KR102585910B1 - Composition for preventing or treating of sepsis comprising Hederacolchiside E - Google Patents

Abstract

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

Description

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

The present invention relates to a composition for preventing or treating sepsis containing 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 the body's abnormal systemic inflammatory response to infection. Severe sepsis and septic shock cause a loss of balance between inflammation, coagulation, and the neuroendocrine system. Even with antibiotics and modern intensive care, the mortality rate of severe sepsis is approximately 30%, resulting in approximately 250,000 deaths annually in the United States.

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

The process of sepsis known to date is somewhat complex. Endotoxins are known to be partially responsible for the progression of sepsis through stimulation and induction of macrophages and/or monocytes, which produce pro-inflammatory mediators such as IL-1β and TNF-α in the early stage and highly mobile cells in the later stage. 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 treatments, but most failed because they could not prevent the progression of sepsis. Current attempts include mechanical ventilation, activated protein C administration, and glucocorticoid treatment. However, several limitations are being pointed out.

High-mobility group box 1, which increases in the late stage of sepsis, is a nuclear protein present in almost all eukaryotic cells and is secreted by both damaged cells and activated immune cells and is known to play an important role as an important mediator of sepsis (Bae et al., 2012). HMGB1 secreted into the extracellular milieu acts as a chemokine or cytokine that stimulates innate immune cells to produce pro-inflammatory cytokines. Cell surface receptors participating in HMGB1 interaction include receptor for advanced glycation end products (RAGE), toll like receptor (TLR)-2, and TLR-4. HMGB1 increases the expression of endothelium's 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). The level of HMGB1 appears to be related to the degree of organ dysfunction (Gibot et al., 2007; Sunden-Cullberg et al., 2005). Unlike early cytokines, HMGB1, which maintains high concentration levels in the circulating blood for 1 to 1.5 days in animal models of endotoxemia and sepsis, may be a potential candidate for the treatment of fatal systemic inflammatory diseases.

On the other hand, hederacolchiside-E (HCE), i.e. 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-glucopyranosyl(1→4)]-α-L-arabinopyranosyl -28-O-[α-L-rhamnopyranosyl (1→4)-β-D- glucopyranosyl(1→6)-β-D-glucopyranosyl ester) was discovered in the leaves of Hedera colchica and Pulsatilla in 1970. It is a non-desmoidic olenan saponin first isolated from the roots of koreana and Anemone raddeana , and anti-inflammatory functions, antioxidant effects, anti-Alzheimer's and neuroprotective activities have been reported (Gepdiremen et al., 2005; Han et al., 2007; Gulcin et al., 2004) . However, the protective effect of HCE against HMGB1-mediated vascular barrier disruption is unknown.

Korean Patent No. 2006659, which is a prior art, describes a composition for preventing or treating sepsis containing ginsenoside Rh1, which reduces the mortality rate due to sepsis by reducing the expression of HMGB1. Additionally, Korean Patent No. 2109744 discloses a composition for preventing or treating sepsis or septic shock containing a decusin derivative. However, these technologies differ in the structure and effect of the present invention. Korean Patent No. 1058587 describes a health food containing Hederacolchiside E with an effect of improving brain function, but this also differs from the present invention in composition and effect.

Korean Patent No. 2006659, Composition for preventing or treating sepsis containing ginsenoside Rh1, registered on July 29, 2019. Korean Patent No. 2109744, Composition for preventing or treating sepsis or septic shock containing a decusin derivative, registered on May 6, 2020. Korean Patent No. 1058587, Oleanane triterpene saponin compound effective in treating dementia and mild cognitive impairment and improving cognitive function, registered on August 16, 2011.

Angus, Derek C. Linde-Zwirble, Walter T., Lidicker, 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. Arcaroli, 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.

The purpose of the present invention is to provide a composition for preventing or treating sepsis containing Hederacolchiside E as an active ingredient.

In order to solve the above problems, the present invention provides a composition for preventing or treating sepsis containing 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 from the group consisting of TLR2 (Toll-like receptor 2), TLR4 (Toll-like receptor 4), and RAGE (receptor for advanced glycation end products), which are receptors for high mobility group box 1 (HMGB1). The expression of one or more selected receptors can be inhibited.

