Application of squalene and pharmaceutical composition thereof in preparation of medicine for preventing or treating endotoxemia

Abstract

The invention discloses an application of squalene or a pharmaceutically acceptable salt thereof in preparation of a medicine for preventing or treating endotoxemia. Experiments show that squalene significantly relieves LPS-induced endotoxemia liver injury through a multi-target mechanism, squalene not only relieves inflammatory response by inhibiting release of inflammatory factors, but also relieves oxidative stress by activating an antioxidant pathway, and the squalene has dual action mechanisms. The invention further provides application of the squalene-containing composition to preparation of the medicine for treating endotoxemia, squalene and glucocorticoid show a synergistic effect in the aspect of inhibiting TNF-alpha, AST can be reduced more remarkably by combining squalene and glucocorticoid, and the effect of dual inhibition of inflammatory mediators is achieved. Besides, the composition of the inducible nitric oxide synthase and squalene is applied to the preparation of the medicine for treating endotoxemia, and the inducible nitric oxide synthase inhibitor can inhibit the activity of iNOS, so that the excessive generation of nitric oxide is reduced, and the inflammatory reaction is further inhibited synergistically.

Description

Technical Field

[0001] The present invention belongs to the technical fields of pharmacology and medicinal chemistry, and particularly relates to the use of squalene and its pharmaceutical compositions in the preparation of drugs for preventing or treating endotoxemia. Background Art

[0002] Endotoxemia is a systemic inflammatory response syndrome caused by the over-activation of the host immune system by lipopolysaccharide (LPS) released by Gram-negative bacteria, often accompanied by multiple organ dysfunction and high mortality. The liver, as the core organ for endotoxin clearance, its injury is a key link in the pathological process of endotoxemia. LPS activates the Toll-like receptor 4 (TLR4) / nuclear factor κB (NF-κB) signaling pathway, induces the explosive release of pro-inflammatory factors (such as IL-1β, TNF-α), and triggers oxidative stress reactions, resulting in hepatocyte lipid peroxidation and mitochondrial dysfunction. This not only causes systemic inflammatory responses, but also disrupts the homeostasis in the brain, induces neuroinflammatory responses. This kind of neuroinflammation will cause the over-activation of immune cells in the brain and release a large number of inflammatory factors, which will further affect synaptic plasticity and neuronal function, etc.

[0003] The application of antibiotics is the main current treatment plan for endotoxemia, which can partially relieve the infection symptoms, but for some severe inflammatory reactions, its treatment effect is not ideal. In addition, the regulatory effect of antibiotics on the inflammatory cascade and oxidative stress is limited, and long-term use is likely to cause drug resistance and immunosuppression. Therefore, developing a new treatment strategy with both anti-inflammatory and antioxidant functions has important clinical significance. Summary of the Invention

[0004] In order to overcome the problems existing in the above-mentioned prior art, one of the purposes of the present invention is to provide the use of squalene or its pharmaceutically acceptable salts. Another purpose of the present invention is to provide the use of a pharmaceutical composition. A third purpose of the present invention is to provide the use of a pharmaceutical composition.

[0005] Squalene (SQ) is a natural triterpenoid compound widely present in the livers of deep-sea fish and plant oils. Research has shown that squalene enhances the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) and reduces the level of malondialdehyde (MDA) by activating the Keap1-Nrf2-ARE pathway, thereby effectively alleviating oxidative stress damage. In addition, its unique all-trans double bond structure can inhibit NF-κB signal transduction and reduce the release of pro-inflammatory factors such as IL-1β and TNF-α. However, the current systematic research on squalene in endotoxemia is still relatively scarce, and its dose-effect relationship, combination drug potential, and molecular mechanism are not yet clear. The present invention applies squalene to drugs for treating endotoxemia and discovers through experiments that squalene has a good effect in treating endotoxemia, expanding the new functions of squalene.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of squalene or a pharmaceutically acceptable salt thereof in any one of a)-b):

[0008] a) Use in the preparation of a drug for preventing or treating endotoxemia;

[0009] b) Use in the preparation of a drug for treating diseases caused by endotoxemia.

[0010] Squalene is mainly derived from shark liver oil, and its content is as high as 95%. Shark liver oil accounts for more than 83% of the mass of deep-sea shark livers. Squalene is a diterpenoid compound composed of 6 non-conjugated double bonds and has strong antioxidant activity. Its antioxidant mechanism lies in that the low ionization threshold of squalene enables it to both provide and receive electrons without destroying the molecular structure of cells.

[0011] Preferably, the pharmaceutically acceptable salt includes at least one of metal salts, ammonium salts, salts formed with inorganic acids, salts formed with organic bases, salts formed with organic acids, salts formed with basic amino acids, and salts formed with acidic amino acids.

