Fullerene combined medicine and preparation method thereof

Abstract

The invention provides a preparation method of a fullerene combined medicine, and belongs to the technical field of nano-medicines. The preparation method specifically comprises the following steps: adding purified C60 fullerene into olive oil to obtain a mixed solution, and sequentially carrying out ball milling operation, centrifugation operation and filtration operation on the mixed solution to obtain the fullerene combined medicine. The fullerene combined medicine can regulate the diversity of intestinal flora, regulate the content of intestinal probiotics and restore the integrity of intestinal barriers so as to relieve acute inflammatory reaction after myocardial infarction. The'flora-intestinal tract-heart 'axis is regulated in a targeted manner through oral administration of the nano material, compared with traditional vein anti-inflammatory treatment, the administration mode is convenient and fast, the local microenvironment of the intestinal tract is precisely regulated and controlled, a complete intervention chain is formed from flora balance and barrier repair to inflammation inhibition through the multi-target-point synergistic effect, and in the aspect of in-vivo inflammatory response, the anti-inflammatory effect is good. The fullerene combined medicine remarkably improves release of inflammatory factors and recruitment of inflammatory cells after myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and in particular relates to a fullerene combination medicine and a preparation method thereof. Background Art

[0002] Acute myocardial infarction remains the leading cause of death worldwide, with inflammation and the repair phase playing a key role in disease progression. Growing evidence suggests that the inflammatory cascade triggered by acute myocardial infarction is considered one of the main mechanisms for poor prognosis in myocardial infarction. Following acute myocardial infarction, various injury-related molecules are released from necrotic cardiac resident cells, triggering an inflammatory cascade within the heart. Although inflammation is essential for converting necrotic tissue into scar, persistent or excessive inflammation may lead to persistent tissue damage and improper healing. However, there is still a lack of safe and effective anti-inflammatory treatments.

[0003] Intestinal commensal microorganisms are the most abundant commensal organisms in the body and have become powerful regulators of the host immune system. Emerging evidence suggests that imbalances in the gut microbiota community are not only associated with intestinal pathologies but also with many extraintestinal diseases, such as cancer, diabetes, neurological diseases, and cardiovascular diseases. Recent studies have revealed an interplay between the gut microbiota and the severity of myocardial infarction (MI). Recent reports have shown that depletion of the gut microbiota in experimental MI models affects cardiac inflammation and outcomes after MI. In addition, circulating lipopolysaccharide (LPS) and bacterial ribosomal DNA (rDNA) levels are significantly increased in patients with ST-segment elevation myocardial infarction (STEMI) and are associated with poor prognosis. These results, for the first time, implicate a role for bacterial invasion and translocation in the inflammatory microenvironment after MI. Intervention of the gut microbiota is a potential therapeutic strategy to reduce post-MI inflammatory responses and improve MI outcomes.

[0004] Therefore, how to develop and verify a therapeutic product that can synergistically regulate the diversity of intestinal flora, precisely control the abundance of probiotics and repair the integrity of the intestinal barrier, and reduce the acute inflammatory response after myocardial infarction by targeted inhibition of the vicious cycle of the gut-heart axis, thereby improving myocardial damage and deterioration of cardiac function, has become a key direction to break through the limitations of existing treatment methods (such as broad-spectrum antibiotics that destroy the balance of the flora or the limited effect of single probiotic intervention), and innovative solutions are urgently needed. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned problems in the existing technology and to propose a fullerene combination drug and a preparation method.

[0006] The specific ideas of the present invention are as follows: Oral fullerene was prepared by ball milling. A myocardial infarction model in mice was established by ligating the left anterior descending artery to study its therapeutic effect and mechanism. The intestinal microenvironment was evaluated by 16S rRNA gene sequencing, pathological analysis, transmission electron microscopy (TEM), immunofluorescence, Western blotting (WB) and other methods. Cardiac pathology was evaluated by echocardiography, TTC staining, pathological analysis, immunofluorescence and other methods. Inflammatory recruitment was evaluated by enzyme-linked immunosorbent assay (ELISA) kit and flow cytometry. Toxic side effects were evaluated by body weight, blood tests and hematoxylin-eosin (HE) staining.

