Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, the research on the substances which can effectively inhibit the activation of NF-kB has important significance for inhibiting the generation and development of dysregulated inflammatory reaction and related diseases. Therefore, the invention provides a short peptide targeting IKK beta to play an inflammation inhibiting function, which comprises an N-terminal amino acid 46-60 region derived from IKIP, and also fuses cell penetrating peptide and polypeptide markers. The N-terminal amino acid 46-60 region derived from IKB kinase binding protein IKIP specifically binds to IKK beta, so that the combination of the IKK beta and IKK gamma is inhibited, the formation of an IKK complex is inhibited, and the activation of a downstream NF-kappa B signal channel is inhibited; obviously reduces the mRNA level and the secretion level of inflammatory cytokines TNF-alpha and IL-6, plays a role in inhibiting inflammatory reaction, and can be applied to inflammatory reaction diseases.
The cell-penetrating peptide enhances the ability of the short peptide to enter cells and improves the absorbability of the short peptide; the polypeptide label facilitates the tracking of the short peptide. Cell-penetrating peptides (which may include various penetrating peptides such as PEP-1, MPG, TAT, Penetratin, Polyarginine, P22N, DPV3, DPV6, and DPV6 to enhance the ability of short peptides to enter cells) and polypeptide markers (such as non-radionuclide markers (C13, H2, N15), fluorescent markers (FAM, FITC), biotin markers, and phosphorylation modifications) may be attached to the N-terminus or C-terminus of the N-terminal amino acid 46-60 region of IKIP. In a preferred embodiment of the present application, the cell-penetrating peptide is TAT and has the sequence: "YGRKKRRQRRR" (SEQ ID NO.4), the polypeptide marker is biotin marker linked to the N-terminus of the N-terminal amino acid 46-60 region of the protein IKIP. Preferably, the short peptide sequence is Biotin-YGRKKRRQRRR-LLSLAMTLGLAWLVF.
The pharmaceutical composition and/or the auxiliary pharmaceutical composition for inhibiting inflammatory reaction or preventing/treating inflammatory reaction diseases are prepared by adding pharmaceutically acceptable auxiliary materials into the provided short peptide which can target IKK beta to play an inflammation inhibiting function as a main active ingredient; preferably, the active ingredients of the pharmaceutical composition are coated with liposomes to facilitate efficient delivery of the pharmaceutical composition to the target site for a stable, sustained release therapeutic effect.
Further, the pharmaceutical composition and/or adjuvant pharmaceutical composition for inhibiting inflammatory response or preventing/treating inflammatory response diseases provided by the present application may be combined with nanotechnology, and the preparation forms include solution preparation, gas preparation, semisolid preparation and solid preparation, further, the preparation forms include tablet, capsule and aerosol, or the preparation forms further include microcapsule, nanocapsule, microsphere, nanosphere or liposome, or the preparation forms further include injection forms, and the injection forms include liquid injection, powder for injection and tablet for injection. Those skilled in the art will recognize that these forms can achieve the functional effects provided by the present application, and those skilled in the art can select different dosage forms and preparation methods as required to improve the stability of the drug, so as to facilitate the preparation, storage and clinical use.
Further, the application provides a use of a short peptide targeting IKK β in preparing a product for inhibiting inflammatory response or preventing or treating inflammatory response diseases, and according to the description of the embodiment of the application, the product comprising a short peptide targeting IKK β provided by the application functions as at least one of the following (a1) to (A5):
(A1) specifically binds to IKK beta, thereby inhibiting the binding of IKK beta to IKK gamma and inhibiting the formation of an IKK complex;
(A2) reducing the expression of inflammatory cytokines;
(A3) inhibiting I κ B phosphorylation or degradation;
(A4) inhibit activation of NF-kB signal channel;
(A5) alleviating pathological damage of inflammatory response diseases.
Further, (A2) reducing the expression level of inflammatory cytokines TNF- α, IL-6, preferably, the mRNA level and secretion level of TNF- α, IL-6;
(A5) obviously reduce the swelling degree of the arthritis affected limb, and/or play an obvious protective role in weight loss, colon length and colon pathological injury caused by colitis.
