CN113797322A

CN113797322A - Application of manganese type high-stability superoxide dismutase in preventing or treating diabetes

Info

Publication number: CN113797322A
Application number: CN202010535206.XA
Authority: CN
Inventors: 孟凡国
Original assignee: Kairui Hengji Biomedical Hangzhou Co ltd
Current assignee: Kairui Hengji Biomedical Hangzhou Co ltd
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2021-12-17

Abstract

The invention relates to application of manganese type high-stability superoxide dismutase in treating diabetes, wherein the amino acid sequence of the manganese type high-stability superoxide dismutase is shown as SEQ ID NO. 4. The manganese type high-stability superoxide dismutase provided by the invention can obviously reduce the blood sugar concentration of diabetes, and has good prevention and treatment effects on hyperglycemia.

Description

Application of manganese type high-stability superoxide dismutase in preventing or treating diabetes

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to prevention or treatment of diabetes.

Background

Diabetes is a metabolic disease and also a very difficult disease to cure, characterized by hyperglycemia. Diabetes is caused by a defect in insulin secretion or an impaired biological action thereof. In diabetes, prolonged hyperglycemia can lead to damage and dysfunction of various tissues, particularly the kidney, heart, blood vessels, nerves. The focus of diabetes therapy is to keep blood glucose levels near normal without causing hypoglycemia. This can be treated by healthy diet, exercise, weight loss and use of appropriate medications (e.g. insulin for type I diabetics, oral medication for type II diabetics, and possibly insulin).

The medicine for treating diabetes is realized by lowering blood sugar level. There are many different classes of antidiabetic drugs: some may be administered orally, such as metformin; while others can only be injected, such as GLP-1 agonists and the like. The injection of insulin for glycemic control remains the most common way to treat diabetes today. However, there are many side effects associated with insulin injections, such as possible hypoglycemia, patient resistance to injections, proliferation of subcutaneous adipose tissue, antibody production, and allergy.

Therefore, the development of an oral medicament or food for reducing blood sugar has very important significance and application value for treating diabetes.

Disclosure of Invention

When the inventor researches manganese type high-stability superoxide dismutase (MS-SOD), the MS-SOD is unexpectedly found to have an effect on controlling blood sugar. Based on this, the present invention provides a new approach for the prevention and treatment of diabetes.

Specifically, the invention provides application of manganese type high-stability superoxide dismutase in preparing products for preventing or treating diabetes, wherein the amino acid sequence of the manganese type high-stability superoxide dismutase is shown as SEQ ID NO. 4.

Preferably, the product is a medicament, food or health product.

Preferably, the medicament is administered by the oral or injectable route; further preferably, the injection is intravenous or intraperitoneal.

Preferably, the medicament is in the form of tablets, capsules, powder injections, injections or aerosols.

Preferably, the medicament further comprises one or more pharmaceutically acceptable adjuvants, such as diluents, binders, wetting agents, disintegrants, solvents, and/or buffers, and the like. These adjuvants are formulated with MS-SOD in a manner well known in the art and in amounts known to those skilled in the art.

The invention also provides a composition for preventing or treating diabetes, which consists of effective components and pharmaceutically acceptable auxiliary materials, and is characterized in that the effective components are manganese type high-stability superoxide dismutase, and the amino acid sequence of the effective components is shown as SEQ ID NO. 4.

Preferably, the activity of superoxide dismutase in said composition is between 200U and 200000U/day.

Preferably, the compositions of the present invention may be used in combination with other antidiabetic agents currently available.

Preferably, the diabetes mellitus described in the present invention is type I diabetes or type II diabetes.

The invention discovers that the MS-SOD is very effective in controlling the blood sugar of mice by oral administration of MS-SOD, and improves the sensitivity of insulin of the mice, thereby showing that the MS-SOD has very positive effect on the treatment of diabetes.

Drawings

FIG. 1 is a graph of mouse weight growth;

FIG. 2 is a graph showing the comparison of body weight and fat content of 9-month-old mice;

FIG. 3 shows the results of glucose tolerance experiments in 9-month-old mice;

FIG. 4 shows the results of the insulin resistance test in 9-month-old mice;

FIG. 5A shows the measurement of blood sugar content in 18-month-old mice;

FIG. 5B shows the result of measuring the insulin content in 18-month-old mice;

FIG. 5C is a graph of HOMA-IR insulin resistance index results;

FIG. 6 shows the results of the insulin resistance test in 18-month-old mice, in which A is the WT and KI results and B is the WT-MS-SOD and KI-MS-SOD results.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments, but the content of the present invention is not limited thereto.