[0007] The objectives of the present invention can be achieved by the following technical solution: a fullerene combination drug comprising fullerene and olive oil, administered orally to alleviate acute inflammatory reactions after myocardial infarction. The fullerene combination drug is named OFO.

[0008] The mechanism is as follows: OFO is primarily distributed in the gastrointestinal tract and regulates the intestinal flora by increasing beneficial bacteria and restoring the intestinal barrier. OFO has also been found to reduce the recruitment of peripheral inflammatory cells and the release of pro-inflammatory cytokines. Consequently, OFO reduces myocardial infarction size by inhibiting cardiac inflammation after myocardial infarction.

[0009] Fullerene combination drugs have little biological toxicity. They can alleviate the acute inflammatory response after myocardial infarction by regulating the diversity of intestinal flora, controlling the content of intestinal probiotics, and restoring the integrity of the intestinal barrier, thereby improving the prognosis of myocardial infarction, fundamentally reducing the infarct size, and improving cardiac function.

[0010] Fullerenes, ground and dissolved in olive oil, are orally administered into the gastrointestinal tract, effectively regulating intestinal flora and improving the intestinal barrier. In the treatment of myocardial infarction, OFO significantly improved intestinal flora disturbances and intestinal barrier function associated with acute myocardial infarction. Furthermore, OFO was shown to protect cardiac function during the acute phase of myocardial infarction, rescue damaged cardiomyocytes, and alleviate inflammation in cardiac tissue. Regarding the in vivo inflammatory response, OFO significantly improved the release of inflammatory factors and the recruitment of inflammatory cells after myocardial infarction. Compared to traditional intravenous nanodrug delivery systems, OFO offers a more convenient drug delivery method during myocardial infarction, alleviating myocardial infarction by improving the gut-heart axis. Fullerenes, ground and dissolved in olive oil, effectively improve intestinal flora disturbances and repair damaged intestinal barrier function, thereby improving cardiac function during the acute phase of myocardial infarction and reducing the inflammatory response. Furthermore, OFO not only provides an effective and safe approach for the treatment of acute myocardial infarction but can also be further expanded to the treatment of other inflammatory diseases.

[0011] A method for preparing a fullerene combination drug comprises the following steps:

[0012] S1 purification: purifying the crude C60 fullerene to obtain purified C60 fullerene;

[0013] S2 mixing: adding purified C60 fullerene to olive oil according to a preset ratio to obtain a mixed solution, and subjecting the mixed solution to ball milling, centrifugation and filtration in sequence to obtain a fullerene combination drug.

[0014] In step S1, the purity of the purified C60 fullerene is not less than 99.9%.

[0015] According to the mass volume ratio, the purified C60 fullerene: olive oil is 1 mg:1 mL.

[0016] In ball milling operations, the milling media is made of agate.

[0017] The ball milling medium is specifically agate grinding balls with a diameter of 10 mm.

[0018] In the filtration operation, a Millipore filter membrane with a pore size of 0.22 μm was used for filtration.

[0019] During ball milling operation, it is required to work in a dark environment.

[0020] In the centrifugal operation, the parameters are as follows: the speed is 5000 r / min and the time is 1 hour.

[0021] The present invention offers the following benefits: an innovative oral delivery system and safety advantages: Olive oil-based fullerene (OFO), prepared by ball milling, overcomes the technical bottleneck of fullerene's poor water solubility, forming a stable, transparent solution that accumulates specifically in the gastrointestinal tract without residue in major organs. Drug administration experiments confirmed that OFO has no toxic effects on the blood system, liver and kidney function, or organ tissues, addressing the potential systemic toxicity issues of traditional nanomaterials and providing a new strategy for the development of safe oral nanomedicines.

[0022] Multi-dimensional regulation of the intestinal microenvironment: OFO can restore the diversity of intestinal flora after myocardial infarction, inhibit the abnormal proliferation of pathogenic bacteria (such as Proteobacteria and Enterobacteriaceae), and promote the colonization of probiotics (such as lactic acid bacteria);

[0023] Repair the integrity of the intestinal mechanical barrier, alleviate colon shortening and mucosal structural damage, and upregulate the expression of tight junction proteins (claudin-1 / occludin);

[0024] Inhibit the translocation of intestinal flora, reduce the release of pro-inflammatory factors such as lipopolysaccharide (LPS) in the circulation, and block the pathological process of "enterogenic inflammation" from the source.

