WO2014012312A1 - 一种抗炎症的脂肽及其制备方法和应用 - Google Patents

一种抗炎症的脂肽及其制备方法和应用 Download PDF

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WO2014012312A1
WO2014012312A1 PCT/CN2012/085171 CN2012085171W WO2014012312A1 WO 2014012312 A1 WO2014012312 A1 WO 2014012312A1 CN 2012085171 W CN2012085171 W CN 2012085171W WO 2014012312 A1 WO2014012312 A1 WO 2014012312A1
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lipopeptide
induced
inflammatory
inflammation
poly
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PCT/CN2012/085171
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English (en)
French (fr)
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李冬青
王玥
李红泉
赖玉平
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华东师范大学
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Priority to US14/440,291 priority Critical patent/US10072051B2/en
Publication of WO2014012312A1 publication Critical patent/WO2014012312A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention relates to the field of bioengineering technology, and in particular to a lipopeptide capable of inhibiting excessive inflammatory reaction, and a preparation method and application thereof. Background technique
  • the inflammatory response is a type of protective response that occurs when the body is damaged by the outside world.
  • the phenotype is red, swollen, hot, painful, and dysfunctional.
  • Proper inflammatory response is beneficial to the body to clear the external antigen and restore the physiological balance of the tissue, but excessive or sustained inflammatory reaction will cause tissue damage and may cause organ dysfunction, such as encephalitis, flitis, nephritis, arthritis, etc. .
  • the basic processes of inflammation include damage, exudation and hyperplasia of local tissues. Since the skin is directly in contact with the external environment, skin inflammation caused by external stimuli is the most common type of inflammation, including psoriasis, atopic dermatitis (eczema), and neurodermatitis. The cause of this type of skin disease is more complicated and difficult to cure. Even if cured, the recurrence rate is high.
  • the lipopeptide is composed of a hydrophilic peptide chain and a lipophilic fatty acid chain, that is, a cyclic or linear lipid raft is formed by about 10 polypeptides and fatty acid chains.
  • Lipopeptides are generally produced by the metabolism of Gram-positive bacteria, which exhibit a variety of biological activities. In the current study, the activity of lipopeptides mainly appears as: surfactant, sterilization, insecticidal and the like. Recently, studies have shown that lipopeptides also have anti-inflammatory activity (Long EM, et al. A subclass of acylated anti-inflammatory mediators usurp Toll-like receptor 2 to inhibit neutrophil recruitment through peroxisome proliferator activated receptor gamma. PNAS, 2011, 108 ( 39): 16357-62)» There are few reports on the anti-inflammatory function of lipopeptides, and it is a brand new field in biopharmaceuticals.
  • Staphylococcus epidermidis is a common group of bacteria that live on the surface of the skin and is a type of commensal bacteria. Staphylococcus epidermidis can produce a variety of active molecules, including bacteriocins, Pep5, PSMs, etc., all of which have bactericidal activity. Recent studies have shown that LTA secreted by S. cerevisiae can inhibit TLR3-mediated inflammation (Lai Y, et ai. Commensal bacteria regulate) Whether lipopeptides of Staphylococcus epidermidis can inhibit inflammation has not been reported.
  • the present invention provides an anti-inflammatory lipopeptide which inhibits poly(LC)-induced inflammatory reaction, prevents inflammation of skin wounds, and reduces inflammation of allergic dermatitis.
  • the invention provides a novel lipopeptide and a preparation method and application thereof.
  • the lipopeptide is obtained by solid phase chemical synthesis, which can inhibit poly(I:C)-induced inflammatory reaction, prevent inflammation of skin wounds, and reduce inflammation of allergic dermatitis.
  • the present invention provides a response of the lipopeptide in inhibiting poly(i:C)-induced inflammation.
  • poly CC induced the expression of a large number of TNF- ⁇ and IL-6
  • lipopeptide inhibited the expression of poly(I:C)-induced TNF- ⁇ and IL-6.
  • lipopeptides significantly inhibit the expression of the inflammatory factor mTNF- (x and m! L-6).
  • a lipopeptide binds to a TLR2 receptor on the cell surface in inhibiting inflammation.
  • keratinocytes (MKC cells) of IIr2 +/ mouse and IlrS ' mice are isolated, and the inhibitory effect of lipopeptide on poiy (I:C)-induced inflammation on both cells is detected at the cellular level.
  • the results showed that lipopeptide significantly inhibited poly(LC)-induced mTNF-a and raIL-6 expression in MKC cells of 73 ⁇ 4 ⁇ 2 +/+ mice, whereas lipids in MKC cells of 7?r2_ mice The peptide does not function as an anti-inflammatory.
  • the present invention provides the use of the lipopeptide for the preparation of a medicament for eliminating inflammation at a wound site which inhibits the occurrence of excessive inflammation in a skin wound.
  • the expression level of inflammatory factor mTNF-(x, m!L-6 in the wound site is significantly increased.
  • the lipopeptide of the present invention can significantly reduce the expression levels of inflammatory factors mTNF-a and mIL-6 at the wound site.
  • the peptide inhibits the inflammatory response at the wound site, speeding up the healing of the wound and promoting healing of the wound.
  • the present invention also provides the use of a lipopeptide for the treatment of atopic dermatitis which is capable of producing DNFB-induced inflammation.
  • DNFB dinitrofluorobenzene
  • the results showed that dinitrofluorobenzene (DNFB) can induce allergic dermatitis in the ears of BALB/c mice, and the expression levels of fflTNF-a and mIL-6 increased significantly.
  • the lipopeptide can significantly inhibit the expression of mlL-4 and mlL-6, and the allergic dermatitis symptoms are alleviated.
  • the present invention also provides the use of the lipopeptide in the preparation of a medicament for treating wound inflammation, and the use in the preparation of a medicament for treating atopic dermatitis.
  • a protein antigen-ovalbumin (OVA) stimulating cell for preparing an allergic dermatitis model is used, for example, NHEK cells are stimulated with PBS, OVA, lipopeptide, lipopeptide and OVA, and RNA is extracted after 24 hours, real- Time RT-PCR was used to detect the expression levels of TNF ⁇ and IL-6. The results showed that OVA can promote the expression of TNF- ⁇ in NHEK cells, lipopeptide inhibition OVA-induced expression of TNF ⁇ . When the PPARy(R) formulation GW9662 was added, the amount of OVA-induced inflammatory factor expression was significantly reduced.
