Application of selenium-rich polypeptide to inhibition of NLRP3 inflammasome-related inflammation and regulation of cholesterol transport protection lipid metabolism abnormality

Abstract

The invention belongs to the field of active peptides, and particularly relates to application of selenium-rich polypeptide in inhibiting NLRP3 inflammasome-related inflammations and regulating cholesterol transport protection lipid metabolism abnormality, the selenium-rich polypeptide is prepared by chelating Adi-Apo fusion protein as shown in SEQ ID NO: 3 and sodium selenite (Na2SeO3); the selenium peptide chelate has the effects of remarkably reducing the concentration of TC / TG in serum, reducing the liver lipid deposition and inflammation level and reducing the atherosclerotic plaque area proportion through high oxidation resistance and high anti-inflammatory activity, and can be used for treating and / or preventing lipid metabolism disorder diseases such as metabolism-related fatty liver diseases and coronary atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of active peptides, and particularly relates to the application of selenium-rich polypeptide in inhibiting NLRP3 inflammasome-related inflammation and regulating cholesterol transport to protect against dyslipidemia. Background Art

[0002] Adiponectin is an adipokine secreted by adipose tissue and has various biological functions such as anti-inflammatory, reducing lipid deposition, anti-atherosclerotic, and regulating insulin sensitivity. Research shows that Adiponectin plays an important metabolic regulatory role by interacting with its receptors AdipoR1 and AdipoR2, can improve insulin sensitivity, promote fatty acid oxidation, and inhibit hepatic gluconeogenesis. In addition, Adiponectin has also been found to have significant anti-inflammatory effects, can inhibit endogenous inflammatory responses, and reduce the risks of metabolic associated fatty liver disease, atherosclerosis, and cardiovascular diseases. However, the decrease in adiponectin levels is closely related to the occurrence of diseases such as obesity, metabolic associated fatty liver disease, diabetes, and metabolic syndrome. Therefore, enhancing the biological function and level of Adiponectin is considered an important direction for regulating dyslipidemia and improving metabolic diseases.

[0003] Apolipoprotein A-I (ApoA-I) is the main protein component of high-density lipoprotein (HDL) and is widely involved in the reverse cholesterol transport process. ApoA-I binds to ATP-binding cassette transporter A1 (ABCA1) to promote the transport of cholesterol from peripheral tissues to the liver, where it is then metabolized or excreted, thereby reducing the total cholesterol levels in liver tissue and blood. Studies have found that the level of ApoA-I is negatively correlated with the risk of (dyslipidemic diseases cardiovascular diseases), and the deficiency or dysfunction of ApoA-I is closely related to the occurrence of metabolic associated fatty liver disease and atherosclerosis. ApoA-I not only has the function of reverse cholesterol transport, but also can reduce the damage of hepatocytes and vascular endothelium through its anti-inflammatory effect, and slow down the progression of fatty liver and arteriosclerosis. Therefore, increasing the level of ApoA-I or enhancing its function is considered an important strategy for slowing down dyslipidemic diseases such as fatty liver and atherosclerosis.

[0004] The NLRP3 inflammasome is an important intracellular immune regulatory structure, and its activation can trigger a series of inflammatory responses, especially being closely related to various metabolic diseases such as metabolic associated fatty liver disease, atherosclerosis, obesity, etc. In recent years, studies have shown that selenium-rich polypeptides have significant anti-inflammatory effects and can reduce the inflammatory responses caused by regulating the activation of the NLRP3 inflammasome. By regulating oxidative stress and anti-inflammatory pathways, selenium-rich polypeptides can effectively inhibit the activation of the NLRP3 inflammasome, thereby reducing chronic low-grade inflammatory responses and contributing to the recovery of lipid metabolism disorders. However, the effects of selenium-rich polypeptides in clinical applications are still limited to a certain extent. How to regulate lipid metabolism disorders while inhibiting the NLRP3 inflammasome remains an important research direction.

