CN105017406B - Novel polypeptide with neuroprotective function - Google Patents
Novel polypeptide with neuroprotective function Download PDFInfo
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- CN105017406B CN105017406B CN201410161234.4A CN201410161234A CN105017406B CN 105017406 B CN105017406 B CN 105017406B CN 201410161234 A CN201410161234 A CN 201410161234A CN 105017406 B CN105017406 B CN 105017406B
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Abstract
The present invention relates to a new kind of polypeptide with nerve protecting function. The invention also relates to the application of the polypeptide and a pharmaceutical composition containing the polypeptide. The polypeptide has small molecular weight and can penetrate various ocular tissue barriers; good water solubility, and can maintain higher concentration in neutral tears, aqueous humor and vitreous humor.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to a novel polypeptide with a neuroprotective function.
Background
Glaucoma (Glaucoma) is a group of optic nerve degenerative diseases caused by various factors such as pathological intraocular pressure (IOP) rise or optic nerve blood flow perfusion pressure reduction, and is the second leading cause of irreversible blinding eye diseases in the world. At present, the main method for clinically treating glaucoma is to reduce intraocular pressure through medicines, lasers and surgical methods so as to relieve mechanical damage caused by pathological high intraocular pressure and improve the low blood flow perfusion state, thereby relieving the apoptosis of retinal ganglion cells. However, clinical studies have shown that a significant proportion of patients still develop disease conditions in which intraocular pressure control is within the normal range, with typical optic disc changes and visual field defects. In addition, the onset and progression of the disease can occur in patients with ocular tension consistently within the normal range, i.e., normal tension glaucoma. Therefore, in addition to active control of intraocular pressure, attention should be paid to the protection of optic nerves for the treatment of glaucoma.
There are various methods of optic nerve protection, such as oral administration, topical eye drop or intravitreal injection to slow, prevent and even reverse nerve cell death; there are also gene therapies, i.e., the reduction of apoptosis of RGCs by recombinant adenovirus-associated virus (rAAV) or lentivirus (lentivirus) -mediated expression of anti-apoptotic proteins or neurotrophic factors; in addition, the preservation of RGCs by stem cell transplantation methods can be achieved by using stem cells to secrete neurotrophic factors and other regulatory factors to reduce neurotrophic factor deprivation, inflammatory responses, oxidative stress, excitotoxicity, etc., associated with the onset of glaucoma. As for medicines, the traditional chemical medicines have large toxic and side effects, and recombinant protein medicines have high production cost and strong immunogenicity, which all prevent the wide application in clinic.
In developing an effective ocular inflammation inhibitor, the particularity of ophthalmic drugs should be fully considered.
First, the eye presents a number of anatomical and functional barriers. Systemic administration often fails to achieve sufficient drug concentrations locally in ocular tissues due to the blood-aqueous and blood-retinal barriers; for topical administration, such as intravitreal injection, macromolecules larger than 76.5kDa are theoretically difficult to penetrate the retina and act on retinal and choroidal neovasculature.
Second, the degree of dissolution of the drug in hydrophilic tears, aqueous humor, and vitreous humor is positively correlated with its effectiveness.
Third, for the above-mentioned primary reasons, ophthalmic drugs have low bioavailability; to increase this, the concentration of the drug to be administered is increased. However, the high-concentration medicine has obvious toxic and side effects, and the high-dose administration cannot be carried out on the whole body or part of the body.
Therefore, there is an urgent need in the art to develop a safe and effective small molecule neuroprotective agent suitable for ocular globe tissues.
Disclosure of Invention
The present invention provides a novel polypeptide having a neuroprotective function, particularly a polypeptide suitable for the tissue of eyeball.
In a first aspect of the invention, there is provided a polypeptide of formula I, or a pharmaceutically acceptable salt thereof
[Xaa0]-[Xaa1]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6]-[Xaa7]-[Xaa8]-[Xaa9]-[Xaa10]-[Xaa12]-[Xaa12]-[Xaa13]-[Xaa14]-[Xaa15]-[Xaa16]-[Xaa17]-[Xaa18]-[Xaa19](I)
In the formula (I), the compound is shown in the specification,
xaa0 is nothing, or 1-5 amino acids form a peptide fragment;
xaa1 is an amino acid selected from the group consisting of: ile, Leu, Val, Met, Ala or Phe;
xaa2 is an amino acid selected from the group consisting of: pen or Hcy;
xaa3 is an amino acid selected from the group consisting of: lys, Arg, Gln, or Asn;
xaa4 is an amino acid selected from the group consisting of: gly, Pro, Ala or;
xaa5 is an amino acid selected from the group consisting of: lys, Arg, Gln, or Asn;
xaa6 is an amino acid selected from the group consisting of: glu or Asp;
xaa7 is an amino acid selected from the group consisting of: val, Ile, Leu, Met, Phe, or Ala;
xaa8 is an amino acid selected from the group consisting of: cys, Hcy, or Pen;
xaa9 is an amino acid selected from the group consisting of: thr or Ser;
xaa10 is an amino acid selected from the group consisting of: acp or beta-Ala;
xaa11 is an amino acid selected from the group consisting of: asn, Gln, His, Lys or Arg;
xaa12 is an amino acid selected from the group consisting of: ala, Val, Leu or Ile;
xaa13 is an amino acid selected from the group consisting of: pro or Ala;
xaa14 is an amino acid selected from the group consisting of: val, Ile, Leu, Met, Phe, or Ala;
xaa15 is an amino acid selected from the group consisting of: ser or Thr;
xaa16 is an amino acid selected from the group consisting of: ile, Leu, Val, Met, Ala or Phe;
xaa17 is an amino acid selected from the group consisting of: pro or Ala;
xaa18 is an amino acid selected from the group consisting of: gln or Asn;
xaa19 is nothing, or 1-5 amino acids form a peptide fragment;
wherein, a disulfide bond is formed between Xaa2 and Xaa8, and the polypeptide has neuroprotective activity.
In another preferred embodiment, the polypeptide is less than or equal to 28 amino acids in length, preferably less than or equal to 25, more preferably less than or equal to 20.
In another preferred embodiment, the polypeptide has at least 12 fixed amino acids, preferably 15, more preferably 16.
In another preferred embodiment, the polypeptide is as shown in SEQ ID NO. 1-10.
