺/PI⁻) were quantitatively detected by FCM. It revealed that OFNS observably improved the proportion of viable cells of H₂O₂-treated HUVECs from 14.45 ± 3.22% to 41.21 ± 3.33%, and early apoptotic cells were decreased from 84.66 ± 3.10% to 58.45 ± 3.41% by OFNS treatment (SI Appendix, Fig. S5).

Subsequently, we examined the regulation effect of OFNS in murine macrophage leukemia cells (RAW264.7), a representative macrophage model. Here, a classical inflammation model in RAW264.7 cells was established using lipopolysaccharide (LPS), one kind of endotoxin (Fig. 1M). The results revealed that OFNS could significantly reduce the content of intracelluar excessive ROS and proinflammatory factors (IL-6 and TNF-α) in RAW264.7 cells treated with LPS using enzyme-linked immunosorbent assay (ELISA) (Fig. 1 N and O). Additionally, WB was performed to quantify the representative proinflammatory protein expression of TNF-α, iNOS, and CD86 (SI Appendix, Fig. S6). It showed that OFNS could strikingly reduce the expression of these proinflammatory proteins in RAW264.7 cells. These collectively indicated that OFNS could enhance cell viability, reverse cellular apoptosis, and resolve the inflammation via regulating cellular redox microenvironment.

The Biodistribution and Acute Toxicity of OFNS in Rats.

Before initiation of the in vivo study, we assessed the biodistribution and acute toxicity of OFNS in rats. After oral administration with OFNS on 1st, 2nd, 3rd, and 5th day, the main organs (heart, liver, spleen, lung, kidney, brain, small intestine, and lymph) and plasma were collected to measure the contents of C₆₀ by liquid chromatography-mass spectrometry. Our results displayed that OFNS mainly accumulated into the intestinal tract, and there was little accumulation into the main organs and circulatory system, suggesting that OFNS were scarcely absorbed into the body (SI Appendix, Table S1). In addition, to detect the excretion rate of OFNS, feces were collected at 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h after oral administration with OFNS (SI Appendix, Table S2). We found that the content of C₆₀ is up to 70.26% at 8 to 12 h, and the excretion rate of OFNS reached to 97.43% within 24 h, which indicated that OFNS were mostly excreted out of the body over time.

To evaluate the acute toxicity of OFNS, an extreme high dose of OFNS (5,000 mg/kg) was orally administered to rats. We found that none of the rats were dead during 2-wk observation. We also statistically analyzed body weights, organ index, and histopathology of main organs (heart, liver, spleen, kidney, and lung) at the end of observation (SI Appendix, Fig. S7), all of which exhibited that OFNS owned little acute toxicity toward rats.

OFNS Prevent Myocardial Injury Induced by MI/RI in Mice.

Encouraged by the above findings, we investigated the effect of OFNS on the myocardial injury induced by MI/RI in mice. Briefly, the left anterior descending artery of mice was ligated with sterile silk thread for 30 min, and unwound the moving knot to cause myocardial injury. We first studied the dose-dependent relationship of OFNS treatment on myocardial injury in MI/RI mice (SI Appendix, Fig. S8A). Three doses of OFNS were used: 50 mg/kg twice daily (OFNS-high, H), 50 mg/kg once daily (OFNS-middle, M), and 25 mg/kg once daily (OFNS-low, L). Echocardiography (ECHO) was used to assess the effects of OFNS on myocardial injury. The main indexes for evaluation of pumping function of the heart include left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) (32). The results of ECHO showed that OFNS could heighten LVEF and LVFS in MI/RI mice after 14 d treatment with a dose-dependent manner (SI Appendix, Fig. S8B). Further, the results of hematoxylin eosin (H&E) staining revealed that the myocardialinfarction areas were notably reduced by OFNS-H treatments (SI Appendix, Fig. S8C).

Then, the other MI/RI bearing mice were randomly divided into four groups: sham group (Sham), MI/RI model group (MI/RI), edaravone (EDA)-treated group (MI/RI+EDA), and OFNS-treated group (MI/RI+OFNS-H), simultaneously using normal mice as a blank control. Respectively, EDA was intravenously injected and OFNS were orally administrated into the MI/RI bearing mice 10 min before moving the knot. Then, it continued dosing with OFNS for 14 d twice daily, and all the mice were killed on the 15th day (Fig. 2A). It revealed that both LVEF and LVFS in ECHO were decreased under MI/RI, which were observably improved after treatment by EDA and OFNS, particularly the latter (Fig. 2B). In addition, Masson staining was conducted to measure the degree of myocardial fibrosis. As seen in Fig. 2C, the sizes of cardiac tissue stained by aniline blue were notably declined by EDA and OFNS treatment. Subsequently, we used tissue TEM to observe ultrastructural morphology of the heart in the infarction region. Our results showed that the abnormities of myocardial mitochondrial including swelling, empty, and crest broke induced by MI/RI were significantly alleviated after OFNS treatment (Fig. 2D). All of those suggested that the damaged myocardium in MI/RI mice were significantly reversed after OFNS treatment.

