CN114702551A

CN114702551A - Polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof

Info

Publication number: CN114702551A
Application number: CN202210358219.3A
Authority: CN
Inventors: 宋燕; 许元生; 张时群; 王晔
Original assignee: Link Health Group
Current assignee: Link Health Group
Priority date: 2018-09-18
Filing date: 2018-09-18
Publication date: 2022-07-05
Anticipated expiration: 2038-09-18
Also published as: CN113307845B; CN109439612B; CN114702551B; CN114644683A; CN114702550A; CN114621324A; CN109439612A; CN114621324B; CN114644683B; CN113307845A; CN114702550B

Abstract

The invention relates to a polypeptide and application thereof, in particular to a polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof. The amino acid sequence of the polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis is selected from at least one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. The polypeptide of the invention has activity which is equivalent to or better than HIP/PAP and can promote hepatocyte proliferation and/or inhibit hepatocyte apoptosis, and the polypeptide of the invention has the advantages of small molecular weight, easy synthesis, low immunogenicity and the like, so the polypeptide of the invention has wide application prospect.

Description

Polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof

The application is a divisional application of the following original divisional applications:

application date of the original divisional application: 09 month and 18 days 2018;

application number of the original divisional application: 2021107606690, respectively;

the invention name of the original divisional application: polypeptides that promote hepatocyte proliferation and/or inhibit hepatocyte apoptosis and uses thereof;

the original divisional application is a divisional application of a parent application:

filing date of parent application: 09 month and 18 days 2018;

application number of the parent application: 2018110919805, respectively;

the invention name of the parent application: polypeptides that promote hepatocyte proliferation and/or inhibit hepatocyte apoptosis and uses thereof; the present application is a divisional re-application of the applicant to the original divisional application in accordance with the review of the examiner.

Technical Field

The invention relates to a polypeptide and application thereof, in particular to a polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof.

Background

The liver is the largest internal organ in the human body (about 2.5% of the total mass of the human body), and is also the central metabolic organ. The major contributors to liver function are parenchymal cells, which account for 80% of the total liver cells. The liver cells play a series of roles including plasma production, carrier protein synthesis, detoxification of digests, conjugation and hormone secretion, regulation of synthesis and metabolism of blood lipids and amino acids, and the like.

Apoptosis (apoptosis) is an active suicide process of cells in specific environments. Apoptosis was two different patterns of apoptosis and death observed by Kerr et al in 1972 when studying liver ischemia. The cell apoptosis is characterized by cell shrinkage, cellular bulge of cell membrane, formation of apoptotic bodies, degradation of nuclear fragments and nuclear DNA and other abnormal phenomena. Apoptosis is a major feature of pathological changes of many diseases in clinic, and particularly in liver diseases, apoptosis of liver cells is an important factor causing liver damage. Hepatocyte apoptosis plays an important role in the mechanism of liver pathology, and disruption of death receptor pathways is considered to be one of the important causes for triggering the development of acute/chronic liver injury. In recent years, researches show that the apoptosis of liver cells is widely involved in pathological processes of various liver diseases, including liver failure, viral hepatitis, hepatic fibrosis, liver cirrhosis, alcoholic liver diseases, non-alcoholic fatty liver diseases, autoimmune liver diseases and the like.

Liver Failure (LF) is a serious liver damage caused by various factors such as virus, drug, alcohol, etc., causing serious dysfunction or decompensation of detoxification, excretion, synthesis, etc., and a group of clinical syndromes mainly manifested by blood coagulation dysfunction, severe jaundice, ascites, hepatic encephalopathy, even digestive tract hemorrhage, etc., with extremely high fatality rate, which is one of the clinical critical conditions of the hepatopathy department. According to the difference of pathological features and disease processes, 2012 edition of guidelines for liver failure diagnosis and treatment classifies liver failure into Acute Liver Failure (ALF), subacute liver failure (SALF), chronic-on-chronic liver failure (ACLF) and Chronic Liver Failure (CLF). Liver failure is clinically critical and still lacks specific drugs with definite curative effects so far, so that the liver failure becomes one of the worldwide intractable diseases, and although artificial liver and liver transplantation are effective treatment methods, the treatment methods cannot be widely popularized and used in China due to the expensive cost, shortage of liver sources, rejection and the like.

The human HIP/PAP protein, also called regenerated protein 3 alpha (regenerated islet-derived protein III-alpha), is a protein consisting of 165 amino acids secreted by pancreas. The study shows that the HIP/PAP protein can protect liver cells from programmed death mediated by tumor necrosis factor, and is a mitogen which can promote mitosis of the liver cells so as to promote the regeneration of the liver cells. At present, the HIP/PAP protein is generally expressed by a recombinant expression system (such as Escherichia coli), the purification method is complex, and the preparation cost is high.

