KR20220111520A - Composition for preventing or treating of sepsis comprising Hederacolchiside E - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of sepsis containing Hederacolchiside E as an active ingredient, and specifically, it is expected to contribute to prevention and treatment of sepsis through a protective effect of Hederacolchiside E against HMGB1-mediated vascular barrier destruction.

Description

Composition for preventing or treating sepsis comprising Hederacolchiside E as an active ingredient

The present invention relates to a composition for preventing or treating sepsis comprising hederacolchiside E as an active ingredient, and to a composition for preventing and treating sepsis mediated by high mobility group box 1 (HMGB1). .

Sepsis is an abnormal systemic inflammatory response of the body to infection. Severe sepsis and septic shock cause an imbalance between inflammation, coagulation, and the neuroendocrine system, and even with antibiotics and modern intensive care, the mortality rate of severe sepsis is about 30%, resulting in about 250,000 deaths each year in the United States.

Multiorgan failure (MOF) is the leading cause of sepsis-induced death (Lee et al., 2019). In particular, most patients die of MOF rather than oxygen deprivation due to severe, life-threatening hypoxemia (Hotchkiss et al., 2016). Although sepsis most commonly affects the lungs and kidneys, acute kidney injury results in systemic damage to multiple organs, including the heart, lungs, brain, spleen, liver, and intestines due to a systemic inflammatory response (Ologunde et al., 2014). .

The progression of sepsis known to date is rather complicated. It is known that endotoxins are partially responsible for the sepsis progression through stimulation and induction of macrophages and/or monocytes, which initially act as pro-inflammatory mediators such as IL-1β and TNF-α, and later with high mobility Group box 1 (high mobility group box 1, HMGB1) is secreted continuously. Initially, anti-inflammatory drugs targeting only the reduction of IL-1β and TNF-α were attempted as therapeutic agents, but most failed because they failed to prevent the progression of sepsis. However, several limitations have been pointed out.

High mobility group box 1, which increases late in sepsis, is a nuclear protein present in almost all eukaryotic cells and is known to play an important mediator role in sepsis as it is secreted by both damaged cells and activated immune cells (Bae et al., 2012). HMGB1 secreted into the extracellular milieu acts as a chemokine or cytokine that stimulates innate immune cells for the production of pro-inflammatory cytokines. Cell surface receptors that participate in the HMGB1 interaction include receptor for advanced glycation end products (RAGE), toll like receptor (TLR)-2, and TLR-4. HMGB1 increases the expression of endothelial intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and cell adhesion molecules (CMA) such as E-Selectin, which promotes inflammation through recruitment of leukocytes ( Bae et al., 2012). This level of HMGB1 appears to be associated with the degree of organ dysfunction (Gibot et al., 2007; Sunden-Cullberg et al., 2005). In contrast to early cytokines, HMGB1, which maintains high concentration levels at days 1-1.5 in circulating blood in animal models of endotoxemia and sepsis, could be a potential candidate for the treatment of lethal systemic inflammatory diseases.

On the other hand, hederacolchiside-E (HCE), that is, 3-O-{α-L-rhamnopyranosyl (1→2)-[β-D-glucopyranosyl (1→4)]-α-L- Arabinopyranosyl}-28-O-[α-L-rhamnopyranosyl (1→4)-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl ester ( 3-O- α-L-rhamnopyranosyl (1→2)-[β-D-glucopyanosyl(1→4)]-α-L-arabinopyanosyl -28-O-[α-L-rhamnopyranosyl (1→4)-β-D- glucopyanosyl(1→6)-β-D-glucopyanosyl ester) was discovered in 1970 in leaves of Hedera colchica and Pulsatilla . As a non-desmoidic olenan saponin first isolated from the roots of koreana and Anemone raddeana , anti-inflammatory, antioxidant, anti-Alzheimer's and neuroprotective activities were reported (Gepdiremen et al., 2005; Han et al., 2007; Gulcin et al., 2004). . However, the protective effect of HCE against HMGB1-mediated vascular barrier destruction of HCE is unknown.

Korean Patent Registration No. 2006659, which is a prior art, describes a composition for preventing or treating sepsis, including ginsenoside Rh1, in which ginsenoside Rh1 reduces the expression of HMGB1 to reduce mortality due to sepsis. In addition, Korea Patent No. 2109744 discloses a composition for preventing or treating sepsis or septic shock containing a deckercin derivative. However, these techniques are different in configuration and effect of the present invention. Korean Patent No. 1058587 discloses a health food having a brain function improvement effect including Hederacolchiside E, but this is also different in the composition and effect of the present invention.

Korean Patent No. 2006659, a composition for preventing or treating sepsis containing ginsenoside Rh1, registered on July 29, 2019. Korean Patent No. 2109744, a composition for preventing or treating sepsis or septic shock containing a deckercin derivative, registered on May 06, 2020. Korean Patent No. 1058587, oleanane-based triterpene saponin compound effective for treating dementia and mild cognitive impairment and improving cognitive function, registered on August 16, 2011.