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

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

The hederacolchiside E increases TNF-α (Tumor necrosis factor-α), IL-1β (Interleukin-1β), IL-6 (Interleukin-6), and NF-α mediated by high mobility group box 1 (HMGB1). It can 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 increases aspartate transaminase (AST), alanine transaminase (ALT), and urea nitrogen (Blood) mediated by high mobility group box 1 (HMGB1). Patients with sepsis by reducing one or more selected from the group consisting of Urea Nitrogen (BUN) creatinine and lactate dehydrogenase (LDH) to protect the liver and kidneys, which are target organs of systemic inflammation that occurs during sepsis, thereby preventing or treating multi-organ failure. can increase the survival rate.

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

The Hederacolchiside E is preferably added at 0.001 to 50% by weight, more preferably at 0.001 to 40% by weight, and most preferably at 0.001 to 30% by weight, based on the total weight of the entire pharmaceutical composition. You can.

The pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, aerosols, external preparations, suppositories, and sterilized injection solutions according to conventional methods. You can. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, 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, etc. These solid preparations contain at least one excipient, such as starch, calcium carbonate, and water, in the solubilized decursinol of the present invention. It is prepared by mixing cross, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, preservatives, and acidulants. may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween-61, cacao, laurin, glycerogeratin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled 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 several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection and dermal application. Hederacolchiside E of the present invention has almost no toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

The present invention provides a health functional food for preventing or improving sepsis containing 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 containing Hederacolchiside E and food chemically acceptable food supplements.

The health functional food contains solubilized Hederacolchiside E, preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, and most preferably 0.001 to 10% by weight, based on the total weight of the entire food. It can be added as a percentage.

The health functional food includes the form of tablets, capsules, pills, or liquid, and foods to which the extract of the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and health products. There are functional foods, etc.

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

Figure 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 HMGB1 release levels in LPS-treated HUVECs (A), CLP animal model (B), TLR2 and TLR4 alc RAGE levels in LPS-treated HUVECs (C), and cell viability (D). am.
Figure 3 confirms the effect of HCE on the activation of SIRT1 (A) and the acetylation of HMGB1 (B). (IP: Immuno Precipitation, WB: Western Blotting)
Figure 4 is a graph showing the effect of HCE on HMGB1-mediated destruction of the vascular barrier. (A) Effect of HCE on LPS-induced permeability in HUVEC. (B) Effect of HCE on HMGB1-induced permeability in HUVEC. (C) Effect of HCE on Evans blue efflux in CLP animal model. (D) Effect of HCE on HMGB1-induced p-38 expression in HUVEC.
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 increase of VCAM, ICAM, and E-Selectin in HUVEC. (B) Effect of HCE on HMGB1-mediated adhesion of neutrophils to HUVEC monolayer. (C) Effect of HCE on HMGB1-mediated neutrophil metastasis to HUVEC monolayer in HUVEC. (D) Effect of HCE on the number of white blood cells migrating to the abdominal cavity in a CLP animal model. (E) Light micrograph showing the effect of HCE on HMGB1-mediated neutrophil metastasis to HUVEC monolayer in HUVEC.
Figure 6 shows the effect of HCE on HMGB1-induced expression of IL-6, TNF-α, and IL-1β and 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 HUVEC. Effect of HCE on HMGB1-mediated activation of NF-κB p65 (E) and ERK 1/2 (F). (G) Intracellular levels of NF-κB in nuclear and cytoplasmic fractions assessed by Western blot analysis.
Figure 7 shows the effect of HCE on mortality and tissue damage after CLP. (A) Inhibitory effect of HCE on mouse lethality induced by CLP. (B) Light micrograph of lung injury induced by CLP (arrows indicate leukocyte infiltration). (C) HCE effect on lung injury induced by CLP. of 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, the content introduced herein is provided to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

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

1.1 Cell culture and reagents

Human umbilical vein endothelial cells (HUVEC) were obtained and maintained from Cambrex Bio Science (Charles City, IA, USA), and all experiments were performed at passages 3 to 5. Gingerone, LPS, crystal violet, Evans blue, MTT, 2-penicillin G and streptomycin, mercaptoethanol and DMSO were purchased from Sigma Chemical Co. (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 ) separation of

HCE is Hedera Leaves of colchica and Pulsatilla First isolated from the roots of koreana and Anemone raddeana , Pulsatilla There are only reports that koreana reduces inflammation induced by Carrageenan, but there are no reports that HCE increases the survival rate of sepsis patients by inhibiting the progression of HMGB1-mediated sepsis.