[0005] Currently, most treatment strategies for lipid metabolism disorders and chronic inflammation focus on single-target therapies. However, due to the complexity of the diseases, single targets often cannot provide ideal treatment effects. Based on this, a fusion protein combining Adiponectin protein and ApoA-I protein has become a new research direction. This fusion protein can not only enhance the functions of Adiponectin in regulating lipid metabolism and anti-inflammation but also utilize the roles of ApoA-I in reverse cholesterol transport, reducing liver lipid deposition, and anti-atherosclerosis. In addition, the fusion protein may have the potential to inhibit the activation of the NLRP3 inflammasome, further reducing the metabolic disorders caused by inflammation. By combining multiple mechanisms of action, this technology can not only improve lipid metabolism disorders but also effectively inhibit the associated chronic inflammation, becoming a treatment strategy with great application prospects. Summary of the Invention

[0006] To solve the deficiencies in the treatment of lipid metabolism disorders and inflammation-related diseases such as metabolic associated fatty liver disease and atherosclerosis in the prior art, the present invention provides a selenium-rich polypeptide that can effectively inhibit the activation of the NLRP3 inflammasome, regulate cholesterol transport, and regulate lipid metabolism disorders.

[0007] Specifically, the selenium-rich polypeptide is prepared by a chelation reaction of an Adi-Apo fusion protein and inorganic selenium. Among them, the Adi-Apo fusion protein is a polypeptide selected from SEQ ID NO:3, and the inorganic selenium is sodium selenite (Na 2 SeO 3 ). This selenium-peptide chelate can not only significantly reduce the concentrations of total cholesterol (TC) and triglyceride (TG) in the serum but also has high antioxidant and anti-inflammatory activities, and can reduce the proportion of fat in liver cells and the proportion of atherosclerotic plaque area, thereby effectively treating and / or preventing lipid metabolism disorders such as metabolic associated fatty liver disease and coronary atherosclerosis.

[0008] In some embodiments, the selenium-rich polypeptide chelates sodium selenite with the Adi-Apo fusion protein, and the formed chelate has excellent biological activity in vivo, can double-regulate cholesterol transport and inflammatory response, and further promote the recovery of lipid metabolism disorders.

[0009] The present invention also provides a preparation method, which includes chelating the Adi-Apo fusion protein shown in SEQ ID NO:3 with sodium selenite (Na 2 SeO 3 ). This method is simple, feasible, and has good application prospects.

[0010] Finally, the present invention provides the application of the selenium-rich polypeptide, and the application includes its application in preparing drugs for treating and / or preventing lipid metabolism disorder diseases such as metabolic associated fatty liver disease and coronary atherosclerosis. By applying the selenium-rich polypeptide to related drugs, it can effectively improve lipid metabolism disorders, reduce inflammatory responses, and slow down the progression of lipid metabolism disorder diseases such as fatty liver and atherosclerosis, thus playing a positive protective role in the health of the body.

[0011] Compared with the prior art, the present invention has at least the following beneficial effects:

[0012] First, the present invention first proposes a selenium-rich polypeptide prepared by chelating an Adi-Apo fusion protein with inorganic selenium, which can simultaneously regulate lipid metabolism and anti-inflammatory functions and provide dual protection;

[0013] Second, the selenium-rich polypeptide has significant antioxidant, anti-inflammatory activities and the effect of improving lipid metabolism, which helps to treat lipid metabolism disorder-related diseases such as metabolic associated fatty liver disease and coronary atherosclerosis, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Ni-NTA column chromatogram and SDS-PAGE detection of the Adi-ApoA fusion protein.

[0015] Figure 2 Potential analysis of the Adi-ApoA fusion protein and its selenium chelate.

[0016] Figure 3 Detection of the lipid-lowering effect of the Adi-ApoA fusion protein and its selenium chelate.

[0017] Figure 4 Detection of the antioxidant effect of the Adi-ApoA fusion protein and its selenium chelate.

[0018] Figure 5 Detection of the anti-inflammatory effect of the Adi-ApoA fusion protein and its selenium chelate.