Further, the inflammatory-responsive diseases include arthritis, inflammatory bowel disease, pancreatitis, pulmonary inflammation, rhinitis, nephritis, tracheitis, bronchitis, asthma, preferably, acute or chronic tracheitis, acute or chronic bronchitis, allergic rhinitis, osteoarthritis, rheumatoid arthritis, inflammatory wrist diseases, acute or chronic colitis, acute or chronic proctitis, acute or chronic pancreatitis, acute or chronic glomerulonephritis. The technical scheme of the present application is particularly excellent, and provides a short peptide targeting IKK β with good inflammation inhibition performance in two completely different diseases, namely arthritis and colitis.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.
Example 1: IKIP action region screening
Research results show that IkB kinase binding protein IKIP and IKK beta have obvious binding effect, and the binding of the IKK beta and the IKK gamma is inhibited, so that the formation of an IKK complex is inhibited, p65 is limited to enter a nucleus, and the expression of inflammatory cytokines is finally inhibited, so that the function of inhibiting inflammatory reaction is exerted; cell experiments and in vivo animal experiments show that the loss of IKIP can cause obvious enhancement of inflammatory response. Applicants have discovered that the function of IKIP is primarily dependent on its N-terminal structure, where the IKK β binding protein IKIP inhibits the formation of the IKK complex by competitively binding IKK α/β with the IKK γ through the 99 amino acids at positions 1-99 of the N-terminus, thereby blocking the phosphorylation process of IKK α/β and negatively regulating the activation of the downstream NF- κ B signaling pathway. Therefore, a series of IKIP truncation bodies are designed, namely IKIP FL-Myc, IKIP 16-374, IKIP31-374, IKIP 61-374, IKIP 76-374 and IKIP 91-374. The N end of the IKIP is screened out a section of functional short peptide which can inhibit inflammatory reaction to synthesize polypeptide with cell permeability, and the polypeptide segments enter cells and are combined with IKK alpha/beta to inhibit NEMO from being combined with the IKK alpha/beta, so that the function of inhibiting NF-kappa B passage is exerted.
Binding experiments of IKIP truncations to IKK β: 293T cells overexpress IKIK beta-HA plasmids and are co-transfected with IKIP-Myc, IKIP 16-374, IKIP31-374, IKIP 61-374, IKIP 76-374 and IKIP 91-374; 24 hours after transfection, the cells were lysed with IP Buffer, proteins were extracted for co-immunoprecipitation, and the binding of IKK β -HA to a series of IKIP truncations was examined.
IKK β phosphorylation assay: 293T cells overexpress IKIK beta-HA plasmids and are co-transfected with IKIP-Myc, IKIP 16-374, IKIP31-374, IKIP 61-374, IKIP 76-374 and IKIP 91-374; at 24 hours after transfection, cells were lysed with RIPA lysate, proteins were extracted, and the phosphorylation level of IKK β was detected by Western Blot.
NF-. kappa.B promoter Activity experiments: 293T cells overexpress NF-kappa-B-luc and IKK beta linker plasmids while co-transfecting IKIP-Myc, IKIP 16-374, IKIP31-374, IKIP 61-374, IKIP 76-374, IKIP 91-374; after 24 hours, the NF-kB activity was detected by a microplate reader.
The results showed that IKIP FL, IKIP 16-374, IKIP31-374 had significant binding to IKK β, while the other truncations did not bind to IKK β, indicating that IKIP might interact with IKK β through the region of amino acids 31-60, as shown in FIG. 1; IKK beta overexpression can spontaneously phosphorylate, and the phosphorylation results of IKK beta show that IKIP FL, IKIP 16-374 and IKIP31-374 can obviously reduce the phosphorylation degree of IKK beta, as shown in figure 2; NF-. kappa.B promoter activity showed that IKIP FL, IKIP 16-374 and IKIP31-374 were able to significantly inhibit NF-. kappa.B promoter activity as shown in FIG. 3. Together, these results indicate that IKIP functions through the region of amino acids 31-60.
IKIP amino acid 31-60 region sequence ARGDAGWADPRTGLSLLSLAMTLGLAWLVF (SEQ ID NO. 5).