  • OVA protein antigen-ovalbumin
  • poiy (I : C induces an inflammatory reaction mechanism; poiy (I: C) binds to TLR3, induces phosphorylation of p65, thereby promoting p65 entry into the nucleus, p65 is incorporated into the nucleus and binds to PMR Y to induce TNF- Expression of alpha and IL-6.
  • a lipopeptide binds to a TLR2 receptor on a cell, promotes phosphorylation of ⁇ -catenui (Tyr654), enhances its stability, and further promotes ⁇ - The transfer of catenin into the nucleus.
  • ⁇ -catenin binds to PPARy, which reduces the binding of p65 to PPARy, thereby inhibiting the development of inflammatory responses.
  • the lipid raft which inhibits the inflammatory reaction of the present invention can be synthesized by a chemical method.
  • This lipopeptide can significantly inhibit the inflammatory response induced by polyi:I::C), and can also inhibit inflammation in the wound site and alleviate the inflammatory response of atopic dermatitis.
  • the mechanism is that lipopeptides bind to the TLR2 receptor and promote the entry of ⁇ -catenin into the nucleus.
  • the ⁇ -caienin nucleus and p65 competitively bind to ⁇ , thereby reducing the binding of ⁇ 65 to PPARy and inhibiting the occurrence of inflammatory reactions.
  • Figure 1 shows that lipopeptide inhibits poly(I:C)-induced expression of TNF ⁇ and IL-6 on NHEK cells.
  • Figure 2 shows that lipid rafts inhibit po!y (:C)-induced inflammatory responses in mice.
  • Figure 3 shows that lipopeptides modulate po!y (i:C; induced expression of mTNF- ⁇ and mIL-6 in mice.
  • Figure 4 shows the effect of lipid rafts on poiy (I : C)-induced mouse ear thickness.
  • Figure 5 shows that NF- ⁇ inhibitor-PDTC inhibits poly(I:C)-induced expression of TNF- ⁇ and IL-6.
  • Figure 6 shows poly(I:C)-induced nuclear entry of p65 into NHEK cells.
  • Figure 7 shows that PPARy inhibitor -GW9662 inhibits poly(I:C)-induced expression of TNF- ⁇ and IL-6.
  • Figure 8 shows that poiy (:i:C) induces binding of p65 to PPARy.
  • Figure 9 shows the effect of lipopeptides on poiy:C)-induced phosphorylation of p65.
  • Figure: 0 indicates the effect of lipopeptide on poiy C)-induced p65 into the nucleus.
  • Figure 1 shows the effect of lipopeptide on pdy (i:C-induced p65 nuclear entry (nuclear separation).
  • Figure 12 shows the effect of lipopeptides on the inflammatory response induced by poiy (I:C) on I7r2 + + and 77 ⁇ 2 mice.
  • Figure 13 shows the effect of lipopeptides on the inflammatory response induced by poiy CC on keratinocytes derived from 7?r2 + A and r2- mice.
  • Figure 14 shows the effect of lipopeptides on ⁇ -catenin phosphorylation ( ⁇ > ⁇ 654).
  • Figure 5 shows that lipopeptide induces nuclear nucleation (nuclear separation) of ⁇ -cateiiin.
  • Figure 6 shows that lipopeptide induces the entry of ⁇ - catenin into the nucleus (immunofluorescence).
  • Figure 17 shows that lipopeptide inhibits poiy (I:C)-induced inflammatory response through ⁇ - cateiiii
  • Figure 18 shows that LiCl induces nuclear nucleation (nuclear separation) of ⁇ -cateiiin.
  • Figure 9 shows that Lia inhibits the poly(I:C)-induced inflammatory response.
  • Figure 20 shows that lipopeptide induces the binding of ⁇ -catenin to ⁇ .
  • Figure 21 shows that lipopeptide promotes wound healing in mouse skin.
  • Figure 22 shows that lipopeptide promotes wound healing rate in mouse skin.
  • Figure 23 shows that lipopeptide inhibits the expression of inflammatory factors niTNF- ⁇ and m!L 6 at the wound site.
  • Figure 24 shows the effect of lipopeptides on DNFB-induced mouse allergic dermatitis.
  • Figure 25 shows the effect of lipopeptides on DNFB-induced mouse ear thickness.
  • Figure 26 shows the effect of lipopeptide on inflammatory factors in DNFB-induced mouse allergic dermatitis.
  • the first amino acid was added to the activated Wang resin, and the condensation reaction was shaken for 2 hours to carry out the ligation reaction. After the connection, the detergent washing resin is added.
  • the cleavage agent is TFA: T]S: H2 0 -25: 1 : l (V A fatty acid is attached to the peptide chain by phthalic acid octyl octyl octyl ester and : methyl morpholine.
  • Lipopeptide inhibits poiy :C) induced inflammatory response in vitro.
  • Human keratinocyte (NHEK) cells were cultured and plated in 24-well plates. When the cells were grown to 70%-80%, 2 ⁇ 8 / ⁇ 1 of poly(I:C) was added, and then different concentrations were added as in Example 1.
  • Example 2 After anesthetizing BALB/c mice, 25 ⁇ 8 of the lipopeptide or PBS as shown in Example 1 was injected intraepithelially into each ear, 22 hours later, and then the same amount of lipopeptide or PBS was injected once, when lipopeptide or After PBS absorption, 25 ⁇ 8 of poly(i:C) was injected into each ear. After 24 hours, the redness of the mouse ears was observed and photographed. A portion of the sleeve RNA was cut from the mouse ear to detect the expression of mTNF-a and mIL-6, and the other part was subjected to HE staining. The experimental results are shown in Fig. 2.
  • NHEK cells were stimulated with PBS, poly(I:C), PDTC (NF- ⁇ inhibitor), poiy (I:C) and PDTC, respectively.
  • RNA was extracted and TNF- ⁇ was detected by real-time RT-PCR.
  • L-6 the expression of L-6.