In another preferred embodiment, the polypeptide is:
xaa0 is none;
xaa1 is an amino acid selected from the group consisting of: ile or Leu;
xaa2 is Pen;
xaa3 is an amino acid selected from the group consisting of: lys or Arg;
xaa4 is an amino acid selected from the group consisting of: gly or Ala;
xaa5 is an amino acid selected from the group consisting of: lys or Arg;
xaa6 is an amino acid selected from the group consisting of: glu or Asp;
xaa7 is an amino acid selected from the group consisting of: val or Leu;
xaa8 is an amino acid selected from the group consisting of: cys or Pen;
xaa9 is an amino acid selected from the group consisting of: thr or Ser;
xaa10 is an amino acid selected from the group consisting of: acp or beta-Ala;
xaa11 is an amino acid selected from the group consisting of: asn or Gln;
xaa12 is an amino acid selected from the group consisting of: ala or Val;
xaa13 is an amino acid selected from the group consisting of: pro or Ala;
xaa14 is an amino acid selected from the group consisting of: val or Leu;
xaa15 is an amino acid selected from the group consisting of: ser or Thr;
xaa16 is an amino acid selected from the group consisting of: ile or Leu;
xaa17 is an amino acid selected from the group consisting of: pro or Ala;
xaa18 is an amino acid selected from the group consisting of: gln or Asn;
xaa19 is none;
wherein a disulfide bond is formed between Xaa2 and Xaa8, and the polypeptide has neuroprotective activity and is substituted with up to 1-5, preferably 1-3, more preferably 1-2 amino acids.
In another preferred embodiment, the Xaa0 is a peptide fragment consisting of 1-3 amino acids; and/or the Xaa19 is a peptide segment consisting of 1-3 amino acids.
In a second aspect of the invention, a derivative polypeptide is provided, wherein the derivative polypeptide is a derivative polypeptide of the polypeptide shown in SEQ ID No. 1 and is selected from the group consisting of:
(a) has an amino acid sequence shown as SEQ ID NO. 1;
(b) 1 through deletion, substitution or addition of 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and has neuroprotective function.
In another preferred embodiment, the polypeptide of (a) has a length of less than or equal to 28 amino acids, preferably less than or equal to 25, more preferably less than or equal to 20.
In another preferred embodiment, the derivative polypeptide is a polypeptide represented by SEQ ID No. 1 substituted with 1-5, preferably 1-3, more preferably 1-2 amino acids; and/or
In another preferred embodiment, the deletion is of 1-3, preferably 1-2, amino acid residues from the C-terminus of the polypeptide; and/or
The two ends of the derivative polypeptide are respectively formed by adding 1-5, preferably 1-4 or 1-3, more preferably 1-2 amino acids.
In another preferred embodiment, said polypeptide is substituted according to the "representative substitution" in table 1.
In another preferred embodiment, said polypeptide is substituted according to "preferred substitutions" in table 1.
In another preferred embodiment, the derivative polypeptide retains > 70% of the neuroprotective activity of the polypeptide of SEQ ID NO. 1.
In another preferred embodiment, the derived polypeptide has a homology of 80% or more, preferably 90% or more, with SEQ ID NO 1; more preferably not less than 95%.
The invention also provides dimeric and multimeric forms of the compounds of formula I that are neuroprotective.
In another preferred embodiment, the present invention also provides an isolated nucleic acid molecule encoding a polypeptide of the present invention as described above.
In a third aspect of the invention, there is provided a pharmaceutical composition comprising:
(a) a polypeptide of any one of the first aspect of the invention, a derivative polypeptide of any one of the second aspect of the invention, or a pharmaceutically acceptable salt thereof; and
(b) a pharmaceutically acceptable carrier or excipient.
In another preferred embodiment, the composition is in the form of an eye drop, injection (e.g., periocular and intraocular injection, especially intravitreal injection), ophthalmic gel, or ophthalmic ointment.
In another preferred embodiment, the composition is in a sustained release dosage form.
In a fourth aspect of the invention, the invention provides a use of the polypeptide of the invention or the derivative polypeptide or the pharmaceutically acceptable salt thereof, which is used for preparing a medicament for neuroprotection or preventing and treating diseases caused by nerve cell injury.
In another preferred embodiment, the subject is a human.
In another preferred embodiment, the nerve cell damage is nerve cell damage associated with nerve cell damage to the eye.
In another preferred embodiment, the disease associated with nerve cell damage is selected from the group consisting of: eye nerve cell injury related diseases, acute or chronic retinal optic nerve cell injury diseases.
In another preferred embodiment, the diseases related to the damage of the nerve cells of the eyes comprise glaucoma, macular degeneration, diabetic retinopathy and retinal artery and vein occlusion.
In a fifth aspect of the invention, there is provided a method of enhancing neuroprotection in a mammal, comprising the steps of: administering a polypeptide of the invention or a pharmaceutically acceptable salt thereof to a subject in need thereof.
In another preferred embodiment, said administration comprises ocular surface administration or intravitreal injection administration
In another preferred embodiment, the subject is a human.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows that NP-17 peptide has a pro-proliferative effect on RGC-5 cells and exhibits dose dependence. NP-17 was most active at a concentration of 1000 ng/ml.
FIG. 2 shows that RGC-5 cells have reduced survival rates in hypoxic environments, and NP-17 is able to increase the survival rate of cells in hypoxic environments, protecting ganglion cells from damage in hypoxic environments.
FIG. 3 shows that NMDA has toxic effects on RGC-5 cells and exhibits dose dependence. The survival rate of RGC-5 cells at 100uM/L in NMDA is about 55% to 60%.
FIG. 4 shows the protective effect of NP-17 on NMDA-induced RGC-5 cell damage: the survival rate of RGC-5 cells is reduced under the condition of NMDA injury, and NP-17 can improve the survival rate of cells under the condition of NMDA induced injury and protect ganglion cells from being damaged by NMDA excitotoxicity.
FIG. 5 shows that RGC-5 apoptosis is increased following NMDA injury, and NP-17 is able to decrease the rate of apoptosis of cells following NMDA-induced injury, protecting ganglion cells.
FIG. 6 shows NP-17 upregulation of the PI3K/Akt pathway: phosphorylation was initially up-regulated 5min after NP-17 action, peaking at 30min and then declining gradually.
FIG. 7 shows the effect of PI3K/Akt pathway inhibitors on NP-17 neuroprotection: after the channel inhibitor is added, the cell death rate is obviously increased; after the signal channel inhibitor is simply added, the inhibitor has no obvious influence on the survival rate of cells; LY294002 was able to significantly reduce the level of p-Akt.
FIG. 8 shows NP-17 upregulation of the ERK pathway: phosphorylation began to increase after 2.5 minutes of NP-17 action, with ERK1 peaking at 7.5 minutes and ERK2 peaking at 10 minutes and then declining gradually.
FIG. 9 shows the effect of ERK pathway inhibitors on NP-17 neuroprotection: after the pathway inhibitor PD098059 is added, the cell death rate is obviously increased; after the signal channel inhibitor is simply added, the inhibitor has no obvious influence on the survival rate of cells; PD098059 was able to significantly reduce the level of p-Akt.