In the following examples, unless otherwise specified, the reagents and apparatus used were those conventional in the art and were obtained commercially; the experimental methods used are also conventional in the art and the person skilled in the art can, without any doubt, carry out the protocol and obtain the corresponding results according to the contents of the examples.

Example 1: preparation of manganese type high stability superoxide dismutase (MS-SOD)

The target gene was obtained by amplifying the genome of Thermus HB27 (purchased from ATCC cell Bank, accession number ATCC BAA-163) as a template with the following primer sequences: a forward primer: 5'-aagaattcatgccgtacccgttcaagct-3' (SEQ ID NO.1) reverse primer: 5'-ctgtcgactcaggctttgttgaagaac-3' (SEQ ID NO. 2); the amplification product was recovered with a recovery kit (purchased from Shanghai (Co.) Ltd. in Biotechnology engineering), double-digested with enzymes EcoRI and SalI, ligated to a plasmid vector pET28a (+) (purchased from Shanghai (Co.) in Biotechnology engineering) Ltd. double-digested with the same enzymes, the recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) (purchased from Shanghai (Co.) in Biotechnology engineering) Ltd. to screen a strain having a correct MS-SOD nucleotide sequence by sequencing, and the strain was cultured to obtain an MS-SOD protein. Wherein, the nucleotide sequence of the MS-SOD coding gene is as follows:

atgccgtacccgttcaagcttcctgacctaggctacccctacgaggccctcgagccccacattgacgccaagaccatggagatccaccaccagaagcaccacggggcctacgtgacgaacctcaacgccgccctggagaagtacccctacctccacggggtggaggtggaggtcctcctgaggcacctcgccgcccttccccaggacatccagaccgccgtgcgcaacaacgggggcgggcacctgaaccacagcctcttctggaggctcctcacccccgggggggccaaggagcccgtgggggagctgaagaaggccattgacgagcagttcgggggcttccaggccctcaaggagaagctcacccaggcggccatgggccggttcggctcgggctgggcctggctcgtgaaggaccccttcggcaagctccacgtcctctccacccccaaccaagacaaccccgtgatggagggcttcacccccatcgtgggcattgacgtctgggagcacgcctactacctcaagtaccagaaccgccgggccgattacctccaggccatctggaacgtcctcaactgggacgtggccgaggagttcttcaataaagcctga(SEQ ID NO.3)。

the amino acid sequence of MS-SOD is as follows:

MPYPFKLPDLGYPYEALEPHIDAKTMEIHHQKHHGAYVTNLNAALEKYPYLHGVEVEVLLRHLAALPQDIQTAVRNNGGGHLNHSLFWRLLTPGGAKEPVGELKKAIDEQFGGFQALKEKLTQAAMGRFGSGWAWLVKDPFGKLHVLSTPNQDNPVMEGFTPIVGIDVWEHAYYLKYQNRRADYLQAIWNVLNWDVAEEFFKKA(SEQ ID NO.4)。

example 2: mouse experiment

1. Animal model:

1)TBC1D1^S231Amutant mice: from the university of Nanjing model animal research center.

The animal model is characterized in that the 231 th serine of the TBC1D1 protein of a mouse is mutated into alanine by means of gene knock-in, the mouse is obese with the age, and severe metabolic syndromes such as high cholesterol, hyperglycemia, hyperinsulinemia, insulin resistance, nonalcoholic fatty liver and the like appear in the old.

2) Wild-type mice: from the university of Nanjing model animal research center.

This example investigated MS-SOD vs. TBC1D1^S231AInfluence of mutant mouse sugar metabolism.

2. The grouping method comprises the following steps:

mice were grouped according to different MS-SOD intervention:

(1) wild control group (WT): 8 wild type mice;

(2) wild + MS-SOD group (WT-MS-SOD): feeding MS-SOD to wild mice, wherein 8 of the mice are intragastrically administered with MS-SOD150U/10g of body weight per day, and 8 mice are intragastrically administered with MS-SOD 300U/10g of body weight;

(3) mutation control group (KI): is TBC1D1^S231AKnock-in mice, 8;

(4) mutation + MS-SOD group (KI-MS-SOD): feeding MS-SOD to mutant mice, wherein 8 mice were gavaged with MS-SOD150U/10g of body weight per day, and 8 mice were gavaged with MS-SOD 300U/10g of body weight per day.