[0012] More preferably, the metal salt includes an alkali metal salt and an alkaline earth metal salt. More preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt. More preferably, the alkaline earth metal salt includes at least one of a calcium salt, a magnesium salt, a barium salt, and an aluminum salt. More preferably, the salt formed with an organic base includes a salt formed with at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine. More preferably, the salt formed with an inorganic acid includes a salt formed with at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid. More preferably, the salt formed with an organic acid includes a salt formed with at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid. More preferably, the salt formed with a basic amino acid includes a salt formed with at least one of the following basic amino acids: arginine, lysine, ornithine. More preferably, the salt formed with an acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid, glutamic acid.

[0013] Preferably, the disease caused by endotoxemia is sepsis.

[0014] Preferably, the drug further includes a pharmaceutically acceptable excipient.

[0015] More preferably, the excipient includes at least one of a desiccant, a stabilizer, an antioxidant, a disintegrant, a lubricant, a colorant, and a dispersant.

[0016] Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins such as human serum albumin; buffering substances such as phosphates; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silicon; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polypropylene-block polymers; lanolin; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol; phosphate buffer solution; and other non-toxic and suitable lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; releasing agents; preservatives and antioxidants.

[0017] Preferably, the drug includes an oral preparation or an injection preparation.

[0018] More preferably, the dosage forms of the oral preparation include granules, tablets, powders, capsules, oral solutions, oral suspensions, dry suspensions, oral emulsions, mucilages, oral liquids, emulsions, colloidal solutions, mixtures, tinctures, drops, suspension drops, syrups, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, sustained-release capsules, controlled-release capsules.

[0019] More preferably, the dosage forms of the injection preparation include injections, injection solutions, injection solutions for injection, injection solutions for intravenous drip, injection suspensions, sterile powders for injection, intravenous injection preparations, injection emulsions, emulsion injections, powder injections, injection preparations, sterile powder injections, lyophilized powder injections, concentrated solutions for injection.

[0020] The second aspect of the present invention provides an application of a pharmaceutical composition in any one of c)-d):

[0021] c) Application in the preparation of a drug for preventing or treating endotoxemia;

[0022] d) Application in the preparation of a drug for treating diseases caused by endotoxemia;

[0023] The pharmaceutical composition includes squalene or a pharmaceutically acceptable salt thereof, and a glucocorticoid.

[0024] Preferably, the mass ratio of squalene to glucocorticoid is 100:(0.1-5).

[0025] Preferably, the glucocorticoid includes at least one of dexamethasone, betamethasone, triamcinolone acetonide, fluticasone, budesonide, clobetasol propionate, and hydrocortisone butyrate.

[0026] Preferably, squalene or a pharmaceutically acceptable salt thereof is used as the active ingredient.

[0027] More preferably, squalene or a pharmaceutically acceptable salt thereof is used as the sole active ingredient.

[0028] Preferably, the drug further includes a pharmaceutically acceptable excipient. The definition of the excipient is as described in the first aspect.

[0029] The third aspect of the present invention provides an application of a pharmaceutical composition in any one of e)-f):

[0030] e) Application in the preparation of a drug for preventing or treating endotoxemia;

[0031] f) Application in the preparation of a drug for treating a disease caused by endotoxemia;

[0032] The pharmaceutical composition includes squalene or a pharmaceutically acceptable salt thereof and an inducible nitric oxide synthase inhibitor.

[0033] Preferably, the mass ratio of squalene to the inducible nitric oxide synthase inhibitor is 100:(8-20).

[0034] Preferably, the inducible nitric oxide synthase inhibitor includes at least one of aminoguanidine, 1400W, L-NIL, and GW274150.

[0035] Preferably, squalene or a pharmaceutically acceptable salt thereof is used as the active ingredient.

[0036] More preferably, squalene or a pharmaceutically acceptable salt thereof is used as the sole active ingredient.

[0037] Preferably, the drug further includes a pharmaceutically acceptable excipient. The definition of the excipient is as described in the first aspect.

[0038] The beneficial effects of the present invention are:

[0039] 1. The present invention proposes the application of squalene in the preparation of drugs for treating endotoxemia. Experiments of the present invention show that squalene significantly alleviates LPS-induced liver injury in murine endotoxemia through a multi-target mechanism, not only by inhibiting the release of inflammatory factors to reduce the inflammatory response, but also by activating the antioxidant pathway to relieve oxidative stress, with a dual mechanism of action. On the one hand, squalene intervention significantly reduces the serum ALT / AST levels (P<0.05), suggesting that it protects hepatocytes by inhibiting lipid peroxidation of the liver cell membrane, enhancing the activities of GSH-PX and SOD, scavenging excessive free radicals induced by LPS (MDA significantly decreases) and mitochondrial dysfunction. This effect may be related to its activation of the Keap1-Nrf2-ARE pathway, which enhances the endogenous antioxidant defense ability by upregulating GSH-PX (significantly increased) and T-SOD (significantly elevated); on the other hand, squalene can inhibit the TLR4 / NF-κB signal transduction, reduce the release of pro-inflammatory factors IL-1β and TNF-α (significantly decreased respectively), and simultaneously upregulate the anti-inflammatory factor IL-10 (significantly increased), thereby remodeling the balance of the inflammatory microenvironment. Therefore, developing squalene into a drug for treating endotoxemia has great application potential.