[0025] Significant cardioprotective and anti-inflammatory effects: OFO effectively reduces myocardial infarction size and improves cardiac ejection function by regulating the intestinal flora and systemic inflammatory response. Its mechanisms include: inhibiting the infiltration of inflammatory cells (neutrophils and monocytes) in cardiac tissue and the release of proinflammatory cytokines (TNF-α and IL-6); reducing myocardial cell apoptosis, hypertrophy, and fibrosis; and relying on immune regulation mediated by the intestinal flora (the efficacy disappears after antibiotic pretreatment), highlighting the specificity of its mechanism of action.

[0026] A groundbreaking therapeutic strategy with potential for clinical application: This approach, for the first time, targets and modulates the "microbiome-gut-heart" axis through oral nanomaterials. Compared to traditional intravenous anti-inflammatory treatments, it offers convenient administration, avoids invasive procedures, and significantly improves patient compliance. It also precisely regulates the local intestinal microenvironment, avoiding the risk of systemic immunosuppression. Its multi-target synergistic approach forms a complete intervention chain, from microbiome balance and barrier repair to inflammation suppression. This strategy can be further expanded to other inflammatory diseases, providing a universal technical platform for interventions in "gut-derived pathologies."

[0027] Clear mechanism validation and technological universality: This systematically validates the molecular basis of OFO's cardioprotective effects through the "gut flora-immunomodulatory" axis, providing a solid theoretical basis for its clinical translation. Furthermore, the fullerene-olive oil composite technology solution offers a simple process and feasibility for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of OFO preparation and safety verification.

[0029] Figure 2 Schematic diagram of the mechanism of OFO in treating myocardial infarction.

[0030] Figure 3 This is a schematic diagram showing the product's efficacy in regulating intestinal flora imbalance induced by myocardial infarction injury.

[0031] Figure 4 This is a schematic diagram showing the product's efficacy in restoring intestinal barrier damage induced by myocardial infarction.

[0032] Figure 5 This is a schematic diagram showing the product's efficacy in improving cardiac function and cardiac inflammation after myocardial infarction.

[0033] Figure 6 This is a schematic diagram showing the product's efficacy in alleviating systemic inflammation after myocardial infarction. DETAILED DESCRIPTION

[0034] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0035] A fullerene combination drug, comprising fullerene and olive oil, is administered orally for alleviating acute inflammatory response after myocardial infarction and is named OFO.

[0036] like Figure 1 As shown in the figure, OFO is prepared by mixing fullerene with olive oil and using ball milling. First, C60 fullerene is separated and purified until the purity reaches 99.9%. Then, solid C60 fullerene is dissolved in olive oil and ball milled in a dark environment at room temperature to form a brown oil solution, as shown in the figure. Figure 1 As shown in Figure A. From a microscopic perspective, there is a clear difference in the size of the transmission electron microscope images before and after ball milling, as shown in Figure A. Figure 1 As shown in Figure B. From a macroscopic perspective, unlike the insoluble particle suspension formed in water, C60 forms a clear transparent solution in olive oil, as shown in Figure 2. Figure 1 In addition, we also conducted a full spectrum scan of olive oil and OFO and found that there was a significant difference between the two at a wavelength of around 350nm, as shown in Figure C. Figure 1 As shown in Figure D. To further explore the metabolic distribution of OFO in the body, we extracted and measured tissue suspensions from the blood, brain, gastrointestinal tract, heart, liver, spleen, lungs, and kidneys at different time points after oral administration of OFO to mice. We found that within 1-4 hours of oral administration, OFO mainly accumulated in the stomach, and after 8-24 hours, OFO was gradually metabolized from the intestine. Interestingly, there was almost no accumulation of OFO in other organs, such as Figure 1 As shown in Figure E;