Fig. 2.

The effects on myocardial injury and intestinal homeostasis of OFNS in MI/RI mice. (A) Schematic diagram depicting the experimental protocol. (B) Assessment of cardiac function with LVEF and LVFS in MI/RI mice after treated with OFNS and EDA by ECHO. (C) The Masson staining of hearts and quantitative statistics of infarction size in different groups. The red dotted line circles the ischemic and infarct areas. (Scale bar, 2.5 mm and 100 μm.) (D) The observation of ultramorphology of heart tissue by TEM. Mit.: mitochondrion. (Scale bar, 500 nm.) (E) The pathological status and histological score of the ileum (n = 5). The blue arrow indicates the shedding of microvilli, and the red arrow indicates the damaged mucosa with lamina propria disintegration. (Scale bar, 500 μm and 100 μm.) (F) Immunostaining for Ki67 (green) on sections from the ileum. (Scale bar, 50 μm.) (G) The observation of the ultramorphology of the ileum by TEM. The red arrow indicates empty mitochondria, and the black arrow refers to damaged microvilli. (Scale bar, 200 nm.) (H) The intracellular ROS levels of intestinal cells by FCM. (I) DHE staining (red) and the relative DHE intensity of the ileum. DAPI (blue) was counterstained for cell nuclei (n = 5). (Scale bar, 100 μm.) (J) Immunostaining for Muc-2 (green) on sections from the ileum and the relative Muc-2 intensity of the ileum. Cell nuclei were counterstained with DAPI. The white arrow indicates the mucous layer. (Scale bar, 100 μm.) (K) The protein expression of ZO-1 and occludin in the ileum. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. MI/RI group; #P < 0.05 vs. sham group).

We then evaluated the intestinal pathological variations in MI/RI mice after OFNS treatment. The shedding of microvilli and lamina propria disintegration in the mucous of the intestine in MI/RI mice were observably improved by OFNS using H&E staining. Specifically, the pathological score of the ileum under MI/RI was reduced from 4.4 ± 0.4 in the model group to 2.6 ± 0.8 in the OFNS-treated group (Fig. 2E). Further, the proliferation of intestinal cells measuring by Ki67 staining was also significantly promoted by OFNS (Fig. 2F). As well, the TEM images of intestine tissues revealed that the damaged subcellular structures (e.g., mitochondria) in intestinal epithelial cells were almost restored to be normal after OFNS treatment (Fig. 2G). To evaluate the effects of OFNS on regulating the redox homeostasis of intestinal microenvironment in MI/RI mice, we further detected the levels of total ROS and O₂^−• in the intestine by DCF and dihydroethidium (DHE), respectively. It displayed that OFNS remarkably decreased the levels of ROS and O₂^−• of the intestine in MI/RI mice (Fig. 2 H and I). The imbalance of redox status in the ileum may cause the disruption of the intestinal barrier. We continued to investigate the effect of OFNS on the intestinal barrier under MI/RI. Our results showed that the incomplete mucus layers in MI/RI mice were reinforced by OFNS (Fig. 2J). In addition, the down-regulated intestinal epithelial tight junction protein, including ZO-1 and occludin, were prominently up-regulated after OFNS treatment (Fig. 2K). These results suggested that OFNS effectively regulate intestinal redox homeostasis and restore the dysfunctional intestinal barrier in MI/RI mice.

The Quantitative Transcriptomics and Proteomics of the Intestine in MI/RI Mice.