[0040] 2. The present invention also proposes the application of a composition containing squalene in the preparation of drugs for treating endotoxemia. Among them, squalene and glucocorticoids show a synergistic effect in inhibiting TNF-α. The combination can make the decrease of AST more significant, achieving the effect of double inhibition of inflammatory mediators, significantly enhancing the anti-inflammatory and antioxidant effects, reducing the dosage of glucocorticoids, and reducing the risk of immunosuppression. In addition, the application of the composition of inducible nitric oxide synthase (iNOS) and squalene in the preparation of drugs for treating endotoxemia. The inducible nitric oxide synthase inhibitor can inhibit the activity of iNOS, thereby reducing the excessive production of nitric oxide (NO), and further synergistically inhibiting the inflammatory response. The compound composition provided by the present invention shows a significant synergistic effect in the prevention and treatment of endotoxemia, can reduce the dosage of a single drug, and reduce the risk of side effects, with high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the experimental procedure for modeling and administering drugs for murine endotoxemia;

[0042] Figure 2 is the pathological morphological observation of liver tissues after modeling in mice; among them, (A) is the normal group, and (B) is the model group; magnification ×100, scale bar: 100μm;

[0043] Figure 3 is the content of ALT in mice of each group after modeling on the 6th day;

[0044] Figure 4The content of AST in mice of each group after modeling on the 6th day;

[0045] Figure 5 The content of ALT in mice of each group after modeling on the 7th day;

[0046] Figure 6 The content of AST in mice of each group after modeling on the 7th day;

[0047] Figure 7 The content of IL-1β in the liver tissues of mice of each group after modeling on the 6th day;

[0048] Figure 8 The content of IL-10 in the liver tissues of mice of each group after modeling on the 6th day;

[0049] Figure 9 The content of TNF-α in the liver tissues of mice of each group after modeling on the 6th day;

[0050] Figure 10 The content of IL-1β in the liver tissues of mice of each group after modeling on the 7th day;

[0051] Figure 11 The content of IL-10 in the liver tissues of mice of each group after modeling on the 7th day;

[0052] Figure 12 The content of TNF-α in the liver tissues of mice of each group after modeling on the 7th day;

[0053] Figure 13 The content of GSH-PX in the liver tissues of mice of each group after modeling on the 6th day;

[0054] Figure 14 The content of SOD in the liver tissues of mice of each group after modeling on the 6th day;

[0055] Figure 15 The content of MDA in the liver tissues of mice of each group after modeling on the 6th day;

[0056] Figure 16 The content of GSH-PX in the liver tissues of mice of each group after modeling on the 7th day;

[0057] Figure 17 The content of SOD in the liver tissues of mice of each group after modeling on the 7th day;

[0058] Figure 18 The content of MDA in the liver tissues of mice of each group after modeling on the 7th day;

[0059] Note: *P < 0.05 compared with the control group; compared with the model group # P < 0.05; compared with the medium-dose prevention and treatment group ▲ P < 0.05. Detailed implementation manners

[0060] The content of the present invention will be further described in detail through specific embodiments below. The raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and separated by simple synthesis unless otherwise specified; the processes adopted, unless otherwise specified, are all conventional processes in the art.

[0061] Example 1

[0062] 1. Experimental supplies and preparations before the experiment

[0063] (1) Experimental animals: 48 male KM mice, weighing 20 - 22 g, purchased from Zhuhai Bestong Biotechnology Co., Ltd. [License number: SCXK(Yue)2020 - 0051], and were housed separately in cages for three days before the experiment, during which they were allowed free movement and diet.

[0064] (2) Experimental reagents are shown in Table 1

[0065] Table 1 Batch numbers and manufacturers of reagents required for the experiment

[0066]

[0067]

[0068] (3) Reagent preparation: Squalene low - dose solution: Take 0.83 mL of squalene solution and add 49.17 mL of soybean oil solution to prepare a solution with a concentration of 0.02 g / mL;

[0069] Dexamethasone solution: Take 1 mL of dexamethasone sodium phosphate injection and add 0.9% sodium chloride solution to 33.3 mL to prepare a solution with a concentration of 0.15 mg / mL;

[0070] Aminoguanidine solution: Weigh 400 mg of AG powder and dissolve it in 8 mL of 0.9% sodium chloride solution to prepare a solution with a final concentration of 50 mg / mL;

[0071] LPS solution: Take 1 mg of lipopolysaccharide powder and add 1 mL of 0.9% sodium chloride solution to prepare a solution with a concentration of 1 mg / mL.

[0072] (4) Grouping of experimental animals and administration methods are shown in Table 2:

[0073] Table 2 Grouping of experimental animals and administration methods

[0074]

[0075] (5) The method for establishing the endotoxemia model is as follows:

[0076] On the 6th day, 2 - 3 hours after administration, except for the control group, the remaining mice were intraperitoneally injected with LPS solution at a single dose of 10 mg / kg to establish an endotoxemia model. If the mice showed symptoms such as huddling due to fear of cold, curling up listlessly, not liking to move, being slow to respond, having dull and expressionless eyes, eye secretions, reduced drinking and eating, diarrhea, difficulty in defecation, feces blocking the anus, and piloerection, it was regarded as successful preparation of the LPS-induced endotoxemia mouse administration method.