3. The treatment method comprises the following steps:

1) and (3) measuring the body weight:

after the wild type mice and the mutant mice were fed with normal diet for 9 months, 8 of the MS-SOD with gavage (150U/10 g of body weight per day), 8 of the MS-SOD with gavage (300U/10 g of body weight per day), and 8 of the MS-SOD without addition were used as controls for 17 months. Changes in mouse body weight were recorded each month. By 18 months, mice were sacrificed.

2) Glucose Tolerance Test (OGTT):

by the ninth month, after the mice were starved overnight for 16 hours, they were subjected to glucose (1.5mg/g body weight) gavage, and tail vein blood of the mice was taken at 0 minute, 15 minutes, 30 minutes, 45 minutes, 60 minutes and 120 minutes after the gavage of glucose, respectively, to examine the basal blood glucose. Blood glucose measurements were performed using a glucometer (Bayer Breeze 2 glucometer).

3) Insulin resistance test (ITT):

by the ninth month, the mice were fasted for 4 hours, were intraperitoneally injected with insulin (0.75mU/g body weight), and were measured for basal blood glucose using a glucometer (Bayer Breeze 2 glucometer) at 0 min, 15 min, 30 min, 45 min, 60 min, and 120 min after the injection of insulin, respectively.

4) Fat and lean content determination:

by the ninth month, the measurement method of Dual-Energy X-ray Absorptionmeter (DEXA) was performed using a PIXimus II densitometer (GE Healthcare) instrument. Specifically, mice were anesthetized with xylazine/ketamine (10. mu.l/g) based on their body weight (xylazine was used at a concentration of 1mg/ml and ketamine was used at a concentration of 10 mg/ml). The body length was measured and the bone densitometer software was used to analyze the fat weight and lean weight of the whole body.

4. The experimental results are as follows:

1) phenotypic differences between mutant and wild-type mice

As the mice grew in month-old, the mutant mice (KI and KI-MS-SOD) showed a significant increase in body weight compared to the wild-type mice (WT and WT-MS-SOD) (FIG. 1). It was found that the increase in body weight of mutant mice was mainly due to the increase in fat content, as measured by the total body fat and lean meat content of the mice (fig. 2). Meanwhile, in 9-month-old mice, the mutant mice developed severe glucose intolerance (fig. 3) and insulin resistance (fig. 4). Phenotypically, mutant mice have a pronounced metabolic syndrome of diabetes.

From the ninth month onwards, MS-SOD (300U/10 g body weight per day) was added to mice in the KI-MS-SOD group and WT-MS-SOD group. From the eleventh month onwards, the body weight of both groups of MS-SOD-fed mice was significantly reduced compared to both the control and model groups. Thus, MS-SOD can reduce the weight of the mice. By 17 months, the body weight of mice in the KI-SOD group was close to that of the normal control group (FIG. 1).

2) Effect of oral administration of MS-SOD on blood glucose and insulin in mice

Venous blood from mice was taken to determine blood glucose (month 18), and mutant mice had significant symptoms of diabetes compared to wild type mice, reaching 7.5mmol/L (FIG. 5A, KI), 6.5ng/mL insulin (FIG. 5B, KI), and 45 HOMA-IR (FIG. 5C, KI). After the MS-SOD is orally taken by both wild type mice and mutant mice, the blood sugar and the insulin content (the insulin content is measured by a mouse insulin enzyme-linked immunoassay kit (Mirabbo)) are obviously reduced. The blood glucose and insulin levels of the mutant mice fed the mice even reached the control group level. It can be seen that oral administration of MS-SOD has significant effect in lowering blood sugar and insulin levels.

The insulin resistance experiment also shows (figure 6) that after the MS-SOD is orally taken, the sensitivity of mouse insulin is obviously improved, which also shows that the control of mouse blood sugar is more effective.