  • poly(i:C) can significantly induce the expression of TNF-a and IL-6
  • poiy (I:C) induces an inflammatory response through the NF- ⁇ signaling pathway.
  • NHEK cells were stimulated with PBS and poiy:C) for 4 h, and after stimulating, 3 ⁇ 4 formaldehyde was fixed at 15 ml1, treated with hydrogen peroxide and Triton X100, and blocked with BSA for half an hour, then p65 antibody was added and incubated. After the overnight addition, the secondary antibody was added to detect the entry of p65 into the nucleus.
  • Poly experimental results shown in FIG. 6 (I: C) induced clearly P 65 into the core, the core group P 65 no.
  • NHEK cells were stimulated with PBS, poly(I:C), GW9662 (PPARy inhibitor), poly(I:C) and GW9662, respectively.
  • RNA was extracted and real-time RT-PCR was used to detect TNF- ⁇ and L-L.
  • Expression of -6 As shown in Figure 7, poly(i:C) can significantly induce the expression of TNF-a and L-6, and poly(I:C)-induced TNF-a and IL-6 are significant after the addition of PPARy inhibits U GW9662. decline. Thus poiy (I:C)-induced TNF-a and IL-6 expression is through ⁇ ⁇ .
  • Example 5 Poly(I:C) induces the binding of ⁇ -catenin to ⁇ .
  • NHEK cells were stimulated with PBS, poiy (I:C), lipopeptide, poiy i:C) lipopeptide, respectively, after stimulating, RIM lysate was used to lyse the cells, and the protein concentration of the lysate was determined by BCA protein quantification kit. 40 f ig was subjected to SDS-PAGE electrophoresis, and the effect of lipopeptide on p65 phosphorylation was detected by Western blot. The experimental results are shown in Figure 9. Poly(I:C) can significantly induce phosphorylation of p65 at 4h and 12h, while lipopeptide has no effect on phosphorylation of p65. Therefore, inhibition of the inflammatory response by lipopeptides is not achieved by affecting the acidification of p65.
  • NHEK cells were stimulated with PBS, poly(I:C), lipopeptide, poly (I:C lipopeptide for 12h, then formaldehyde was fixed for 15mm, treated with hydrogen peroxide and Triton X100, blocked with BSA for half an hour, then The p65 antibody was added and incubated overnight at 4 ° C. After overnight, a secondary antibody was added and the p65 nucleus was detected on a fluorescence microscope. The results are shown in Figure 10. Compared with the control group, the lipopeptide had no effect on the entry of p65 into the nucleus. Poly(I:C) significantly induced the incorporation of p65 into the nucleus, and the poly(I:C) fatliquoring group also showed significant p65 entry into the nucleus.
  • the nucleoproteins of NHEK cells were isolated by PBS, poly(I:C), lipopeptide and poly(I:Q lipopeptides, respectively), and the content of p65 in the nucleus was detected by Western blot.
  • the experimental results are shown in the figure. 11: Poiy (I:C)
  • the content of p65 in the nucleus of the stimulation group significantly increased the content of p65 in the poly(LC)-stimulated nucleus after the addition of lipopeptide. Therefore, the lipopeptide induced by poiy (I:C) The entry of p65 into the nucleus has no effect.
  • Example 7 Lipid peptide inhibits poiyO::C) induced inflammatory response by TLR2
  • the keratinocytes (MKC cells) of C57BLZ6 mice and ⁇ 7 ⁇ 2 ⁇ ⁇ mice were isolated, and MKC cells were stimulated with PBS, poly(I:C)- «lipopeptide, poty (I:C) lipopeptide, and extracted 24 hours later.
  • RNA, real-time RT-PCR was used to detect the expression of mTNF-a and mIL-6 in cells.
  • lipopeptides significantly inhibited poly(I:C)-induced mTNF-a and mIL-6 expression in MKC cells of C57BL/6 mice, but in MKC cells of I7r2-mouse
  • the upper lipopeptide does not inhibit the inflammatory response.
  • the above results indicate that lipopeptide inhibits ix>iya:c-induced inflammatory response through Th'2. Implementation of two: lipopeptide induces ⁇ -cateiiiii phosphorylation and nucleation
  • j3 ⁇ 4 6 .g ml of lipopeptide stimulated NHEK cells, and after stimulation, RIPA lysate was added to collect proteins. After determination of protein concentration, take 40 ⁇ ⁇ SDS- PAGE protein electrophoresis, the phosphorylation of ⁇ -catenin detected by Western blot. The results showed that lipopeptide can induce the phosphorylation of Tyr654 in ⁇ -catenin, which is most obvious at 2 h, as shown in Fig. 14. 1 ⁇ 654 phosphorylation can enhance the stability of ⁇ - cateni «, which in turn promotes the entry of catenin into the nucleus.
  • NHEK cells were stimulated with PBS and lipopeptide for 12 h, then fixed with formaldehyde for 15 miii, treated with hydrogen peroxide and Triton XI 00, blocked with BSA for half an hour, and then added with ⁇ -catena antibody, and incubated overnight at 4 °C. After overnight, a secondary antibody was added and the nucleus of ⁇ -cateniii was detected on a fluorescence microscope. The results of the experiment demonstrated that the lipopeptide significantly promoted the entry of ⁇ - catemii into the nucleus, while the ⁇ -cateniii of the control group was outside the nucleus, as shown in Fig. 16.
  • lipopeptide promoted the phosphorylation of 3 ⁇ 4n 654 of ⁇ - catenin, enhanced its stability, and promoted the entry of ⁇ -cateiiin into the nucleus.
  • Example 9 Lipopeptide inhibits poiy(::C)-induced inflammatory response by ⁇ -catenin
  • Example 8 I demonstrated that lipopeptide promotes phosphorylation of ⁇ -cateiiii and promotes its entry into the nucleus.
  • LiCl is an inhibitor of GSfGp, and activation of GSK3P can lead to degradation of ⁇ -cateiiin.
  • LiCl can be Induction of ⁇ -cateiiiii into the nucleus on vascular smooth muscle cells.
  • the present inventors conducted experimental studies on NHEK cells and found that LiC can also induce the entry of ⁇ -catenin into the nucleus of NHEK cells, as shown in FIG.