[0077] (6) The procedures for experimental animal modeling and administration are as Figure 1 shown, and the specific steps are as follows:

[0078] Normal control group: Intraperitoneally inject 0.5 mL of normal saline once a day for 7 consecutive days. No modeling treatment was carried out in the middle. After the injection of normal saline for mice on the 6th day, 3 animals were sacrificed immediately in the afternoon to collect blood, freeze various fresh tissues, and fix various fresh tissues with paraformaldehyde. On the morning of the 7th day, continue to intraperitoneally inject 0.5 mL of normal saline for 1 treatment. 3 - 5 hours after the treatment, that is, in the afternoon of the same day, the remaining 3 animals were sacrificed to collect blood and freeze 2 portions of various fresh tissues and fix 2 portions of various fresh tissues with paraformaldehyde.

[0079] Model group: Intraperitoneally inject 0.5 mL of normal saline once a day for 6 consecutive days. 2 hours after the injection of normal saline on the 6th day, modeling administration was carried out. 3 hours after the modeling administration, 3 animals were sacrificed immediately according to the above method. On the morning of the 7th day (the 2nd day after the end of modeling), continue to intraperitoneally inject 0.5 mL of normal saline for 1 treatment. 3 - 5 hours after the treatment, that is, in the afternoon of the same day, the remaining 3 animals were sacrificed according to the above method.

[0080] Squalene medium-dose preventive administration group: Gavage at a dose of 0.2 g / kg body weight once a day for 6 consecutive days before modeling. 2 hours after the gavage administration on the 6th day, modeling administration was carried out according to the pre-experiment conditions. 3 hours after the modeling administration, 3 animals were sacrificed immediately according to the above method. On the morning of the 7th day (the 2nd day after the end of modeling), intraperitoneally inject 0.5 ml of normal saline for 1 treatment. 3 - 5 hours after the treatment, that is, in the afternoon of the same day, the remaining 3 animals were sacrificed according to the above method.

[0081] Squalene low-dose / medium-dose / high-dose preventive and treatment groups: Gavage at doses of 0.1 g / kg, 0.2 g / kg, and 0.4 g / kg body weight once a day for 6 consecutive days before modeling. 1 - 2 hours after the gavage administration on the 6th day, modeling administration was carried out. 3 hours after the modeling administration, 3 animals in each group were sacrificed immediately according to the above method. On the morning of the 7th day (the 2nd day after the end of modeling), each group was given a treatment once. 3 - 5 hours after the treatment, that is, in the afternoon of the same day, the remaining animals in each group were sacrificed according to the above method.

[0082] Squalene medium dose + dexamethasone prevention and treatment group: Squalene was intragastrically administered at a dose of 0.2 g / kg body weight, once a day. At the same time, dexamethasone was intraperitoneally injected at a dose of 1.5 mg / kg, once a day. Before modeling, the two drugs of squalene medium dose + dexamethasone were continuously administered for 6 days. After the combined administration on the 6th day, modeling administration was carried out after an interval of 1 - 2 h. 3 h after the modeling administration, 3 animals were immediately sacrificed according to the above method. On the morning of the 7th day (i.e., the 2nd day after the end of modeling), the two drugs of squalene medium dose + dexamethasone were continued to be administered for treatment once. 3 - 5 h after the treatment, the remaining 3 animals were sacrificed according to the above method on the same afternoon.

[0083] Squalene medium dose + aminoguanidine prevention and treatment group: Squalene was intragastrically administered at a dose of 0.2 g / kg body weight, once a day. At the same time, aminoguanidine was intravenously injected at a dose of 25 mg / kg, once a day. Before modeling, the two drugs of squalene medium dose + aminoguanidine were continuously administered for 6 days. After the combined administration on the 6th day, modeling administration was carried out after an interval of 1 - 2 h. 3 h after the modeling administration, 3 animals were immediately sacrificed according to the above method. On the morning of the 7th day (i.e., the 2nd day after the end of modeling), the two drugs of squalene medium dose + aminoguanidine were continued to be administered for treatment once. 3 - 5 h after the treatment, the remaining 3 animals in each group were sacrificed according to the above method on the same afternoon.

[0084] 2. Index test methods

[0085] (1) After sacrificing the mice, the liver tissue was immediately taken out and stored at -80 °C. It was taken out when tested. Before detection, the liver tissue needed to be cut and weighed first. Then an appropriate amount of PBS (PH 7.4) was added. Then it was quickly refrigerated in liquid nitrogen for standby. After the sample melted, it still needed to be stored at a temperature of 2 - 8 °C. An appropriate amount of PBS (PH 7.4) was added, and the sample was homogenized evenly using a homogenizer. The centrifugation time was about 20 min (2000 - 3000 rpm / min). The supernatant after centrifugation was collected and aliquoted. One aliquot was used for detection, and the rest was refrigerated for standby. The contents of IL-1β, IL-10, TNF-α, T-SOD, MDA, and GSH-PX in the liver tissue were detected by enzyme-linked immunosorbent assay (ELISA). The specific operation was carried out strictly according to the instructions of the relevant kit.

