A biomarker and therapeutic target for type 2 diabetes and an application method of enteromorpha oligosaccharide

Abstract

The present invention discloses a biomarker and a therapeutic target for type 2 diabetes and an application method of enteromorpha oligosaccharides. In this scheme, OASL protein or its encoding gene or transcription product is used as a biomarker and a therapeutic target for type 2 diabetes caused by a high-sugar and high-fat diet; the biomarker is detected by specifically amplifying RNA transcribed from the OASL gene using RT-qPCR technology; for the target, the present invention also develops an enteromorpha oligosaccharide drug, which can specifically increase the expression of OASL protein and improve fasting blood sugar, insulin and glycosylated hemoglobin levels, and is of great significance for the rapid identification and treatment of type 2 diabetes.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to biomarkers and therapeutic targets for type 2 diabetes and an application method of enteromorpha oligosaccharide. Background Art

[0002] Type 2 diabetes is a heterogeneous, hormonal and metabolic disease characterized by hyperglycemia and insulin resistance. It is currently spreading among the population at a certain incidence rate. With the rapid development of medical and health technologies in recent decades, as well as the continuous improvement of various drug treatment and detection methods, great progress has been made in the treatment of diabetes, but diabetes and its complications still threaten people's health.

[0003] A high-sugar, high-fat diet is one of the important risk factors for obesity and type 2 diabetes. It is also prone to induce other serious complications such as lipid metabolism disorders, atherosclerosis, heart disease and cardiovascular disease. With the continuous deepening of metabolomics, proteomics, and transcriptomics research and the application of high-throughput sequencing technology, many diabetes-related biomarkers have been discovered. However, the causes of type 2 diabetes are complex and the physiological indicators and responses are variable during treatment. We need more targeted biomarkers for the treatment of type 2 diabetes caused by various factors.

[0004] 2′,5′-Oligadenylate Synthetase L (OASL) is an antiviral enzyme that counteracts viral attacks by degrading viral and host RNA. The enzyme uses ATP in a 2'-specific nucleotide transfer reaction to synthesize 2'-5'-oligoadenylate, which can bind to ribonuclease L to activate it, degrade RNA, and inhibit protein synthesis. The enzyme is formed under the induction of interferon and is therefore a key enzyme in the antiviral and anti-tumor processes of interferon.

[0005] Chinese patent CN201510122371.1 discloses a method for silencing the OASL gene in a DF-1 cell line, which is used to increase the proliferation titer of avian influenza virus in the cell line, and belongs to the field of biotechnology; US patent US20090611746 discloses a method for preventing or treating negative-strand RNA virus infection using the innate immune gene OASL, that is, a method for treating a disease, condition or condition caused by a negative-sense single-stranded RNA virus in an individual in need, including at least using an isolated 2',5'-oligoadenylate synthetase-like protein or an isolated polynucleotide encoding the 2'-5'-oligoadenylate synthetase-like protein to the individual in need. The above patents are the discovery and application of OASL and virus-related diseases, and there has been no report on the relationship between OASL and the intestine and type 2 diabetes. Summary of the invention

[0006] In view of this, the purpose of the present invention is to propose a biomarker and therapeutic target for type 2 diabetes and an application method of Enteromorpha oligosaccharide that is reliable, efficient and has no toxic side effects.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] Use of the OASL gene or its transcription product or OASL protein in the preparation of a biomarker and therapeutic target reagent for type 2 diabetes, wherein the OASL gene or its transcription product or OASL protein is used as a biomarker and therapeutic target for type 2 diabetes, the nucleotide sequence of the OASL gene is shown in SEQ ID NO.1, and the amino acid sequence of the OASL protein is shown in SEQ ID NO.2.

[0009] As a possible implementation method, this scheme further applies RT-qPCR technology to specifically amplify the RNA transcribed from the OASL gene for detection;

[0010] Wherein, the amplification primer sequence includes:

[0011] Forward primer sequence, SEQ ID NO.3: 5'-AGAGTCAAGAGATAGGCAGGACAGTA-3',

[0012] The rear primer sequence, SEQ ID NO. 4: 3′-CTACACAGCAAGGATGGTCAGAACT-5′;

[0013] In addition, the RT-qPCR amplification procedure is as follows:

[0014] (1) 95℃ for 60s, one cycle;

[0015] (2) 95°C and 60°C were set to run for 20 s and 60 s respectively, and run for 40 cycles;

[0016] (3) Melting curve analysis: 95°C, 15 s; 60°C, 30 s; 95°C, 15 s; one cycle.

