Application of water-soluble fullerene nano-material in preparation of medicine for treating and / or relieving endotoxemia

Abstract

The invention discloses an application of a water-soluble fullerene nano material in preparation of a medicine for treating and / or relieving endotoxemia. The method for treating and / or relieving the endotoxemia comprises the following steps: 1) inhibiting inflammation caused by the endotoxemia; 2) the intestinal epithelial barrier is protected or intestinal epithelial barrier injury caused by endotoxemia is relieved; the inventor of the invention discovers that the water-soluble fullerene blocks the pyroptosis of macrophages induced by inflammation by down-regulating the expression of gasdermin D (N-GSDMD) after cutting for the first time. The AFRM repairs epithelium connection and extracellular matrix, and reduces oxidative damage to intestinal epithelial barrier. The AFRM with broad-spectrum and self-cascade redox regulation characteristics can be used for remodeling the intestinal microenvironment in three aspects of relieving excessive inflammation, inhibiting pyroptosis of macrophages and maintaining the integrity of the intestinal epithelial barrier, so that multi-target synergistic efficient treatment on the LPS-induced endotoxemia is realized.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of a water-soluble fullerene nanomaterial in the preparation of a drug for treating and / or alleviating endotoxemia. Background Art

[0002] Endotoxemia is a severe systemic inflammatory response syndrome complicated by multiple organ failure, with extremely high mortality rates worldwide. Acute inflammation, cytokine storm, and epithelial / endothelial damage caused by pathogen infection are the most important pathophysiological characteristics of endotoxemia. The intestinal microenvironment (including the intestinal immune system and intestinal epithelial barrier) plays a vital role in the progression of endotoxemia. Specifically, the intestinal immune system is the body's first line of defense against the invasion of external pathogens, and the destruction of the intestinal epithelial barrier is more likely to lead to pathogen invasion, causing secondary infection and further severe inflammatory response. At present, the clinical treatment of endotoxemia mainly relies on antibiotics, active fluid resuscitation, and supportive care, but the mortality rate is still high. Although a large number of potential anti-inflammatory therapeutic targets have been discovered, the treatment effect is generally poor. One of the main reasons is that single-target therapy is difficult to simultaneously control the inflammatory storm and multiple organ damage of endotoxemia. Therefore, there is an urgent need to develop new therapies that can act on multiple targets to effectively reduce the mortality rate of endotoxemia.

[0003] In the early stages of endotoxemia, the abnormal intestinal microenvironment manifests as acute, dynamic, and uncontrolled inflammation. It is often stimulated by inflammatory mediators from pathogens, such as lipopolysaccharide (LPS), which recruit innate immune cells (such as neutrophils and macrophages) for a short period of time and release inflammatory cytokines (such as interleukin-1β [IL-1β], interleukin-6 [IL-6], and tumor necrosis factor-α [TNF-α]) and reactive oxygen species (ROS). Overactivation of innate immune cells further leads to cell pyroptosis. All inflammatory cytokines produced during inflammation and pyroptosis can jointly induce an inflammatory storm and are rapidly released into the extracellular space, ultimately triggering an imbalance in the intestinal microenvironment and causing acute damage including epithelial / endothelial cells. Therefore, inhibiting the overactivation and pyroptosis of innate immune cells and effectively protecting the integrity of the intestinal epithelium in the intestinal microenvironment may be the prospect of multi-target treatment of endotoxemia.

[0004] So far, the booming nanotechnology has provided innovative technical solutions for the treatment of endotoxemia. Fullerene and its derivatives, as effective carbon nanomaterials, have been widely studied in the past decade due to their excellent performance in regulating the redox microenvironment to treat major diseases. However, there are no reports on the use of fullerene nanomaterials in alleviating endotoxemia. Summary of the invention

[0005] The purpose of the present invention is to provide a new medical application of water-soluble fullerene nanomaterials.

[0006] In a first aspect, the present invention provides: use of water-soluble fullerene nanomaterials in the preparation of drugs for treating and / or alleviating endotoxemia.

[0007] As mentioned above, the treatment and / or alleviation of endotoxemia is manifested in the following aspects:

[0008] 1) Inhibit inflammation caused by endotoxemia;

[0009] 2) Protect the intestinal epithelial barrier or reduce intestinal epithelial barrier damage caused by endotoxemia.