1.0 kg of dried roots of Anemone raddeana were mixed with 1.5 L of distilled water in a mixer and placed in an incubator at 37°C for 4 hours. The mixture was suspended in 80% MeOH (6L), stirred for 6 hours and then filtered. The filter cake was extracted three times in succession with equal amounts of 80% MeOH. The resulting filtrates were combined and concentrated in vacuo to obtain brown syrup (88.86 g). Thereafter, the dark brown syrup (88.86g) was suspended in distilled water (2L) and fractionated with ethyl ether (0.8L × 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%) and analyzed by TLC (BuOH :AcOH:HO = 5:1:1), seven major fractions (R1-R7) were obtained and compared with standard HCE (Wuhan Chemnorn 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 × 4.6 mm). Mobile phase conditions were set as gradient solvent system A (acetonitrile) and B [(0.1% phosphoric acid in water (v/v)]. The detailed gradient elution was 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. The flow rate of mobile phase was 1.0 mL/min, wavelength was 203 nm, column temperature was 30℃, injection volume was 10 μL, physicochemical data of separated HCE were confirmed by referring to previous reports. HCE was used at 1, 2, 5, 10, or 20 μM for in vitro analysis and at 0.2, 0.5, 1, or 2 mg/kg for in vivo analysis.

1.3 Sepsis animal model

Male C57BL/6 mice (6–7 weeks old, 27 g) were purchased from Orient Bio Co. (Sungnam, Republic of Korea). The animal model of CLP-induced sepsis was prepared according to the method of Jung et al. (2016), and the cecum of a group of sham control animals was exposed but no ligation or perforation was made. Samples such as HCE compounds or GR were injected intravenously 24 h after the CLP procedure and assessed for their effects on HMGB1 secretion, cell permeability and leukocyte migration, or were injected intravenously at 12 and 50 h after CLP to assess survival. This protocol was approved by the Kyungpook National University Animal Care Committee before 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 statistical significance. 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 About 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 treated with 0, 1, 2, 5, 10, 20 μM of HCE and 20 μM of ZGR, respectively, and the amount of HMGB1 released for 16 hours was analyzed by ELISA. It was measured and shown in Figure 2A. As shown in Figure 2A, the secretion of HMGB1 in HUVECs was significantly increased by LPS, and this increase was suppressed in a concentration-dependent manner by HCE treatment.

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

Next, the effect of HCE on the expression levels of the well-known HMGB1 receptors TLR2, TLR4, and RAGE was confirmed. HUVECs prepared in Example 1.1 were treated with HMGB1 (1 μg/mL), then treated with 5, 10, 20 μM of HEC, and 20 μM of ZGR, respectively, and left for 16 hours. Afterwards, the expression levels of TLR2, TLR4, and RAGE were confirmed using whole-cell ELISA and are shown in Figure 2C. The 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 Figure 2C, the expression levels of TLR2 (white), TLR4 (gray), and RAGE (black) were rapidly increased by HMGB1 treatment, and this increase was suppressed by HCE treatment in a concentration-dependent manner.

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

< Example 3. HCE of SIRT1 activation and of HMGB1 In acetylation See the impact >

It is known that the ability of HMGB1 to bind DNA in the cytoplasm is due to hyperacetylation of HMGB1 (Bonaldi et al., 2003). In other words, hyperacetylation of the serine residue of HMGB1 promotes cytoplasmic secretion of HMGB1 (Youn et al., 2006). At this time, 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 treating HUVECs with HCE (20 μM) for 0, 2, 4, 6, 8, 10, and 12 hours, the cells were lysed and analyzed through Western blotting to determine the expression level of SIRT1, as shown in Figure 3A. . As shown in Figure 3A, the expression of SIRT1 was evident after 4 hours of incubation, peaked after 6 hours and was maintained for up to 8 hours, and then gradually decreased.

Additionally, the effects of HCE on deacetylation of HMGB1 and induction of SIRT1 expression were 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), 10mM, for 1 hour before HCE treatment. After incubation for 6 h, cells were lysed, immunoprecipitated with anti-HMGB1 antibody, and acetylated HMGB1 and total HMGB1 protein levels were determined 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 and shown in Figure 3B. As shown in Figure 3B, stimulation with LPS increased HMGB1 acetylation, and this increase was significantly reduced by HCE. Additionally, sirtinol treatment was shown to significantly increase acetylation of HMGB1, reversing the effect of HCE. These results suggest that HCE significantly reduced LPS-induced HMGB1 release from HUVECs through SIRT1-mediated deacetylation of HMGB1.