To thoroughly uncover the molecular mechanism of OFNS treatment on intestinal homeostasis, we explored both quantitative transcriptomics and proteomics of intestinal tissues. The mRNA and protein were extracted from the ileum and then sequenced and analyzed to quantify the expressions of mRNA and protein, respectively (Fig. 3A). For transcriptome, there were 17,739 genes identified and quantified, and 5,629 mRNA were significantly changed (Q < 0.05). There were 320 differential mRNA [fold change (FC) > 2 and P value < 0.05] in the group of MI/RI+OFNS vs. MI/RI. These relevant differential mRNA were further annotated into three categories by gene ontology (GO) enrichment analysis: cellular component, molecular function, and biological process. From the cellular component analysis, it could see that these differential mRNA mainly located in the extracelluar region and intracellular membrane-bounded organelles such as the endoplasmic reticulum (SI Appendix, Fig. S9A). In addition, we found that the molecular function of differential mRNA was primarily related to oxidoreductase activity (SI Appendix, Fig. S9B). Particularly, the differential mRNA were closely associated with biological processes such as innate immune response, response to bacterium, blood coagulation, metabolic process, and so on (Fig. 3B). To comprehend the advanced biological functions of these differential mRNA, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. As shown in Fig. 3C, we found that the differential mRNA were highly correlated with the metabolic pathways (e.g., retinol metabolism and MAPK signaling pathway), response to bacterium pathways (e.g., NOD-like receptor signaling pathway), as well as complement and coagulation cascades. To further screen the crucial differential mRNA, we continued to analyze the interaction between the above differential mRNA. It could see that there are ca. 8 networks in interaction network diagram of differential mRNA (SI Appendix, Fig. S10). Notably, we found that there were several highly interconnected nodes among these three processes: metabolic pathways, response to bacterium, complement and coagulation cascades (Fig. 3D).

Fig. 3.

Quantitative transcriptomics and proteomics profiling. (A) The workflow of transcriptomic and proteomic analysis. (B) GO analysis of related differential mRNA for the biological process in transcriptomics. (C) KEGG pathway in transcriptomics. (D) PPI network analysis of related differential mRNA by STRING with median confidence (P < 0.400) in transcriptomics. (E) GO analysis of related differential proteins for the biological process in proteomics. (F) KEGG pathway in proteomics. (G) PPI network analysis of related differential proteins by STRING with median confidence (P < 0.400) in proteomics. (H) The heatmap of related differential proteins of proteomics.

For proteomics, there were 6,448 proteins identified, and 5,572 proteins were quantified. The proteins with FC >1.2 and P value < 0.05 were regarded as differential expression proteins. After filtering, there were 765 differential expression proteins between normal and MI/RI group, 918 differential expression proteins between MI/RI and OFNS+MI/RI group, and 412 differential expression proteins in both above comparison groups (SI Appendix, Fig. S11). Similar to transcriptomics, we subjected these related differential expression proteins with three categories (cellular component, molecular function, and biological process) based on GO enrichment analysis (SI Appendix, Fig. S12 and Fig. 3E). It found that the differential expression proteins were mainly involved in immune response, complement activation (alternative pathway and classical pathway), blood coagulation, and so forth. Subsequently, the KEGG functional analysis revealed that these differential expression proteins mainly enriched into metabolic pathways, complement and coagulation cascades (Fig. 3F). In addition, to highlight key differential proteins, the protein-protein interactions (PPI) analysis was carried out by the database of STRING (Fig. 3G and SI Appendix, Fig. S13). We could find that all of differential proteins were divided into five networks. Particularly, there were two main networks: metabolic pathways along with complement and coagulation cascades. Specially, several highly interconnected nodes among the differential proteins are complement C3 (C3), plasminogen (Plg), glutathione peroxidase 3 (Gpx3), poly (ADP-ribose) polymerase1 (Parp1), factor XII (F12), complement C4b (C4b), and fatty acid-binding protein 1 (Fabp-1), cytochrome C oxidase subunit 5A (Cox5a), lactate dehydrogenase (LDHa), etc.

All of differential proteins in PPI were further shown in a heat map (Fig. 3H). It revealed that most of the differential proteins in processes of metabolic pathways along with complement and coagulation cascades were observably down-regulated in the MI/RI group compared with the normal group. Of note, these differential proteins were significantly up-regulated after OFNS treatment. Together, combined with the above high throughput screening of transcriptomics and proteomics, it indicated that metabolic pathway as well as complement and coagulation cascades pathway were most greatly affected by OFNS treatment. Therefore, we further detected the expressions of these proteins using WB in the following study.

OFNS Maintain Intestinal Immunity Homeostasis to Resolve Systemic Inflammation.