IKIP amino acid 31-75 region sequence ARGDAGWADPRTGLSLLSLAMTLGLAWLVFQQSEKFAKVEKQYRL (SEQ ID NO. 6); according to the sequence, the inventor designs three short peptides respectively containing IKIP amino acid 31-45, 46-60 and 61-75 region sequences (the IKIP amino acid 31-45 region sequence is SEQ ID NO.1, the 46-60 region sequence is SEQ ID NO.2 and the 61-75 region sequence is SEQ ID NO.3), the TAT transmembrane peptide sequence at the fusion N end is YGRKKRRQRRR and carries a biotin label, and short peptides F0, F1 and F2 are obtained:
F0(31-45)Biotin-YGRKKRRQRRR-ARGDAGWADPRTGLS;
F1(46-60)Biotin-YGRKKRRQRRR-LLSLAMTLGLAWLVF;
F2(61-75)Biotin-YGRKKRRQRRR-QQSEKFAKVEKQYRL;
after the design is finished, Shanghai Biotech engineering Limited company is entrusted to carry out short peptide synthesis, purification and analysis based on the sequence.
Mouse peritoneal macrophages were cultured, and the fluorescence intensity and distribution in cells of the short peptides were measured by confocal microscopy at different time points with the addition of the short peptides (5 μ M), showing that: the short peptides F0 and F2 are uniformly distributed in the cells; while short peptide F1 is more concentrated in the cell, it is likely that this region is the endoplasmic reticulum localization sequence of IKIP, and thus short peptide F1 is likely to be concentrated in the endoplasmic reticulum, as shown in FIG. 4.
Example 2: verification of the Effect of short peptides on inflammatory cytokine expression
(1) Short peptides (5. mu.M) F0, F1 and F2 are added into mouse abdominal cavity macrophages in advance for pretreatment, LPS, PGN, TNF alpha, IL-1 beta and the like are added for stimulation respectively after 2 hours, and the mRNA levels of inflammatory cytokines TNF-alpha and IL-6 are detected by RT-PCR at different time points.
(2) Short peptides (5 mu M) F0, F1 and F2 are added into mouse abdominal cavity macrophages in advance for pretreatment, LPS and PGN are added respectively after 2 hours for stimulation, and after 24 hours of culture, the secretion levels of inflammatory cytokines TNF-alpha and IL-6 are detected by ELISA.
(3) Short peptides (5. mu.M) F0, F1 and F2 were pre-treated by pre-addition to mouse peritoneal macrophages, LPS, PGN, TNF α and IL-1 β were added for 2 hours later for stimulation, and the level of adaptor protein phosphorylation in the signal pathway was detected by Western Blot at different time points.
The results show that: under the stimulation of various stimulators of NF-kB signal pathway, short peptide F1 can significantly reduce the mRNA level and the secretion level of TNF-alpha and IL-6, as shown in FIGS. 6 and 7; and can obviously reduce the phosphorylation levels of IKK beta and IkB, and short peptides F0 and F2 do not have the function, as shown in figure 8.
The IKIP46-60 amino acid sequence is compared with the anti-inflammatory activity of the IKIP full-length short peptide: the amino acid at the position 46-60 of the IKIP or the IKIP full-length short peptide pretreats the macrophage for 1h, then LPS stimulates the cells (0h, 4h), and qRT-PCR technology is used for detecting the expression of inflammatory cytokines TNF-alpha and IL-6 mRNA.
The results showed that the amino acid sequence at positions 46-60 of IKIP inhibited inflammatory cytokine expression more strongly than the full-length sequence, as shown in FIG. 5.
Example 3: verification of the Effect of short peptides on the IKK Complex
(1)293T cells, over-expressing IKK β -HA plasmid, 24 hours after transfection, adding short peptides F0, F1, F2(5 μ M), 2 hours after the addition of IP Buffer lysis cells. mu.L of cell lysate was used as Input, 50. mu.L of Streptavidin Beads were added to the remaining sample, and after shaking 4H in a silent mixer, the Beads were washed 1000g 4min 4 times with IP Buffer. Western Blot detects the binding of the short peptide to IKK beta.
(2)293T cells overexpress IKK α -Myc, IKK β -Myc, IKK γ -Myc, 24 hours after transfection, short peptide F1(5 μ M) was added. After further 2 hours of culture, the cells were lysed by IP Buffer. mu.L of cell lysate was used as Input, 50. mu.L of Streptavidin Beads were added to the remaining sample, and after shaking 4H in a silent mixer, the Beads were washed 1000g 4min 4 times with IP Buffer. Western Blot detects the binding of the short peptide to each member of the IKK complex.