Angus, Derek C. Linde-Zwirble, Walter T., Lidicer, Jeffrey, Clermont Gilles, Carcillo Joseph, Pinsky, Michael R., Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care, Critical Care Medicine, July 2001, Volume 29, Issue 7, p 1303-1310 W. Lee, S. K. Ku, J.E. Kim, G. E. Cho, G.Y. Song, J.S. Bae, Pulmonary protective functions of rare ginsenoside rg4 on particulate matter-induced inflammatory responses, Biotechnol. Bioprocess Eng. 24 (2019) 445-453. R.S. Hotchkiss, L.L. Moldawer, S. M. Opal, K. Reinhart, I.R. Turnbull, J. L. Vincent, Sepsis and septic shock, Nat. Rev. Dis. Primers 2 (2016) 16045. [2] M.T. Ziesmann, J. C. Marshall, Multiple organ dysfunction: the defining R. Ologunde, H. Zhao, K. Lu, D. Ma, Organ cross talk and remote organ damage following acute kidney injury, Int. Urol. Nephrol. 46 (2014) 2337-2345. J.S. Bae, Role of high mobility group box 1 in inflammatory disease: focus on sepsis, Arch. Pharm. Res. 35 (2012) 1511-1523. A. Gepdiremen, V. Mshvildadze, H. Suleyman, R. Elias, Acute anti-inflammatory activity of four saponins isolated from ivy: alpha-hederin, hederasaponin-c, hederacolchiside-e and hederacolchiside-f in carrageenan-induced rat paw edema , Phytomedicine 12 (2005) 440-444. C.K. Han, W. R. Choi, K. B. Oh, Cognition-enhancing and neuroprotective effects of hederacolchiside-e from pulsatilla koreana, Planta Med. 73 (2007) 665-669. I. Gulcin, V. Mshvildadze, A. Gepdiremen, R. Elias, Antioxidant activity of saponins isolated from ivy: alpha-hederin, hederasaponin-c, hederacolchiside-e and hederacolchiside-f, Planta Med. 70 (2004) 561-563. Jonas Sunden-Cullberg, Anna Norrby-Teglund, Carl Johan Treutiger, The role of high mobility group box-1 protein in severe sepsis, Curr Opin Infect Dis., 2006 Jun;19(3):231-6. Riedemann NC, Guo RF, Ward PA. The enigma of sepsis. J Clin Invest 2003; 112:460-467. H. Wang, O. Bloom, M. Zhang, J. M. Vishnubhakat, M. Ombrellino, J. Che, A. Frazier, H. Yang, S. Ivanova, L. Borovikova, K.R. Manogue, E. Faist, E. Abraham, J. Andersson, U. Andersson, P.E. Molina, N.N. Abumrad, A. Sama, K.J. Tracey, Hmg-1 as a late mediator of endotoxin lethality in mice, Science 285 (1999) 248-251. E. Abraham, J. Arcaoli, A. Carmody, H. Wang, K.J. Tracey, Hmg-1 as a mediator of acute lung inflammation, J. Immunol. 165 (2000) 2950-2954. Gibot, S., Massin, F., Cravoisy, A., Barraud, D., Nace, L., Levy, B., Bollaert, P.E., 2007. High-mobility group box 1 protein plasma concentrations during septic shock. Intensive Care Med 33, 1347-1353. Sunden-Cullberg, J., Norrby-Teglund, A., Rouhiainen, A., Rauvala, H., Herman, G., Tracey, K.J., Lee, M.L., Andersson, J., Tokics, L., Treutiger, C.J. , 2005. Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med 33, 564-573. B. Jung, H. Kang, W. Lee, H.J. Noh, Y.S. Kim, M.S. Han, M.C. Baek, J. Kim, J.S. Bae, Anti-septic effects of dabrafenib on hmgb1-mediated inflammatory responses, BMB Rep. 49 (2016) 214-219. T. Bonaldi, F. Talamo, P. Scaffidi, D. Ferrera, A. Porto, A. Bachi, A. Rubartelli, A. Agresti, M.E. Bianchi, Monocytic cells hyperacetylate chromatin protein hmgb1 to redirect it towards secretion, EMBO J. 22 (2003) 5551-5560. J.H. Youn, J.S. Shin, Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion, J. Immunol. 177 (2006) 7889-7897. M.M. Rabadi, S. Xavier, R. Vasko, K. Kaur, M.S. Goligorksy, B.B. Ratliff, High-mobility group box 1 is a novel deacetylation target of sirtuin1, Kidney Int. 87 (2015) 95-108. S.M. Weis, Vascular permeability in cardiovascular disease and cancer, Curr. Opin. Hematol. 15 (2008) 243-249. W. Lee, S. K. Ku, Y.M. Lee, J.S. Bae, Anti-septic effects of glyceollins in hmgb1-induced inflammatory responses in vitro and in vivo, Food Chem. Toxicol. 63 (2014) 1-8. B.S. Lee, C. Lee, S. Yang, S.K. Ku, J.S. Bae, Renal protective effects of zingerone in a mouse model of sepsis, BMB Rep. 52 (2019) 271-276. Y.H. Qin, S. M. Dai, G.S. Tang, J. Zhang, D. Ren, Z. W. Wang, Q. Shen, Hmgb1 enhances the proinflammatory activity of lipopolysaccharide by promoting the phosphorylation of mapk p38 through receptor for advanced glycation end products, J. Immunol. 183 (2009) 6244-6250. R. Palumbo, B.G. Galvez, T. Pusterla, F. De Marchis, G. Cossu, K. B. Marcu, M. E. Bianchi, Cells migrating to sites of tissue damage in response to the danger signal hmgb1 require nf-kappab activation, J. Cell Biol. 179 (2007) 33-40. B. Jung, H. Kang, W. Lee, H.J. Noh, Y.S. Kim, M.S. Han, M.C. Baek, J. Kim, J.S. Bae, Anti-septic effects of dabrafenib on hmgb1-mediated inflammatory responses, BMB Rep. 49 (2016) 214-219.