[0037] In order to conduct in-depth in vivo studies, we first tested the safety of OFO. We tested the liver and kidney function of the serum of mice that had been given OFO for a long time (7 days, xx mg / day). After OFO treatment, ALT, AST, BUN, and CRE all showed normal levels. Figure 1 As shown in Figure F. The number of white blood cells (WBC), red blood cells (RBC) and platelets (PLT) in the blood routine test also showed normal levels compared with the control mice, as shown in Figure Figure 1 As shown in Figure G, these indicate that OFO has no detectable damage to blood cells. Major organs such as the heart, liver, spleen, lungs, and kidneys were stained with hematoxylin-eosin (HE) and showed no obvious inflammation, cell necrosis, and apoptosis. Figure 1As shown in Figure H, these results show that OFO had no detectable damage to blood cells. Hematoxylin-eosin (HE) staining of major organs such as the heart, liver, spleen, lungs, kidneys, and brain showed no obvious inflammation, cell necrosis, or apoptosis. These results indicate that OFO has virtually no toxic side effects on the test mice.

[0038] The OFO product preparation process is as follows: C60 fullerene is isolated and purified to a purity of 99.9%. 100 mg of the purified C60 fullerene is dissolved in 100 mL of olive oil. The mixture is stirred in a ball mill (Model QM-QX2L, MITR) at 600 rpm for 30 minutes using 10 mm diameter agate balls. The stirred mixture is centrifuged at 5,000 rpm for 1 hour. The supernatant is filtered through a 0.22 μm pore size Millipore filter.

[0039] exist Figure 1 middle, Figure 1 (A) Preparation of OFO. (B) TEM images of fullerenes before and after ball milling (scale bar = 200 nm). (C) Photographs of C60 in water, olive oil, and OFO. (D) Full spectra of olive oil and OFO. (E) Biodistribution of OFO measured by absorbance measurement. (F) Biochemical markers related to liver and kidney function (n = 6, mean ± SD). (G) Typical hematological parameters (n = 6, mean ± SD). (H) Hematoxylin-eosin (H&E) stained pathological sections of major organs of mice in the saline group and OFO-treated group (scale bar = 200 μm)

[0040] Product Usage: OFO product can reduce the acute inflammatory response after myocardial infarction by regulating the diversity of intestinal flora, controlling the content of intestinal probiotics and restoring the integrity of the intestinal barrier, thereby improving the prognosis of myocardial infarction. The specific mechanism is as follows: Figure 2 As shown, after oral administration of fullerene olive oil (OFO) to mice with myocardial infarction, OFO mainly accumulated in the gastrointestinal tract, regulated the intestinal microbiota, restored the intestinal barrier, improved systemic inflammation, and then reduced cardiac inflammation, thereby alleviating myocardial infarction.

[0041] Product function verification:

[0042] OFO can regulate myocardial infarction-induced intestinal flora imbalance: Figure 3To determine the effect of OFO on the intestinal microbiota after acute myocardial infarction, we performed either ligation of the anterior descending artery or sham surgery on mice and performed 16S ribosomal RNA (rRNA) gene sequencing on the colon contents of the mice 3 days after surgery. The results of principal coordinate analysis (PCoA) showed that the intestinal microbial community structure of the myocardial infarction group was significantly different from that of the sham surgery group, as shown in Figure 2. Figure 3 As shown in Figure A; α diversity analysis using Chao1 index and Shannon index showed that OFO increased the richness and diversity of intestinal flora after myocardial infarction, as shown in Figure 4. Figure 3 As shown in Figures B and C of the 3D images, it is important to analyze the composition of the microbiota at different taxonomic levels. Significant changes in microbiota characteristics were observed at the phylum level, especially the level of Proteobacteria was significantly higher than that of sham-operated mice. Figure 3 As shown in Figures D and F in Figure 3, the main groups of Proteobacteria, such as Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria, were significantly increased in the myocardial I / R group compared with the sham group. Figure 3 As shown in Figures E and G.

[0043] In mice with myocardial I / R injury, the proportion of pathogenic bacteria Gammaproteobacteria in the Proteobacteria phylum was significantly higher in the myocardial I / R group than in the sham operation group. Figure 3 As shown in Figure 3H, at the family and genus levels, markers of post-myocardial I / R bacterial dysbiosis include an increase in Escherichia coli within the Enterobacteriaceae family and a decrease in Lactobacillus, a recognized probiotic.