(3)293T cells over-express IKK β -HA and IKK γ -Myc plasmids, 24H transfection, short peptides F0, F1, F2(5 μ M), 2 hours after the addition of IP Buffer lysis cells. Taking 50 mu L of cell lysate as Input, adding HA antibody into the rest samples to carry out co-immunoprecipitation experiment, and detecting the combination condition of IKK beta and IKK gamma by Western Blot.
The results show that only the short peptide F1 can have significant binding effect with IKK beta in three short peptides of F0, F1 and F2, and the short peptide F1 only has binding effect with IKK alpha/beta in an IKK complex and does not have binding effect with IKK gamma; further results indicate that short peptide F1, upon binding to IKK β, inhibits the binding of IKK β to IKK γ, thereby affecting the formation of the IKK complex, as shown in figures 9-11.
Example 4: verification of the function of short peptides in DSS-induced acute colitis model
4.1 construction of DSS-induced acute colitis model
The male mice of SPF grade, C57BL/6 mice of 7-8 weeks old, with the weight of 20-22g, purchased from Beijing Huafukang company, were fed to the SPF animal center of Shandong university and divided into four groups, which were a normal control group, a DSS model group, a F1 group, and a F2 group, each group consisting of 6 mice. Normal control mice were given normal drinking water and the remaining groups were fed 3% DSS solution for one week. The DSS model group is injected with PBS solution at the tail vein every day, the F1 group is injected with short peptide F1(10mg/kg) at the tail vein every day, and the F2 group is injected with short peptide F2(10mg/kg) at the tail vein every day;
at the beginning of DSS modeling, the diet, hair, and mobility of mice were observed daily, the weights of each group of mice were recorded daily, the feces of the mice were collected, the behavior of the feces and occult blood of the mice were observed daily, and the severity of colitis symptoms in the mice was evaluated based on the Disease Activity Index (DAI), which is (weight loss score + stool behavior score + stool blood score)/3. As shown in fig. 13 and 14.
DAI scoring is as follows:
DAI score
Taking the colon of the mouse after one week, and counting the length of the colon of each group; and (3) intercepting the colon near-rectal side, washing by PBS, fixing by 4% paraformaldehyde solution, and analyzing pathological damage of the colon in each group by HE staining.
4.2 histopathological Observation and evaluation
The colon tissue to be tested for pathology was fixed in 4% paraformaldehyde, stained by hematoxylin-eosin staining (HE), and the pathological changes of the colon tissue were observed under a microscope, and evaluated according to the methods of evaluation of colon histopathology by Rachmilewitz et al.
1) Dehydrating colon tissue fixed by paraformaldehyde with ethanol of different concentrations;
2) mixing a dehydrating agent, paraffin and a clearing agent, and carrying out clearing treatment on the tissues;
3) immersing the tissue with paraffin;
4) embedding the paraffin-soaked colon tissue;
5) slicing the paraffin blocks with the thickness of about 5 mu m, and baking;
6) xylene dewaxing was performed using different concentrations of xylene and alcohol;
7) hematoxylin staining, eosin counterstaining;
8) after being soaked in absolute ethyl alcohol and dimethylbenzene, the gel is sealed by neutral resin;
9) the slice structure of the colon tissue was observed under a microscope.
Colon histopathology score: rachmilewitz et al used four parameters for assessment of colon pathology, i.e., depth of ulcer, extent of ulcer, degree of inflammation, location of fibrosis. Each parameter is graded by severity on a scale of 0-4, with higher grades resulting in more pathological damage to the colon tissue. As shown in fig. 15.
4.3 occult blood test
Dissolving 1g of o-tolidine in a mixed solution of 50ml of glacial acetic acid and 50ml of absolute ethanol, preparing no o-tolidine solution, and storing at 4 ℃ in the dark.
② the feces are dissolved in double distilled water (500 or 1000ul), and centrifuged for 3000r/min and 5 min.
③ Add 40ul of the supernatant into a 96-well plate, drop 40ul of o-tolidine solution, and drop 40ul of 3% H2O2In (1).