[0086] (2) Prepare pathological sections by conventional HE staining method and observe the pathological changes of the liver under a light microscope.

[0087] (3) Statistical analysis was performed using SPSS 26.0 statistical software, and graphs were plotted using GraphPad Prism 10.0 software. One-way ANOVA was used for paired comparison analysis. Measurement data conformed to a normal distribution, and experimental data were expressed as "mean ± standard deviation". The t-test was used for comparison between two groups; one-way ANOVA was used for comparison among multiple groups. Post hoc tests were performed based on the results of the homogeneity of variance test. If the P value of the homogeneity of variance test was > 0.05, the LSD method was used for post hoc tests; when P < 0.05, Tamhane's T2 (heterogeneous variance) was used for analysis. The criterion for statistical significance was P < 0.05.

[0088] 3. Experimental Results

[0089] (1) Pathological changes after mouse modeling

[0090] After mouse modeling, the pathological morphology of liver tissue was observed as Figure 2 shown. In the normal group, the hepatocytes were arranged regularly, of normal size, with no obvious hemorrhage, necrosis, or inflammatory cell infiltration; in the model group, a large number of hepatocyte edema was visible around the central vein, some hepatocytes were necrotic, and scattered macrophages and increased KUFFER cells were observed.

[0091] (2) Contents of ALT and AST in mice on the 6th day after modeling

[0092] The contents of ALT and AST in mice on the 6th day after modeling were as Figure 3 and Figure 4 shown. The specific data are shown in Table 3.

[0093] Compared with the normal group, the ALT and AST in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone group, and medium-dose + aminoguanidine group were significantly increased (P < 0.05), indicating abnormal liver function in the LPS experimental group.

[0094] Compared with the model group, the ALT and AST in the medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone group, and medium-dose + aminoguanidine group were significantly decreased (P < 0.05), suggesting a preventive effect in the medicated groups.

[0095] Compared with the medium-dose prevention and treatment group, the AST in the medium-dose + dexamethasone group was significantly decreased (P < 0.05), suggesting a better preventive effect of combined medication.

[0096] Table 3 Contents of ALT and AST in mice on the 6th day after modeling

[0097]

[0098]

[0099] Note: *P < 0.05 compared with the normal group; compared with the model group # P < 0.05; compared with the medium-dose prevention and treatment group ▲ P < 0.05.

[0100] (3) Contents of ALT and AST in mice after modeling on the 7th day

[0101] The contents of ALT and AST in mice after modeling on the 7th day were as Figure 5 and Figure 6 shown. The specific data are shown in Table 4.

[0102] Compared with the normal group, ALT and AST in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone group, and medium-dose + aminoguanidine group were significantly increased (P < 0.05), indicating abnormal liver function in the LPS experimental group.

[0103] Compared with the model group, ALT and AST in the medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone group, and medium-dose + aminoguanidine group were significantly decreased (P < 0.05), suggesting a therapeutic effect in the drug-treated groups.

[0104] Compared with the medium-dose prevention and treatment group, AST in the medium-dose + dexamethasone group was significantly decreased (P < 0.05), which was statistically significant, suggesting a better therapeutic effect of the combination of squalene and dexamethasone.

[0105] Table 4 Contents of ALT and AST in mice after modeling on the 7th day

[0106] Group ALT AST Normal group (A) 26±2.65 113.3±3.76 Model group (B) <![CDATA[93.03±8.72 * > <![CDATA[457.7±19.51 * > Medium-dose prophylaxis (C) <![CDATA[76.63±13.35 * > <![CDATA[374.8±18.67 * > Low-dose pretreatment (D) <![CDATA[95.07±4.85 * > <![CDATA[320.5±13.23 * > Medium-dose pretreatment (E) <![CDATA[64.1±5.25 *# > <![CDATA[324±27.58 *# > High-dose pretreatment (F) <![CDATA[53.87±8.17 *# > <![CDATA[278.1±12.31 *# > Medium-dose + dexamethasone (G) <![CDATA[47.97±2.68 *# > <![CDATA[196±31.86 *#▲ > Medium-dose + aminoguanidine (H) <![CDATA[50.43±9.21 *# > <![CDATA[194.2±6.34 *# >

[0107] Note: Compared with the normal group * P < 0.05; compared with the model group # P < 0.05; compared with the medium-dose prevention and treatment group ▲ P < 0.05.

[0108] (4) Contents of IL-1β, IL-10, and TNF-α in the liver tissue of mice after modeling on the 6th day

[0109] The contents of IL-1β, IL-10, and TNF-α in mice after modeling on the 6th day were respectively as Figures 7 - 9 shown. The specific data are shown in Table 5.