FIG. 10 shows that NP-17 inhibits the expression of Caspase 3: NP-17 can reduce the expression of Caspase3 in RGC-5 cells after NMDA damage, and its inhibiting ability is stronger than BDNF.
Figure 11 shows that inhibitors of Caspase3 inhibit apoptosis: the NMDA group is not added, after the Caspase3 inhibitor Z-DEVD-FMK is added, the death rate of cells is not obviously influenced, and the inhibitor has no cytotoxicity on the surface; with the addition of the NMDA group, cell death rates were significantly reduced and dose-dependent with the inhibitor.
FIG. 12 shows that NP-17 increases Bcl-2 expression, inhibits the expression of Bad and Bax: NP-17 is capable of decreasing the expression of Bad and Bax in RGC-5 cells after NMDA injury, increasing the expression of Bcl-2.
FIG. 13 shows that NP-17 protected ganglion cells the number of ganglion cells in the NMDA group decreased significantly, and the number of ganglion cells remaining after NP-17 injection was in excess of that in the NMDA group.
FIG. 14 shows that NP-17 inhibits apoptosis of ganglion cells: the number of the apoptosis of the ganglion cell layer of the NMDA group is obviously increased, and the number of the apoptosis of the RGC cell is obviously reduced after NP-17 is added.
FIG. 15 shows that NP-17 decreased Caspase3 levels in retinal tissues, NMDA significantly increased Caspase3 levels in retinal tissues, and Caspase3 levels in retinal tissues significantly decreased after intravitreal NP-17 injection.
FIG. 16 shows the effect of polypeptide N1 on RGC-5 cell proliferation: polypeptide N1 from different concentration groups had no pro-proliferative effect on RGC-5 cells, p > 0.05.
FIG. 17 shows the effect of polypeptide N2 on RGC-5 cell proliferation: polypeptide N1 from different concentration groups had no pro-proliferative effect on RGC-5 cells, p > 0.05.
Detailed Description
The present inventors have conducted extensive and intensive studies to prepare, for the first time, a small-molecule polypeptide derived from a brain-derived neurotrophic factor, having neuroprotective activity, and having a molecular weight of less than 5kD (e.g., only about 2 kD). Specifically, the inventor designs a plurality of candidate sequences based on homology analysis, biological characteristic analysis and other analysis by applying a bioinformatics method, synthesizes and modifies the candidate sequences by adopting a solid phase method, separates and purifies the candidate sequences to obtain high-purity polypeptide NP-17, identifies the polypeptide NP-17 by using HPLC (high performance liquid chromatography) and MS (Mass Spectrometry), and screens RGC (human dendritic cells) induced by in-vitro NMDA (N-methyl-DA) to obtain a novel small-molecule polypeptide with neuroprotective effect, wherein the activity degree of the small-molecule polypeptide can reach 80% of BDNF (brain derived neurotrophic factor), and the optimal effective concentration of the polypeptide is obtained. In addition, the inventor also finds out through experiments that the polypeptide NP-17 is realized by increasing the concentration of Bcl-2 protein and inhibiting the expression of Caspase 3.
The polypeptide of the invention has small molecular weight and can permeate various eye tissue barriers; the water solubility is good, and the high concentration can be kept in neutral tears, aqueous humor and vitreous humor; the safety is high, and the toxic and side effects on biological tissues are small; the eye local medicine has high bioavailability and can reduce dosage, thereby reducing systemic side effect. The present invention has been completed based on this finding.
Brain-derived neurotrophic factor (BDNF)
Brain-derived neurotrophic factor (BDNF) is a macromolecular protein with neurotrophic and neuroprotective effects, which was first discovered by Barde et al in 1982. The BDNF molecular monomer consists of 119 amino acids, the protein isoelectric point is 9199, the molecular weight is 13.15kD, and a plurality of Loop structures can be formed in the molecule. BDNF is a neurotrophic factor with the highest content in vivo, is widely distributed in a nervous system, an endocrine system, bone tissues and cartilage tissues, and can activate a downstream pathway and regulate the behaviors and functions of nerve cells by combining with tropomyosin receptor kinase (TrkB), such as increasing synaptic plasticity, promoting neurogenesis, promoting neuron differentiation, repairing damaged neurons and the like, thereby playing an important role in the growth and development of the nerve tissues and the repair of the damaged neurons.
BDNF has very strong biological activity, but is difficult to pass through various physiological barriers such as blood brain barrier, blood retina barrier and the like due to large molecular weight, and is very short in half-life in vivo, so that the BDNF is difficult to be applied to clinic at present. The objective group utilizes bioinformatics technology to screen out biological peptides with hairpin-like LOOP structures similar to BDNF, and the biological peptides can be combined with TrkB receptors to exert activities similar to BDNF. In the prior art, a plurality of biological peptides with LOOP structures have been synthesized, but the highest activity is only 60% of BDNF, and the activity is difficult to meet the clinical requirement.
Active polypeptide
In the present invention, the terms "polypeptide of the present invention", "NP-17 polypeptide", "short peptide NP-17", or "peptide NP-17" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of peptide NP-17 (I (Pen) KGKEVCT (Acp) NAPPVSIPQ, as shown in SEQ ID NO: 1) having neuroprotective activity, and the polypeptide of the present invention is a loop polypeptide having an intrachain disulfide bond. Furthermore, the term also includes the neuroprotective function of SEQ ID NO:1 variant of the sequence. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 5 (usually 1 to 4, more preferably 1 to 3, most preferably 1 to 2) amino acids, and addition or deletion of one or several (usually within 5, preferably within 3 to 4, more preferably within 1 to 2) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention.
The invention also includes active fragments, derivatives and analogs of the NP-17 polypeptide. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains neuroprotective function or activity. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the NP-17 polypeptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a protein which is then fused to a leader sequence, a secretory sequence or a tag sequence such as 6 His). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 5, preferably up to 3-4, more preferably up to 1-2 amino acids by amino acids of similar or analogous nature compared to the amino acid sequence of formula I. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.
TABLE 1
| Initial residue(s) | Representative substitutions | Preferred substitutions |
| Ala(A) | Val;Leu;Ile | Val |
| Arg(R) | Lys;Gln;Asn | Lys |
| Asn(N) | Gln;His;Lys;Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro;Ala | Ala |
| His(H) | Asn;Gln;Lys;Arg | Arg |
| Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
| Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
| Lys(K) | Arg;Gln;Asn | Arg |
| Met(M) | Leu;Phe;Ile | Leu |
| Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr;Phe | Tyr |
| Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
| Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
The invention also provides analogs of the NP-17 polypeptides. These analogs may differ from the native NP-17 polypeptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
Some of the commonly used unnatural amino acids are listed in Table 1b below.
TABLE 1b
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.
The polypeptides of the invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".