  • Example 8 demonstrates that lipopeptides can promote the entry of ⁇ -catenin into the nucleus, and LiCl can also promote the entry of ⁇ - cateniii into the nucleus.
  • Example 4 has demonstrated that pojy (I:C) induces an inflammatory response through ⁇ , a transcription factor located in the nucleus.
  • Example 8 demonstrates that lipopeptides can promote the entry of ⁇ - cateniii into the nucleus. This example demonstrates the interaction between ⁇ -catenin and ⁇ ⁇ . After immunizing NHEK cells with lipopeptide for 12 h, cells were lysed with NP40, ⁇ -catenin antibody was added to the cell lysate, and incubated overnight at 4 °C. After addition of Protein AG, incubation was continued for 1 to 2 h, and the protein was collected by centrifugation.
  • Lipopeptide inhibits skin wound inflammation and promotes wound healing
  • lipopeptides can inhibit poly(i:C)-induced inflammation in vitro or in vivo.
  • This example further demonstrates that lipopeptides inhibit inflammatory responses on skin wounds.
  • C57BL / 6 mice depilation back between 50 ⁇ ⁇ intradermal injection per mouse of formula (1) shown lipopeptide, 24h after doing the wound position of the injection lipopeptide daily wound healing wounds calculated photographing condition .
  • the skin around the wound was cut, and a part of the RNA was extracted, and part of it was stained with HE. The results of the experiment are shown in Fig. 21 and Fig. 22.
  • the lipopeptide After injection of the lipopeptide, the lipopeptide can significantly promote the healing of the wound, improve the healing speed of the wound, and at the same time reduce the inflammatory reaction at the wound site, as shown in Fig. 23. Lipid peptides promote healing of wounds by inhibiting excessive inflammatory responses at the wound site.
  • Example 12 Lipopeptide inhibits inflammatory response to atopic dermatitis
  • DNFB dinitrofluorobenzene
  • mTNF- ⁇ and mlL-6 were significantly increased by DNFB, and the expression of m: L-6 and mIL-4 was significantly inhibited after the addition of lipopeptide (P ⁇ 0.05), as shown in Fig. 26(AB); mTNF-ou mIL-13 and mIL-33 also inhibited, as shown in Figure 26 (CE).

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Abstract

本发明公开了一种抗炎症的脂肽，包括肽链和脂肪链，肽链和脂肪链通过肽键相连，脂肪酸接在肽链N端。该脂肽抑制polyI：C诱导的炎症反应，防止皮趺伤口的发炎，减轻过敏性皮炎等皮趺炎症的发炎反应。本发明还公开了该脂肽的制备方法。

Description

技术领域
本发明涉及生物工程技术领域， 尤其是涉及一种可以抑制过度炎症反应的脂肽及其制备 方法和应用。 背景技术
炎症反应是机体受到外界损伤时产生的一类保护性应答反应， 與型特征是红、 肿、 热、 痛和功能障碍。 适当的炎症反应有利于机体清除外界抗原， 恢复组织生理平衡， 但是过度的 或持续的炎症反应就会造成组织损伤， 并可能造成器官功能性障碍， 比如脑炎、 fl 炎、 肾炎、 关节炎等。 炎症的基本过程包括局部组织的损伤、 渗出和增生。 由于皮肤直接与外面环境接 触， 由外界刺激引起的皮肤炎症是最常见的一类炎症， 包括银屑病、 过敏性皮炎 （湿疹）、 神 经性皮炎等。 这类皮肤性疾病的病因比较复杂， 比较难治愈， 即使治愈， 复发率也很高。