An object of the present invention is to provide a composition for preventing or treating sepsis comprising hederacolchiside E (Hederacolchiside E) as an active ingredient.

In order to solve the above problems, the present invention provides a composition for preventing or treating sepsis comprising hederacolchiside E as an active ingredient.

The hederacolchiside E can inhibit the release of high mobility group box 1 (HMGB1) from damaged cells or activated immune cells in high mobility group box 1 (HMGB1) mediated sepsis.

In addition, the hederacolchiside E is a receptor for high mobility group box 1 (HMGB1), TLR2 (Toll-like receptor 2), TLR4 (Toll-like receptor 4) and RAGE (receptor for advanced glycation end products) from the group consisting of Inhibit expression of one or more selected receptors.

The hederacolchiside E can prevent or treat sepsis by inducing activation of sirtuin 1 protein or deacetylation of high mobility group box 1 (HMGB1).

In addition, the hederacolchiside E can maintain vascular barrier integrity by inhibiting high mobility group box 1 (HMGB1) mediated increase in vascular permeability, and maintaining the vascular barrier integrity. By inhibiting the expression of one or more endothelial cell adhesion molecules (CAM) selected from the group consisting of vascular endothelial E-selectin, ICAM-1 (Intercellular Adhesion Molecule-1) and VCAM-1 (Vascular Cell Adhesion Molecule-1), It can be achieved by inhibiting the adhesion and migration of monocytes or leukocytes to the vascular endothelium.

The hederacolchiside E is high mobility group box 1 (HMGB1) mediated increased TNF-α (Tumor necrosis factor-α), IL-1β (Interleukin-1β), IL-6 (Interleukin-6), NF- It is possible to inhibit the expression of one or more selected from the group consisting of κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and ERK 1/2 (Extracellular signal-regulated kinases 1/2).

In addition, the hederacolchiside E is high mobility group box 1 (HMGB1) mediated increased aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (Blood) Urea Nitrogen, BUN) Reduces one or more selected from the group consisting of creatinine and lactate dehydrogenase (LDH) to protect the liver and kidney, target organs for systemic inflammation that occurs during sepsis, to prevent or treat multi-organ failure in sepsis patients can increase the survival rate of

The composition for preventing or treating sepsis comprising Hederacolchiside E of the present invention as an active ingredient may be provided as a pharmaceutical composition.

The hederacolchiside E (Hederacolchiside E) is preferably added in an amount of 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, and most preferably 0.001 to 30% by weight based on the total weight of the total pharmaceutical composition. can

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively can Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, and acidulants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, starch, calcium carbonate, and water in the solubilized deckercinol of the present invention. It is prepared by mixing cross or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, preservatives, and acidifying agents are included. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween-61, cacao butter, laurin fat, glycerogelatin, and the like can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 500 mg/kg/day. A preferred dosage is 0.1 mg/kg/day to 200 mg/kg/day, and a more preferred dosage is 1 mg/kg/day to 200 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection and dermal application. Hederacolchiside E (Hederacolchiside E) of the present invention is a drug that can be safely used even when taken for a long period for the purpose of prevention because it has almost no toxicity and side effects.

The present invention provides a health functional food for preventing or improving sepsis comprising hederacolchiside E (Hederacolchiside E) as an active ingredient.

The hederacolchiside E can inhibit the release of high mobility group box 1 (HMGB1) from damaged cells or activated immune cells in high mobility group box 1 (HMGB1) mediated sepsis.

The health functional food may be provided as a health functional food comprising hederacolchiside E (Hederacolchiside E) and a food pharmaceutically acceptable supplementary additive.

The health functional food is preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, most preferably 0.001 to 10% by weight, based on the total weight of the whole food, solubilized hederacolchiside E (Hederacolchiside E) % can be added.

The health functional food includes the form of tablets, capsules, pills or liquids, and the like, and the food to which the extract of the present invention can be added, for example, various foods, beverages, gum, tea, vitamin complex, health Functional foods, etc.