Polypeptides are compounds formed from multiple amino acids by the dehydrocondensation of the amino group of one amino acid with the carboxyl group of another amino acid, and may also be derived from intermediates of proteolysis. Polypeptides generally refer to compounds formed by dehydration condensation of less than 50 amino acids, and polypeptides of more than 50 amino acids are generally referred to as proteins. The polypeptide medicine has the characteristics of small molecular weight, easy synthesis, small side effect, strong specificity and the like, and rarely causes serious immune reaction.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that: the polypeptide for promoting the proliferation of the liver cells and/or inhibiting the apoptosis of the liver cells has an amino acid sequence selected from at least one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

The polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis can be a polypeptide shown by any one amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, or can be a combination of any two or more (including two) of the 6 polypeptides.

The polypeptides shown in SEQ ID NO. 1-SEQ ID NO. 6 are derived from human HIP/PAP protein. The inventor finds that most of polypeptide fragments derived from the human HIP/PAP protein have NO biological activity in a large amount of research, but unexpectedly finds that the polypeptides shown in SEQ ID NO: 1-SEQ ID NO:6 have the activity of promoting the proliferation of liver cells and/or inhibiting the apoptosis of the liver cells which is equivalent to or better than the HIP/PAP.

As a preferred embodiment of the polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 4. Research shows that in the polypeptide shown as SEQ ID NO. 1-SEQ ID NO. 6 and the HIP/PAP, the polypeptide shown as SEQ ID NO. 4 has the strongest effect of promoting hepatocyte proliferation and inhibiting hepatocyte apoptosis, and the activity of the polypeptide is obviously higher than that of the HIP/PAP.

In addition, the invention also provides the application of the polypeptide in preparing products for promoting hepatocyte proliferation or treating diseases caused by hepatocyte apoptosis.

As a preferred embodiment of the use according to the invention, the product is a medicament.

As a preferred embodiment of the use according to the invention, the medicament is in liquid form.

As a preferred embodiment of the use of the present invention, the promotion of hepatocyte proliferation is promotion of liver regeneration in vivo or promotion of hepatocyte growth in vitro.

In a preferred embodiment of the use of the present invention, the disease caused by apoptosis of hepatocytes is liver failure, viral hepatitis, liver fibrosis, liver cirrhosis, alcoholic liver disease, non-alcoholic fatty liver disease or autoimmune liver disease. More preferably, the liver failure is acute liver failure.

The inventor of the invention proves that the polypeptide shown by SEQ ID NO 1-SEQ ID NO 6 has the activities of promoting hepatocyte proliferation and inhibiting hepatocyte apoptosis through in vitro cell experiments, so that the polypeptide can be used for promoting hepatocyte proliferation or treating diseases caused by hepatocyte apoptosis. Research shows that the diseases of hepatocyte apoptosis include liver failure, viral hepatitis, cholestatic liver injury, alcoholic liver injury, non-alcoholic steatohepatitis, cirrhosis, toxin-or drug-mediated liver injury and the like, and the polypeptide of the present invention has the activity of inhibiting hepatocyte apoptosis, so the polypeptide can be used for treating the diseases. The polypeptide of the invention shows good treatment effect on acute liver failure in an animal model of the acute liver failure, and further proves that the polypeptide can be used for treating diseases related to hepatocyte apoptosis.

Finally, the invention also provides a medicament for promoting the proliferation of liver cells and/or inhibiting the apoptosis of the liver cells, which comprises the polypeptide.

As a preferred embodiment of the medicament for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis according to the present invention, the medicament further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be routinely selected by those skilled in the art according to the dosage form of the drug and the like. The dosage form of the drug includes, but is not limited to, a liquid dosage form, a gas dosage form, a solid dosage form, etc., and the drug is a liquid dosage form as a preferred embodiment of the drug for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis according to the present invention. The medicament of the present invention may be administered by intravenous injection or subcutaneous injection.

As a preferred embodiment of the drug for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis according to the present invention, the promotion of hepatocyte proliferation is promotion of hepatocyte regeneration in vivo or promotion of hepatocyte growth in vitro.

Compared with the prior art, the invention has the beneficial effects that: the invention provides polypeptides for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis, the length of the polypeptides is 9-27 amino acids, the length of the HIP/PAP protein is 165 amino acids, the length of the polypeptide of the invention is significantly shorter than the HIP/PAP protein, but the polypeptide of the invention has the activity of promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis which is equivalent to or better than the HIP/PAP protein, and the polypeptide of the invention has the advantages of small molecular weight, easy synthesis, low immunogenicity and the like, so the polypeptide of the invention has wide application prospect.