[0110] The content of inflammatory factors in cells stimulated by LPS for 3 - 5 h was detected by ELISA method. The results showed that compared with the normal group, the levels of IL-1β in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly increased (P<0.05); there were no significant changes in IL-10 between the model group and other drug treatment groups, and there was no statistical significance; the levels of TNF-α in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly increased (P<0.05), indicating that an inflammatory response occurred in the LPS experimental group.

[0111] Compared with the model group, the levels of IL-1β in the medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly decreased (P<0.05); there were no significant changes in IL-10 in other drug treatment groups, and there was no statistical significance; the levels of TNF-α in the medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly decreased (P<0.05), indicating that the drug treatment groups had preventive effects.

[0112] Compared with the medium-dose prevention and treatment group, there were no significant differences in IL-1β, IL-10, and TNF-α among other drug treatment groups, and there was no statistical significance.

[0113] Table 5 Contents of IL-1β, IL-10, and TNF-α in the liver tissues of mice after modeling on the 6th day

[0114] Group IL-1β IL-10 TNF-α Normal group (A) 40.75±5.75 36.38±13.34 165.78±29.63 Model group (B) <![CDATA[99.04±7.16 * > 23.69±4.65 <![CDATA[466.15±25.25 * > Medium-dose prophylaxis (C) <![CDATA[86.99±6.47 * > 25.67±7.58 <![CDATA[388.03±50.74 * <!-- 8 -->]]> Low-dose pretreatment (D) <![CDATA[86.55±6.12 * > 25.1±3.25 <![CDATA[385.48±21.24 * > Medium-dose pretreatment (E) <![CDATA[63.35±5.85 # > 37.06±3.87 <![CDATA[336.69±66.85 *# > High-dose pretreatment (F) <![CDATA[54.03±16.9 # > 35.75±2.54 <![CDATA[317.46±47.18 *# > Medium-dose + dexamethasone (G) <![CDATA[64.86±3.76 *# > 32.14±0.48 <![CDATA[286.11±50.60 *# > Medium-dose + aminoguanidine (H) <![CDATA[73.56±7.02 *# > 37.73±1.83 <![CDATA[315.4±19.99 *# >

[0115] Note: Compared with the normal group * P<0.05; compared with the model group # P<0.05; compared with the medium-dose prevention and treatment group ▲ P<0.05.

[0116] (5) Contents of IL-1β, IL-10, and TNF-α in the liver tissues of mice after modeling on the 7th day

[0117] The contents of IL-1β, IL-10, and TNF-α in mice after modeling on the 7th day were respectively as Figures 10 - 12 shown, and the specific data are shown in Table 6.

[0118] The content of inflammatory factors in cells stimulated with LPS for 24 h was detected by ELISA method. The results showed that, compared with the normal group, the levels of IL-1β in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly increased (P<0.05); the levels of IL-10 and TNF-α in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly decreased (P<0.05), indicating that there was still an inflammatory response in the LPS experimental group after 24 h.

[0119] Compared with the model group, the levels of IL-1β in the high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly decreased (P<0.05); the levels of IL-10 in the medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly increased (P<0.05); the levels of TNF-α in the medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly decreased (P<0.05), indicating that there was a therapeutic effect in the medicated groups.

[0120] Compared with the medium-dose prevention and treatment group, the level of IL-1β in the medium-dose + dexamethasone prevention and treatment group was significantly decreased (P<0.05); there were no significant changes in IL-10 between the single-drug administration groups and the combined-drug administration groups at other doses, and there was no statistical difference; the levels of TNF-α in the medium-dose + dexamethasone prevention and treatment group and medium-dose + aminoguanidine prevention and treatment group were significantly decreased (P<0.05), indicating that the combined-drug administration group had a better therapeutic effect than the single-drug administration group.

[0121] Table 6 Contents of IL-1β, IL-10, and TNF-α in the liver tissues of mice after modeling on the 7th day

[0122] Group IL-1β IL-10 TNF-α Normal group (A) 41.6±4.3 62.28±1.47 158.5±15.78 Model group (B) <![CDATA[92.02±4.64 * > <![CDATA[28.6±2.76 * > <![CDATA[467.5±23.87 * > Medium-dose prophylaxis (C) <![CDATA[82.97±3.81 * > <![CDATA[35.38±6.57 * > <![CDATA[347.3±11.53 *# > Low-dose pretreatment (D) <![CDATA[78.92±7.98 * > <![CDATA[27.65±6.42 * > <![CDATA[358±18.97 *# > Medium-dose pretreatment (E) 75.67±9.1 <![CDATA[39.98±4.18 *# > <![CDATA[345.4±25.14 *# > High-dose pretreatment (F) <![CDATA[74.15±8.97 # > <![CDATA[41.49±0.62 *# > <![CDATA[317.5±25.00 *# > Medium-dose + dexamethasone (G) <![CDATA[54.41±4.12 *#▲ > <![CDATA[42.5±3.67 *# > <![CDATA[261.9±8.95 *#▲ > Medium-dose + aminoguanidine (H) <![CDATA[65.76±3.73 *# > <![CDATA[40.22±1.22 *# > <![CDATA[238.7±15.37 *#▲ >

[0123] Note: Compared with the normal group * P<0.05; compared with the model group # P<0.05; compared with the medium-dose prevention and treatment group ▲ P<0.05.