[0044] exist Figure 3 (A) Principal coordinate analysis (PCoA) plot based on unweighted_unifrac (Beta diversity). (B) Alpha diversity using the Chao1 index. (C) Alpha diversity using the Shannon index. (D) Relative abundance at the phylum level. (E) Relative abundance at the genus level. (F) Heatmap of gut microbiota composition at the genus level. (G) LEfSe analysis at the genus level.

[0045] OFO restores myocardial infarction-induced intestinal barrier damage: Figure 4As shown in Figure 4, in order to further explore the effects of OFO on the intestine, intestinal phenotype detection was performed on mice on the third day after myocardial infarction. We found that after myocardial infarction, the length of the colon was reduced by about 2 mm compared with the control group. Oral administration of OFO can effectively prevent the shortening of the colon, as shown in Figures A and B in Figure 4. Since the integrity of the intestinal barrier is closely related to changes in intestinal flora, changes in the intestinal barrier after myocardial infarction were further evaluated. The morphology and histological staining of the entire intestine showed that the intestinal mechanical barrier of mice with myocardial I / R injury was significantly deteriorated compared with the control group mice, as shown by a higher Chiu pathological score, as shown in Figure 4. Figure 4 Consistent with these changes, transmission electron microscopy detected disorganized mucosal microvilli and disrupted intestinal epithelial structure after myocardial I / R injury, as shown in Figures C-E. Figure 4 Figure F in Figure 2. Tight junction proteins are key components of the intestinal epithelial barrier. Claudin-1 and occludin are very important tight junction proteins. Through quantitative detection of these two proteins, we found that claudin-1 and occludin were significantly downregulated in the intestine of the myocardial infarction group, while the OFO treatment group improved the loss of tight junction proteins. At the same time, further immunofluorescence found that the tight junction protein claudin-1 in the myocardial infarction group was significantly downregulated in the jejunum and colon, while OFO effectively alleviated the decrease of claudin-1, as shown in Figure 2. Figure 4 G in Figure 2. Taken together, these results indicate that infarction caused significant intestinal mucosal damage and intestinal barrier dysfunction. After OFO treatment, intestinal mucosal damage and intestinal barrier function were significantly improved.

[0046] exist Figure 4 (A) Overall morphology of the gastrointestinal tract of sham-operated mice, MI-injured mice, and OFO-treated MI-injured mice on day 3 after surgery. (B) Colon length of sham-operated mice, MI-injured mice, and OFO-treated MI-injured mice (n = 6). (C) Representative images of hematoxylin and eosin (HE) staining of the intestine of sham-operated mice, MI-injured mice, and OFO-treated MI-injured mice on day 3 after surgery. Scale bar = 100 μm. (D) and (E): Injury assessment based on intestinal HE staining in (C), expressed as the Chiu score (n = 6). (F) Representative transmission electron microscopy images of intestinal epithelial sections from sham-operated mice, MI-injured mice, and OFO-treated MI-injured mice. Scale bar = 1 μm. (G) and (H): Relative protein expression of occludin and claudin1 in colonic tissues was determined by Western blotting (n = 3). (I) Fluorescence microscopy images of occludin (red) and 4',6-diamidino-2-phenylindole (DAPI, blue) in the colon. Scale bars = 200 μm (colon), 50 μm (jejunum).

[0047] OFO improves cardiac function and cardiac inflammation after myocardial infarction: Figure 5 Next, we investigated whether OFO treatment had a therapeutic effect on myocardial infarction-induced cardiac injury. Figure 5 The results are shown in Figure A. OFO treatment effectively reversed the decline in ejection fraction (EF%) and fractional shortening (FS%) of the heart in mice with acute myocardial infarction. Figure 5 As shown in Figures B and C. In the acute phase of myocardial I / R injury, OFO-treated mice showed reduced infarct size and cardiac fibrosis compared with the control group. Cardiac TTC staining also supported that OFO can effectively reduce myocardial infarction size after myocardial infarction. Figure 5 Further cardiac pathological staining showed that OFO could not only alleviate the infiltration of inflammatory cells and LPS in cardiac tissue after myocardial infarction, but also effectively reduce cardiac tissue cell apoptosis and myocardial cell hypertrophy after myocardial infarction. Figure 5 These results indicate that OFO can protect cardiac function in the acute phase of myocardial infarction, rescue damaged cardiomyocytes, and improve cardiac tissue inflammation.