And fourthly, observing the result, and scoring according to the color depth.
The results show that tail vein injection short peptide F1 can significantly reduce the weight loss of mice, and the fecal characters and occult blood conditions of the mice are obviously reduced compared with the DSS model group, while the tail vein injection short peptide F2 has no obvious difference from the DSS model group as shown in figures 12, 13 and 14; histopathological results show that the length of the colon is obviously shortened by feeding DSS, and HE staining results show structural changes such as crypt atrophy and distortion, inflammatory cell infiltration such as lymphocyte and plasma cell appears on the intestinal wall, and chronic inflammatory changes such as thinning of the intestinal wall; after the short peptide F1 is injected, the colon of the mouse is obviously longer than that of the DSS model group, pathological damage is obviously relieved, and the short peptide F2 is not obviously different from the DSS model group, as shown in figures 15 and 16.
Example 5: verification of short peptide function in Zymosan-induced acute arthritis model
Construction of Zymosan-induced acute arthritis model
SPF-grade, 7-8 week old C57BL/6 male mice, weighing 20-22g, purchased from Beijing Huafukang, were fed to the SPF animal center of Shandong university and divided into four groups, which were a normal control group, a Zymosan model group, a F1 group, and a F2 group, each group consisting of 6 mice.
A. Preparing a Zymosan solution: dissolving 15mgZymosan and 1mL sterile PBS, boiling twice, and performing ultrasonic treatment;
B. preparing 4% chloral hydrate solution, injecting 10 mul Zymosan into the ankle joint after injecting anesthetized mice into the abdominal cavity, and injecting PBS solution into a normal control group; after induction of inflammation, mice in the Zymosan model group, F1 group, and F2 group were injected with PBS, short peptide F1, and short peptide F2(10mg/kg) daily, respectively, and the ankle diameter of the mice was measured at a fixed position daily. The measurement is carried out for four days continuously, and the change of the ankle joint swelling degree of the mice is counted.
And C, injecting PBS (phosphate buffer solution), short peptide F1 and short peptide F2(10mg/kg) into mice of a Zymosan model group, a F1 group and a F2 group respectively in tail vein, injecting 4% chloral hydrate anesthetizing mice into abdominal cavities after 2 hours, injecting Zymosan Zymosan (15mg/ml)10ul into ankle joints, killing the mice after 24H, fixing the ankle joints in 4% formaldehyde, performing HE staining after EDTA decalcification, and analyzing inflammatory injury, inflammatory cell infiltration and chondrocyte injury conditions of the ankle joints. The inventors used the following parameters for the histopathological change score: the degree of inflammation of the tissue inflammation was evaluated by grade, and infiltration of inflammatory cells into the synovium (infiltration) and infiltration of inflammatory cells into the secretions of the joint cavity (exudate) were counted. The scoring scale was from 0 (no inflammation) to 3 (severe inflammation). As shown in fig. 17.
D. And (3) after the mice are treated by the same experiment as the experiment B, taking ankle joints, weighing, shearing, adding steel balls, fully grinding by using a grinder, centrifuging by using a 12000rpm 4-degree centrifuge for 10 minutes, then extracting supernatant, and detecting the expression conditions of IL-1 beta, TNF-alpha, IL-6, chemotactic factors CXCL1 and CXCL2 by ELISA.
The results show that: in the Zymosan induced acute arthritis model, the inflammation reaches the most serious degree on the first day of modeling, the swelling of the affected limb is the most serious, and then the inflammation is gradually recovered. Compared with a Zymosan model group, the development of inflammation of the mouse can be obviously relieved after the short peptide F1 is injected, and the swelling of the joint of the affected limb of the mouse is light; the HE staining results show that, compared with the Zymosan model group, the injection of the short peptide F1 can obviously relieve the symptoms of synovitis and obviously reduce the cell infiltration; whereas injection of short peptide F2 had no significant effect. The above results indicate that short peptide F1 can significantly alleviate Zymosan-induced acute arthritis, alleviating the development of inflammation, as shown in fig. 17-20.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Shandong university
<120> IKK beta-targeting short peptides and application thereof in inflammatory diseases
<130> 202022150
<160> 6
<170> PatentIn version 3.3
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