脂肽是由亲水的肽链和亲脂的脂肪酸链两部分组成，即由 10个左右的多肽和脂肪酸链形 成环形或线性的脂駄。 脂肽类物质一般是由革兰氏阳性细菌代谢产生， 其表现出多种生物活 性。 在目前的研究中， 脂肽的活性主要表现为： 表面活性剂、 杀菌、 杀虫等。 最近有研究表 明脂肽还具有抗炎症的活性 (Long EM, et al. A subclass of acylated anti-inflammatory mediators usurp Toll-like receptor 2 to inhibit neutrophil recruitment through peroxisome proliferator activated receptor gamma.PNAS, 2011, 108(39): 16357-62)» 目前关于脂肽的抗炎症功能的研究 报道不多， 在生物制药方面是一个全新的领域。
表皮葡萄球菌是生存在皮肤表面的一类常见菌群， 是一类共生菌。 表皮葡萄球菌可以产 生多种活性分子， 包括细菌素， Pep5, PSMs等， 这些物质都具有杀菌活性。 最近研究证明表 皮葡萄球菌分泌的 LTA能抑制 TLR3介导的炎症 (Lai Y， et ai.Commensal bacteria regulate 关于表皮葡萄球菌属的脂肽是否能够抑制炎症， 目前尚未有相关的报道。
本发明提供一种抗炎症的脂肽， 可以抑制 poly(LC)诱导的炎症反应， 防止皮趺伤口的发 炎， 减轻过敏性皮炎的炎症反应。
发明内容
本发明提出了一种新型脂肽及其制备方法和应用。 该脂肽是采用固相化学合成的方法获 得， 可以抑制 poly(I:C)诱导的炎症反应， 防止皮肤伤口的发炎， 减轻过敏性皮炎的炎症反应。 本发明的发明目的之一是提供一种抗炎症的脂肽， 所述脂肽包括肽链和脂肪酸链， 肽链 和脂肪酸链通过肽键相连； 其结构如式 （1 ) 所示， 成线状；
Figure imgf000004_0001
本发明的另一发明目的是提供一种所述抗炎症的脂肽的制备方法， 所述多肽链采用固相 化学合成的方法， 脂肪酸链通过邻苯二甲酰丁辛酯和 N-甲基吗琳接在所述肽链上。
本发明提供了所述脂肽在抑制 poly(i:C)诱导的炎症中的应] ¾。 体外实验表明 poly CC)诱 导大量的 TNF- α和 IL- 6的表达， 脂肽抑制了 poly(I:C)诱导的 TNF- α和 IL- 6的表达。 体内实 验表明脂肽明显抑制炎症因子 mTNF-(x和 m!L-6的表达。
本发明中提供了脂肽在抑制炎症中与细胞表面的 TLR2受体相结合。
本发明中， 在野生型 （TIr2+/ ) 和 7Ir2基因敲除 (I7r2^小鼠上同时检測脂肽对 poly(I:C) 诱导的炎症的抑制作用， 结果表明在 27r2^小鼠上， 脂肽明显抑制 poly(I:C)诱导的炎症， mTNF-α和 mIL- 6的表达量明显 降。 37r2敲除后脂肽则不能抑制炎症反应。
本发明中， 分离 IIr2+/小鼠和 IlrS '小鼠的角质形成细胞 (MKC细胞)， 在细胞水平检测 脂肽在这两种细胞上对 poiy(I:C)诱导的炎症的抑制作用。 结果表明在 7¾~2+/+小鼠的 MKC细 胞上脂肽可以显著性的抑制 poly(LC)诱导的 mTNF- a、 raIL-6的表达；而在 7?r2_ 小鼠的 MKC 细胞上脂肽不能起到抗炎症作用。
本发明提供了所述脂肽在制备消除伤口部位炎症药物中的应用， 所述脂肽抑制皮肤伤口 中过度炎症的发生。
通常伤口部位的炎症因子 mTNF-(x、 m!L-6表达量呈显著上升。 实验表明， 本发明脂肽可 以使伤口部位的炎症因子 mTNF- a、 mIL-6表达量明显下降， 于脂肽抑制了伤口部位的炎 症反应， 使得伤口的愈合速度加快， 促进伤口的愈合。
本发明还提供脂肽在治疗过敏性皮炎方面的应用，所述脂肽能够 ίΦ制 DNFB诱导的炎症。 实验结果表明，二硝基氟苯 (DNFB)可以在 BALB/c小鼠耳朵上诱导过敏性皮炎， fflTNF- a 和 mIL-6的表达量明显上升。但加入本发明脂肽后，脂肽可明显抑制 mlL-4和 mlL- 6的表达， ϋ过敏性皮炎症状减轻。
本发明还提供所述脂肽在制备治疗伤口炎症药物中的应用， 以及在在制备治疗过敏性皮 炎药物中的应用。
此外， 用一种制备过敏性皮炎模型的蛋白质抗原-卵清蛋白 （OVA)剌激细胞， 例如， 用 PBS、 OVA, 脂肽、脂肽加 OVA刺激 NHEK细胞， 24h后抽提 RNA, real-time RT-PCR检测 TNF α和 IL- 6的表达量。 实验结果发现 OVA可以促进 NHEK细胞 TNF- α的表达， 脂肽抑制 OVA诱导的 TNF α的表达。 当加入 PPARy裨制剂 GW9662之后， OVA诱导的炎症因子表达 量明显降低。
本发明中， poiy(I:C诱导炎症反应的机理是； poiy(I:C)结合 TLR3 , 诱导 p65的磷酸化， 进而促进 p65的入核， p65入核后结合 PMRY, 共同诱导 TNF-α和 IL-6的表达。
本发明中， 脂肽抑制 poly(LC)诱导的炎症反应的机理是： 脂肽结合细胞上的 TLR2受体， 促进 β-catenui的磷酸化（Tyr654), 增强其稳定性， 进而促进了 β-catenin 向细胞核内的转移。 在细胞核内 β-catenin结合 PPARy, 减少了 p65与 PPARy的结合， 从而抑制了炎症反应的发 生。
本发明抑制炎症反应的脂駄可通过化学方法合成。 该类脂肽可以明显抑制 polyi:I::C)诱导 的炎症反应， 还可以抑制伤口部位的炎症， 缓解过敏性皮炎的炎症反应。 其机理是脂肽通过 结合 TLR2受体， 促进 β-catenin的入核， β-caienin 入核与 p65竞争性地与 ΡΡΑΚγ结合， 进 而减少 ρ65与 PPARy的结合， 抑制了炎症反应的发生。
跗图说明
图 1表示脂肽在 NHEK细胞上抑制 poly(I:C)诱导的 TNF α和 IL-6的表达。
图 2表示脂趺在小鼠体内抑制 po!y( ::C)诱导的炎症反应。