The present invention relates to a composition for preventing or treating sepsis comprising hederacolchiside E as an active ingredient. It is expected to contribute to prevention and treatment.

1 is an HPLC chromatogram of standard hederacolchiside E (A) and purified hederacolchiside E (B).
Figure 2 is a graph showing the effect of HCE on LPS-treated HUVECs (A), HMGB1 release levels of CLP animal models (B), TLR2, TLR4 alc RAGE levels (C) and cell viability in LPS-treated HUVECs (D) to be.
Figure 3 confirms the effect of HCE on the activation of SIRT1 (A) and acetylation of HMGB1 (B). (IP: Immuno Precipitation, WB: Western Blotting)
4 is a graph showing the effect of HCE on the destruction of the HMGB1-mediated vascular barrier. (A) Effect of HCE on LPS-induced permeability in HUVECs. (B) Effect of HCE on HMGB1-induced permeability in HUVECs. (C) Effect of HCE on Evans blue efflux in CLP animal model. (D) Effect of HCE on HMGB1-induced p-38 expression in HUVECs.
Figure 5 shows the effect of HCE on HMGB1-mediated expression of CAM, neutrophil adhesion and leukocyte migration. (A) Effect of HCE on HMGB1-mediated increases in VCAM, ICAM, and E-Selectin in HUVECs. (B) Effect of HCE on HUVEC monolayer adhesion of HMGB1-mediated neutrophils in HUVECs. (C) Effect of HCE on HUVEC monolayer transition of HMGB1-mediated neutrophils in HUVECs. (D) Effect of HCE on the number of leukocytes migrating into the abdominal cavity in an animal model of CLP. (E) Light micrographs showing the effect of HCE on HUVEC monolayer transition of HMGB1-mediated neutrophils in HUVECs.
Figure 6 shows the effect of HCE on the expression of HMGB1-induced IL-6, TNF-α and IL-1β and the activation of the NF-κB / ERK 1/2 pathway. ELISA (AC) or Western blotting analysis (D) showing the effect of HCE on HMGB1-mediated production of TNF-α (A, D), IL-6 (B, D) and IL-1β (C) in HUVECs (D). Effect of HCE on HMGB1-mediated activation of NF-κB p65 (E) and ERK 1/2 (F). (G) Western blot analysis evaluation of intracellular levels of NF-κB in nuclear and cytoplasmic fractions.
7 shows the effect of HCE on mortality and tissue damage after CLP. (A) Inhibitory effect of HCE on CLP-induced mouse lethality. (B) Light micrograph of lung injury induced by CLP (arrows indicate leukocyte infiltration). (C) Effect of HCE on lung injury induced by CLP. HCE for (D) aspartate transaminase (AST) and alanine transaminase (ALT), (E) creatinine, (F) blood urea nitrogen (BUN) and (G) lactate dehydrogenase (LDH). effect.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

< Example 1. Cells, animal models and Hederacolchiside -E preparation>

1.1 Cell Culture and Reagents

Human umbilical vein endothelial cells (HUVECs) were obtained and maintained from Cambrex Bio Science (Charles City, IA, USA), and all experiments were used in the 3-5 passage culture steps. Gingeron, LPS, Crystal Violet, Evans Blue, MTT, 2-penicillin G and streptomycin, mercaptoethanol and DMSO were obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). DMSO was used as a control or solvent for HCE or ZGR. HCE was purchased from Abcam (Cambridge, MA, USA) and human recombinant HMGB1 was purchased from Abnova (Taipei City, Taiwan).

1.2 Hederacolchiside -E ( HCE ) isolate

HCE is Hedera Colchica leaves and Pulsatilla It was first isolated from the roots of koreana and Anemone raddeana , Pulsatilla There is a report that koreana reduces inflammation induced by Carrageenan, but there is no report that HCE inhibits HMGB1-mediated sepsis progression and increases the survival rate of sepsis patients.

Pheasant's wind flower ( Anemone raddeana ) 1.0 kg of dried roots were ground in a mixer with 1.5 L of distilled water and placed in an incubator at 37 ° C for 4 hours. The mixture was suspended in 80 % MeOH (6L), stirred for 6 h and then filtered. The filter cake was extracted three consecutive times with the same amount of 80% MeOH. The resulting filtrates were combined and concentrated in vacuo to give a brown syrup (88.86 g). Then, the dark brown syrup (88.86 g) was suspended in distilled water (2 L) and partitioned with ethyl ether (0.8 L × 2). The remaining water portion (910 mL) was chromatographed on 670 g of Diaion HP20 resin eluting with a gradient of aqueous EtOH (EtOH-H ₂ O 0%, 10%, 30%, 50%, 70% and 90%) by TLC (BuOH) Seven major fractions (R1-R7) based on :AcOH:H2O=5:1:1) were obtained and compared with standard HCE (Wuhan Chem Norn Biotech Co., Ltd.), respectively, the purity of HCE was determined by BDS Hypersil It was confirmed to be 96.9% by analytical HPLC on a C18 column (250 x 4.6 mm). Mobile phase conditions were set to A (acetonitrile) and B [(0.1% phosphoric acid (v/v) in water] as gradient solvent system. Detailed gradient elution is as follows: 0-28 min, 23%-36% A 28-42 min, 36%-56% A; 42-52 min, 56%-90% A; 52-54 min, 90%-90% A; 54-58 min, 90%-23% A; 58 -67 min, 23% -23% A. Flow rate of mobile phase is 1.0 mL/min, wavelength is 203 nm, column temperature is 30 °C, injection volume is 10 µL, physicochemical data of separated HCE is confirmed by referring to previous reports HCE was used at 1, 2, 5, 10 or 20 μM for in vitro assays and 0.2, 0.5, 1 or 2 mg/kg for in vivo assays.