[0017] As a possible implementation method, the present invention further proposes the use of Enteromorpha oligosaccharides in the preparation of drugs for type 2 diabetes, wherein the drugs are used to increase the expression of OASL protein and improve fasting blood glucose, insulin and glycosylated hemoglobin levels.

[0018] As a preferred implementation option, preferably, the method for preparing Enteromorpha oligosaccharides described in this scheme comprises:

[0019] The 80-mesh Enteromorpha powder is mixed with pure water at a ratio of 1:30-1:50 g / mL, and then ultrasonic-assisted extraction is performed for 1-2 hours, and then centrifuged at 4000-5000 rpm for 8-12 minutes, and then vacuum filtration is performed to obtain the supernatant, and then rotary evaporation is performed at a temperature of 50-75°C, and then 3.5-4.5 times the volume of 90%-100% ethanol is added for alcohol precipitation for more than 8 hours; then, after being treated at a centrifugal speed of 4000-5000 rpm for 8-12 minutes, the precipitate is taken and placed at 50-70°C for drying, and then the product is dissolved in pure water at a ratio of 1:80-120 (g:mL), and then protease is added to the solution to make the enzyme activity 350-450 U / mL, and then heated at 45-50°C. The method comprises the following steps: enzymolysis for 1.5-2.5 hours under the condition of 100-100 ℃ water bath for 10-30 minutes to inactivate the enzyme, and then treating at 4000-5000 rpm centrifugal speed for 8-12 minutes, taking the supernatant, and then dialyzing with a dialysis bag with a molecular weight of 15kDa for 36-60 hours, collecting the liquid in the bag, concentrating and freeze-drying to obtain Enteromorpha polysaccharide; adding concentrated sulfuric acid to a 0.8%-1.2% Enteromorpha polysaccharide aqueous solution, wherein 2.5-3ml concentrated sulfuric acid is added per liter of solution, acid hydrolyzing at 90-100 ℃ water bath for 1-2 hours, cooling to room temperature, adding 0.5-2mol / L NaOH to adjust the solution to neutrality, and then adding 90%-100% ethanol solution at a volume ratio of 1:1 for alcohol precipitation for 8-12 hours, taking the supernatant and dialyzing it in a 3kDa dialysis bag, collecting the solution outside the bag, concentrating and freeze-drying to obtain Enteromorpha oligosaccharide.

[0020] By adopting the above-mentioned technical scheme, the present invention has the following beneficial effects compared with the prior art: the OASL protein, gene and transcription product of the type 2 diabetes biomarker provided by the scheme of the present invention can be used as a biomarker for the rapid diagnosis of type 2 diabetes and a new target for clinical treatment. Compared with the traditional blood sugar and glycosylated hemoglobin detection methods, the present scheme can provide more accurate and direct reference data for the detection of the biomarker, which helps to improve the accuracy of clinical diagnosis; in addition, the therapeutic target provided by the scheme of the present invention has opened up a new path for the treatment of type 2 diabetes, and this target can promote the development and application of a series of new drugs for type 2 diabetes. At the same time, the Enteromorpha oligosaccharide provided by the present invention is a green bioactive substance extracted and prepared from natural seaweed as a therapeutic drug for type 2 diabetes, which has the advantages of rapidity, high efficiency, and no toxic side effects. The raw materials come from Enteromorpha, a common marine biological pollutant, and its development and utilization are of great significance for maintaining the ecological balance of the ocean. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The differential expression of OASL protein between the rats in the model group and the Enteromorpha oligosaccharide-administered group described in Example 1 of the present invention was found through proteomics and bioinformatics analysis: the interaction network association diagram of differentially expressed proteins in jejunum tissue was constructed through the String website, in which the yellow nodes and blue nodes respectively represent significant upper and lower regulation (E / M).

[0023] Figure 2 This is a process flow chart of Enteromorpha oligosaccharide prepared in Example 3 of the present invention.

[0024] Figure 3 This is Example 4 of the present invention, Enteromorpha oligosaccharides improve the mRNA expression of OASL in diabetic rats.

[0025] Figure 4 The changes in OASL protein expression, fasting blood glucose, insulin and glycosylated hemoglobin in the model group and the treatment group after administration of Enteromorpha oligosaccharides described in Example 4 of the present invention; wherein a is the change in fasting blood glucose in rats; b is the change in oral glucose tolerance in rats; c is the change in C-peptide in rats; d is the change in serum insulin in rats; and e is the change in glycosylated hemoglobin in rats.