For metabolic pathways, we detected the protein expressions including Gpx-3, Parp1, Cox5a, LDHa, Cytochrome C, Fabp1, etc. It could find that the expressions of these protein were not significantly changed after OFNS treatment (SI Appendix, Fig. S14). For complement and coagulation cascades, we detected the protein expressions including C3, C4b, F12, Plg, F13a1, and so forth. We found that the expressions of C3, the most core molecule in both classical and alternative pathways of the complement system, were highly reduced in the MI/RI model groups. Notably, the abnormal descent of C3 was reversed by OFNS (Fig. 4A). However, the expression of F12 and C4b protein (belong with classical pathways in complement system) were no significant changes after OFNS treatment (SI Appendix, Fig. S15). In addition, we found the content of complement C3 convertase (C3bBb, assigned to the alternative pathway in the complement system) was reduced in MI/RI mice. Interestingly, it was enhanced after OFNS treatment (SI Appendix, Fig. S16). For coagulation cascades, two crucial molecules, Plg, and F13a1, were observably declined under MI/RI. Notably, OFNS boosted the expressions of these proteins. These data suggested that OFNS markedly up-regulated complement and coagulation cascades in the MI/RI mice ileum.

Fig. 4.

Measurements of intestinal immunity and systemic inflammation after OFNS treatment. (A) The protein expression of C3, Plg, and F13a1 in the ileum. (B) Immunostaining and the relative intensity of Ly6G (red) and CD68 (green) in the ileum (n = 5). DAPI was counterstained for cell nuclei. (Scale bar, 50 μm.) (C) The expression of inflammatory cytokines (TNF-α, IFN-γ, and IL-6) in the intestine by PCR. (D) The analysis of T cells (CD3⁺T) in the ileum by FCM. (E) The levels of NEUT, MONO, and LY at the end of treatment in peripheral blood by blood routine examination (n = 5). (F) The analysis of white blood cells (WBCs) including NEUT and MONO in peripheral blood by FCM. (G) The inflammatory indicators (TNF-α, IL-1β, and IL-6) in the serum. (H) The analysis of T cells (CD3⁺T) including CD4⁺T and CD8⁺T cells in peripheral blood by FCM. (I and J) WB analysis of the proinflammatory protein (COX-2, IL-6, and iNOS) and anti-inflammatory protein (TGF-β1 and Foxp3) expression in relative to GAPDH in the cardiac tissue. (K) WB analysis of p-GSK-3β, p-ERK, and p-p38 protein expression in relative to GAPDH in the cardiac tissue. (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. MI/RI group).

The complement and coagulation cascades are closely related to innate immunity and adaptive immunity (33, 34). Thus, we further investigated the levels of neutrophils, macrophages, and associated cytokines, as well as the contents of T cells in the intestine of MI/RI mice after OFNS treatment using immunofluorescence staining or FCM. Our data revealed that OFNS could reduce the contents of neutrophils and macrophages along with proinflammatory cytokines, including TNF-α, IFN-γ, and IL-6 in MI/RI mice, indicating the inflammation induced by MI/RI was observably resolved after OFNS treatment (Fig. 4 B and C). Additionally, OFNS notably enhanced the total contents of T cells in the intestine of MI/RI mice (Fig. 4D). Together, these results suggested that OFNS could resolve inflammation and restore immune homeostasis of the intestine in MI/RI mice.

Subsequently, we investigated the regulation effects of OFNS on systemic inflammation and adaptive immunity. First, we detected the blood routine of MI/RI mice after the OFNS treatment. We found that the counts of neutrophil (NEUT) and monocyte (MONO) in the peripheral blood were increased in MI/RI mice and reduced by OFNS (Fig. 4E). Meanwhile, the counts of lymphocyte (LY) were decreased in MI/RI mice and enhanced by OFNS. The results in FCM of NEUT and MONO were greatly consistent with those in blood routine (Fig. 4F and SI Appendix, Fig. S17). In addition, the abnormalities of inflammatory cytokines, including TNF-α, IL-1β, and IL-6 were also reversed almost to normal in the serum of MI/RI mice after OFNS treatment (Fig. 4G). Simultaneously, CD4⁺T and CD8⁺T cells, which are the two large subgroups of T lymphocytes (SI Appendix, Fig. S18), were reduced in MI/RI mice and enhanced by OFNS (Fig. 4H). All the above results reflected that OFNS could inhibit systemic inflammation and maintain systemic immunity homeostasis.