1.3 Sepsis Animal Model

Male C57BL/6 mice (6-7 weeks old, 27 g) were obtained from Orient Bio Co., Ltd. (Sungnam, Republic of Korea). An animal model of CLP-induced sepsis was prepared according to the method of Jung et al. (2016), and the sham control group of animals exposed the cecum but did not make ligation or perforation. Samples such as HCE compounds or GR were injected intravenously for 24 h after CLP procedure and their effects on HMGB1 secretion, cell permeability and leukocyte migration were evaluated, or viability was assessed by intravenous injection at 12 and 50 h after CLP. This protocol was approved by the Animal Care Committee of Kyungpook National University prior to conducting the study (IRB No. KNU2017-102).

1.4 Statistical processing

Data are expressed as mean ± standard deviation (SD) from three independent experiments. ANOVA and Tukey's post-hoc test were performed to compare different groups. P values less than 0.05 were considered statistically significant. To evaluate the outcome of CLP-induced sepsis, a Kaplan-Meier curve was created to compare survival differences.

< Example 2. of HMGB1 emission and HMGB1 for the expression level of the receptor HCE's Check the effect >

It was confirmed whether HMGB1 release induced by LPS was regulated by HCE treatment.

HUVECs prepared in Example 1.1 were stimulated with LPS (100 ng/mL) and HCEs were treated with 0, 1, 2, 5, 10, 20 μM, and ZGR 20 μM, respectively, and the amount of HMGB1 released for 16 hours was measured by ELISA. was measured and shown in FIG. 2A. As shown in FIG. 2A , the secretion of HMGB1 in HUVECs was significantly increased by LPS, and this increase was inhibited in a concentration-dependent manner by HCE treatment.

The sepsis model mouse prepared in Example 1.3 was injected with HCE or ZGR intravenously after 12 hours of CLP, and then 24 hours later, the serum of the mouse was obtained and measured by ELISA, which is shown in FIG. 2B. As shown in FIG. 2B , the concentration of HMGB1 in mouse serum was significantly increased by CLP, and this increase was inhibited in a concentration-dependent manner by HCE treatment.

Next, the effect of HCE on the expression level of the well-known HMGB1 receptors TLR2, TLR4 and RAGE was confirmed. The HUVECs prepared in Example 1.1 were treated with HMGB1 (1 μg / mL), then treated with HEC 5, 10, 20 μM, and ZGR 20 μM, respectively, and then left for 16 hours. Then, the expression levels of TLR2, TLR4 and RAGE were confirmed using whole cell ELISA, and it is shown in FIG. 2C. Expression levels of TLR2, TLR4 and RAGE were tested by purchasing specific antibodies for each receptor (A-9, H-80 and A-9, respectively; Santa Cruz Biotechnology Inc., Dallas, Texas, USA). As shown in FIG. 2C , the expression levels of TLR2 (white), TLR4 (grey) and RAGE (black) were rapidly increased by HMGB1 treatment, and this increase was inhibited in a concentration-dependent manner by HCE treatment.

In addition, cell viability following HCE administration was evaluated by MTT assay using HUVECs. HUVECs were incubated with LPS (100 ng/mL) or HCE for 48 h for 16 h and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as shown in Figure 2D. indicated. As shown in Figure 2D, HCE at 100 ng/mL LPS for 16 h or 10, 20 or 50 μM concentration for 48 h did not affect the viability of the cells of HUVECs, indicating no toxicity. These results show that HCE may be effective in early intervention to prevent the release of HMGB1 and progression to severe sepsis.

< Example 3. HCE of SIRT1 activation and of HMGB1 on acetylation Check the impact >

It is known that the ability of HMGB1 to bind DNA to the cytoplasm is due to hyperacetylation of HMGB1 (Bonaldi et al., 2003). That is, hyperacetylation of serine residues in HMGB1 promotes cytoplasmic secretion of HMGB1 (Youn et al., 2006), and sirtuin 1 (SIRT1) is known to activate deacetylation of HMGB1 (Rabadi et al., 2015). Therefore, the present inventors confirmed the effect of HCE on the activation of SIRT1 and the acetylation of HMGB1.