< Example 4. HCE's of the blood vessel barrier HMGB1 Check the effect on vector destruction >

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

The monolayer of HUVEC was treated with LPS (100 ng/mL) or HMGB1 (1 μg/mL), respectively, and treated with 0, 1, 2, 5, 10, 20 μM of HCE or 20 μM of ZGR for 6 hours, respectively. Barrier integrity was assessed by monitoring the flux of Evans blue-bound 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 Figures 4A and 4B, LPS and HMGB1 significantly increased the permeability of the HUVEC monolayer, and HCE inhibited both LPS- and HMGB1-mediated hyperpermeability in a concentration-dependent manner.

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

The HMGB1-induced vascular destruction response 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 is shown in Figure 4D. As shown in Figure 4D, HMGB1 increased phosphorylation of p38, and HCE inhibited HMGB1-mediated phosphorylation of p38. HMGB1-mediated barrier disruption, increased permeability and phosphorylation of p38 were restored by HCE, suggesting that HCE has the potential as a preservative.

< Example 5. HCE's CAM's HMGB1 Determination of effects on mediator 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 leukocyte migration across the vascular endothelium to sites of inflammation. Therefore, we analyzed the effect of HCE on HMGB1-mediated regulation of CAM in HUVEC, adhesion of human neutrophils to HUVEC, and migration of neutrophils through HUVEC monolayer.

Migration of human neutrophils into HUVEC was performed in 6.5-mm-sized Transwell plates containing a filter with a pore size of 8 μm, applying the method of Lee et al. (2019). To obtain confluent endothelial monolayers, HUVECs were cultured for 3 days, before adding neutrophils to the upper compartment, HUVECs were stimulated with HMGB1 (1 μg/mL) for 16 h and then incubated with ZGR at 20 μM or HCE for 6 h at each concentration. cultured for a while. Next, the Transwell plate was incubated at 37°C for 2 hours and floating or non-migrating neutrophils and HUVECs in the upper chamber and filter membrane were removed. Neutrophils that migrated to the bottom of the filter were fixed with 8% glutaraldehyde and stained with 0.25% crystal violet in 20% methanol (w/v). Nine randomly selected high-power microscopic fields (200X) were then counted. All experiments were repeated twice per well in duplicate wells, and the results were expressed as migration index and are shown in FIGS. 5A to 5C and 5E. As shown in Figures 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 upregulated by HCE. It was suppressed in a concentration-dependent manner. In addition, HMGB1-mediated adhesion of neutrophils to HUVEC monolayers (Figure 5B) and increased neutrophil migration through HUVEC monolayers (Figures 5C and 5E) were also inhibited by HCE in a concentration-dependent manner.

The expression of CAM upregulated by HMGB1 was suppressed by HCE in a concentration-dependent manner, and in addition to the expression level of CAM, HCE also reduced the adhesion of human neutrophils to HUVEC 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 into the breathing chamber and then animals were anesthetized with a face mask via a small rodent gas anesthesia machine (RC2; Vetequip, Pleasanton, CA, USA). Afterwards, mice were treated with HMGB1 (2 μg/mouse, i.v.) for 16 hours and then intravenously injected with HCE (0.2, 0.5, 1.0, 2.0 mg/kg) or ZGR (0.7 mg/kg). Six hours later, mice were sacrificed and peritoneal fluid (20 μl) samples obtained by washing the abdominal cavity with saline (5 mL) were stained with 0.38 mL of Turk's solution (0.01% crystal violet in 3% acetic acid) for leukocyte count under light microscopy. are counted and shown in Figure 5D. As shown in Figure 5D, treatment with HMGB1 increased the migration of leukocytes, 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 for inhibition of α production>

HMGB1 aggravates the pathological state of sepsis by inducing the production of inflammatory cytokines through various signaling pathways, including NF-κB and ERK 1/2 (Jung et al., 2016). Therefore, we evaluated the inhibitory effect of HCE on 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, then treated with HCE for 6 h, and incubated with 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), TNF-α, IL-6, and IL-1β levels in cell culture supernatants were analyzed with ELISA kits and are shown in Figures 6A to 6A. Shown in 6F. As shown in Figures 6A to 6F, the production of inflammatory cytokines enhanced by HMGB1 in HUVEC was suppressed by HCE treatment. Additionally, HMGB1 induced nuclear localization of p65 NF-κB in HUVEC, which was inhibited by HCE treatment, as shown in Figure 6G.