Coding sequence
The present invention also relates to polynucleotides encoding NP-17 polypeptides. It encodes short peptide NP-17 shown in SEQ ID NO. 1.
The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be the coding strand or the non-coding strand. The full-length NP-17 nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination or artificial synthesis. At present, DNA sequences encoding the polypeptides of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to vectors comprising the polynucleotides of the invention, and to genetically engineered host cells produced with the vectors of the invention or the coding sequence for the NP-17 polypeptide.
In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the NP-17 polypeptide.
Preparation method
The polypeptides of the invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptides of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.
A preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques, such as Boc solid phase method, Fmoc solid phase method or a combination of both. The solid phase synthesis can quickly obtain samples, and can select proper resin carriers and synthesis systems according to the sequence characteristics of target peptides. For example, the preferred solid support in the Fmoc system is Wang resin with C-terminal amino acid attached to the peptide, Wang resin is polystyrene in structure, and the arm between the Wang resin and the amino acid is 4-alkoxybenzyl alcohol; the Fmoc protecting group was removed by treatment with 25% piperidine/dimethylformamide for 20 minutes at room temperature and extended from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After completion of the synthesis, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protecting groups are removed, optionally by filtration and isolated as a crude peptide by ether precipitation. After lyophilization of the resulting solution of the product, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. When the solid phase synthesis is performed using the Boc system, it is preferable that the resin is a PAM resin to which a C-terminal amino acid in a peptide is attached, the PAM resin has a structure of polystyrene, and an arm between the PAM resin and the amino acid is 4-hydroxymethylphenylacetamide; in the Boc synthesis system, after the cycle of deprotection, neutralization and coupling, Boc of the protecting group is removed with TFA/Dichloromethane (DCM) and diisopropylethylamine (DIEA/dichloromethane neutralization. peptide chain condensation is completed, the peptide chain is cleaved from the resin by treatment with Hydrogen Fluoride (HF) containing p-cresol (5-10%) at 0 ℃ for 1 hour while removing the protecting group, the peptide is extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), the solution is lyophilized and then further separated and purified with molecular sieves Sephadex G10 or Tsk-40f, followed by high pressure liquid phase purification to obtain the desired peptide, various coupling agents and coupling methods known in the field of peptide chemistry can be used to couple each amino acid residue, for example, Dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3, 3-tetraurea Hexafluorophosphate (HBTU) can be used for direct coupling of the synthesized short peptide, the purity and structure of the product can be confirmed by reversed-phase high performance liquid chromatography and mass spectrometry.
In a preferred embodiment, the polypeptide NP-17 of the invention is prepared by a solid phase synthesis method according to the sequence, and is purified by high performance liquid chromatography to obtain high-purity target peptide freeze-dried powder which is stored at the temperature of-20 ℃.
Another method is to produce the polypeptide of the invention by recombinant techniques. The polynucleotides of the present invention may be used to express or produce recombinant NP-17 polypeptides by conventional recombinant DNA techniques. Generally, the following steps are performed:
(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding an NP-17 polypeptide, or with a recombinant expression vector containing the polynucleotide;
(2) a host cell cultured in a suitable medium;
(3) isolating and purifying the protein from the culture medium or the cells.
The recombinant polypeptide may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
Because the polypeptide of the invention is short, a plurality of polypeptides can be considered to be connected in series, a multimeric expression product is obtained after recombinant expression, and then the required polypeptide is formed by enzyme digestion and other methods.
Pharmaceutical compositions and methods of administration
In another aspect, the present invention provides a pharmaceutical composition comprising (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient. The amount of the polypeptide of the present invention is usually 10. mu.g to 100 mg/dose, preferably 100. mu.g to 1000. mu.g/dose.
For the purposes of the present invention, an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg, of the polypeptide of the invention to a subject. In addition, the polypeptides of the invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
Pharmaceutically acceptable carriers in therapeutic compositions can comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.
Once formulated, the compositions of the present invention may be administered by conventional routes including, but not limited to: ocular surface, periocular, intraocular (especially intravitreal), intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated may be an animal; especially a human.
When the pharmaceutical composition of the present invention is used for practical treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, eyedrops, ampoules (in particular, intravitreal injections), ophthalmic gels and ophthalmic ointments are mentioned.
These pharmaceutical compositions may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may be carried out in a conventional manner according to the dosage form.
For example, the formulation of eye drops may be carried out by: the short peptide NP-17 or a pharmaceutically acceptable salt thereof is dissolved in sterile water (in which a surfactant is dissolved) together with a basic substance, the osmotic pressure and the pH value are adjusted to physiological conditions, and suitable pharmaceutical additives such as a preservative, a stabilizer, a buffer, an isotonizing agent, an antioxidant and a tackifier may be optionally added and then completely dissolved.
The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the short peptide NP-17 or a salt thereof can be incorporated into a pellet or microcapsule carried by a slow release polymer and then surgically implanted into the tissue to be treated. In addition, the short peptide NP-17 or a salt thereof can be used by inserting an intraocular lens previously coated with a drug. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.
When the pharmaceutical composition of the present invention is used for practical treatment, the dosage of the short peptide NP-17 or a pharmaceutically acceptable salt thereof as an active ingredient can be determined reasonably according to the body weight, age, sex, degree of symptoms of each patient to be treated. For example, when the eye drops are topically applied, the concentration is usually about 0.1 to 10wt%, preferably 1 to 5wt%, and the administration is carried out 2 to 6 times per day, 1 to 2 drops each time.
Experimental animal model
NMDA-induced apoptotic RGC cells
The invention adopts RGC cells induced by NMDA to be used as a model for in vivo and in vitro experiments. Glutamate is the major excitatory neurotransmitter in the central nervous system, whereas accumulation of high concentrations of glutamate in the extracellular fluid can produce toxic effects on cells via NMDA receptors. Excitotoxicity is considered to be a common pathogenic pathway for many neurological diseases, including acute ischemic diseases as well as chronic neurodegenerative diseases. Excessive glutamate activates NMDA receptors, so that Ca2+ is greatly infused, an intracellular apoptosis program is started, and apoptosis is induced. NMDA receptor mediated cell death is also an important factor in the death of retinal ganglion cells in a variety of ocular diseases. Thus, NMDA-induced retinal ganglion cell layer damage was experimentally selected to mimic the pathological features of a variety of ocular diseases.
Industrial applicability
The pharmaceutical composition containing the peptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient has a significant neuronal protection function. Animal experiments prove that the polypeptide can protect RGC cells induced by apoptosis by NMDA and improve the survival rate of the RGC cells, thereby obtaining better nerve cell protection effect.