Next, we continued to explore the mechanism on relieving myocardial injury under MI/RI after OFNS treatment. It showed that protein expressions of proinflammatory factors, such as cyclooxygenase-2 (COX-2), IL-6, and iNOS in the myocardium under MI/RI were greatly reduced by OFNS treatment (Fig. 4I). Accordingly, the protein expression of anti-inflammatory proteins, such as transforming growth factor β1 (TGF-β1) and forkhead box protein p3 (Foxp3) were increased after OFNS treatment (Fig. 4J). Subsequently, the expressions of glycogen synthase kinase (GSK)-3β and extracellular signal-regulated protein kinase (ERK) in ischemia–reperfusioninjury rescue kinase (RISK) signaling pathway were observably up-regulated after treatment with OFNS (Fig. 4K), which also down-regulated the abnormal expression of mitogen-activated protein kinases p38 (mediates proapoptotic or cardiomyogenic differentiation of cardiac cells). All of these suggested that the excessive inflammatory microenvironment and injured myocardial protection-related proteins in MI/RI mice were significantly reversed after OFNS treatment to restrain the cardiac injury.

The Biosafety Assessment after OFNS Treatment in MI/RI Mice.

The biosafety of OFNS treatment was evaluated by the histopathological analysis. The main organs including the liver, spleen, kidney, and lung were harvested and stained with H&E after the treatment with OFNS (SI Appendix, Fig. S19). It showed that there was no obvious inflammation, cellular necrosis, and apoptosis in the OFNS treatment group, which suggested that there was scarcely any side-effect of OFNS toward MI/RI mice.

OFNS Treat MI in Mice and Mini-pigs.

Then, we further evaluated the effects of OFNS on ischemic myocardial injury using MI models in mice and in mini-pigs. The MI-bearing mice were obtained by tightly ligating the left anterior descending coronary artery with sterile silk thread until the end of the experiments on the 3rd day (Fig. 5A). Then, they were randomly divided into three groups: sham group (Sham), MI model group (MI), and OFNS-treated group (MI+OFNS). The OFNS were orally administrated into the MI-bearing mice 2 h before the ligation, and 6 h after the ligation, respectively. Then, OFNS were continuously administrated for 3 d twice daily, and all the mice were euthanized on the 4th day. Based on the pathological images of the heart in Fig. 5B, we observed that OFNS notably decreased the infarction area in MI mice compared with the sham group. Furthermore, the myocardial injury markers including creatine kinase myocardial band isoenzymes and cardiac troponin T were increased abnormally in mice under MI (Fig. 5 C and D). Of note, both of those were significantly reduced after OFNS treatment. In addition, the abnormal oxidative stress-related indicators of MI mice including manganese superoxide dismutase (Mn-SOD) and malondialdehyde (MDA) were almost reversed to the normal levels by OFNS treatment (Fig. 5 E and F).

Fig. 5.

Anti-MI effects of OFNS in mice and -mini-pigs. (A) Schematic diagram depicting the experimental protocol of OFNS attenuating the damage of the myocardium and regulating the level of oxidative stress after MI in mice. (B) The pathological status of the heart treated with OFNS after MI (n = 5). (Scale bar, 2.5 mm and 100 μm.) (C and D) The markers of myocardial injury treated with OFNS after MI (n = 5). (E and F) The representative indicators of oxidative stress in vivo. (G) Schematic diagram depicting the experimental protocol of OFNS protecting mini-pigs from myocardial ischemia injury. (H) Representative ECHO imaging by the modified Simpson method of short-axis views for each treatment group at 45 min, 3 d, 14 d, and 28 d after MI (n = 5). (I) Representative PET-CT cardiac images of the OFNS-treated mini-pig heart after MI. White arrows indicate the changes between the MI group and the MI+OFNS group. SA is the short axis, HLA represents the horizontal long axis, and VLA is the vertical long axis. (J) The metabolic defect area of MI mini-pigs after OFNS treatment by PET-CT. (K) Representative images of the heart and heart sections of OFNS-treated mini-pigs 28 d after MI. White arrows indicate the location of the surgical embolism. Quantification of the infarct sizes as percentages is also shown in the Right panel. (Scale bar, 1.0 cm.) (L) The pathological status of the apical position of the mini-pig heart treated with OFNS after MI. (Scale bar, 2.5 mm and 100 μm.) (M) The observation of the ultramorphology of the infarction region and the border region of the mini-pig heart by TEM. The red arrow indicates empty mitochondria, and the yellow arrow refers to damaged or distorted mitochondria. (Scale bar, 500 nm.) (*P < 0.05 and **P < 0.01 vs. MI group).