[0010] Furthermore, the inhibition of inflammation caused by endotoxemia is embodied in at least one of the following aspects:

[0011] a1) It inhibited the increase in the percentage of neutrophils caused by endotoxemia and restored the proportion of neutrophils to normal;

[0012] a2) It inhibited the decrease in lymphocyte percentage caused by endotoxemia and restored the lymphocyte ratio to normal trend;

[0013] a3) inhibited the increase of pro-inflammatory cytokine levels;

[0014] a4) inhibit macrophage inflammation;

[0015] a5) inhibit inflammation-induced macrophage pyroptosis;

[0016] a6) Scavenging of a broad spectrum of reactive oxygen species (ROS).

[0017] Furthermore, the pro-inflammatory cytokines include TNF-α, IL-1β and IL-6.

[0018] Furthermore, the protection of the intestinal epithelial barrier or the alleviation of intestinal epithelial barrier damage caused by endotoxemia is embodied in at least one of the following aspects:

[0019] b1) Inhibit endotoxemia-induced superoxide anion (O2 ·- ) levels are elevated;

[0020] b2) Inhibition of endotoxemia-induced decrease in the expression levels of tight junction-associated proteins, zonula occludens 1 (ZO-1) and occludin in the ileum.

[0021] Optionally, the drug preparation comprises one or more of a solution, a suspension, a granule, a lyophilized powder, an emulsion, an oil, a syrup, an aerosol, and a nanoformulation. Preferably, the solution is an injection.

[0022] In the above applications, when preparing the medicine, AFRM can be used as one of the active ingredients, or as the only active ingredient. In the above applications, when preparing the medicine, a carrier material can also be added.

[0023] Carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, flavors, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal administration; respiratory tract administration, such as nasal cavity; mucosal administration.

[0024] In a second aspect, the present invention provides the use of water-soluble fullerene nanomaterials in the preparation of products for inhibiting macrophage inflammation.

[0025] Furthermore, the inhibition of macrophage inflammation is embodied in at least one of the following aspects:

[0026] 1) Inhibited the increase of pro-inflammatory cytokines TNF-α and IL-1β in inflammatory macrophages;

[0027] 2) downregulate LPS / TLR4 / NF-κB and MAPK / ERK signaling pathways in inflammatory macrophages;

[0028] 3) Reduced the level of ROS in inflammatory macrophages.

[0029] In a third aspect, the present invention provides the use of water-soluble fullerene nanomaterials in the preparation of products for inhibiting inflammation-induced macrophage pyroptosis.

[0030] Furthermore, the inhibition of inflammation-induced macrophage pyroptosis is embodied in at least one of the following aspects:

[0031] 1) Reduced the proportion of macrophage death and inhibited the increase of ROS in pyroptotic cells;

[0032] 2) Inhibited the activity of Caspase-1 and the activation of GSDMD in the classical inflammasome pathway of cell pyroptosis;

[0033] 3) Reduced the levels of proinflammatory cytokines IL-1β, interleukin 18 (IL-18), and late proinflammatory mediator high-mobility group protein B1 (HMGB-1).

[0034] In a fourth aspect, the present invention provides the use of water-soluble fullerene nanomaterials in the preparation of products for alleviating intestinal epithelial barrier damage caused by oxidative damage.

[0035] Furthermore, the reduction of intestinal epithelial barrier damage caused by oxidative damage is embodied in at least one of the following aspects:

[0036] 1) Reduce the level of ROS in the extracellular environment and thus reduce the level of ROS in cells;

[0037] 2) Inhibited the decrease in the expression levels of zonula occludens 1 (ZO-1), collagen 1 (Col 1) and fibronectin (Fn1) caused by oxidative damage.

[0038] In a fifth aspect, the present invention also provides a pharmaceutical composition for treating and / or alleviating endotoxemia, wherein the active ingredient of the pharmaceutical composition comprises a water-soluble fullerene nanomaterial.

[0039] In a sixth aspect, the present invention also provides a method for treating and / or alleviating endotoxemia, comprising administering an effective amount of a water-soluble fullerene nanomaterial or a pharmaceutical composition to a subject suffering from endotoxemia.

[0040] In the present invention, the term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0041] In the present invention, the term "subject" may refer to a patient or other animal that receives the composition of the present invention to treat, prevent, alleviate and / or relieve the diseases or conditions described in the present invention, particularly mammals, such as humans, dogs, monkeys, cows, horses, etc.

[0042] The water-soluble fullerene nanomaterial described in the present invention has a general structural formula of C 2n (OH) x (Amino Acid) y ; wherein, 20≤n≤60, 0≤x<50, 0<y. Amino Acid is a water-soluble amino acid.

[0043] The above structural formula indicates that both the hydroxyl group and the water-soluble amino acid are connected to the fullerene.

[0044] In the general structural formula of the water-soluble fullerene nanomaterial, n can specifically be 30 or 35.