[0124] (6) Contents of GSH-PX, SOD, and MDA in the liver tissues of mice after modeling on the 6th day

[0125] The contents of GSH-PX, SOD, and MDA in the liver tissues of mice after modeling on the 6th day were as Figures 13 - 15 shown, and the specific data are shown in Table 7.

[0126] The oxidative stress level of cells stimulated by LPS for 3 - 5 h was detected by ELISA method. The results showed that compared with the normal group, the GSH-PX in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, and medium-dose + dexamethasone prevention and treatment group was significantly decreased (P<0.05); the T-SOD in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group was significantly decreased (P<0.05), and the MDA was significantly increased (P<0.05); indicating that an inflammatory response occurred in the LPS experimental group.

[0127] Compared with the model group, the GSH-PX in the medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group was significantly increased (P<0.05); the T-SOD in the medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group was significantly increased (P<0.05), and the MDA was significantly decreased (P<0.05); indicating that there was a preventive effect in the medicated groups.

[0128] Compared with the medium-dose prevention and treatment group, there were no significant changes in GSH-PX, T-SOD, and MDA in the single-dose administration groups and combined medication groups with other doses, and there was no statistical difference.

[0129] Table 7 Contents of GSH-PX, SOD, and MDA in the liver tissues of mice after modeling on the 6th day

[0130] Group GSH-PX SOD MDA Normal group (A) 1 8.56±0.26 81.76±2.11 2.8±0.17 Model group (B) <![CDATA[11.16±0.37 * > <![CDATA[26.82±3.87 * > <![CDATA[8.10±0.78 * > Medium-dose prophylaxis (C) <![CDATA[12.62±1.39 * > <![CDATA[49.46±4.43 *# > <![CDATA[5.76±0.66 *# > Low-dose pretreatment (D) <![CDATA[12.7±1.35 * > <![CDATA[49.35±8.00 *# > <![CDATA[5.79±0.11 *# > Medium-dose pretreatment (E) <![CDATA[14.89±0.17 *# > <![CDATA[53.25±5.47 *# > <![CDATA[5.68±0.57 *# > High-dose pretreatment (F) <![CDATA[15.77±0.12 # > <![CDATA[54.03±7.62 *# > <![CDATA[5.75±1.62 *# > Medium-dose + dexamethasone (G) <![CDATA[14.33±0.93 *# > <![CDATA[51.5±5.74 *# > <![CDATA[5.32±0.51 *# > Medium-dose + aminoguanidine (H) <![CDATA[15.85±2.04 # > <![CDATA[57.97±14.35 *# > <![CDATA[5.12±0.52 *# >

[0131] Note: *P<0.05 compared with the normal group; compared with the model group # P<0.05; compared with the medium-dose prevention and treatment group ▲ P<0.05.

[0132] (7) Contents of GSH-PX, SOD, and MDA in the liver tissues of mice after modeling on the 7th day

[0133] The contents of GSH-PX, SOD, and MDA in the liver tissues of mice after modeling on the 7th day were respectively as Figures 16 - 18 shown, and the specific data are shown in Table 8.

[0134] The oxidative stress level was detected by ELISA method after LPS-stimulated cells for 24 h. The results showed that compared with the normal group, GSH-PX and T-SOD in the model group, medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group were significantly decreased (P<0.05); MDA in the model group and low-dose prevention and treatment group was significantly increased (P<0.05); it indicated that there was still an inflammatory response in the LPS experimental group after 24 h.

[0135] Compared with the model group, GSH-PX in the medium-dose prevention group, low-dose prevention and treatment group, medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group was significantly increased (P<0.05); T-SOD in the medium-dose prevention and treatment group, high-dose prevention and treatment group, medium-dose + dexamethasone prevention and treatment group, and medium-dose + aminoguanidine prevention and treatment group was significantly increased (P<0.05); MDA in other medicated groups had no significant change, without statistical difference.

[0136] Compared with the medium-dose prevention and treatment group, GSH-PX, T-SOD, and MDA in other single-dose and combined medication groups had no significant change, without statistical difference.