[0048] exist Figure 5 (A) Schematic diagram of the experimental process. (B) Representative ultrasound images of mouse hearts. (C) Quantitative analysis of left ventricular ejection fraction (EF%) and fractional shortening (FS%) by echocardiography. (D) Representative images of hematoxylin-eosin (HE) and Masson's trichrome staining of heart tissues from sham-operated mice, myocardial infarction-injured mice, and OFO-treated mice 3 days after surgery (n=6; scale bar=200 μm). (E) Representative images of TTC staining of heart tissues from sham-operated mice, myocardial infarction-injured mice, and OFO-treated mice 3 days after surgery (n=6). (F) Quantitative analysis of the percentage of fibrosis area and the percentage of myocardial infarction area in heart tissues from sham-operated mice, myocardial infarction-injured mice, and OFO-treated mice 3 days after surgery (n=6). (HL) Representative images and quantitative analysis of HE, LPS, Tunnel, and WGA staining of heart tissues from sham-operated mice, myocardial infarction-injured mice, and OFO-treated mice 3 days after surgery (n=6)

[0049] OFO relieves systemic inflammation after myocardial infarction: Figure 6 As shown in the results, in order to clarify the association between the beneficial effects of OFO and intestinal barrier function, inflammation-related biomarkers in mouse serum were detected. It was found that OFO intervention significantly reduced the concentrations of LPS, TNF-α, IL-1β, IL-6, and CCL2 in serum after myocardial infarction ( Figure 5AE). Flow cytometry analysis was further performed, showing that circulating neutrophils and monocytes, especially pro-inflammatory Ly6C-high monocytes, were significantly increased in myocardial infarction model mice. OFO intervention significantly reduced the number of neutrophils and monocytes in peripheral blood ( Figure 5 FG). In addition, flow cytometry analysis showed that the spleen ( Figure 5 HI) and bone marrow (BM) ( Figure 5 JK). As expected, myocardial infarction induced a significant increase in the recruitment of neutrophils and monocytes to the spleen and bone marrow of mice. Notably, OFO intervention resulted in a significant attenuation of infarction-induced inflammatory cell recruitment. Together, these data suggest that OFO intervention restored intestinal barrier integrity, thereby significantly reducing endotoxin-mediated inflammatory responses. Furthermore, the data suggest that OFO intervention inhibited the recruitment of inflammatory cells to the spleen and bone marrow by inhibiting the mobilization of monocytes from the spleen.

[0050] exist Figure 6 (A) Serum lipopolysaccharide (LPS) levels in sham-operated mice, MI-injured mice, and OFO-treated MI-injured mice (n=6).

[0051] (BE) Enzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and C-C motif ligand 2 (CCL2) in mouse plasma samples (n = 6). (FG) Representative flow cytometric analysis and quantification of neutrophils and monocytes in mouse blood. Data are shown normalized to the control group (n = 6). (HI) Representative flow cytometric analysis and quantification of neutrophils and monocytes in mouse spleen. Data are shown normalized to the control group (n = 6). (JK) Representative flow cytometric analysis and quantification of neutrophils and monocytes in mouse bone marrow. Data are shown normalized to the control group (n = 6).

[0052] The experiments were conducted as follows: Animals: Male C57BL / 6J mice (aged 6-8 weeks) were purchased from Weitong Lihua Co., Ltd. (China). Mice were housed in a standardized, temperature-controlled, well-ventilated animal room with a 12-h light / dark cycle and free access to food and water. All experimental protocols involving live animals were approved by the Animal Ethics Committee.

[0053] Myocardial Infarction Model: Biodistribution Study: Male C57BL / 6J mice were orally treated with OFO (6.5 mg / kg, n=3). Mice were sacrificed 1, 4, 8, 24 hours, and 7 days after treatment to collect blood and organs (heart, liver, spleen, lung, kidney, brain, stomach, and intestine).