To investigate the treatment effect of OFNS on MI in preclinical species, we established porcine MI models using an interventional embolization method. Simply, the balloon and embolus were used to block the left anterior descending coronary artery 1-2 cm below the first diagonal branch in mini-pigs, and coronary angiography was conducted to confirm each occlusion at the same location (SI Appendix, Fig. S20). After operation, it could be seen that the ST segment was elevated and the QRS amplitude was widened in the electrocardiography (ECG) (SI Appendix, Fig. S21), which identified that we successfully obtained porcine MI models. Then, MI-bearing pigs were randomly divided into two groups: MI model group (MI, n = 5) and OFNS-treated group (MI+OFNS, n = 5, 10 mg/kg) (Fig. 5G). The LVEF was used to evaluate the therapeutic effect of OFNS treatment by ECHO. It could see that the LVEF in the MI group remained in a lower level all the while on the 45th min, 3rd day, 14th day, and 28th day (Fig. 5H). Delightedly, they were significantly improved in the MI+OFNS group (61.05 ± 2.89%) compared with the MI group (52.04 ± 2.81%) on the 3rd day. On the 28th day, the LVEF was reached to 64.88 ± 3.02% after OFNS treatment. These results suggested that OFNS had a positive effect on mitigating the negative LV remodeling and boosting the morphology and pumping effectiveness of the heart after MI in mini-pigs. To further detect the myocardial viability, the ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography–computed tomography (PET-CT) tests were conducted on the 29th day (Fig. 5I). The more vigorous glucose metabolism, the more uptake of ¹⁸F-FDG in PET-CT. It showed both in bullet graph and coronal section view that the metabolic defect areas were significantly reduced after treatment with OFNS (Fig. 5J). Taken together, we confirmed that OFNS effectively restored myocardial activity after myocardial ischemia in mini-pigs. After harvesting, it could visually observe that the infarct sizes were notably declined by OFNS treatment (Fig. 5K). From the histopathological observation on the position of the heart apex in mini-pigs (Fig. 5L), we found that there was a significant decrease in the area of myocardial fibrosis and myocardialinfarction, and an increase in the normal myocardial fibers after treatment with OFNS. Further, we observed the hyperfine structure of the heart both in the infarction region and border region by TEM (Fig. 5M). It showed that OFNS alleviated the pathological phenomena such as abnormal mitochondria, empty mitochondria, and broken myocardial fibers in myocardial tissues of MI-bearing mini-pigs. All in all, OFNS remarkably prevented against the ischemic myocardial injury under MI both in mice and in mini-pigs.

Discussion

In this study, we proposed a strategy to protect against myocardial injury through bidirectional communication along the heart–gut immune axis (Fig. 6). Specifically, we explored antioxidative OFNS to regulate intestinal redox homeostasis to reduce systematic inflammation for preventing remote myocardial injury. In addition, we developed this principle into treating acute MI both in mice and in mini-pigs. Our work offers a general and potent route to alleviate excessive inflammation and remote tissue injury by addressing the small intestine immune axis.

Fig. 6.

The mechanism diagram of OFNS on preventing myocardial injury in MI/RI mice. MI/RI induces hypoperfusion of intestinal tissues, which leads to the intestinal redox imbalance, resulting in the damages of the intestinal barrier, intestinal immunity disorders, and systemic inflammation. All of these further aggravate the myocardial injury. After OFNS treatment, the intestinal redox homeostasis was recovered, inducing to restore the integrity of intestinal epithelial barrier and intestinal immunity homeostasis. Ultimately, these contribute to systemic inflammation attenuation and myocardial injury mitigation. Created by BioRender.com.

This strategy is motivated by the close communication between the intestine and the heart. The intestinal tissue needs abundant blood supply to maintain its normal function, accounting for 40% of the total body blood supply. During ischemic stress in the myocardium, it could decrease cardiac pump function and cause insufficient intestinal perfusion (35, 36), leading to serious intestinal dysfunctions. Specifically, the intestinal redox homeostasis would be destroyed, resulting in intestinal inflammation and immune imbalance (37, 38). Once excessive inflammation and immune cell depletion, the pathogens and toxic substances translate into the systemic circulation (39). Subsequently, this leads to the amplification of the systemic inflammatory response, further weakening adaptive immunity and then increasing the risk of cardiovascular events after MI or MI/RI.

Given the role of fullerenes in protecting cells and tissues against inflammation and immune disruptions, emerging studies have reported their application in the treatment of multiple inflammatory-related diseases, including tumor, diabetes, atherosclerosis, and so forth (40–43). Traditional attempts have been made to deliver fullerenes by intravenous or local injection (24, 25). However, most of the injected fullerenes are captured by the reticuloendothelial system, resulting in poor retention in the heart (26–28). Currently, we developed a way by oral delivery of fullerenes instead of intravenous administration to prevent against myocardial injury induced by both ischemia and reperfusion in mice and mini-pigs.