The main advantages of the invention include:
(a) the polypeptide NP-17 of the invention has small molecular weight and can penetrate through an eye tissue barrier;
(b) the water solubility is good, and the high concentration can be kept in neutral tears, aqueous humor and vitreous humor;
(c) the safety is high, and the toxic and side effects on biological tissues are small; the bioavailability of the eye local medicine is high, and the activity can reach 80 percent of BDNF, so that the dosage can be reduced, and the side effect of the whole body can be reduced;
(d) the preparation method can be used for preparing the compound through solid phase synthesis, and has high purity, large yield and low cost;
(e) the polypeptide of the invention has good stability.
Therefore, the polypeptide is expected to be developed into a medicament for treating eye diseases related to nerve cell injury and other related diseases.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations.
EXAMPLE 1 Synthesis and characterization of the polypeptide
NP-17 (SEQ ID: I (Pen) KGKEVCT (Acp) NAPPSIPQ) polypeptide was synthesized using a commercially available symphony polypeptide synthesizer and purified by HPLC and MS methods. After synthesis and purification, the purity of the polypeptide is more than 95 percent, and the polypeptide is stored at the temperature of minus 20 ℃ for later use.
Example 2 Effect of NP-17 on RGC-5 cells in hypoxic Environment
1. Materials and methods
1.1 Experimental cells and materials: rat retinal ganglion cell-5 (RGC-5), purchased from the university of Dan college of medicine; DMEM high-glucose medium, purchased from Invitrogen, usa; cell LIVE/DEAD Assay Kit, available from Invitrogen, usa; MTS was purchased from Promega corporation, USA.
1.2 screening for optimal concentration of NP-17: RGC-5 cells were cultured in high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and streptomycin double antibody at 37 deg.C and 5% CO2Taking cells in logarithmic growth phase, and adjusting the density to 2 × 104Perwell in 96-well plates. When the cells adhere well and grow and fuse to 60%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. Cells were randomly divided into Blank control (Blank), NP-17(1ng/ml), NP-17(10ng/ml), NP-17(100ng/ml), NP-17(1000ng/ml) and NP-17(5000ng/ml) groups, each of which had six replicate wells placed at 37 ℃ in 5% CO2Culturing in an incubator by a conventional method. After 24h, MTS20ul was added to each well, and after 2h, the OD was measured by spectrophotometry at 490 nm.
1.3 model establishment and intervention RGC-5 cells were cultured in the same manner as above, cells in logarithmic growth phase were taken and density was adjusted to 2 × 104Perwell was seeded in 2 96-well plates. When the cells adhere well and grow and fuse to 60%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The cells were randomly divided into a Control group (Control), an anoxic + blank group, an anoxic + NP-17 group, and an anoxic + BDNF group, each group having six multiple wells. Control group was incubated at 37 ℃ with 5% CO2Culturing in incubator, and culturing the rest 3 groups at 37 deg.C and 5% CO2、5%O2Culturing in an anoxic box, and detecting by using CELL LIVE/DEAD Assay Kit after 24 hours.
1.4 statistical analysis: experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
2.1 the proliferative effects of NP-17 on RGC-5 cells: the NP-17 peptide has a pro-proliferative effect on RGC-5 cells and appears dose-dependent. NP-17 was most active at a concentration of 1000 ng/ml. As shown in FIG. 1, p <0.001 for NP-17 at 1ug/ml and p <0.01 for NP-17 at 100 and 5000 ng/ml.
2.2 Effect of NP-17 on RGC-5 cells in hypoxic environments: under the anoxic environment, the survival rate of RGC-5 cells is reduced, and NP-17 can improve the survival rate of cells under the anoxic environment and protect ganglion cells from being damaged by the anoxic environment. See figure 2 (. beta. denotes p <0.05,. beta.denotes p < 0.01).
3. Small knot
Through cell proliferation experiments, the optimal activity concentration of the NP-17 effect is screened out, and through further establishing an RGC-5 hypoxia injury model and observing the influence on the survival rate of the RGC-5 cell, the NP-17 is proved to be capable of improving the survival rate of the RGC-5 cell in a hypoxia environment and protecting nerve cells.
Example 3 Effect of NP-17 on NMDA-induced RGC-5 cell injury
1. Materials and methods
1.1 Experimental cells and materials: rat retinal ganglion cell-5 (RGC-5), purchased from the university of Dan college of medicine; DMEM high-glucose medium, purchased from Invitrogen, usa; cell LIVE/DEAD Assay Kit, available from Invitrogen, usa; MTS, available from Promega corporation, USA; the Tunel apoptosis detection kit is purchased from Promega corporation, USA; NMDA was purchased from Sigma, USA.
1.2 model building and intervention: RGC-5 cells were cultured in high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and streptomycin double antibody at 37 deg.C and 5% CO2The amplification culture was performed in the incubator of (1), and the following experiments were performed:
1.2.1 cells in logarithmic growth phase were taken and the density was adjusted to 2 × 104Perwell in 96-well plates. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The cells were randomly divided into a Control group (Control), an NMDA (10 uM/L) group, an NMDA (25 uM/L) group, an NMDA (50 uM/L) group, an NMDA (100 uM/L) group, an NMDA (200 uM/L) group, an NMDA (400 uM/L) group, an NMDA (800 uM/L) group, and an NMDA (1600 uM/L) group, each of which was provided with six wells. Placing at 37 ℃ and 5% CO2Culturing in an incubator by a conventional method, and detecting by using CELL LIVE/DEAD Assay Kit after 24 hours.
1.2.2 cells from logarithmic growth phase were taken and adjusted to a density of 2 × 104Well inoculationIn 96-well plates. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The cells were randomly divided into a Control group (Control), an NMDA + blank group, an NMDA + NP-17 group, and an NMDA + BDNF group, each group having six multiple wells. Placing at 37 ℃ and 5% CO2Culturing in an incubator by a conventional method, and detecting by using CELL LIVE/DEAD Assay Kit after 24 hours.
1.2.3 cells in logarithmic growth phase were taken and the density was adjusted to 2 × 105Perwell in 24-well plates. When the cells adhere well and grow and fuse to 60%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The cells were randomly divided into a Control group (Control), an NMDA + blank group, an NMDA + NP-17 group (NP-17 concentration of 1ug/ml), and an NMDA + BDNF group, each group having 3 multiple wells. Placing at 37 ℃ and 5% CO2Culturing in an incubator by a conventional method, and detecting by using a Tunel kit after 24 hours.
1.3 statistical analysis: experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
2.1 NMDA action on RGC-5 cell injury: NMDA has toxic effects on RGC-5 cells and appears dose-dependent. The survival rate of RGC-5 cells at 100uM/L in NMDA is approximately 55% to 60%, as shown in FIG. 3. NMDA was chosen as the experimental concentration at a concentration of 100 uM/L.