After HUVECs were treated with HCE (20 μM) for 0, 2, 4, 6, 8, 10 and 12 hours, the cells were lysed and analyzed through Western blotting to measure the expression level of SIRT1, which is shown in FIG. 3A. . As shown in FIG. 3A , the expression of SIRT1 was evident after 4 hours of incubation, peaked after 6 hours, maintained for up to 8 hours, and then gradually decreased.

In addition, the effect of HCE on the deacetylation of HMGB1 and the induction of SIRT1 expression was investigated. HUVECs were treated with LPS (100 ng/mL) with or without HCE (20 μM). Some HUVECs were treated with the SIRT1 inhibitor sirtinol (Srtnl), 10 mM, for 1 hour before HCE treatment. After incubation for 6 hours, cells were lysed, immunoprecipitated with anti-HMGB1 antibody, and acetylated HMGB1 and total HMGB1 protein levels were measured by immunoblot analysis using anti-acetyl-lysine (K) or anti-HMGB1 antibody, respectively. Meanwhile, the same volume of medium was collected and the released HMGB1 was detected by western blotting, as shown in Fig. 3B. As shown in Figure 3B, stimulation by LPS increased HMGB1 acetylation, and this increase was significantly reduced by HCE. It was also shown that sirtinol treatment reversed the effect of HCE and significantly increased the acetylation of HMGB1. These results suggest that HCE significantly reduced HMGB1 release from HUVECs by LPS through SIRT1-mediated deacetylation of HMGB1.

< Example 4. HCE's vascular barrier HMGB1 Check the effect on mediated destruction >

Maintaining vascular barrier integrity plays an important role in severe vascular inflammatory diseases, and LPS and HMGB1 disrupt this vascular barrier integrity (Weis et al., 2008; Lee et al., 2014). Therefore, we analyzed vascular permeability to evaluate the vascular barrier protective efficacy of HCE in HUVECs.

A monolayer of HUVEC was treated with LPS (100 ng/mL) or HMGB1 (1 μg/mL), respectively, and HCE was treated with 0, 1, 2, 5, 10, 20 μM or ZGR 20 μM for 6 hours, respectively. Barrier integrity was assessed by monitoring the flux of Evans blue-bound albumin albumin across the monolayer of HUVECs according to the method of Lee et al. (2019) and shown in Figures 4A and 4B. As shown in FIGS. 4A and 4B , LPS and HMGB1 significantly increased the permeability of a monolayer of HUVECs, and HCE inhibited both LPS and HMGB1-mediated permeability in a concentration-dependent manner.

For in vivo experiments, according to the method of Lee et al. (2019), HCE 0.2, 0.5, 1.0, 2.0 mg/kg or ZGR 0.7 mg/kg or HMGB1 (2 μg/mouse) was administered to male mice 12 h after CLP. intravenous injection. After 12 hours, the mice were euthanized, and then, the amount of Evans blue dye was measured by peritoneal lavage, and the vascular permeability was measured and shown in FIG. 4C. As shown in FIG. 4C , vascular permeability was rapidly increased by CLP, and this increase was decreased in a concentration-dependent manner by HCE.

The vascular destruction response by HMGB1 is mediated by phosphorylation of p38 (Qin et al., 2009; Palumbo et al., 2007). Therefore, to confirm the effect of HCE on HMGB1-mediated phosphorylation of p38, HUVECs treated with HCE (1,2, 5, 10, 20 μM) or ZGR (20 μM) were stimulated with HMGB1. After 6 hours, the effect of HCE or ZGR on the phosphorylation level of p38 was analyzed by ELISA and shown in FIG. 4D. As shown in FIG. 4D , HMGB1 increased p38 phosphorylation, and HCE inhibited HMGB1 mediated p38 phosphorylation. HMGB1-mediated barrier disruption, increased permeability and phosphorylation of p38 were restored by HCE, suggesting that HCE has potential as a preservative.

< Example 5. HCE's CAM's HMGB1 Confirmation of effect on mediated expression, neutrophil adhesion and leukocyte migration >

HMGB1 increases the surface expression levels of various endothelial CAMs, including E-selectin, ICAM-1 and VCAM-1, which are required for leukocytes to cross the vascular endothelium to the site of inflammation. Therefore, we analyzed the effect of HCE on HMGB1-mediated regulation of CAM in HUVECs, adhesion of human neutrophils to HUVECs, and metastasis of neutrophils through HUVEC monolayers.

Migration of human neutrophils to HUVECs was performed in a 6.5-mm Transwell plate containing a filter with a pore size of 8 μm by applying the method of Lee et al. (2019). HUVECs were cultured for 3 days to obtain confluent endothelial monolayers, and HUVECs were stimulated with HMGB1 (1 μg/mL) for 16 h before adding neutrophils to the upper compartment, followed by 6 h at each concentration of 20 μM or HCE with ZGR. incubated during Next, the Transwell plate was incubated at 37 °C for 2 h, and suspended or non-floating neutrophils and HUVECs in the upper chamber and filter membrane were removed. Neutrophils migrating down the filter were fixed with 8% glutaraldehyde and stained with 0.25% crystal violet in 20% methanol (w/v). Nine randomly selected high-power microscopy fields (200X) were then counted. All experiments were repeated twice per well in duplicate wells, and the results were expressed as migration indix and are shown in FIGS. 5A to 5C and 5E. 5A to 5C and 5E, HMGB1 increased the expression of E-selectin (black bars), VCAM-1 (white bars) and ICAM-1 (grey bars) in HUVECs, which were was inhibited in a concentration-dependent manner. In addition, HMGB1 increased neutrophil adhesion to HUVEC monolayers (Fig. 5B) and neutrophil migration through HUVEC monolayers (Figs. 5C, 5E) were also concentration-dependently inhibited by HCE.