图 3表示脂肽在小鼠体内捭制 po!y(i:C;诱导的 mTNF- α和 mIL- 6的表达。
图 4表示脂駄对 poiy(I:C)诱导的小鼠耳朵厚度的影响。
图 5表示 NF- κΒ抑制剂 - PDTC抑制 poly(I:C)诱导的 TNF- α和 IL- 6的表达。
图 6表示 poly(I:C)诱导 NHEK细胞 p65的入核。
图 7表示 PPARy抑制剂 -GW9662抑制 poly(I:C)诱导的 TNF-α和 IL-6的表达。
图 8表示 poiy(:i:C)诱导 p65与 PPARy的结合。
图 9表示脂肽对 poiy :C)诱导的 p65磷酸化的影响。
图 : 0表示脂肽对 poiy C)诱导的 p65入核的影响 ί免疫荧光
图】1表示脂肽对 pdy(i:C诱导的 p65入核的影响 (核质分离)。
图 12表示脂肽对 I7r2+ +和 77Γ2 ·小鼠上 poiy(I:C)诱导的炎症反应的影响。
图 13表示脂肽对来源于 7?r2+A和 r2-小鼠的角质形成细胞上 poiy CC)诱导的炎症反应 的影响。
图 14表示脂肽对 β- catenin磷酸化 （Ί>τ654) 的影响。
图】5表示脂肽诱导 β- cateiiin的入核 （核质分离）。
图】6表示脂肽诱导 β- catenin的入核 （免疫荧光）。
图 17表示脂肽抑制 poiy(I:C)诱导的炎症反应通过 β- cateiiii 图 18表示 LiCl诱导 β-cateiiin的入核 （核质分离）。
图】9表示 Lia抑制 poly(I:C)诱导的炎症反应。
图 20表示脂肽诱导 β- catenin与 ΡΡΑΚγ的结合。
图 21表示脂肽促进小鼠皮肤伤口愈合。
图 22表示脂肽促进小鼠皮肤伤口愈合率。
图 23表示脂肽抑制伤口部位炎症因子 niTNF-α和 m!L 6的表达。
图 24表示脂肽对 DNFB诱导的小鼠耳朵过敏性皮炎的影响。
图 25表示脂肽对 DNFB诱导的小鼠耳朵厚度的影响。
图 26表示脂肽对 DNFB诱导的小鼠耳朵过敏性皮炎的炎症因子影响。
具体实施方式
结合以下具体实施例和附图， 对本发明作进一步的详细说明， 本发明的保护内容不局限 于以下实施例。 在不背离发明构思的精神和范围下， 本领域技术人员能够想到的变化和优点 都被包括在本发明中， 并且以所^的权利要求书为保护范围。 实施本发明的过程、 条件、 试 剂、 实验方法等， 除以下专门提及的内容之外， 均为本领域的普遍知识和公知常识， 本发明 没有特别限制内容。
实施例】， 脂肽的合成。
委托吉尔生化（上海）有限公司合成如式 （1 )结构的脂肽。 其中多肽链的合成采用固相 合成的方法， 脂肪酸通过邻苯二甲醜丁辛酯和 N-甲基吗琳接在肽链上。
Figure imgf000006_0001
以上多肽链的固相合成采用 FMOC 合成法。 具体歩骤为：
1.在活化的王氏树脂中加入第一个氨基酸， 加入缩合剂震荡 2h进行连接反应。 连接后 加入洗涤剂洗涤树脂。
2.将第一个氨基酸的氨基保护基 Frax>c脱保护。
3.加入第二个氨基酸及缩合剂进行缩合。
4.重复歩骤 2和歩骤 3， 直到所有氨基酸縮合完毕。
5. 加入切割剂将多肽^树脂上切割下来。 切割剂为 TFA: T】S: H2 0 -25: 1 : l(V A 脂肪酸通过邻苯二 ^酰丁辛酯和: ^甲基吗啉接在肽链上。
6. 采用高效液相色谱将合成的粗品进行纯化， 最终得到目的产物。 实施俩 2: 脂肽在体外抑制 poiy :C)诱导的炎症反应。 培养人角质形成细胞 (NHEK)细胞， 接种在 24 孔板内， 当细胞长至 70%- 80%时， 加入 2μ8/ίη1的 poly(I:C), 然后加入不同浓度的如实施例 1式（1 )所示的脂肽。 刺激 24小^, 然 后提取细胞的总 R A, 反转录成 cDNA后， real-time RT-PCR检测 TNF-α和 IL-6的表达， 实 验结果表明， 如图 i 所示， poly(I:Cy可以诱导 TNF- e 和 IL- 6的大量表达;加入脂肽后， 脂肽 抑制了 po!y(i:C)诱导的 TNF- a和 [L- 6的表达。 实施例 3: 脂肽在体内抑制 poly(LC)诱导的炎症反应。
将 BALB/c小鼠麻醉后， 在每只耳朵上皮间注射 25μ8如实施例 1所示的脂肽或 PBS, 22 小 '后再-一次注射相同量的脂肽或 PBS， 当脂肽或 PBS 吸收后， 每只耳朵再注射 25μ8 的 poly(i:C), 24h后观察小鼠耳朵的红肿现象， 拍照。 将小鼠耳朵剪下一部分袖提 RNA, 检测 mTNF-a和 mIL-6的表达， 另外一部分做 HE染色。实验结果如图 2 所示， p0ly(I:C)组的小鼠 耳朵可以看到很明显的红 ϋ中现象， 打入脂肽后， 耳朵的红肿现象减弱。 同样 real-time R PCR 结果证明， 注射脂肽后可以很明显地抑制炎症因子 mTNF- n mlL- 6的表达， 见图 3。 注射 脂肽后小鼠耳朵的厚度明显变薄， 耳朵上的白细胞数量明显下降， 见图 4。 实施例 4: poiy(: :C)通过 NF- κΒ信号通路及 PPAR- γ诱导炎症反应。
用 PBS、 poly(I:C), PDTC (NF-κΒ抑制剂)、 poiy(I:C)加 PDTC分别剠激 NHEK细胞， 24h 后， 抽提 RNA, real-time RT-PCR检测 TNF-α和〗L-6的表达。 如图 5所示， poly(i:C)可以显 著地诱导 TNF-a和 IL- 6的表达，加入 NF-κΒ抑制剂 PDTC后， poiy(I:C)诱导的 TNF-a和 IL- 6 表达下降。 由此可见， poiy(I:C)诱导炎症反应是通过 NF-κΒ信号通路。
用 PBS和 poiy :C)分别刺激 NHEK细胞 4h,剌激之后 ]¾甲醛固定 15m½,用过氧化氢和 Triton XlOO处理后， 用 BSA封阻半小时后， 加入 p65抗体， 孵育。 过夜后加入二抗， 检 测 p65的入核。 实验结果如图 6所示， poly(I:C)很明显地诱导 P65的入核， 对照组未见 P65 的入核。