[0026] Figure 5 The expression of OASL mRNA in Example 5 of the present invention, the changes in the expression of OASL in IR-HepG2 cells after interference, and the changes in the glucose consumption of cells (compared with the normal group, p<0.05: # , p<0.01: ## ; Compared with the model group, p<0.05: * , p<0.01: ** ). DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Similarly, the following examples are only partial embodiments of the present invention rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0028] Example 1

[0029] Evidence for OASL as a biomarker for type 2 diabetes

[0030] The differential expression of OASL between model group rats (high-sugar and high-fat diet induced type 2 diabetes) and normal rats was found through proteomics and bioinformatics technology analysis.

[0031] Through 4D Laber free proteomics analysis, all 6810 proteins were detected. Combined with bioinformatics analysis, the P value below 0.05 was set as the threshold to judge significant differences. Among them, 265 proteins showed differential expression in the comparison between the model group and the normal group. Furthermore, through the differential protein network interaction diagram, it was found that OASL was in a core position with a large number of functional network nodes in the protein interaction network, ranked high in VIP prediction, and its difference multiple was large and significant, indicating that this protein plays an extremely important role in type 2 diabetes and is involved in maintaining normal glucose metabolism function (reference Figure 1 ).

[0032] Example 2

[0033] OASL gene detection method

[0034] (1) Extraction of RNA

[0035] The total RNA in the jejunum tissues was extracted according to the instructions of the total RNA extraction kit, and the concentration was measured by a micro-UV spectrophotometer and stored in a -80°C refrigerator for later use.

[0036] (2) Reverse transcription

[0037] According to the instructions of the reverse transcription kit with gDNA Eraser, the successfully extracted jejunal total RNA was reverse transcribed to obtain cDNA. After measuring the concentration, it was appropriately diluted 3-5 times according to experimental requirements and stored at -□80℃ for future use.

[0038] (3) RT-qPCR

[0039] The first strand of cDNA obtained after reverse transcription of RNA was used as a template to design primers (primers are shown in Table 1 below). The expression of the target gene was detected by SYBR qPCR SuperMix Plus kit. The PCR amplification program was as follows: 95°C for 60 s, one cycle; 95°C and 60°C for 20 s and 60 s, respectively, for 40 cycles; and finally melting curve analysis, 95°C for 15 s; 60°C for 30 s; 95°C for 15 s; one cycle.

[0040] Table 1 Primers used for gene detection

[0041]

[0042] Note: The leading sequence of OASL is shown as SEQ ID NO.3, and the trailing sequence is shown as SEQ ID NO.4. The leading sequence of Gapdh is shown as SEQ ID NO.5, and the trailing sequence is shown as SEQ ID NO.6.

[0043] Example 3

[0044] Preparation of Enteromorpha oligosaccharide drugs targeting OASL

[0045] Weigh 100g Enteromorpha powder (80 mesh) and dissolve it at a solid-liquid ratio of 1:40. Ultrasonic assisted extraction was performed at 60℃, 45kHZ, and 200W power for 2h. After centrifugation at 4500rpm for 10min, the precipitate was discarded and the supernatant was removed. The supernatant was vacuum filtered to obtain the filtrate, which was then concentrated to 500mL by rotary evaporation. 2000mL of 95% ethanol was added at a ratio of 1:4 and precipitated overnight (more than 8h). Then, it was centrifuged at 4500rpm for 10min to obtain a precipitate, which was placed in a 50℃ oven for drying. Pure water was added to the dried precipitate at a ratio of 1:100 and an electric stirrer was used to assist dissolution until it was completely dissolved. Subsequently, neutral protease was added to the solution to make the enzyme activity 400U / mL, and enzymolysis was performed at the optimal temperature for 2h. After the enzymolysis was completed, the solution was placed in a 100℃ water bath, inactivated for 20min, centrifuged at 4500rpm for 10min, and the precipitate was removed and the supernatant was taken. The obtained liquid was dialyzed for 48 hours with a dialysis bag with a molecular weight of 15kDa, the solution in the bag was collected and concentrated to 10-20mL, and freeze-dried to obtain the green algae Enteromorpha polysaccharide. Accurately weigh 15g of green algae Enteromorpha polysaccharide and dissolve it in pure water to form a 1% polysaccharide solution. Concentrated sulfuric acid was added until the solution concentration reached 0.05mol / L. After acid hydrolysis in a 100℃ water bath for 1.5h, it was allowed to stand and cool. After cooling, 1.0mol / L NaOH was added to adjust the solution to neutrality, and the solution was concentrated. 95% ethanol solution was added at a ratio of 1:1 for more than 8h, and the supernatant was concentrated and freeze-dried to finally obtain Enteromorpha oligosaccharide (EPO) (reference Figure 2 ).