[0045] When multiple amino acids participate in the reaction, the number of amino acids and hydroxyl groups modified on the surface of the carbon cage will also be different due to the different affinities of different amino acids to the fullerene carbon cage in the reaction.

[0046] In the above-mentioned nanomaterial, the water-soluble amino acid is selected from at least one of alanine, glycine, serine, arginine, lysine and aspartic acid.

[0047] In the above-mentioned nanomaterial, the hydrated particle size of the nanomaterial is 1 to 1000 nm.

[0048] The water-soluble fullerene nanomaterial of the present invention can be prepared by existing methods. For example, when it contains amino acid groups, it can be prepared by referring to the method described in the literature (Carbon, 2006 (44): 496-500).

[0049] According to one embodiment of the present invention, the water-soluble fullerene nanomaterial is alanine fullerene derivative (AFRM), and the structural formula is: 60 (OH) ～12 (NHCH2CH2COOH) ～5 .

[0050] The alanine fullerene derivative is prepared by a one-pot liquid-liquid method, wherein a 60% ethanol aqueous solution containing 0.25 g of β-alanine (β-Ala) and 0.67 g of NaOH is slowly dripped into 50 mg of C 60 The reaction mixture was added to a saturated toluene solution and refluxed at 80°C for 48 hours. After the reaction, the aqueous phase was separated and the solid was precipitated with anhydrous ethanol. After centrifugation, the solid was re-dissolved and dialyzed with ultrapure water to obtain a water-soluble AFRM.

[0051] Furthermore, the AFRM has a spherical shape with a diameter of 24.64±1.88 nm.

[0052] Water-soluble fullerene nanomaterials have superoxide dismutase (SOD)-like and peroxidase (POD)-like activities and hydroxyl radical (·OH) scavenging capabilities, and can simulate the complex multi-enzyme cascade reactions involved in redox regulation in cells. Water-soluble fullerene nanomaterials can effectively regulate the redox balance of inflammatory macrophages and achieve anti-inflammatory effects by downregulating the LPS / TLR4 / NF-κB and MAPK / ERK signaling pathways.

[0053] The inventors of the present invention first discovered that water-soluble fullerene nanomaterials blocked inflammation-induced macrophage pyroptosis by downregulating the expression of cleaved gasdermin D (N-GSDMD). AFRM repairs epithelial junctions and extracellular matrix, reducing oxidative damage to the intestinal epithelial barrier. AFRM, which has broad-spectrum and self-cascading redox regulation properties, can reshape the intestinal microenvironment by alleviating excessive inflammation, inhibiting macrophage pyroptosis, and maintaining the integrity of the intestinal epithelial barrier, achieving multi-target synergistic and efficient treatment of LPS-induced endotoxemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The morphology (left) and hydrated particle size and zeta potential (right) of AFRM are shown;

[0055] Figure 2 The infrared spectrum (A), thermogravimetric analysis spectrum (B) and elemental analysis (C) of AFRM are shown;

[0056] Figure 3 Shows O2 before and after AFRM treatment ·- EPR spectrum of (left), POD-like enzyme activity (middle) and EPR spectrum of ·OH (right);

[0057] Figure 4 Shown are DCF fluorescence images (A) and flow cytometry results (B) of inflammatory macrophages under different treatments;

[0058] Figure 5 Shown are the levels of TNF-α in inflammatory macrophages (A) and IL-1β in cell culture supernatant (B) under different treatments;

[0059] Figure 6 The expression of marker proteins in the NF-κB signaling pathway in inflammatory macrophages under different treatments is shown;

[0060] Figure 7 The expression of marker proteins in the MAPK / ERK signaling pathway in inflammatory macrophages under different treatments is shown;

[0061] Figure 8 The flow cytometric analysis and quantitative statistics of BMDMs death caused by activation of typical pathways under different treatments are shown;

[0062] Fig. 9 Fluorescence microscopic images showing BMDMs pyroptosis caused by activation of typical pathways under different treatments;

[0063] Fig.10 Shown are the DCF flow cytometry analysis (left) and Caspase 1 enzyme activity (right) of BMDMs pyroptosis caused by typical pathway activation under different treatments;

[0064] Fig.11 The expression of GSDMD protein in cells that induce pyroptosis of BMDMs under typical pathway activation under different treatments is shown;

[0065] Fig.12 The levels of IL-1β, IL-18, and HMGB-1 in the cell culture supernatant of BMDMs pyroptosis induced by typical pathway activation under different treatments are shown;

[0066] Fig.13 Shown are the viability of epithelial cells treated with AFRM under oxidative damage induced by H2O2 (left) and menadione (right);