用 PBS、 poly(I:C)、 GW9662(PPARy抑制剂)、 poly(I:C)加 GW9662分别刺激 NHEK细胞， 24h后, 抽提 RNA, real-time RT- PCR检测 TNF-α和〗L-6的表达。 如图 7所示， poly(i:C)可 以显著诱导 TNF- a和】L- 6的表达， 加入 PPARy抑制齐 U GW9662后， poly(I:C)诱导的 TNF- a 和 IL-6显著下降。 因此 poiy(I:C)诱导的 TNF-a和 IL-6的表达是通过 ΡΡΑΙ γ。 实施例 5: poly(I:C)诱导 β- catenin与 ΡΡΑΛγ的相互结合。
实施俩 4证明 poiy( ::C)可以诱导 p65的磷酸化并且诱导其入核， ¾证明 poly C)诱导炎 症反应是通过 PPARy,本实施例证明入核的 p65与存在于细胞核内的 ΡΜΚγ是否存在相互作 用。 用 po¼: :C)刺激 ΝΗΕΚ细胞 4h、 〗2h后， 用 NP40裂解细胞， 在细胞裂解液中加入 p65 抗体， 4°C孵育过夜。 加入 ProteinAG后， 继续孵育 l-2h， 离心收集 Protein AG, 洗涤缓冲 液洗两次后， 加入 20μ1 2χ的上样缓冲液, 上样到 SDS-PAGE上, 用 Western blot检测 ΡΡΑΙΙγ 的表达。 实验结果如图 8所示， poly(I:C)可以显著诱导 ρ65与 ΡΡΑΚγ的结合， 即 po!y(i:C)诱 导炎症反应可通过 p65与 ΡΡΑΚγ共同实现的。 实施例 6： 脂肽不影响 poly(I:C)诱导的 ρ65磯酸化以及 ρ65的入核。
用 PBS、 poiy(I:C), 脂肽、 poiy i:C)加脂肽分别刺激 NHEK细胞， 剠激之后加入 RIM裂 解液裂解细胞，用 BCA蛋白质定量试剂盒测定裂解液蛋白质浓度后，取 40fig进行 SDS- PAGE 电泳，通过 Western blot检测脂肽对 p65磷酸化的影响。实验结果如图 9所示， poly(I:C)在 4h、 12h可以明显地诱导 p65的磷酸化， 而脂肽对 p65的磷酸化没有影响。 因此脂肽抑制炎症反 应不是通过影响 p65的磯酸化来实现的。
用 PBS、 poly(I:C), 脂肽、 poly(I:C加脂肽分别刺激 NHEK细胞 12h， 然后 甲醛固定 15mm, 用过氧化氢和 Triton X100处理后, 用 BSA封阻半小时, 然后加入 p65抗体， 4°C孵 育过夜。 过夜后， 加入二抗， 在荧光显微镜上检测 p65的入核。 实验结果如图 10所示， 与对 照组相比， 脂肽对 p65的入核没有影响， poly(I:C)很明显地诱导 p65的入核， poly(I:C)加脂駄 组也可以看到很明显的 p65的入核。
用 PBS、 poly(I:C), 脂肽、 poly(I:Q加脂肽分别刺激 NHEK细胞 12h后， 分离 NHEK细 胞的核蛋白， 然后用 Western blot检测细胞核内 p65的含量。实验结果如图 11所示： poiy(I:C) 刺激组细胞核内 p65的含量显著上 加入脂肽后对 poly(LC)刺激的细胞核内 p65的含量没 有影响。 因此脂肽对 poiy(I:C)诱导的 p65的入核是没有影响的。
综上所述脂駄不影响 poly(I:C)诱导的 p65磷酸化以及 P65的入核， 脂肽抑制炎症反应并 不是通过抑制 p65磷酸化以及 p65的入核来实现的。 实施例 7: 脂肽通过 TLR2抑制 poiyO::C)诱导的炎症反应
在野生型 (..蕭 /÷、和 r2-小鼠上同时检测脂肽对 pdy(: :C)诱导的炎症的抑制作用。 将 野生型 （27r2 A ) C57BL/6小鼠和 27r2-小鼠麻醉后， 在每只耳朵上皮间注射 25μ8的脂肽或 PBS, 22小时后再一次注射相同量的脂肽或 PBS,当脂趺或 PBS吸收后，每只耳朵再注射 25μβ 的 po!y(i:C), 24h后观察小鼠耳朵的红肿现象， 将小鼠耳朵剪下抽提 RNA, 检测 mTNF- α和 mIL-6的表达。实验结果如图 12所示，在野生型（27r2 )C57BL/6组，脂肽明显地抑制 poiy(I:C) 诱导的 mTNF-α和 mIL-6 的表达， mTNF- 和 mIL-6 的表达量明显下降， 即脂肽明显 ίΦ制 poly(LC)诱导的炎症。 而 T 2敲除后脂肽不能抑制炎症反应。
分离 C57BLZ6小鼠和 Ι7Γ2 ·Λ小鼠的角质形成细胞 （MKC细胞）， 分别用 PBS、 poly(I:C) -« 脂肽、 poty(I:C)加脂肽刺激 MKC细胞, 24h后提取 RNA, real-time RT-PCR检测细胞的 mTNF-a 和 mIL-6的表达。如图 13所示，在 C57BL/6小鼠的 MKC细胞上,脂肽可以显著地抑制 poly(I:C) 诱导的 mTNF-a和 mIL-6的表达， 但是在 I7r2-小鼠的 MKC细胞上脂肽不能抑制炎症反应。 以上结果表明， 脂肽抑制 ix>iya:c诱导的炎症反应是通过 Th'2。 实施俩 8: 脂肽诱导 β-cateiiiii磷酸化及入核
在不同的时间 （0h、 2h、 4h、 8h、 12hu 24h) j¾ 6 .g ml的脂肽刺激 NHEK细胞， 刺激之 后加入 RIPA裂解液， 收集蛋白。 将蛋白测定浓度后， 取 40μ§蛋白进行 SDS- PAGE电泳， 通 过 Western blot检测 β-catenin的磷酸化。 实验结果表明, 脂肽可以诱导 β-catenin的 Tyr654位 的磷酸化， 在〗 2h最明显， 如图 14所示。 1 Γ654位磷酸化可以增强 β- cateni«的稳定性， 进 而促进 catenin的入核。 同样 )¾ 6μ§/ηι1的脂肽刺激 ΝΗΕΚ细胞不同的时间， 然后提取细胞 的核蛋白， Western biot结果证明脂肽可以明显的促进 NHEK细胞中 β- cateiiiri的入核， 如图 15所示。
又 PBS、脂肽刺激 NHEK细胞 12h，然后用甲醛固定 15miii后，用过氧化氢和 Triton XI 00 处理后， 用 BSA封阻半小时， 然后加入 β- catena抗体， 4°C孵育过夜。 过夜后， 加入二抗， 在荧光显微镜上检测 β-cateniii的入核。 实验结果证明脂肽明显地促进 β- catemii的入核， 而对 照组的 β-cateniii在核外， 如图 16所示。