[0054] Biodistribution study: Male C57BL / 6J mice were orally administered OFO (6.5 mg / kg / d, n=8). Mice were sacrificed 1, 4, 8, 12, and 24 hours and 2, 3, and 7 days after treatment, and blood and organs (heart, liver, spleen, lung, kidney, brain, stomach, and intestine) were collected.

[0055] 16S rRNA gene sequencing analysis of the intestinal microbiome: After administration, fecal samples were collected using metabolic cages and immediately frozen at -80°C. Subsequently, total genes were extracted and quantified using a Qubit2.0 fluorometer (Invitrogen, USA). 30-50 ng of DNA and the V3 and V4 regions of 16SrRNA were selected to generate amplicons. The forward primer sequence "XXX" and the reverse primer sequence "XXX" were used to amplify microbial genes. After purification, the amplified DNA library was loaded onto the Illumina MiSeq instrument for sequencing according to the manufacturer's instructions (Illumina, USA). Sequencing used a 2x300 paired end (PE) configuration. Data analysis was performed by professionals using the QIIME software package.

[0056] Transmission electron microscopy: Mouse colon tissue was cut into 3mm 3 The sample was fixed with electron microscopy fixative for 4 hours, washed 3 times with PBS, and dehydrated with acetone. The sample was placed in a mixture of epoxy resin (such as Epon812) and acetone (1:1, 2:1) and soaked at room temperature for 2–4 hours, and finally soaked in pure resin overnight. The sample was transferred to an embedding mold and filled with pure resin for embedding. The solidified resin block was cut into ultrathin sections with a thickness of 50–90 nm using an ultramicrotome (such as Ultramicrotome). The sections were placed on a copper or nickel grid. 2% uranyl acetate stain (UranylAcetate) was used to stain the sample for 15–30 minutes. Then it was stained with lead citrate (LeadCitrate) for 5–15 minutes. The stained sample was placed in a transmission electron microscope for observation and photography.

[0057] Western blotting: Total protein was extracted from intestinal tissue using RIPA lysis buffer containing PMSF and quantified using a BCA kit. Protein samples were mixed with loading buffer, denatured by heating at 100°C for 5 minutes, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked in 5% skim milk powder in TBS-T buffer at room temperature for 2 hours. Specific primary antibodies against NLRP3, caspase-1, IL-1β, GAPDH, and β-actin were then added and incubated overnight at 4°C. Subsequently, the membrane was incubated with the corresponding secondary antibody at 37°C for 30 minutes, and the signal was detected using an ECL luminescence kit and an Amersham Imager 600 system to record the protein bands.

[0058] Histopathology: Intestinal and cardiac tissues were excised and fixed in 4% paraformaldehyde for 48 hours, then dehydrated in various concentrations of sucrose solution and embedded in OCT. Intestinal and cardiac tissue sections (5 μm thick) were stained with hematoxylin-eosin, EVG, and Masson's trichrome according to standard protocols. After staining, changes in aortic tissue were observed and photographed using a light microscope (Olympus, Tokyo, Japan). Intestinal and cardiac tissue sections were fixed and permeabilized with 0.1% Triton X-100. After blocking, sections were incubated with anti-CD68 and anti-Ly6G polyclonal antibodies, respectively, at 4°C overnight. Subsequently, the treated Raw264.7 cells were washed three times with PBS and stained with Alexa Fluor 488 and Alexa Fluor 555 secondary antibodies for 1 hour. Finally, nuclei were stained with DAPI. Stained aortic tissues were observed using confocal laser scanning microscopy (CLSM). All histological evaluations were independently scored by two pathologists who were blinded to the experimental procedures. ImageJ software was used to evaluate the inflammatory cell infiltration, fibrosis area and fluorescence intensity.

[0059] ELISA: According to the instructions of the ELISA kit, the main factors detected include tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), chemokine 2 (CCL2), and interleukin 6 (IL-6).

[0060] Flow cytometry: Harvest mouse hearts and spleens. Prepare a single-cell suspension and add red blood cell lysis buffer. Incubate at room temperature for 10 minutes, wash with PBS, and centrifuge to remove the supernatant. Add fluorescently labeled monoclonal antibodies to the samples and incubate at 4°C in the dark for 1 hour. Wash the samples with PBS.