Drawings

FIG. 1 is a graph showing the results of comparing the activity of the polypeptide of the present invention in promoting hepatocyte proliferation with HIP/PAP;

FIG. 2 is a graph showing the results of comparing the activity of the polypeptide of the present invention in inhibiting hepatocyte apoptosis with HIP/PAP;

FIG. 3 is a graph showing the results of comparing the survival rates of the polypeptides of the present invention and HIP/PAP-treated mice in the treatment of acute liver failure.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

Example 1 Polypeptides

An example of the polypeptide of the present invention that promotes hepatocyte proliferation and/or inhibits hepatocyte apoptosis is shown in table 1.

TABLE 1

Name (R)	Serial number	Sequence of
			IGL9	SEQ ID NO:1	IGLHDPTQG
PTQ9	SEQ ID NO:2	PTQGTEPNG
			LHD9	SEQ ID NO:3	LHDPTQGTE
SLS27	SEQ ID NO:4	SLSRSTAFLRWKDYNCNVRLPYVCKFT
			AYG25	SEQ ID NO:5	AYGSHCYALFLSPKSWTDADLACQK
VSV19	SEQ ID NO:6	VSVLSGAEGSFVSSLVKSI

The polypeptide of this embodiment is synthesized by Shenzhen HongLiao assist science and technology Limited, with purity > 98%, and 50mg each.

Example 2 detection of Activity to promote hepatocyte proliferation in vitro

In this example, the human liver cell line HL-7702 was purchased from ATCC.

The method for detecting the activity of 6 polypeptides described in example 1 and HIP/PAP in promoting hepatocyte proliferation in vitro comprises the following steps:

taking out the frozen HL-7702 cells, and thawing in a water bath at 37 ℃. Centrifuging at 800rpm for 5 minutes on a low speed centrifuge; the supernatant was aspirated and the cells were then resuspended in 1ml of medium.

Resuspended cells were transferred to a fresh flask, added with the appropriate amount of MEM media, and allowed to CO₂Culturing in an incubator (5% CO culture conditions)₂Saturated humidity, 37 ℃).

When the cells grow to 80%, digesting the cells, blowing to disperse the cells, counting, and adjusting the cell concentration to 1 × 10⁵Adding into 96-well plate at a concentration of 100 μ l per well, i.e. 1 × 10 cells per well⁴And (4) respectively.

After the cells were adherent, different drugs were added at two concentrations (1. mu.g/ml, 10. mu.g/ml) for each drug, IGL9, PTQ9, LHD9, SLS27, AYG25, VSV19, HIP/PAP, respectively, and PBS was used as a negative control.

After drug treatment for 0, 2, 3, 4, 5 days, cellTiter96AQ single solution cell proliferation assay reagent (Promega, cat. No. g3582) was added at a ratio of 1/10. Namely, 10. mu.l of the test solution was added to 100. mu.l of the culture solution.

After 4 hours of incubation, the plate was read by microplate reader and OD490 data was measured.

The results are shown in FIG. 1, and it can be seen from FIG. 1 that HIP/PAP and the polypeptide of the present invention both have the effect of promoting hepatocyte proliferation, and the dose-effect relationship is obvious that the activity of 6 polypeptides of the present invention is equivalent to or better than HIP/PAP, wherein SLS27 has the strongest effect of promoting hepatocyte proliferation.

Example 3 detection of in vitro inhibitory Activity on apoptosis of hepatocytes

The method for detecting the activity of 6 polypeptides and HIP/PAP in vitro inhibiting the hepatocyte apoptosis described in example 1 comprises the following steps:

human liver cell HL-7702 with cell concentration of 1 × 10 laid on 96-well plate⁵Each cell/ml, 100. mu.l per well, i.e. 1X 10 cells per well⁴And (4) respectively.

20ng/mL Act D2 is added into each hole for processing for 30min, 80ng/mL TNF-alpha is processed for 48h, and an apoptosis model is established.

The corresponding drug treatments, IGL9, PTQ9, LHD9, SLS27, AYG25, VSV19, HIP/PAP, respectively, were added in a dosing volume of 10. mu.l, two concentrations of each drug (final concentration 1. mu.g/ml, 10. mu.g/ml, respectively) in a volume of 10. mu.l, and the same volume of PBS was used as a negative control.

24h after drug treatment, 100. mu.l Caspase-

Reagent (caspase-

3/7Assay promega, Cat number: g8090) The contents of each well were gently mixed on a shaker at 300-. Incubation was carried out at room temperature (18-22 ℃) for 30 minutes to 3 hours.