After treatment with OFNS, the excessive ROS due to intestinal injury were greatly reduced, and the disrupted intestinal barriers under MI/RI were observably restored. To deepen the pathophysiological understanding of OFNS in myocardial injury, we applied the quantitative transcriptomics as well as proteomics, and found that OFNS balanced intestinal immunity to resolve systemic inflammation by regulating complement and coagulation cascades pathways. Complement, as a major component of immunity, is required for the clearance of invading pathogens, destroying them directly by activating innate and adaptive immune cells (44, 45). Under some circumstances, effective adaptive immunity depends on an intact complement system (39, 46). Complement system also interacts with coagulation cascades to maintain immune homeostasis (47, 48). In MI/RI mice, the complement system is overused to defend against invading pathogens resulted from intestinal barrier dysfunction. This imbalance in complement and coagulation cascades further leads to immune disorder and excessive inflammation in the ileum. Interestingly, OFNS repaired the intestinal barrier, activating complement and coagulation cascades, avoiding the massive invasion by pathogens, which further contributed to the immune homeostasis and the decreased inflammation. Accompanied by resolved systemic inflammation and strengthened systemic adaptive immunity after OFNS treatment, the myocardial protective signaling pathway was up-regulated to improve the myocardial function and decrease the infarction area of MI/RI mice. Given that both MI and MI/RI are mainly caused by excessive inflammation, we further investigated the anti-MI effect of OFNS. It demonstrated that OFNS could restrain MI-induced ischemic cardiac injury both in rodents and nonrodents animal models.

In summary, we have explored the role of OFNS in regulating intestinal redox and resolving systemic inflammation to restrain remote myocardial injury in multiple preclinical species. We confirmed the close communication between the intestine and the heart, especially the small intestine. We demonstrated that OFNS could effectively regulate cellular redox balance resulting in protecting cells against oxidative injury and reducing excessive inflammation in vitro. Of note, OFNS inhibited myocardial injury under MI/RI although they do not enter the circulatory system. Mechanically, we observed that OFNS addressed intestinal redox unbalance and further restored intestinal immunity homeostasis, thereby reducing systematic inflammation. Particularly, we found that the epithelial barrier as well as the complement and coagulation cascades in intestinal tissues of MI/RI mice were significantly potentiated by OFNS treatment. Notably, OFNS also prevent ischemic cardiac injury under MI both in mice and in mini-pigs. Together, we provided a promising concept for tissue injury repair by focusing on the small intestine immune axis.

Materials and Methods

Preparation of OFNS.

Forty grams of C₆₀ powder was mixed with 280 g MCC, 8 g CMC, 48 g HPC, and other pharmaceutical excipients (6 g silica gel, 12 g copovidone, and 6 g magnesium stearate) by a high-speed mixer granulator (GHL-1L/10L, Zhiyang Machinery, China). Then, they were pressed into 1,000 tablets (400 mg/tablet each) by rotary tablet press.

Cell Culture.

Small intestinal crypt-like cells (IEC-6), HUVECs, and mouse macrophage (RAW264.7) cells were from the National Infrastructure of Cell Line Resource (Shanghai, China). HUVECs were cultivated in minimum essential medium (MEM, Invitrogen, California, USA). IEC-6 and RAW264.7 were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, California, USA).

The Cellular Uptake of OFNS In Vitro.

The 1 mL of IEC-6 cells (1 × 10⁵/mL) was incubated in a 6-well plate for 24 h. Afterward, the medium was replaced by DMEM with OFNS (100 μg/mL) added for 24 h. Subsequently, the cells were collected for TEM observation.

The Antioxidant Capacity of OFNS in IEC-6 Cells.

The 200 μL of IEC-6 (5 × 10⁴/mL) was cultivated in a 96-well plate for 24 h and then replaced with 200 μL 800 μM H₂O₂ for 1 h. Afterward, the medium were replaced with OFNS (0, 10, 20, 40, 80, 100 μg/mL) for 3 h. Subsequently, the cell viability was assessed according to the instruction manual of CCK-8. The 1 mL of IEC-6 (3 × 10⁵/mL) was incubated in a 6-well plate for 24 h and replaced with DMEM (1 mL) or DMEM with H₂O₂ (800 μM, 1 mL) added for 1 h. Afterward, the substrate was updated with/without OFNS (100 μg/mL, 1 mL) for 3 h. Subsequently, DCFH-DA (10 μM) was acted as a probe for ROS detection. The fluorescence of DCF was observed by laser confocal microscopy and quantified by a flow cytometer (FCM, Invitrogen, AttuneTM NxT, USA).