The expression of CAM up-regulated by HMGB1 was suppressed in a concentration-dependent manner by HCE, and it was confirmed that, in addition to the expression level of CAM, HCE also reduced the adhesion of human neutrophils to HUVECs and subsequent metastasis.

For in vivo experiments, male mice were anesthetized with oxygen containing 2% isoflurane (Forane; JW Pharmaceutical, Seoul, South Korea). Oxygen was first administered to the breathing chamber and then anesthetized with a face mask via a small rodent gas anesthesia machine (RC2; Vetequip, Pleasanton, CA, USA). Thereafter, mice were treated with HMGB1 (2 μg/mouse, i.v.) for 16 hours, and then HCE (0.2, 0.5, 1.0, 2.0 mg/kg) or ZGR (0.7 mg/kg) was injected intravenously. After 6 h, mice were sacrificed and a sample of peritoneal fluid (20 μl) obtained by washing the abdominal cavity with physiological saline (5 mL) was stained with 0.38 mL of Turk's solution (0.01% crystal violet in 3% acetic acid) to count leukocytes under an optical microscope. was counted and shown in FIG. 5D. As shown in FIG. 5D , leukocyte migration was increased by HMGB1 treatment, and HCE decreased it in a concentration-dependent manner.

< Example 6. HCE's NF - κB / ERK Signal and IL- 1β , IL-6 and TNF -Check to inhibit the production of α>

HMGB1 exacerbates the pathological condition of sepsis by inducing inflammatory cytokine production through various signaling pathways, including NF-κB and ERK 1/2 (Jung et al., 2016). Therefore, the present inventors evaluated the inhibitory effect of HCE on the HMGB1-induced production of TNF-α, IL-1β and IL-6 and activation of NF-κB and ERK1/2 signaling.

HUVECs were stimulated with HMGB1 1 μg/mL for 16 h, followed by HCE treatment for 6 h, followed by extracellular signal-regulated kinase (ERK) 1/2 (R&D Systems, Minneapolis, MN, USA), p65 subunit of nuclear. Total and phosphorylation levels of factor kappa B (NF-κB) (Cell Signaling Technology, Danvers, MA, USA), and TNF-α, IL-6 and IL-1β levels in cell culture supernatants were analyzed with an ELISA kit to analyze FIG. 6A to FIG. 6F. 6A to 6F, the production of inflammatory cytokines enhanced by HMGB1 in HUVECs and was inhibited by HCE treatment. HMGB1 also induced the nuclear localization of p65 NF-κB in HUVECs, as shown in Fig. 6G, which was inhibited by HCE treatment.

< Example 7. HCE dosing CLP Confirmation of effect on survival rate and tissue damage reduction in mice with induced sepsis>

The present inventors actually confirmed the effect of HCE on the survival rate of sepsis animal model. A single injection of HCE or ZGR did not protect against CLP-induced lethality (data not shown). Therefore, HCE (1.0 or 2.0 mg/kg) or ZGR (0.7 mg/kg) was injected intravenously at 12 and 50 hours after CLP, and the survival rate of the animals was evaluated every 12 hours until 132 hours after CLP, as shown in FIG. 7A . As shown in FIG. 7A , double administration of HCE significantly improved the survival rate of mice after CLP, and the HCE 2.0 mg/kg administration group showed a higher survival rate than the positive control, ZGR administration group (p <0.00001).

Systemic inflammation that occurs during sepsis progression frequently triggers MOFs, with the liver and kidneys being the main target organs. Therefore, we evaluated the potential protective effect of HCE on lung damage due to CLP. Male mice (n = 5) were subjected to CLP followed by intravenous administration of HCE (1.0 or 2.0 mg/kg) or ZGR (0.7 mg/kg) at 12 and 50 hours post CLP. After 4 days of CLP, mice were sacrificed, and blind observation was performed by optical microscopic analysis of the lung specimens, and the lung structures, tissue edema, and infiltration of inflammatory cells of the lung specimens were evaluated and shown in FIGS. 7B and 7C. 7B and 7C , HCE improved CLP-induced interstitial lung edema and severe damage to lung tissue. In addition, plasma levels of ALT and AST (FIG. 7D), which are indicators of liver damage, and BUN and creatinine, which are indicators of kidney damage, were confirmed. Plasma levels of blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) were measured using a commercial kit (Pointe Scientific, Lincoln Park, MI, USA). was used to determine As a result, ALT and AST, BUN, and creatinine significantly increased by CLP were inhibited by HCE ( FIGS. 7D to 7F ), and all these increases and an increase in LDH, another important indicator of tissue damage, were also inhibited ( FIG. 7D to 7F ). 7G).