[0067] Fig.14 Shown are the extracellular ROS levels in epithelial cells before and after AFRM treatment under oxidative damage induced by H2O2 (left) and menadione (right);

[0068] Fig.15 Shown are flow cytometric analysis and quantitative statistics of intracellular ROS in epithelial cells before and after AFRM treatment under oxidative damage induced by H2O2 (A) and menadione (B);

[0069] Fig.16 The expression of Col 1 and Fn1 in epithelial cells before and after AFRM treatment under H2O2-induced oxidative damage is shown;

[0070] Fig.17 Shown is the expression of ZO-1 in epithelial cells before and after AFRM treatment under H2O2-induced oxidative damage;

[0071] Fig.18 The ratios of neutrophils (left) and lymphocytes (right) in whole blood of mice under different treatments are shown;

[0072] Fig.19 The levels of proinflammatory cytokines in the serum of mice under different treatments are shown;

[0073] Fig. 20 The fluorescence images and quantitative statistics of DHE in mouse ileum tissue under different treatments are shown;

[0074] Fig.21 The fluorescence images and quantitative statistics of tight junction proteins ZO-1 and Occludin in mouse ileum tissue under different treatments are shown;

[0075] Fig. 22 Shown are H&E pathological sections of mouse ileum tissues under different treatments. DETAILED DESCRIPTION

[0076] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0077] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0078] Example 1: Preparation of water-soluble fullerene material-alanine fullerene nanomaterial (AFRM)

[0079] Synthesis method: Alanine fullerene nanomaterial (AFRM) was prepared by a one-pot liquid-liquid method: 60% ethanol aqueous solution containing 0.25 g β-Ala (β-alanine) and 0.67 g NaOH was slowly dripped into 50 mg C 60The reaction mixture was added to a saturated toluene solution and refluxed at 80°C for 48 hours. After the reaction, the aqueous phase was separated and the solid was precipitated with anhydrous ethanol. After centrifugation, the solid was reconstituted with ultrapure water and dialyzed to obtain a water-soluble AFRM.

[0080] Structural characterization: The morphology of AFRM was characterized by transmission electron microscopy (TEM, HT7700, Hitachi, Japan). The hydration diameter and zeta potential of AFRM were measured at 25 °C by dynamic light scattering (DLS, NanoZS ZEN3600, Malvern, UK). The content of crystalline water in AFRM freeze-dried solid was analyzed by thermogravimetric analysis (TGA, Pyris-1, PerkinElmer, USA) under nitrogen atmosphere. The ratio of C, H and N elements in AFRM freeze-dried solid and the solid surface functional groups were determined by elemental analysis (EA, Flash Smart, Thermo Fisher, USA) and Fourier transform infrared spectroscopy (FTIR, iN10-iZ10, Thermo Fisher, USA).

[0081] Conclusion: Transmission electron microscopy showed that the morphology of AFRM was spherical with a diameter of 24.64±1.88nm; the hydrodynamic diameter of AFRM was 37±0.34nm and the zeta potential was -40.70±0.34mV (see Figure 1 ). Infrared spectrum (see Figure 2 ) in the O(N)-H stretching vibration band (3400-3300cm -1 ) and C=O stretching vibration peak (1700cm -1 ) showed that β-Ala was covalently linked to C 60 The water content and the ratio of C, H, and N elements in AFRM solid were determined by thermogravimetric analysis and elemental analysis, and the average structural formula of AFRM was calculated as: 60 (OH) ～12 (NHCH2CH2COOH) ～5 9H2O (see Figure 2 ).

[0082] Example 2: Broad-spectrum ROS scavenging ability of AFRM

[0083] Experimental Methods: Electron paramagnetic resonance (EPR, E500, Bruker, Germany) was used to evaluate the O2 scavenging ability of AFRM. ·- Activity: KO2-crown ether-DMSO produces O2 ·-BMPO (Dojindo, Japan, B568) was used as a spin trapping agent, and the final concentration of BMPO (50 mM), diethylenetriaminepentaacetic acid (DTPA, 100 μM), KO2-crown-DMSO (10 mM) and various concentrations of AFRM or water were mixed, and the EPR signal was detected after 2 minutes of reaction. A 100 mM KO2-crown-DMSO working solution was prepared as follows: 71.1 mg KO2 and 600 mg 18-crown-6 were dissolved in 10 mL of ultra-dry DMSO until a transparent yellow solution was formed. The peroxidase-like (POD) activity of AFRM was detected using 3,3',5,5'-tetramethylbenzidine (TMB) as a color indicator to determine the ability to scavenge H2O2: TMB (20mM), H2O2 (22mM) and AFRM (200μM) were mixed in 50mM NaAc-HAc buffer solution (pH=4.5), and the color change was observed after 5 minutes of reaction, and the absorbance change was recorded at 652nm every 5 minutes using a microplate reader. The hydroxyl radical scavenging activity of AFRM was evaluated using EPR: DMPO (Dojindo, Japan, D048) was used as a spin trap, 25μL H2O2 (100mM), 10μL DMPO (200mM) and 25μL AFRM of different concentrations were mixed, and then the sample was irradiated with ultraviolet light. After 15s, the EPR signal was collected.