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

The present inventors actually confirmed the effect of HCE on the survival rate of a sepsis animal model. A single injection of HCE or ZGR did not protect against CLP-induced mortality (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 Figure 7A. As shown in Figure 7A, double administration of HCE significantly improved the survival rate of mice after CLP, and the HCE 2.0 mg/kg administered group showed a higher survival rate than the positive control ZGR administered group (p <0.00001).

Systemic inflammation that occurs during the course of sepsis frequently induces MOF, with liver and kidney being the main target organs. Therefore, we evaluated the potential protective effect of HCE against lung damage caused by CLP. Male mice (n = 5) were subjected to CLP and then administered HCE (1.0 or 2.0 mg/kg) or ZGR (0.7 mg/kg) intravenously at 12 and 50 h after CLP. Mice were sacrificed 4 days after CLP and blinded observation was performed with light microscopy analysis of lung specimens to evaluate lung structure, tissue edema and infiltration of inflammatory cells, as shown in Figures 7B and 7C. As shown in Figures 7B and 7C, HCE improved CLP-induced interstitial pulmonary edema and severe damage to lung tissue. In addition, plasma levels of ALT and AST, indicators of liver damage (Figure 7D), and BUN and creatinine, 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 commercial kits (Pointe Scientific, Lincoln Park, MI, USA). It was determined using As a result, ALT, AST, BUN, and creatinine, which were significantly increased by CLP, were suppressed by HCE (Figures 7D to 7F), and all of these increases were also suppressed, as well as the increase in LDH, another important indicator of tissue damage (Figures 7G).

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

< example 1. Pharmaceutical preparations>

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. A 10% gelatin solution was added to this mixture, then pulverized and passed through a 14 mesh sieve. This was dried and 160 g of potato starch, 50 g of activator, and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.

example 1-2. Manufacturing of injectable solutions

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.

< example 2. Manufacturing of health functional foods>

example 2-1. Manufacturing of health functional foods

Hederacolchiside E of the present invention 100 mg, vitamin mixture appropriate amount, vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg, vitamin C 10 mg, Biotin 10㎍, nicotinic acid amide 1.7㎎, folic acid 50㎍, calcium pantothenate 0.5㎎, appropriate amount of mineral mixture, ferrous sulfate 1.75㎎, zinc oxide 0.82㎎, magnesium carbonate 25.3㎎, potassium phosphate monobasic 15㎎, calcium phosphate dibasic. It was manufactured by mixing 55 mg of potassium citrate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride into granules, but it can be modified and manufactured into various dosage forms depending on the purpose. In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily modified, and the above ingredients may be mixed according to a typical health functional food manufacturing method.

example 2-2. Manufacturing of health functional beverages

A beverage was prepared by mixing 100 mg of Hederacolchiside E of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water and stirring, heating, filtering, sterilizing, and refrigerating according to a typical beverage production method.

Claims (10)

A composition for preventing or treating sepsis containing Hederacolchiside E as an active ingredient. According to paragraph 1,
The hederacolchiside E is characterized in that it inhibits 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. Composition for prevention or treatment. According to claim 1 or 2,
The hederacolchiside E is selected from the group consisting of TLR2 (Toll-like receptor 2), TLR4 (Toll-like receptor 4), and RAGE (receptor for advanced glycation end products), which are receptors for 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. According to claim 1 or 2,
The hederacolchiside E is a composition for preventing or treating sepsis, characterized in that it induces sirtuin 1 protein activation or deacetylation of high mobility group box 1 (HMGB1). According to 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. According to clause 5,
Maintaining the vascular barrier integrity involves 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). A composition for preventing or treating sepsis, characterized in that it consists of inhibiting the expression of CAM (cell adhesion molecule). According to claim 1 or 2,
The hederacolchiside E increases TNF-α (Tumor necrosis factor-α), IL-1β (Interleukin-1β), IL-6 (Interleukin-6), and NF-α mediated by high mobility group box 1 (HMGB1). Prevention or treatment of sepsis, characterized by inhibiting 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) Composition for. According to claim 1 or 2,
The hederacolchiside E increases aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen mediated by high mobility group box 1 (HMGB1). , BUN) A composition for preventing or treating sepsis, characterized in that it reduces one or more selected from the group consisting of creatinine and lactate dehydrogenase (LDH). A health functional food for preventing or improving sepsis containing Hederacolchiside E as an active ingredient. According to paragraph 1,
The hederacolchiside E is characterized in that it inhibits 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. Health functional food for prevention or improvement.