[0046] Example 4

[0047] Effects of Enteromorpha oligosaccharide drugs targeting OASL

[0048] The present invention specifically verifies the effect of OASL downregulation on fasting insulin and glycosylated hemoglobin in the model group and the drug-treated group. It is found that the downregulation of OASL in the model group caused a significant shift in fasting blood glucose, insulin and glycosylated hemoglobin levels, while after restoring OASL expression, the two levels returned to normal, indicating that the inhibition of OASL worsens the body's ability to regulate glucose metabolism (reference Figure 4 ), the specific steps are as follows:

[0049] (1) Changes in OASL protein expression

[0050] Protein extraction and enzymatic hydrolysis: The jejunal tissue was placed in a pre-cooled mortar, and after adding liquid nitrogen to grind it into powder, 4 times the volume of lysis buffer (1% protease inhibitor, 1% SDS, 1% phosphatase inhibitor, 50mM NAM, 3μM TSA) was added for lysis. Centrifuge at 12000r for 10min at 4℃, discard the precipitate, and determine the protein concentration of the supernatant. The protein of each sample was hydrolyzed with an equal amount of trypsin, and the volume was adjusted to be consistent with the lysis buffer. Slowly add TCA with a final concentration of 20%, vortex mix, precipitate at 4℃ for 2h, 4500r, centrifuge for 5min, discard the supernatant, and wash the precipitate with pre-cooled acetone 2-3 times. After drying the precipitate, add TEAB with a final concentration of 200mM, break the precipitate with ultrasound, and add trypsin at a ratio of 1:50 (protease: protein, m / m), and enzymatic hydrolysis overnight. Then add DTT to a final concentration of 5mM and reduce it at 56℃ for 30min. Finally, IAA was added to a final concentration of 11 mM and incubated at room temperature in the dark for 15 min.

[0051] Liquid chromatography-mass spectrometry analysis: The peptides were dissolved in liquid chromatography mobile phase A and separated using the NanoElute ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was a solution containing 0.1% formic acid and 100% acetonitrile. Liquid phase gradient settings: 0-70min, 6% to 24% B; 70-84min, 24% to 35% B; 84-87min, 35% to 80% B; 87-90min, 80% B, and the flow rate was maintained at 450nL / min. After separation by the ultra-high performance liquid phase system, the peptides were injected into the Capillary ion source for ionization and then entered the timsTOF Pro mass spectrometer for analysis. The ion source voltage was set to 1.75kV, and the peptide parent ions and their secondary fragments were detected and analyzed using high-resolution TOF. The secondary mass spectrometry scan range was set to 100-1700. The data acquisition mode used was parallel accumulation serial fragmentation (PASEF) mode. After one primary mass spectrum was acquired, 10 secondary spectra with parent ion charge numbers in the range of 0-5 were acquired in PASEF mode. The dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scanning of parent ions.

[0052] For protein difference analysis, the ratio of the average values ​​of all biological replicate quantitative values ​​of each protein in the comparison sample pair was selected as the fold change (FC), among which the EPL / Mod Ratio was 2.93, with a significance of p < 0.05 (p = 0.0476). The expression of OASL protein in the Enteromorpha oligosaccharide treatment group showed a significant up-regulation trend (see Table 2).

[0053] Table 2

[0054]

[0055]

[0056] (2) Changes in OASL gene expression

[0057] Based on the method in Example 2, RNA was extracted from jejunal tissue for reverse transcription, and the cDNA obtained after RNA reverse transcription was used as a template for PCR amplification. The specific parameters and primer sequences were the same as in Example 2.

[0058] like Figure 3 The test results showed that the mRNA expression level of OASL in the jejunum tissue of rats in the low-dose Enteromorpha oligosaccharide (EPL) group was significantly higher than that in the model group (p<0.05).

[0059] (3) Changes in fasting blood glucose and oral glucose tolerance

[0060] After 28 days of continuous gavage, all rats were fasted for 12 hours (during which they were allowed to drink water freely), and the fasting blood glucose (FBG) and glucose tolerance (OGTT) were measured by OMRON blood glucose meter. The ability of experimental rats to handle blood glucose can be directly measured by the results of OGTT. Blood was taken from the tail vein of rats at 0 hours to measure fasting blood glucose. After the measurement, all rats were gavaged with glucose (2g / kg), and blood was taken from the tail vein at 0.5, 1 and 2 hours to measure blood glucose.