[0084] Conclusion: EPR results show that AFRM exhibits excellent O2 ·- Clearing ability (see Figure 3 AFRM exhibits POD-like enzyme activity, catalyzing the oxidation of TMB by H2O2 to produce H2O and TMB blue oxidation product (see Figure 3 AFRM significantly scavenged ·OH in a concentration-dependent manner (see Figure 3 In conclusion, AFRM itself has an inherent self-cascading ability to remove O2 ·- , H2O2 and ·OH characteristics, and has excellent broad-spectrum effect of removing excess ROS.

[0085] Example 3: AFRM inhibits inflammation in macrophages

[0086] Cells:RAW 264.7 cells.

[0087] Grouping: The blank control group was filled with high-glucose DMEM medium (Gibco, USA, C11995500BT) (complete medium) containing 10% fetal bovine serum (Gibco, USA, 10099141C), the model control group was filled with complete medium containing 100 ng / mL LPS, and the experimental group was filled with complete medium containing 200 μM AFRM and 100 ng / mL LPS.

[0088] Experimental methods: RAW 264.7 cells were cultured using the above complete medium. RAW 264.7 cells were grown to an appropriate density and treated according to group requirements for 24 hours. Then they were stained with 2,7-dichlorofluorescein diethyl ester (DCFH-DA, 10 μM, Beyotime, China, S0033S) for 30 minutes in the dark. Subsequently, the cells were detected by flow cytometry (FCM, Attune NXT, Thermo Fisher, USA) and laser scanning confocal microscopy (CLSM, FV1000-IX81, Olympus, Japan). The expression level of intracellular tumor necrosis factor-α (TNF-α) was analyzed by immunoblotting (WB). The concentration of interleukin-1β (IL-1β) in the cell culture suspension was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Jianglai, China, JL18442). Western Blot analysis was used to detect the expression levels of intracellular Toll-like receptor 4 (TLR4), p65 protein (p65), Nfκb inhibitor α (IκBα), extracellular regulated protein kinase (ERK) and its phosphorylated proteins.

[0089] Conclusion: See Figure 4-7 . Compared with the control group, ROS increased significantly in the model group, and AFRM treatment reduced the level of ROS in inflammatory macrophages. AFRM inhibited the increase in the levels of proinflammatory cytokines TNF-α and IL-1β in inflammatory macrophages. AFRM downregulated the LPS / TLR4 / NF-κB and MAPK / ERK signaling pathways in inflammatory macrophages. In short, AFRM inhibits macrophage inflammation by clearing ROS in inflammatory macrophages and downregulating the LPS / TLR4 / NF-κB and MAPK / ERK signaling pathways.

[0090] Example 4: AFRM inhibits inflammation-induced macrophage pyroptosis

[0091] Cells: Primary bone marrow-derived macrophages (BMDMs).

[0092] Grouping: The blank control group was filled with high-glucose DMEM medium (Gibco, USA, C11995500BT) (complete medium) containing 10% fetal bovine serum (Gibco, USA, 10099141C), the model control group was filled with complete medium containing LPS and ATP, and the experimental group was filled with complete medium containing 200 μM AFRM, LPS and ATP.

[0093] Isolation and culture of BMDMs: Under sterile conditions, take the tibia and femur of 3-week-old C57BL / 6J mice. After sterilization, cut the tibia and femur with scissors to expose the bone marrow. Use a syringe to draw phosphate buffered saline (PBS), blow out the bone marrow cells, pass through a 200-mesh cell strainer, and collect by centrifugation. Resuspend the cells in DMEM complete medium containing 30 ng / mL macrophage colony stimulating factor (M-CSF, Nearshore Protein, China, CK02) to induce bone marrow cells to differentiate into BMDMs. Carry out cell experiments when the cell density is cultured to more than 60%.