2.2 protective effects of NP-17 on NMDA-induced RGC-5 cell injury: the survival rate of RGC-5 cells is reduced under the condition of NMDA injury, and NP-17 can improve the survival rate of cells under the condition of NMDA induced injury and protect ganglion cells from being damaged by NMDA excitotoxicity. See figure 4 (. beta. denotes p <0.05,. beta.denotes p < 0.01).
2.3 Effect of NP-17 on NMDA-induced apoptosis of RGC-5 cells: after NMDA injury, RGC-5 apoptosis is increased, and NP-17 can reduce apoptosis rate of NMDA-induced injury cells and protect ganglion cells. See fig. 5.
3. Small knot
Through an NMDA (N-methyl-D) damage experiment, the concentration for establishing an NMDA damage model is screened, then, NP-17 is used for intervention, the influence of NP-17 on the survival rate and apoptosis of RGC-5 cells is observed, and the fact that NP-17 can improve the survival rate of the RGC-5 cells after NMDA damage, inhibit the apoptosis of the cells and protect nerve cells is proved.
Example 4 role of PI3K/Akt pathway in NP-17 neuroprotection
1. Materials and methods
1.1 Experimental cells and materials: rat retinal ganglion cell-5 (RGC-5), purchased from the university of Dan college of medicine; DMEM high-glucose medium, purchased from Invitrogen, usa; p-Akt antibody was purchased from CST, USA; beta-Actin antibodies were purchased from Epitomic, USA; LY294002 and Wortmannin were purchased from MERK, USA; western blot related instruments were purchased from Bio-Rad, USA; western Blot related reagents were purchased from Biyuntian, China.
1.2 Experimental methods: RGC-5 cells were cultured in high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and streptomycin double antibody at 37 deg.C and 5% CO2The amplification culture was performed in the incubator of (1), and the following experiments were performed:
1.2.1 Effect of NP-17 on the PI3K-Akt pathway cells in logarithmic growth phase were taken and adjusted to a density of 5 × 105The cells were seeded in large dishes, and 6 cells were seeded in total. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. Medium containing NP-17 biological peptide was added to each of the large dishes, and cellular protein was extracted at the following time points, starting timing after addition: 0min, 5min, 10min, 30min, 1h and 6 h. The western blot experiment was performed after protein extraction.
1.2.2 Effect of PI3K-Akt pathway inhibitors on cell mortality by taking cells in logarithmic growth phase and adjusting the density to 2 × 104Perwell in 96-well plates. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. Cells were randomized into Blank control, NP-17+ LY294002 (30 uM), NP-17+ Wortmannin (100 nM), LY294002 (30 uM) and Wortmannin (100 nM) with six replicate wells per set. Placing at 37 ℃ and 5% CO2IncubatorThe culture was routinely performed and after 24 hours the Assay was performed using the cellive/DEAD Assay Kit.
1.2.3 Effect of inhibitors on PI3K-Akt pathway cells in logarithmic growth phase were harvested and adjusted to a density of 5 × 105The cells were seeded in large dishes, and 4 cells were added in total. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The following groups were randomized: blank group, NP-17 group, LY294002 group, and NP-17+ LY294002 group. Inhibitor + NP-17 group inhibitor LY294002 was added first and acted for 15 minutes, followed by NP-17 for 10 minutes.
1.3 statistical analysis: and (4) scanning the gray values of the strips of the group by using a gray scanning software Bandscan, and carrying out quantitative analysis and statistics. Experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
2.1 NP-17 upregulates the PI3K/Akt pathway: phosphorylation was initially up-regulated 5min after NP-17 action, peaking at 30min and then declining gradually, as shown in FIG. 6.
2.2 Effect of PI3K/Akt pathway inhibitors on NP-17 neuroprotection: after the channel inhibitor is added, the cell death rate is obviously increased; after the signal channel inhibitor is simply added, the inhibitor has no obvious influence on the survival rate of cells; LY294002 was able to significantly reduce the level of p-Akt. See figure 7 for details.
3. Small knot
NP-17 can up-regulate the expression of PI3K/Akt pathway and show time dependence; by using PI3K/Akt pathway inhibitors LY294002 and Wortmannin, the inhibitors were found to increase cell death rate after NMDA injury; the inhibitor can effectively inhibit the phosphorylation of a PI3K/Akt pathway, and has no obvious influence on the cell survival rate.
This experiment shows that NP-17 may play a neuroprotective role by activating PI 3K/Akt.
Example 5 role of the ERK pathway in NP-17 neuroprotection
1. Materials and methods
1.1 Experimental cells and materials: rat retinal ganglion cell-5 (RGC-5), purchased from the university of Dan college of medicine; DMEM high-glucose medium, purchased from Invitrogen, usa; p-ERK antibody was purchased from CST, USA; beta-Actin antibodies were purchased from Epitomic, USA; PD098059 was purchased from MERK, USA; western blot related instruments were purchased from Bio-Rad, USA; western Blot related reagents were purchased from Biyuntian, China.
1.2 Experimental methods: RGC-5 cells were cultured in high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and streptomycin double antibody at 37 deg.C and 5% CO2The amplification culture was performed in the incubator of (1), and the following experiments were performed:
1.2.1 Effect of NP-17 on the ERK pathway cells in the logarithmic growth phase were harvested and adjusted to a density of 5 × 105The cells were seeded in large dishes, and 7 cells were seeded in total. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. Medium containing NP-17 biological peptide was added to each of the large dishes, and cellular protein was extracted at the following time points, starting timing after addition: 0min, 2.5min, 5min, 7.5min, 10min, 30min, 1 h. Western Blot experiments were performed after protein extraction.
1.2.2 Effect of inhibitors of the ERK pathway on cell death rates cells in the logarithmic growth phase were harvested and adjusted to a density of 2 × 104Perwell in 96-well plates. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. Cells were randomly divided into Blank control group of Blank, NP-17 group, NP-17+ PD098059 (20 uM) group and PD098059 (20 uM) group, each group having six duplicate wells. Placing at 37 ℃ and 5% CO2The cells were cultured routinely in an incubator and after 24 hours were tested using the cellive/DEAD Assay Kit.
1.2.3 Effect of inhibitors on the ERK pathway cells in logarithmic growth phase were harvested and adjusted to a density of 5 × 105The cells were seeded in large dishes, and 4 cells were added in total. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The following groups were randomized: blank group, NP-17 group, PD098059 group, and NP-17+ PD098059 group. Inhibitor + NP-In group 17, inhibitor PD098059 was added first, and after 15 minutes of action, NP-17 was added for 10 minutes of action.