As can be seen from the above results, it was confirmed that hederacolchiside E (HCE) reduced HMGB1-mediated vascular barrier destruction by decreasing HMGB1 release of LPS-activated HUVECs, inhibiting CLP-mediated release of HMGB1, and increasing barrier integrity. In addition, the barrier protective effect of HCE was confirmed using a sepsis mouse model. Treatment with HCE in this model significantly reduced CLP mortality and multiple organ failure. This shows that HCE serves as a preventive or therapeutic agent for severe vasculitic diseases including sepsis and septic shock.

< Formulation example 1. Pharmaceutical preparations>

Formulation example 1-1. manufacture of tablets

20 mg of hederacolchiside E of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. After adding a 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This was dried and the mixture obtained by adding 160 g of potato starch, 50 g of activity and 5 g of magnesium stearate to this was made into tablets.

Formulation example 1-2. Preparation of injectable solution

10 mg of hederacolchiside E of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20° C. for 30 minutes.

< Formulation example 2. Manufacturing of health functional food>

Formulation example 2-1. Manufacturing of health functional food

Hederacolchiside E of the present invention 100mg, vitamin mixture appropriate amount, vitamin A acetate 70㎍, vitamin E 1.0mg, vitamin B1 0.13mg, vitamin B2 0.15mg, vitamin B6 0.5mg, vitamin B12 0.2㎍, vitamin C 10mg, Biotin 10㎍, nicotinic acid amide 1.7mg, folic acid 50㎍, calcium pantothenate 0.5mg, mineral mixture appropriate amount, ferrous sulfate 1.75mg, zinc oxide 0.82mg, magnesium carbonate 25.3mg, potassium monophosphate 15mg, dicalcium phosphate 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, and magnesium chloride 24.8 mg were mixed to prepare granules, but it can be prepared by modifying various formulations according to the use. In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily modified, and it may be prepared by mixing the above ingredients according to a conventional health functional food manufacturing method.

Formulation example 2-2. Manufacture of health functional beverages

100 mg of hedera colchiside E of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed and stirred, heated, filtered, sterilized, and refrigerated according to a conventional beverage preparation method to prepare a beverage.

Claims (10)

A composition for preventing or treating sepsis comprising Hederacolchiside E (Hederacolchiside E) as an active ingredient. The method of claim 1,
The hederacolchiside E is sepsis, characterized in that it inhibits the release of high mobility group box 1 (HMGB1) of damaged cells or activated immune cells in high mobility group box 1 (HMGB1) mediated sepsis. A composition for prophylaxis or treatment. 3. The method of claim 1 or 2,
The hederacolchiside E is selected from the group consisting of Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), and receptor for advanced glycation end products (RAGE), which are receptors of high mobility group box 1 (HMGB1). A composition for preventing or treating sepsis, characterized in that it inhibits the expression of one or more receptors. 3. The method of claim 1 or 2,
The hedera colchiside E is a composition for preventing or treating sepsis, characterized in that it induces activation of sirtuin 1 protein or deacetylation of high mobility group box 1 (HMGB1). 3. The method of claim 1 or 2,
The hederacolchiside E is a composition for preventing or treating sepsis, characterized in that it maintains vascular barrier integrity by inhibiting high mobility group box 1 (HMGB1)-mediated increase in vascular permeability. 6. The method of claim 5,
Maintaining the vascular barrier integrity is at least one endothelium selected from the group consisting of E-selectin, ICAM-1 (Intercellular Adhesion Molecule-1) and VCAM-1 (Vascular Cell Adhesion Molecule-1) of vascular endothelium. A composition for preventing or treating sepsis, characterized in that it consists of suppressing the expression of CAM (cell adhesion molecule). 3. The method of claim 1 or 2,
The hederacolchiside E is high mobility group box 1 (HMGB1) mediated increased TNF-α (Tumor necrosis factor-α), IL-1β (Interleukin-1β), IL-6 (Interleukin-6), NF- Prevention or treatment of sepsis, characterized in that it suppresses the expression of one or more selected from the group consisting of κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and ERK 1/2 (Extracellular signal-regulated kinases 1/2) for composition. 3. The method of claim 1 or 2,
The hederacolchiside E is high mobility group box 1 (HMGB1) mediated increased aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (Blood Urea Nitrogen) , BUN) a composition for preventing or treating sepsis, characterized in that it reduces at least one selected from the group consisting of creatinine and lactate dehydrogenase (LDH). Health functional food for preventing or improving sepsis containing Hederacolchiside E as an active ingredient. The method of claim 1,
The hederacolchiside E is sepsis, characterized in that it inhibits the release of high mobility group box 1 (HMGB1) of damaged cells or activated immune cells in high mobility group box 1 (HMGB1) mediated sepsis. Health functional food for prevention or improvement.

Images