Sequence listing

<110> Kai Rui constant biopharmaceutical (Hangzhou) Co., Ltd

<130> DSP1F200879JW

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aagaattcat gccgtacccg ttcaagct 28

<210> 2

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctgtcgactc aggctttgtt gaagaac 27

<210> 3

<211> 615

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgccgtacc cgttcaagct tcctgaccta ggctacccct acgaggccct cgagccccac 60

attgacgcca agaccatgga gatccaccac cagaagcacc acggggccta cgtgacgaac 120

ctcaacgccg ccctggagaa gtacccctac ctccacgggg tggaggtgga ggtcctcctg 180

aggcacctcg ccgcccttcc ccaggacatc cagaccgccg tgcgcaacaa cgggggcggg 240

cacctgaacc acagcctctt ctggaggctc ctcacccccg ggggggccaa ggagcccgtg 300

ggggagctga agaaggccat tgacgagcag ttcgggggct tccaggccct caaggagaag 360

ctcacccagg cggccatggg ccggttcggc tcgggctggg cctggctcgt gaaggacccc 420

ttcggcaagc tccacgtcct ctccaccccc aaccaagaca accccgtgat ggagggcttc 480

acccccatcg tgggcattga cgtctgggag cacgcctact acctcaagta ccagaaccgc 540

cgggccgatt acctccaggc catctggaac gtcctcaact gggacgtggc cgaggagttc 600

ttcaataaag cctga 615

<210> 4

<211> 204

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Pro Tyr Pro Phe Lys Leu Pro Asp Leu Gly Tyr Pro Tyr Glu Ala

1 5 10 15

Leu Glu Pro His Ile Asp Ala Lys Thr Met Glu Ile His His Gln Lys

20 25 30

His His Gly Ala Tyr Val Thr Asn Leu Asn Ala Ala Leu Glu Lys Tyr

35 40 45

Pro Tyr Leu His Gly Val Glu Val Glu Val Leu Leu Arg His Leu Ala

50 55 60

Ala Leu Pro Gln Asp Ile Gln Thr Ala Val Arg Asn Asn Gly Gly Gly

65 70 75 80

His Leu Asn His Ser Leu Phe Trp Arg Leu Leu Thr Pro Gly Gly Ala

85 90 95

Lys Glu Pro Val Gly Glu Leu Lys Lys Ala Ile Asp Glu Gln Phe Gly

100 105 110

Gly Phe Gln Ala Leu Lys Glu Lys Leu Thr Gln Ala Ala Met Gly Arg

115 120 125

Phe Gly Ser Gly Trp Ala Trp Leu Val Lys Asp Pro Phe Gly Lys Leu

130 135 140

His Val Leu Ser Thr Pro Asn Gln Asp Asn Pro Val Met Glu Gly Phe

145 150 155 160

Thr Pro Ile Val Gly Ile Asp Val Trp Glu His Ala Tyr Tyr Leu Lys

165 170 175

Tyr Gln Asn Arg Arg Ala Asp Tyr Leu Gln Ala Ile Trp Asn Val Leu

180 185 190

Asn Trp Asp Val Ala Glu Glu Phe Phe Lys Lys Ala

195 200

Claims

1. The application of the manganese type high-stability superoxide dismutase in preparing products for preventing or treating diabetes mellitus is disclosed, wherein the amino acid sequence of the manganese type high-stability superoxide dismutase is shown as SEQ ID NO. 4.

2. The use according to claim 1, wherein the product is a medicament, a food or a nutraceutical.

3. Use according to claim 2, wherein the medicament is administered by the oral or injectable route.

4. Use according to claim 3, wherein the injection is intravenous or intraperitoneal.

5. The use of any one of claims 2-4, wherein the medicament is in the form of a tablet, capsule, powder for injection, or aerosol.

6. The use according to any one of claims 2 to 4, wherein the medicament further comprises one or more pharmaceutically acceptable excipients.

7. The use according to claim 6, wherein the one or more pharmaceutically acceptable excipients are selected from diluents, binders, wetting agents, disintegrants, solvents and buffers.

8. The composition for preventing or treating diabetes comprises an effective component and pharmaceutically acceptable auxiliary materials, and is characterized in that the effective component is manganese type high-stability superoxide dismutase, and the amino acid sequence of the effective component is shown as SEQ ID NO. 4.

9. The composition as claimed in claim 8, wherein the activity of superoxide dismutase in the pharmaceutical composition is 200U-200000U/day.