1.3 statistical analysis: and (4) scanning the gray values of the strips of the group by using a gray scanning software Bandscan, and carrying out quantitative analysis and statistics. Experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
2.1 NP-17 upregulates the ERK pathway: phosphorylation began to upregulate after 2.5 minutes of NP-17 action, with ERK1 peaking at 7.5 minutes and ERK2 peaking at 10 minutes and then declining, as shown in detail in FIG. 8.
2.2 Effect of ERK pathway inhibitors on NP-17 neuroprotection: after the pathway inhibitor PD098059 is added, the cell death rate is obviously increased; after the signal channel inhibitor is simply added, the inhibitor has no obvious influence on the survival rate of cells; PD098059 was able to significantly reduce the level of p-Akt. See figure 9 for details.
3. Small knot
NP-17 is able to up-regulate the expression of the ERK pathway and exhibits a temporal dependence; by using the ERK pathway inhibitor PD098059, the inhibitor was found to increase cell death rate following NMDA injury; the inhibitor can effectively inhibit phosphorylation of ERK pathway without significant influence on cell survival rate.
This experiment suggests that NP-17 may exert neuroprotective effects by activating ERK.
Example 6 Effect of NP-17 on Caspase3 expression in RGC-5 cells
1. Materials and methods
1.1 Experimental cells and materials: rat retinal ganglion cell-5 (RGC-5), purchased from the university of Dan college of medicine; DMEM high-glucose medium, purchased from Invitrogen, usa; caspase3/7Assay Kit was purchased from Ivitrogen, USA; inhibitor Z-DEVD-FMK was purchased from Sigma.
1.2 Experimental methods: RGC-5 cells were cultured in high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and streptomycin double antibody at 37 deg.C and 5% CO2Culture of (2)Amplification culture is carried out in a box, and the following experiments are respectively carried out:
1.2.1 Effect of NP-17 on Caspase3 expression cells in logarithmic growth phase were harvested and adjusted to a density of 2 × 104The cells were plated in 96-well plates. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The medium containing the NP-17 polypeptide was replaced, the incubation was carried out in an incubator for 24 hours, the medium containing NMDA was replaced for 15 minutes, and the medium before replacement was then carried out for another 6 hours. The expression of the Caspase3 was detected in each group using Caspase3/7Assay Kit according to the Kit instructions.
1.2.2 Effect of Caspase3 inhibitor Z-DEVD-FMK on cell mortality by taking cells in logarithmic growth phase and adjusting the density to 2 × 104Perwell in 96-well plates. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The cells were randomly divided into Blank control group of Blank, Z-DEVD-FMK (50uM), Z-DEVD-FMK (100uM), NMDA + Z-DEVD-FMK (50uM) and NMDA + Z-DEVD-FMK (100uM), each of which was set with six replicates. Placing at 37 ℃ and 5% CO2The cells were cultured routinely in an incubator and after 24 hours were tested using the cellive/DEAD Assay Kit.
1.3 statistical analysis: experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
2.1 NP-17 inhibits the expression of Caspase 3: NP-17 can reduce the expression of Caspase3 in RGC-5 cells after NMDA damage, and its inhibiting ability is stronger than BDNF. See figure 10 for details.
2.2 Caspase3 inhibitors inhibit apoptosis: the NMDA group is not added, after the Caspase3 inhibitor Z-DEVD-FMK is added, the death rate of cells is not obviously influenced, and the inhibitor has no cytotoxicity on the surface; with the addition of the NMDA group, cell death rates were significantly reduced and dose-dependent with the inhibitor. See figure 11 for details.
3. Small knot
NP-17 can inhibit expression of Caspase 3; by using Caspase3 inhibitor Z-DEVD-FMK, it was found that the use of the inhibitor reduces cell death rate following NMDA injury; the inhibitor is not cytotoxic by itself.
Through the experiment, Caspase3 plays an important role in the neuroprotective effect of NP-17.
Example 7 Effect of NP-17 on Bcl-2 family protein expression in RGC-5 cells
1. Materials and methods
1.1 Experimental cells and materials: rat retinal ganglion cell-5 (RGC-5), purchased from the university of Dan college of medicine; DMEM high-glucose medium, purchased from Invitrogen, usa; bcl-2, Bad and Bax were purchased from CST, USA; western blot related instruments were purchased from Bio-Rad, USA; western Blot related reagents were purchased from Biyuntian, China.
1.2 Experimental methods: RGC-5 cells were cultured in high-glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and streptomycin double antibody at 37 deg.C and 5% CO2The culture box of (2) is used for carrying out amplification culture, cells in logarithmic growth phase are taken, and the density is adjusted to be 5 × 106The seeds were inoculated in large dishes. When the cells adhere well and grow and fuse to 80%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The experimental groups were as follows: blank, NP-17, and BDNF groups. The medium containing the NP-17 polypeptide was replaced, the incubation was carried out in an incubator for 24 hours, the medium containing NMDA was replaced for 15 minutes, and the medium before replacement was then carried out for another 6 hours. Each histone is extracted and Western Blot experiment is carried out.
1.3 statistical analysis: and (4) scanning the gray values of the strips of the group by using a gray scanning software Bandscan, and carrying out quantitative analysis and statistics. Experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
NP-17 increased Bcl-2 expression, inhibited Bad and Bax expression: NP-17 is capable of decreasing the expression of Bad and Bax in RGC-5 cells after NMDA injury, increasing the expression of Bcl-2. See figure 12 for details.
3. Small knot
NP-17 can inhibit the expression of Bad and Bax, and increase the expression of Bcl-2.
Example 8 protective Effect of NP-17 on NMDA-induced retinal Damage
1. Materials and methods
1.1 Experimental animals and materials: healthy male Wister rats, about 200g, were purchased from the animal center of the Chinese academy of medical sciences; NMDA was purchased from Sigma, USA; other related immunohistochemical reagents were purchased from Biyuntian corporation.
1.2 model preparation and intervention: the rats were randomly divided into 5 groups: control, Vehicle group, NMDA + NP-17 group and NMDA + BDNF group, 10 rats per group, and the right eye was selected as the experimental eye. The Vehicle group was injected with 4uL of PBS buffer; injecting NMDA with the concentration of 5mM at 4uL in a vitreous cavity of an NMDA group, wherein the concentration is 20 nmol/eye; injecting a mixed solution of 4uLNMDA and NP-17 into the NMDA + NP-17 group, wherein the NMDA content in the solution is 20nmol, and the NP-17 content in the solution is 10 ug; the NMDA + BDNF group is injected with 4uL of mixed solution of NMDA and BDNF, the NMDA content in the solution is 20nmol, and the BDNF content in the solution is 5 ug. 7 days after injection, animals were sacrificed and rat eye or retinal tissue was removed for the following experiments:
1.2.1 protective Effect of NP-17 on rat retinal ganglion cells: after injecting the medicine into the vitreous cavity for 7 days, the rat eyeballs are taken out, the fixing liquid is fixed for 24 hours, and the eyeballs are sliced in the sagittal position, wherein the section needs to contain optic nerves. Conventional HE staining. And after dyeing is finished, observing and taking a picture under a light mirror.