The Effect of OFNS on Intercellular Connection in the High ROS Environment.

The 1 mL of IEC-6 cells (3 × 10⁵/mL) was incubated in a 6-well plate for 24 h. Then, the medium was replaced by DMEM with H₂O₂ (800 μM) added or DMEM with H₂O₂ (800 μM) and OFNS (100 μg/mL) added for 6 h. Subsequently, the IEC-6 cells were collected for the detection of intercellular connection.

Animals.

Male C57BL/6 mice (22 ± 2 g) and female ICR mice (24 to 26 g) were maintained from Beijing Vital River Laboratory Animal Technology Co., Ltd., China. SD Rat (6 to 8 w) and KM mice (20 ± 2 g) were acquired from Beijing HFK Bioscience Co., Ltd, China. Bama mini-pigs (30 to 35 kg) were purchased from Tianjin Bainong Laboratory Animal Breeding Technology Co., Ltd, China. Mice, rat, and mini-pigs were acclimatized to the laboratory condition for 7 d before every experiment. And all the mice and mini pigs received food and water ad libitum.

The Mouse MI/RI Model.

We used silk thread (6-0 sterile) to bind the moving knot rapidly, which caused myocardial ischemia in the anterior wall of the left ventricle for 30 min. Then, the moving knot was removed to allow reperfusion of the myocardium in mice. MI/RI modeling was monitored by recording electrocardiogram (ECG, ADInstruments Powerlab 8/35, ADInstruments, AUS) changes before and after myocardial ischemia. The mice were randomly assigned to five groups (n = 7) to explore the therapeutic effect of OFNS (50 mg/kg, twice/day, 14 d) on MI/RI mice: 1) the normal control group was normal mice without further treatment; 2) the sham group was only threading, no ligation, and i.g. saline (200 μL, twice/day) on days 1 to 14; 3) the MI/RI group was i.g. saline (200 μL, twice/day) on days 1 to 14; 4) the MI/RI+EDA group was i.v. EDA 10 min before moving knot and i.g. saline (200 μL, twice/day) on days 1 to 14; and 5) the MI/RI+OFNS group was i.g OFNS 10 min before moving knot and i.g. OFNS (200 μL, twice/day) on days 1 to 14. Echocardiogram (ECHO, Vevo2100, VisualSonics, CAN) was conducted to evaluate the effect of OFNS on MI/RI mice. LVEF is calculated by end-diastolic volume and end-systolic volume of LVFS is calculated by internal diameter at end-diastole (LVIDd) and end-systole (LVIDs) of left ventricular.

The Mouse MI Model.

We used silk thread (6-0 sterile) to quickly tie the knot and observed that the bottom of the mouse heart turned white. We adopted a purse suture to quickly close the thoracic cavity and squeeze to prevent pneumothorax, resulting in myocardial ischemia in the anterior wall of the left ventricle. Mice were randomly assigned to three groups (n = 5) and treated with OFNS (50 mg/kg, twice/day, 3 d) for MI to evaluate the effect of OFNS on MI.

The Mini-pig MI Model.

The balloon and embolus were used to block the left anterior descending coronary artery 1 to 2 cm below the first diagonal branch in miniature pigs. Coronary angiography was conducted to confirm that the occlusion was at the same location to ensure the relative uniformity of the MI model. ECG was used to determine that the mini-pig MI model was acute ST-segment elevation myocardialinfarction. Then, the mini-pigs were randomly assigned to two groups (n = 5) and treated with/without OFNS (10 mg/kg) for a month. ECHO imaging was conducted at 45 min and on 3 d, 14 d, and 28 d after MI to evaluate the cardiac morphology as well as pumping effectiveness. To identify and quantitatively evaluate the viable myocardium in the area of myocardialinfarction, we performed the PET-CT on the 29th day. At the end of the experiment, we harvested the mini-pig heart and quantified the infarct sizes of the heart. The pathological status of the apical position of the mini-pig heart was conducted by H&E staining, and the ultramorphology of the infarction area and noninfarction zone of the mini pig heart was observed by TEM.

Statistical Analysis.

Statistical analysis was performed using GraphPad Software 8.2.1. Data reported were means ± SEM. Student’s t test or one-way ANOVA was performed to assess statistical significance of the results between two groups or several groups. Differences were considered statistically significant when the P value < 0.05.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix. The data, materials, and software that support the findings of this study are all available for referrence.