以上实验证明脂肽促进 β- catenin的 ¾n 654位的磷酸化，增强其稳定性，进而促进 β- cateiiin 的入核。 实施例 9： 脂肽通过 β-catenin抑制 poiy(: :C)诱导的炎症反应
实施例 8中我^证明脂肽促进 β -cateiiiii的磷酸化进而促进其入核，本实施例中我们用基 因沉默技术将 β- catenin基因沉默， 然后 ]¾检测脂趺是否还会抑制 poly^C)介导的炎症反应。 实验结果证明 β- catenin基因沉默后发现脂肽不能抑制 poly(I:C)诱导的炎症反应， 而对照组中 脂肽可以抑制 poiy(I:C)诱导的炎症反应， 如图 17所示。 因此脂肽可以促进 catenin的入核， β-catenin入核后抑制 poiyC C)诱导的炎症反应， 将 β- cate n基因沉默后脂肽不能抑制炎症反 应。
LiCl是 GSfGp的抑制剂， GSK3P的活化可以导致 β-cateiiin的降解。有文章报道 LiCl可 以在血管平滑肌细胞上诱导 β-cateiiiii的入核。本发明在 NHEK细胞上进行实验研究发现， LiC 也可以诱导 NHEK细胞中 β-catenin的入核,如图 18所示。实施例 8证明脂肽可以促进 β-catenin 的入核， LiCl也可以促进 β- cateniii的入核。我们进一步验证 LiCi对 poly(I:C)诱导的炎症反应 的影响,用 PBS、 LiCk poty(I:C)、 PBS加 LiCl分别剌激 NHE 细胞, 24h后提取细胞的 RNA, real-time RT-PCR结果证明 LiCl可以抑制 poly(I:C)诱导的炎症反应, 如图 19所示。 实施例 10: 脂肽诱导 β- cateiiiri与 PPAR'y结合
实施例 4已经证明 pojy(I:C)诱导炎症反应是通过 ΡΡΑΚγ, PPARy是一种位于细胞核内的 转录因子。 实施例 8证明脂肽可以促进 β- cateniii的入核。 本实施倒证实 β- catenin与 ΡΡΑΙ γ 之间存在的相互作用。 用脂肽刺激 NHEK细胞 12h后， 用 NP40裂解细胞， 在细胞裂解液中 加入 β-catenin抗体， 4°C孵育过夜。 加入 Protein AG后， 继续孵育 1- 2h，离心收集 Protein AG 用洗涤缓冲液洗两次后， 加入 20μ1 2χ的上样缓冲液， SDS- PAGE电泳后， 用 Western biot检 测 ΡΡΑΚγ的表达。 实验结果如图 20所示， 脂肽可以显著地诱导 β- catenin与 ΡΡΑϋγ的结合。 脂肽抑制炎症反应可通过促进 β-catenin与 ΡΡΑΚγ的相互结合来实现的。 实施例】1 : 脂肽抑制皮肤伤口炎症并促进伤口的愈合
上述实施俩已经证明脂肽可以在体外或体内抑制 poly(i:C)诱导的炎症。 本实施例进一步 证实脂肽会抑制皮肤伤口上的炎症反应。将 C57BL/6小鼠背部脱毛后，每只老鼠皮间注射 50μβ 的式 （1 ) 所示脂肽， 24h后， 在注射脂肽的位置做伤口， 每天对伤口进行拍照计算伤口的愈 合状况。 3天后， 将伤口周围的皮肤剪下， 一部分抽提 RNA,—部分做 HE染色。 实验结果如 图 21 , 图 22所示， 注射入脂肽后， 脂肽可以明显地促进伤□的愈合， 提高伤口的愈合速度， 同时使得伤口部位的炎症反应下降， 如图 23所示。脂肽促进伤口的愈合可通过抑制伤口部位 的过度炎症反应实现的。 实施例 12: 脂肽抑制过敏性皮炎的炎症反应
将 BALBZc小鼠腹部脱毛后， 在腹部涂抹： 100μ§的二硝基氟苯（DNFB, 5rag/ml), DNFB 溶解在体积比等于 4:1的丙酮和橄榄油混合液内。 DNFB连续涂抹 2次， 5天后， 在小鼠耳朵 上注射 25μ8的脂肽或 PBS, 22h后再注射一次， 待脂肽或 PBS被耳朵吸收后， 每只耳朵涂抹 20μ§的 DNFB(2mg/mi), 2天后测量小鼠耳朵的厚度，剪下一部分抽提 RNA, real-time Ι 'Γ-PCR 检测 mTNF- a、 mIL- 6、 tm】:L4、 miL-13和 mIL- 33的表达量， 另一部分做 HE染色， 观察小 鼠耳朵部分白细胞的浸润以及厚度。 实验结果如图 24所示表明， DNFB可以诱导小鼠耳朵上 产生过敏性皮炎， 耳朵部位出现明显的红肿现象， 加入脂肽后， 红肿现象减弱， 耳朵厚度变 薄（图 25 )。DNFB诱导 mTNF- α和 mlL- 6的表达量明显上升，加入脂肽后明显地抑制了 m: L-6 和 mIL-4的表达 (P<0.05), 如图 26(A-B)所示; 对 mTNF-ou mIL-13、 mIL-33也有抑制作用， 如图 26 (C-E) 所示。

Claims

权 利 要 求 书
1. 一种抗炎症的脂肽， 其特征在于， 所述脂肽包括肽链和脂肪链， 肽链和脂肪链通过肽键 相连， 脂肪酸接在肽链 N端； 其结构如式 （1 ) 所示， 成线状；
Figure imgf000012_0001
(式 1 )。
2. 如权利要求 1 所述脂肽的制备方法， 其特征在于， 肽链采 固相化学合成的方法， 脂肪 链通过邻苯二甲醜丁辛酯和 N-甲基吗琳接在所述肽链上。
3. 如权利要求 1所述脂肽在捭制 poiy(I:C)诱导的炎症中的应用。
4. 如权利要求 1所述脂肽在抑制皮肤伤口过度炎症发生中的应用。
5. 如权利要求 1所述脂肽在抑制 DNFB诱导的炎症中的应用。
6. 如权利要求 1所述脂肽在制备治疗伤口炎症药物中的应用。
7. 如权利要求 1所述脂趺在制备治疗过敏性皮炎药物中的应用。
8. 如权利要求 3-7任一项所述的应用，其特征在于所述脂肽与 TLR2受体结合，诱导 β-catenin 的入核， β catenin与 p65竞争性结合 PPAR γ, 进而起到抗炎症的作用。
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