[0094] Experimental methods: BMDMs in the model group were stimulated with LPS (1 μg / mL) for 5 h and then with ATP (5 mM, dissolved in serum-free medium) for 1 h to establish a typical inflammasome pathway model of cell pyroptosis. In the experimental group, 200 μM AFRM was incubated with LPS and ATP. Cells were collected and stained with propidium iodide (PI, 200 ng / mL, Beyotime, China, ST1569) in the dark, and the proportion and morphology of pyroptotic cells were detected by flow cytometry (Attune NXT, Thermo Fisher, USA) and laser scanning confocal microscopy (FV1000-IX81, Olympus, Japan), respectively. Cells were stained with DCFH-DA (10 μM) in the dark for 30 min, and the mean fluorescence intensity was measured by flow cytometry (Attune NXT, Thermo Fisher, USA) to determine the intracellular ROS level. The expression levels of gasdermin D (GSDMD) and N-GSDMD (N-terminal domain after cleavage of GSDMD) were analyzed by WB. The concentrations of IL-1β, interleukin 18 (IL-18) and high-mobility group protein B1 (HMGB-1) in the culture supernatant of BMDMs were measured by ELISA kits (Jianglai, China). The enzyme activity of Caspase-1 in BMDMs was determined by the cysteine-containing aspartate proteolytic enzyme 1 (Caspase-1) activity assay kit (Biyuntian, China, C1101).

[0095] Conclusion: See Figure 8-12 . Compared with the blank control group, the cell death rate in the model control group was significantly increased, the cells were swollen, and the intracellular ROS increased. After AFRM treatment, the cell death rate was reduced, the cell swelling was alleviated, and the increase of ROS was inhibited. In addition, AFRM inhibited the activity of Caspase-1 and the activation of GSDMD in the classical inflammasome pathway of cell pyroptosis, and reduced the levels of proinflammatory cytokines IL-1β, IL-18 and late proinflammatory mediator HMGB-1. AFRM can inhibit inflammation-induced macrophage pyroptosis.

[0096] Example 5: AFRM reduces intestinal epithelial barrier damage caused by oxidative damage

[0097] Cells: IEC-6 cells.

[0098] Grouping: The blank control group was composed of high-glucose DMEM medium (Gibco, USA, C11995500BT) (complete medium) containing 10% fetal bovine serum (Gibco, USA, 10099141C), the model control group 1 was composed of complete medium containing H2O2, the model control group 2 was composed of complete medium containing menadione, and the experimental groups were composed of complete medium containing AFRM, H2O2 or menadione.

[0099] Experimental methods: IEC-6 cells were cultured in the above complete medium. IEC-6 cells were induced with exogenous and endogenous oxidative stress using H2O2 and menadione, respectively. IEC-6 cells were cultured with 1.2×10 4 Cells / well were seeded in 96-well plates. After overnight incubation, cells were damaged with H2O2 (600 μM) or menadione (20 μM) for 1 hour. The old culture medium was then discarded, and culture medium containing different concentrations of AFRM was added to treat IEC-6 cells for 6 hours. Cell viability was measured using a CCK-8 kit (Dojindo, Japan, CK04). IEC-6 cells were cultured at 3.6×10 4 Cells were inoculated into 6-well plates and cultured overnight. The cells were damaged with H2O2 (1 mM) or menadione (60 μM) for 1 hour. In the experimental group, cells were incubated with AFRM and H2O2 or menadione. The cells were collected and stained with DCFH-DA (10 μM) in the dark, and the intracellular ROS level was detected by flow cytometry (Attune NXT, Thermo Fisher Scientific, USA). The culture supernatant of the same cell samples was tested for extracellular ROS level using an in vitro ROS / RNS detection kit (Cell Biolabs, USA, STA-347). The expression levels of zonula occludens 1 (ZO-1), collagen 1 (Col 1) and fibronectin (Fn1) in H2O2-induced oxidative damage in IEC-6 cells were analyzed by WB.

[0100] Conclusion: See Figure 13-17 . The results of exogenous oxidative damage induced by H2O2 showed that AFRM, which is mainly distributed in the extracellular microenvironment of epithelial cells, reduced the level of ROS in the extracellular environment and significantly increased cell viability in a concentration-dependent manner. In the endogenous model induced by menadione, AFRM could not improve the cell viability of damaged IEC-6 cells under the same treatment. In the H2O2-induced IEC-6 injury model, AFRM treatment inhibited the decrease in the expression levels of ZO-1, Col 1 and Fn1 caused by oxidative damage. In summary, AFRM can restore collagen, fibronectin and cellular tight junction proteins by regulating extracellular redox homeostasis and protect the integrity of the epithelial cell barrier.