1.2.2 Effect of NP-17 on retinal ganglion cell apoptosis following NMDA-induced injury: after injecting the medicine into the vitreous cavity for 7 days, the rat eyeballs are taken out, the fixing liquid is fixed for 24 hours, and the eyeballs are sliced in the sagittal position, wherein the section needs to contain optic nerves. Cadaveric wax sections were prepared routinely, stained according to Turnel kit instructions, and photographed under confocal microscopy.
1.3 statistical analysis: counting the number of HE stained segment cells and Tunel stained segment apoptosis, and carrying out quantitative analysis and statistics. Experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
2.1 NP-17 protects ganglion cells the number of ganglion cells in the NMDA group decreased significantly, and the number of ganglion cells remaining after NP-17 injection was excessive in the NMDA group. See figure 13 for details.
2.2 NP-17 inhibits apoptosis of ganglion cells: the number of the apoptosis of the ganglion cell layer of the NMDA group is obviously increased, and the number of the apoptosis of the RGC cell is obviously reduced after NP-17 is added. See figure 14 for details.
3. Small knot
NP-17 protects the retina, reduces NMDA toxicity to the retina, and reduces apoptosis of retinal RGC cells.
Example 9 Effect of NP-17 on Caspase3 expression in rat retinas
1. Materials and methods
1.1 Experimental animals and materials: healthy male Wister rats, about 200g, were purchased from the animal center, Chinese academy of medical sciences, Caspase3/7Assay Kit was purchased from Ivitrogen, USA; cell lysates were purchased from MERK, USA.
1.2 Each of 3 rat eyeballs in example 7 was collected, retinal tissue was separated, 200. mu.l of cell lysate was added to each eyeball, disrupted by ultrasonication and lysed, and centrifuged at 12000rpm for 5 minutes to obtain a supernatant. The content of Caspase3 in each group was determined according to the Caspase3/7Assay Kit operating manual. Each set was replicated 6 wells.
1.3 statistical analysis: experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
The NP-17 reduces the content of Caspase3 in the retinal tissue, namely the NMDA can obviously increase the content of Caspase3 in the retinal tissue, and the content of Caspase3 in the retinal tissue is obviously reduced after the NP-17 is injected in a vitreous cavity. See figure 15 for details.
3. Small knot
The content of Caspase3 in retinal tissues can be obviously increased by injecting NMDA in a vitreous cavity, and the content of Caspase3 can be reduced by NP-17.
EXAMPLE 10 preparation and characterization of the derived Polypeptides
The following polypeptides were prepared and identified by substituting, adding or deleting a part of amino acids within a reasonable range according to the method of example 1 and table 1:
TABLE 2
Example 11 Activity test of the polypeptide of example 10
The effect of each of NP-17 derived polypeptides 1-9(SEQ ID NO: 2-10) on NMDA-induced apoptosis of RGC-5 cells at a concentration of 1ug/ml was determined as described in example 3, and the results are shown in Table 3.
The results showed that the number of RGC cells induced by NMDA apoptosis was increased in the treated group of the above-mentioned derivative polypeptides 1 to 9. It can be seen that the polypeptides derived from SEQ ID No. 1 all have a certain neuroprotective effect because they increase RGC-5 activity compared to NMDA. It is considered that the derived polypeptides obtained by substituting, adding or deleting the polypeptide of the present invention within a reasonable range have a certain neuroprotective effect, although the activity is different.
Comparative example 1
Activity identification of two polypeptides
2. Materials and methods
1.1 Experimental cells and materials: rat retinal ganglion cell-5 (RGC-5), present at the college of medicine, university of Compound Dane; DMEM high-glucose medium, purchased from Invitrogen, usa; MTS was purchased from Promega corporation, USA.
1.2 polypeptide activity identification: RGC-5 cells are obtained from 10% fetal bovineSerum (FBS), 100U/ml high-sugar DMEM medium with penicillin and streptomycin double antibody, and placing at 37 deg.C and 5% CO2Taking cells in logarithmic growth phase, and adjusting the density to 2 × 104Perwell in 96-well plates. When the cells adhere well and grow and fuse to 60%, the DMEM medium without fetal calf serum is replaced to carry out serum starvation culture for 24 hours. The cells were randomly divided into Blank control (Blank), polypeptide (1ng/ml), polypeptide (10ng/ml), polypeptide (100ng/ml), polypeptide (1000ng/ml) and polypeptide (5000ng/ml) groups, each group was set with six replicate wells and placed at 37 ℃ in 5% CO2Culturing in an incubator by a conventional method. After 24h, MTS20ul was added to each well, and after 2h, the OD was measured by spectrophotometry at 490 nm.
1.3 statistical analysis: experimental data are expressed as mean ± standard deviation and statistically analyzed using the SPSS17.0 statistical software package. The difference was statistically significant with P <0.05 using independent sample t test.
2. Results of the experiment
2.1 the proliferative Effect of the polypeptide N1 on RGC-5 cells: polypeptide N1 from different concentration groups had no pro-proliferative effect on RGC-5 cells, p > 0.05. See fig. 16.
2.2 the proliferative Effect of the polypeptide N2 on RGC-5 cells: polypeptide N1 from different concentration groups had no pro-proliferative effect on RGC-5 cells, p > 0.05. See fig. 17.
3. Small knot
Through cell proliferation experiments, the polypeptide N1 and the polypeptide N2 cannot promote the RGC-5 cell proliferation and are inactive.
Claims (9)
1. A polypeptide, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NOs: 1-10, and the polypeptide has neuroprotective activity.
2. A pharmaceutical composition comprising:
(a) the polypeptide of claim 1, or a pharmaceutically acceptable salt thereof; and
(b) a pharmaceutically acceptable carrier or excipient.
3. The composition of claim 2, wherein the composition is in the form of an eye drop, injection, ophthalmic gel, or ophthalmic ointment.
4. The composition of claim 2, wherein the composition is a periocular injection or an intraocular injection.
5. The composition of claim 2, wherein the composition is a vitreous intra-cavity injection.
6. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for neuroprotection or prevention of a disease associated with neuronal cell damage.
7. The use of claim 6, wherein the disease associated with nerve cell damage is selected from the group consisting of: eye nerve cell injury related diseases, acute or chronic retinal optic nerve cell injury diseases.
8. The use of claim 7, wherein the diseases associated with nerve cell damage to the eye comprise glaucoma, macular degeneration, diabetic retinopathy, and retinal arteriovenous obstruction.
9. An isolated nucleic acid molecule encoding the polypeptide of claim 1.
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