[0137] Table 8 Contents of GSH-PX, SOD, and MDA in liver tissues of mice after modeling on the 7th day

[0138] Group GSH-PX SOD MDA Normal group (A) 19.31±0.31 95.05±1.09 4.68±0.58 Model group (B) <![CDATA[8.757±1.13 * > <![CDATA[40.76±6.81 * > <![CDATA[7.89±0.38 * > Medium-dose prophylaxis (C) <![CDATA[13.73±0.72 *# > <![CDATA[55.01±6.01 * > 6.37±1.04 Low-dose pretreatment (D) <![CDATA[13.11±0.70 *# > <![CDATA[56.68±2.79 * > <![CDATA[7.39±0.43 * > Medium-dose pretreatment (E) <![CDATA[14.21±1.52 *# > <![CDATA[58.85±3.02 *# > 6.45±0.6 High-dose pretreatment (F) <![CDATA[14.01±1.96 *# > <![CDATA[54.92±2.09 *# > 6.22±0.62 Medium-dose + dexamethasone (G) <![CDATA[13.42±1.82 *# > <![CDATA[59.26±1.09 *# > 5.95±0.76 Medium-dose + aminoguanidine (H) <![CDATA[14.45±1.39 *# > <![CDATA[66.47±6.03 *# > 6.26±1.06

[0139] Note: *P<0.05 compared with the normal group; compared with the model group # P<0.05; compared with the medium-dose prevention and treatment group ▲ P<0.05.

[0140] 4. Summary of experimental results

[0141] This invention demonstrates that squalene (SQ) significantly alleviates LPS-induced endotoxemic liver injury in mice through a multi-target mechanism. First, SQ intervention significantly reduces the serum ALT / AST levels (P<0.05), indicating that it protects hepatocytes by inhibiting lipid peroxidation of the liver cell membrane, enhancing the activities of GSH-PX and SOD, scavenging the excessive free radicals induced by LPS (MDA significantly decreases), and mitochondrial dysfunction. This effect may be related to its activation of the Keap1-Nrf2-ARE pathway, which enhances the endogenous antioxidant defense ability by upregulating GSH-PX (significantly increased) and T-SOD (significantly enhanced). Second, SQ remodels the balance of the inflammatory microenvironment by inhibiting the TLR4 / NF-κB signal transduction, reducing the release of pro-inflammatory factors IL-1β and TNF-α (significantly decreased respectively), and simultaneously upregulating the anti-inflammatory factor IL-10 (significantly increased). Dexamethasone, as a glucocorticoid, can rapidly inhibit inflammatory signal transduction (such as blocking the release of IL-1β). The combination of SQ and dexamethasone (SQ+Dex) shows a synergistic effect in inhibiting TNF-α, and the decrease in AST in the combination group is more significant, achieving a dual inhibitory effect on inflammatory mediators. This may stem from the complementarity of the rapid blockade of NF-κB nuclear translocation by dexamethasone and the long-term regulation of oxidative stress by SQ. Inducible nitric oxide synthase (iNOS) drives the inflammatory process by abnormally upregulating the biosynthesis of nitric oxide (NO), and aminoguanidine (AG), as its specific inhibitor, can significantly inhibit the IL-1β-induced inflammatory cascade reaction by blocking the iNOS / NO signal axis, thereby playing an anti-inflammatory therapeutic role.

[0142] This invention not only provides an experimental basis for squalene as an adjuvant therapeutic drug for endotoxemia, but also reveals the potential advantages of the combination of natural products and synthetic drugs. The SQ+Dex regimen can reduce the dosage of glucocorticoids (reducing the risk of immunosuppression), and at the same time alleviate drug-induced liver injury through antioxidant effects. This strategy is worthy of further verification in preclinical models of sepsis.

[0143] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of squalene or a pharmaceutically acceptable salt thereof in any one of a) - b): a) Use in the preparation of a drug for preventing or treating endotoxemia; b) Use in the preparation of a drug for treating a disease caused by endotoxemia.

2. The application according to claim 1, wherein The disease caused by endotoxemia is sepsis.

3. The application according to claim 1, characterized in that, The drug further comprises a pharmaceutically acceptable excipient.

4. The application according to claim 1, characterized in that The drug comprises an oral preparation or an injection preparation.

5. Use of a pharmaceutical composition in any one of c) - d): c) Use in the preparation of a drug for preventing or treating endotoxemia; d) Use in the preparation of a drug for treating a disease caused by endotoxemia; It is characterized in that The pharmaceutical composition comprises squalene or a pharmaceutically acceptable salt thereof, and a glucocorticoid.

6. The application according to claim 5, wherein The mass ratio of squalene to the glucocorticoid is 100: (0.1 - 5).

7. The application according to claim 5, wherein The glucocorticoid comprises at least one of dexamethasone, betamethasone, triamcinolone acetonide, fluticasone, budesonide, clobetasol propionate, hydrocortisone butyrate.

8. Use of a pharmaceutical composition in any one of e) - f): e) Use in the preparation of a drug for preventing or treating endotoxemia; f) Use in the preparation of a drug for treating a disease caused by endotoxemia; It is characterized in that The pharmaceutical composition comprises squalene or a pharmaceutically acceptable salt thereof, and an inducible nitric oxide synthase inhibitor.

9. The application according to claim 8, wherein The mass ratio of squalene to the inducible nitric oxide synthase inhibitor is 100: (8 - 20).

10. The application according to claim 8, characterized in that, The inducible nitric oxide synthase inhibitor comprises at least one of aminoguanidine, 1400W, L-NIL, GW274150.

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