[0101] Example 6: Therapeutic effect of AFRM on endotoxemia mouse model

[0102] Animals and treatment: Male C57BL / 6J mice (wild type, 6 weeks old) were purchased from Beijing Huafukang Bioscience Co., Ltd. (Beijing, China). All mice were housed in a temperature-controlled specific pathogen-free (SPF-grade) environment with a 12-h light and 12-h dark cycle. In addition, all mice were fed with sterile water and standard ordinary food, which was purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China).

[0103] Grouping: blank control group (5 rats, intraperitoneal injection of normal saline, tail vein injection of normal saline), model control group (5 rats, intraperitoneal injection of LPS, tail vein injection of normal saline), experimental group (5 rats, intraperitoneal injection of LPS, tail vein injection of AFRM).

[0104] Experimental protocol for inhibiting inflammation in endotoxemia mice: mice were intraperitoneally injected with LPS (2 mg / kg) to induce endotoxemia, and mice in the blank control group were intraperitoneally injected with normal saline. Half an hour after LPS injection, normal saline or AFRM (3 mM, 200 μL) was injected into the tail vein of endotoxemia mice. Six hours after LPS injection, mice were euthanized. 20 μL of blood was taken in the anticoagulation catheter, and blood routine analysis was performed using an automatic hematology analyzer (BC-5000VET, Mindray, China), and the percentages of neutrophils and lymphocytes were counted. The whole blood was placed at room temperature for 2 hours and centrifuged at 1000 g for 15 minutes to separate the serum. The levels of early proinflammatory cytokines (TNF-α and IL-1β) in serum were measured by ELISA kits (Invitrogen, USA, 88-7324-22 and 88-7013-22). The level of IL-6 in serum was detected using an ELISA kit (Jianglai, China, JL20268).

[0105] Conclusion: See Figure 18-19 . Compared with the blank control group, the percentage of neutrophils in endotoxemic mice was significantly increased, and the percentage of lymphocytes was decreased. In addition, the levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum were significantly increased. After AFRM treatment, the ratio of neutrophils and lymphocytes returned to the normal trend, and the increase in the levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 was inhibited. In conclusion, AFRM inhibits systemic inflammation in endotoxemic mice.

[0106] Experimental protocol for protecting the intestinal epithelial barrier in endotoxemic mice: mice were intraperitoneally injected with LPS (2 mg / kg) to induce endotoxemia, and mice in the blank control group were intraperitoneally injected with saline. Half an hour after LPS injection, saline or AFRM (3 mM, 200 μL) was injected into the tail vein of endotoxemic mice. After 36 hours of treatment, the mice were anesthetized and killed. Ileal tissue was collected. For DHE staining, the collected intestinal tissue was rinsed with pre-cooled saline. The samples were flattened, frozen in liquid nitrogen, embedded in OCT (Sakura, USA), and cut into 8 μm thick slices (CM1950, Leica, Germany) with a freezing microtome and placed on a slide. The samples were washed with PBS and stained with DHE solution in the dark at 37 ° C for 30 minutes. Subsequently, the slides were rinsed with PBS and mounted with anti-fluorescence quenching mounting solution containing DAPI (Biyuntian, China, P0131). For immunofluorescence staining, the intestinal tissue was fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The intestinal samples were cut into 4 μm thick paraffin-embedded tissue sections. The samples were dewaxed and hydrated with xylene and gradient ethanol, respectively. Then, heat-induced antigen retrieval was performed and blocked with bovine serum albumin (BSA) for 30 minutes at room temperature. Next, the sections were incubated with ZO-1 (Proteintech, China, 21773-1-AP) or Occludin primary antibody (Abcam, UK, ab216327) solution at 4 ° C overnight, washed, and incubated with AlexaFluor 488-conjugated goat anti-rabbit IgG antibody (Thermo, USA, A32731) at room temperature for 1 hour. After washing, the sections were mounted with anti-fluorescence quenching mounting solution containing DAPI (Biyuntian, China, P0131). Immunofluorescence imaging was performed using a laser scanning confocal microscope (FV1000-IX81, Olympus, Japan). The ileum was stained with hematoxylin and eosin (H&E), and the mouse ileum tissue was collected and fixed with 4% paraformaldehyde. The intestine was then embedded in paraffin, sliced ​​and dewaxed. Next, the slices were dehydrated with different concentrations of ethanol. Finally, the slices were stained with H&E dye. Histological examination and microscopic imaging were performed using a pathological slice scanner (KF-PRO-005, Jiangfeng Bio, China).