[0061] For whole blood, EDTA was used for anticoagulation. A 100-μl whole blood sample was added to red blood cell lysis buffer, incubated at room temperature for 10 minutes, washed with PBS, and centrifuged to remove the supernatant. For bone marrow, the mouse femur was carefully removed, both ends cut with sterilized scissors, and irrigated with PBS. Fluorescently labeled monoclonal antibodies were then added to the sample and incubated at 4°C in the dark for 1 hour. The sample was then washed with PBS. Samples were detected using LSRFortessa. FlowJo was used for flow cytometry data analysis.

[0062] In the present invention, fullerenes are dissolved and ground in olive oil and then introduced into the gastrointestinal tract by gavage, effectively regulating the intestinal flora and improving the intestinal barrier. The in vivo safety of OFO was first observed. After administration of OFO, OFO was mainly metabolized in the gastrointestinal tract, and there were no obvious long-term safety issues in major organs. In the treatment of myocardial infarction, OFO significantly improved the intestinal flora disorder and intestinal barrier function caused by acute myocardial infarction. In addition, OFO can also protect cardiac function in the acute phase of myocardial infarction, rescue damaged myocardial cells and improve cardiac tissue inflammation. In terms of in vivo inflammatory response, OFO significantly improved the release of inflammatory factors and the recruitment of inflammatory cells after myocardial infarction.

[0063] During myocardial infarction, compared with traditional intravenous nanodrug delivery systems, the present invention presents a more convenient way of administration, alleviating myocardial infarction by improving the gut-heart axis. In the present invention, fullerenes dissolved and ground in olive oil can effectively improve intestinal flora disorders and repair damaged intestinal barriers, thereby improving cardiac function in the acute phase of myocardial infarction and reducing inflammatory responses in the body. In addition, the present invention not only provides an effective and safe method for the treatment of acute myocardial infarction, but can also be further expanded to the treatment of other inflammatory diseases.

[0064] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Those skilled in the art of the present invention may make various modifications or additions to the described specific embodiments or replace them in a similar manner, but will not deviate from the spirit of the present invention or exceed the defined scope. Although the present invention has been described and illustrated in detail in the foregoing description, such illustrations and descriptions are considered to be illustrative or exemplary rather than restrictive. It should be understood that within the scope of the following claims, those of ordinary skill in the art may make changes and modifications. Specifically, the present invention encompasses additional embodiments having any combination of features from the different embodiments described above.

Claims

1. A fullerene combination drug, characterized in that: The fullerene combination medicine consists of fullerene and olive oil, is orally administered, and is used to alleviate acute inflammatory response after myocardial infarction.

2. A method for preparing a fullerene combination drug, for preparing the fullerene combination drug according to claim 1, characterized in that: The specific steps include: S1 purification: purifying the crude C60 fullerene to obtain purified C60 fullerene; S2 mixing: adding purified C60 fullerene to olive oil according to a preset ratio to obtain a mixed solution, and sequentially subjecting the mixed solution to ball milling, centrifugation, and filtration to obtain a fullerene combination drug.

3. The method for preparing a fullerene combination drug according to claim 2, wherein: In step S1, the purity of the purified C60 fullerene is not less than 99.9%.

4. The method for preparing a fullerene combination drug according to claim 2, wherein: According to the mass volume ratio, the purified C60 fullerene: olive oil is 1 mg:1 mL.

5. The method for preparing a fullerene combination drug according to claim 2, wherein: In ball milling operations, the milling media is made of agate.

6. The method for preparing a fullerene combination drug according to claim 5, wherein: The ball milling medium is specifically agate grinding balls with a diameter of 10 mm.

7. The method for preparing a fullerene combination drug according to claim 2, wherein: In the filtration operation, a Millipore filter membrane with a pore size of 0.22 μm was used for filtration.

8. The method for preparing a fullerene combination drug according to claim 1, wherein: During ball milling operation, it is required to work in a dark environment.

9. The method for preparing a fullerene combination drug according to claim 2, wherein: In the centrifugal operation, the parameters are as follows: the speed is 5000 r / min and the time is 1 hour.

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