[0061] The test results showed that the experimental rats in the Model group had a significantly decreased ability to absorb and process high levels of glucose in the blood, and were unable to regulate the high-sugar environment in the blood normally. After 4 weeks of intervention with low-dose Enteromorpha (EPL) and high-dose Enteromorpha (EPH), compared with the model group, the FBG and oral glucose tolerance (OGTT) levels of rats treated with Enteromorpha oligosaccharides were significantly reduced (p < 0.5), and low-dose Enteromorpha treatment had a better improvement effect on the OGTT level of rats in the model group (p < 0.1). In summary, it can be shown that both the high- and low-dose Enteromorpha oligosaccharide groups can alleviate the glucose tolerance damage caused by the high-sugar environment in the model rats.

[0062] (4) Changes in serum C-peptide, insulin, and glycosylated hemoglobin

[0063] Part of the whole blood of the rats was collected, and the glycated hemoglobin content of each group of rats was measured and analyzed according to the operating instructions of the glycated hemoglobin kit of Nanjing Jiancheng Bioengineering Institute. In addition, a portion of the blood of the rats was collected, and after standing at room temperature for 1 hour, it was centrifuged at 4°C, 3000r / min for 10 minutes, and the upper serum was taken and stored at -20°C for the determination of other indicators. The C-peptide (CP) and insulin (INS) rat ELISA kits were used to determine the CP and INS content in rat serum.

[0064] The test results showed that in the diabetic rat model induced by STZ, the fasting blood glucose FBG value increased significantly, while the serum insulin and CP levels showed a downward trend. After 4 weeks of treatment with Enteromorpha oligosaccharides, the FBG of the experimental rats decreased significantly, and the serum insulin content and CP level were improved. The ability of Enteromorpha oligosaccharides to maintain the stability of glucose in the blood can be determined by detecting the glycosylated hemoglobin (Ghb) in the whole blood of rats. As shown in Figure 4-2c&d, after 28 days of treatment, the Enteromorpha oligosaccharide treatment groups achieved good results: the Chb content of rats in the Enteromorpha low-dose group was significantly lower than that in the model group (p<0.01). In summary, the intake of Enteromorpha oligosaccharides has a good effect on stabilizing blood sugar and improving pancreatic β-cell function in aging diabetic rats.

[0065] Example 5

[0066] The expression of OASL in IR-HepG2 cells was interfered by transfection experiments to verify the effect of OASL as a therapeutic target

[0067] In this embodiment, IR-HepG2 cells were used as the transfection object. The experiment set up positive and negative controls. A meaningless blank sequence interference was used as the blank control, which was set as the NC group. The classic GAPDH interference sequence was used as the positive control, which was set as the GAPDH group. After the transfection complex was prepared, the 12-well plate was replaced with 0.8 ml of preheated serum-free medium in each well during the static period. After the static period, the transfection complex was immediately added to the well, and 200 μL was added to each well. After the addition, the plate was gently shaken to evenly distribute the complex, and then placed at 37°C, 5% CO 2 The cells were cultured statically under the conditions of RT-PCR and the complete culture medium was used for 24 hours after 6 hours. Total RNA of the cells was extracted and the expression of mRNA was determined after reverse transcription. The inhibition rate of the target gene OASL after cell transfection was verified by RT-qPCR experiment, and the glucose content in the culture medium was determined.

[0068] like Figure 5 As shown in the figure, the induction of insulin resistance HepG2 cell model (IR-HepG2) by dexamethasone at a concentration of 2 μM for 72 hours was successful. The glucose consumption of the model group cells was significantly lower than that of the blank control group (p<0.01), and the inhibition rate reached 23.6%. After the siRNA interference sequence interfered with the OASL gene in the IR-HepG2 cells of the model group, the glucose consumption of each group decreased by 28.11% (reference Figure 5 ), which was close to the NC group. The changes in glucose consumption in each group after interference were compared with the mRNA expression, and it was found that the glucose consumption in the group with high inhibition rate of OASL gene mRNA expression decreased accordingly, indicating that the expression of OASL gene would affect the ability of IR-HepG2 cells to consume glucose, and it was judged that it had the effect of regulating sugar metabolism.

[0069] The above descriptions are only some embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of OASL protein in the preparation of a product for diagnosing type 2 diabetes, characterized in that: The amino acid sequence of the OASL protein is shown in SEQ ID NO.2.

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