The fluorescence value of each sample was measured on a fluoroscope (Luminometer) (Promega, GloMax bioluminescence detector).

The results are shown in FIG. 2, and it can be seen from FIG. 2 that HIP/PAP and the polypeptide of the present invention both have the effect of inhibiting hepatocyte apoptosis, and the dose-effect relationship is obvious that the activity of 6 polypeptides of the present invention is equivalent to or better than HIP/PAP, wherein the SLS27 has the strongest effect of inhibiting hepatocyte apoptosis.

Example 4 testing the efficacy of treatment of acute liver failure in animal models

The method for detecting the 6 polypeptides described in example 1 and the effect of HIP/PAP on the treatment of acute liver failure comprises the following steps:

the polypeptide SLS27 with the highest activity in examples 2 and 3 was selected for in vivo activity studies to examine the therapeutic effect on a mouse model of acute liver failure.

80 male BALB/C mice at 8 weeks of age were randomly divided into four groups, namely a normal control group, a negative control group, an SLS 27-treated group and an HIP/PAP-treated group, and were administered with physiological saline, SLS27 polypeptide (2.5mg/kg), HIP/PAP (2.5mg/kg), respectively, by tail vein injection in an injection volume of 100. mu.l. After 2 days, 5% of D-galactosamine (D-Gal, sigma) is given to establish an acute liver failure model, the dose is 1.0g/kg, and normal control groups are given with the same dose of physiological saline.

After the mice are injected with D-Gal for 6h, the negative control group mice begin to have the phenomena of reduced activity and reduced diet, and the normal control group, SLS27 and HIP/PAP treatment groups have no obvious change; after 48 hours, the activity of the negative control group mice is obviously reduced and the mice begin to die, and the death rate reaches 100% within 96 hours. After 168h (7 days) of treatment, the survival rate of mice in the normal control group is 100%, the survival rate of mice in the SLS27 treated group and the HIP/PAP treated group is 60% and 40%, respectively, and the survival rate of mice in the negative control group is 0. Compared with the negative control group, the animal survival rate of the SLS27 treated group is obviously higher (P < 0.01) and is better than that of the HIP/PAP group, which indicates that the activity of the SLS27 polypeptide is higher than that of the HIP/PAP.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Guangzhou Zhicheng medical science and technology Limited

<120> polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and use thereof

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 9

<212> PRT

<213> Artificial Synthesis

<400> 1

Ile Gly Leu His Asp Pro Thr Gln Gly

1 5

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<211> 9

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<213> Artificial Synthesis

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Pro Thr Gln Gly Thr Glu Pro Asn Gly

1 5

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Leu His Asp Pro Thr Gln Gly Thr Glu

1 5

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Ser Leu Ser Arg Ser Thr Ala Phe Leu Arg Trp Lys Asp Tyr Asn Cys

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Asn Val Arg Leu Pro Tyr Val Cys Lys Phe Thr

20 25

<210> 5

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<212> PRT

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Ala Tyr Gly Ser His Cys Tyr Ala Leu Phe Leu Ser Pro Lys Ser Trp

1 5 10 15

Thr Asp Ala Asp Leu Ala Cys Gln Lys

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<212> PRT

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Val Ser Val Leu Ser Gly Ala Glu Gly Ser Phe Val Ser Ser Leu Val

1 5 10 15

Lys Ser Ile

Claims

1. The polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis is characterized in that the amino acid sequence of the polypeptide is SEQ ID NO. 6.

2. Use of a polypeptide according to claim 1 in the manufacture of a product for promoting hepatocyte proliferation or treating a disease resulting from hepatocyte apoptosis.

3. Use according to claim 2, wherein the product is a medicament.

4. The use of claim 3, wherein the medicament is in a liquid dosage form.

5. The use of claim 2, wherein the promotion of hepatocyte proliferation is the promotion of liver regeneration in vivo or the promotion of hepatocyte growth in vitro.

6. The use according to claim 2, wherein the disease due to hepatocyte apoptosis is liver failure, viral hepatitis, liver fibrosis, cirrhosis, alcoholic liver disease, non-alcoholic fatty liver or autoimmune liver disease.

7. The use of claim 6, wherein the liver failure is acute liver failure.

8. A medicament for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis, comprising the polypeptide of claim 1.

9. The agent for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis according to claim 8, wherein the agent is in liquid form.

10. The agent for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis according to claim 8, wherein the promotion of hepatocyte proliferation is the promotion of liver regeneration in vivo or the promotion of hepatocyte growth in vitro.