[0107] Conclusion: See Figure 20-22 Compared with the blank control group, the O2 ·- The level increased by about 1.5 times. ·-The level decreased significantly, and there was almost no difference from the blank control group. The expression of ZO-1 and Occludin proteins in the ileum tissue of endotoxemia mice was significantly reduced. After AFRM treatment, the green fluorescence intensity representing ZO-1 or Occludin in intestinal epithelial cells was significantly enhanced, which was almost indistinguishable from the blank control group mice. Pathological sections showed that LPS modeling caused the villi to fall off in the ileum of mice and the epithelial barrier was severely damaged. AFRM treatment inhibited the serious damage of LPS to the ileum tissue of mice.

Claims

1. Use of a water-soluble fullerene nanomaterial in the preparation of a drug for treating and / or alleviating endotoxemia; the water-soluble fullerene nanomaterial has a general structural formula of C 2n (OH) x (Amino Acid) y ;in, 20≤n≤60, 0≤x<50, 0<y; Amino Acid is a water-soluble amino acid.

2. The use according to claim 1, characterized in that: The treatment and / or alleviation of endotoxemia is embodied in the following aspects: 1) Inhibit inflammation caused by endotoxemia; 2) Protect the intestinal epithelial barrier or reduce intestinal epithelial barrier damage caused by endotoxemia.

3. The use according to claim 2, characterized in that: The inhibition of inflammation caused by endotoxemia is embodied in at least one of the following aspects: a1) It inhibited the increase in the percentage of neutrophils caused by endotoxemia and restored the proportion of neutrophils to normal; a2) It inhibited the decrease in lymphocyte percentage caused by endotoxemia and restored the lymphocyte ratio to normal trend; a3) inhibited the increase of pro-inflammatory cytokine levels; a4) inhibit macrophage inflammation; a5) inhibit inflammation-induced macrophage pyroptosis; a6) Eliminate a broad spectrum of reactive oxygen species; Alternatively, the protection of the intestinal epithelial barrier or the alleviation of intestinal epithelial barrier damage caused by endotoxemia is embodied in at least one of the following aspects: b1) Inhibits O2 in the ileum caused by endotoxemia · -Elevated levels; b2) Inhibition of endotoxemia-induced decrease in the expression levels of tight junction protein-related proteins, zonula occludens 1 (ZO-1) and occludin in the ileum.

4. Application of water-soluble fullerene nanomaterials in the preparation of products for inhibiting macrophage inflammation; the water-soluble fullerene nanomaterials have a general structural formula of C 2n (OH) x (Amino Acid) y ;in, 20≤n≤60; 0≤x<50; 0<y, Amino Acid is a water-soluble amino acid.

5. The use according to claim 4, characterized in that: The inhibition of macrophage inflammation is embodied in at least one of the following aspects: 1) Inhibited the increase of pro-inflammatory cytokines TNF-α and IL-1β in inflammatory macrophages; 2) downregulate LPS / TLR4 / NF-κB and MAPK / ERK signaling pathways in inflammatory macrophages; 3) Reduced the level of ROS in inflammatory macrophages.

6. Application of water-soluble fullerene nanomaterials in the preparation of products for inhibiting inflammation-induced macrophage pyroptosis; The water-soluble fullerene nanomaterial has a general structural formula of C 2n (OH) x (Amino Acid) y ;in, 20≤n≤60, 0≤x<50, 0<y; Amino Acid is a water-soluble amino acid.

7. The use according to claim 6, characterized in that: The inhibition of inflammation-induced macrophage pyroptosis is embodied in at least one of the following aspects: 1) Reduced the proportion of macrophage death and inhibited the increase of ROS in pyroptotic cells; 2) Inhibited the activity of Caspase-1 and the activation of GSDMD in the classical inflammasome pathway of cell pyroptosis; 3) Reduced the levels of proinflammatory cytokines IL-1β, IL-18 and late proinflammatory mediator HMGB-1.

8. Use of water-soluble fullerene nanomaterials in the preparation of products for reducing intestinal epithelial barrier damage caused by oxidative damage; the water-soluble fullerene nanomaterials have a general structural formula of C 2n (OH) x (Amino Acid) y ;in, 20≤n≤60, 0≤x<50, 0<y; Amino Acid is a water-soluble amino acid.

9. The use according to claim 8, characterized in that: The reduction of intestinal epithelial barrier damage caused by oxidative damage is embodied in at least one of the following aspects: 1) Reduce the level of ROS in the extracellular environment and thus reduce the level of ROS in cells; 2) Inhibited the decrease in the expression levels of zonula occludens 1 (ZO-1), collagen 1 (Col 1) and fibronectin (Fn1) caused by oxidative damage.

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