[0019] Figure 6 Detection of the proportion of hepatic adipocyte degeneration of the Adi-ApoA fusion protein and its selenium chelate.

[0020] Figure 7 Detection of the proportion of atherosclerotic plaque area of the Adi-ApoA fusion protein and its selenium chelate. Detailed implementation manners

[0021] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0022] Example 1 Preparation of the Adi-ApoA fusion protein

[0023] According to the record in NCBI, the mature peptide sequence without signal peptide of Adiponectin precursor [Homo sapiens] (NCBI Reference Sequence: NP_001171271.1):

[0024] GHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN (SEQ ID NO: 1).

[0025] According to the record in NCBI, the mature peptide sequence without signal peptide of ApoA-I [Homo sapiens] (GenBank: CAA30377.1):

[0026] DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ(SEQ ID NO:2).

[0027] Design the Adi-Apo fusion protein sequence according to the structural characteristics of the Adiponectin protein and the ApoA-I protein, with GGGGSGGGGS as the linker:

[0028] GHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTNGGGGSGGGGSGGGGSGGGGSDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ(SEQ ID NO:3).

[0029] The expression plasmid pET-28a(+)-Adi-ApoA was synthesized by GenScript Biotech Corporation, and the constructed plasmid (pET-28a(+)-Adi-ApoA) was transformed into Escherichia coli BL21(DE3) strain. The plasmid was introduced into Escherichia coli by chemical transformation method. The competent Escherichia coli was mixed with the recombinant plasmid, incubated on ice and then heat shocked (42 °C, 45 seconds), and then placed on ice for 5 minutes. The transformed Escherichia coli colonies were selected by ampicillin. The positive clones were picked and cultured on a small scale, and the plasmid was extracted using a plasmid extraction kit for verification. The selected positive clones were inoculated into 5 mL of LB medium containing antibiotics and cultured overnight (16 - 18 hours) on a shaker at 37 °C. The next day, the overnight culture was inoculated into 1 L of fresh LB medium and continued to be cultured at 37 °C until the OD600 reached 0.6 - 0.8; when the cell growth reached the logarithmic phase, IPTG (concentration 0.5 mM) was used for induction, and the temperature was adjusted to 18 °C for low-temperature culture (to reduce the formation of inclusion bodies). The culture was continued for 12 - 16 hours to promote the expression of the fusion protein. The induced cells were collected by centrifugation (4000g, 4 °C, 10 minutes), and the culture medium was discarded. The cell pellet was resuspended in lysis buffer (PBS buffer containing 0.1% TritonX-100). The cells were lysed using an ultrasonic cell disruptor (on ice). After cell lysis, the cell debris was removed by centrifugation (12000g, 4 °C, 30 minutes), and the supernatant was collected. The lysate was washed through a Ni-NTA column, and the fusion protein was eluted with PBS buffer containing 250 mM imidazole. The purity of the fusion protein was detected by SDS-PAGE to ensure that it was the target protein, see Figure 1 .

[0030] Figure 1 The results showed that the molecular weight of the Adi-ApoA fusion protein was about 54.31 kDa, which was in line with expectations, and the protein purity was high enough for subsequent bioactivity experiments.

[0031] Example 2 Preparation and Identification of Selenium Chelate of Adi-ApoA Fusion Protein

[0032] The purified Adi-ApoA fusion protein was dissolved in PBS buffer, the protein concentration was controlled at 8 mg / mL, and the pH value of the buffer was set to 7.4 to maintain the stability of the protein. An appropriate amount of sodium selenite (Na 2 SeO 3) Dissolve it with deionized water to make the concentration of the sodium selenite solution 2 mM, ensuring that pH = 7.0, which helps to ensure the good solubility and reactivity of sodium selenite. Slowly add the sodium selenite solution to the Adi-ApoA fusion protein solution and stir. Ensure gentle stirring throughout the process so that sodium selenite is evenly distributed and reacts with the protein. The reaction temperature is set at 20 - 25 °C and the time is 24 hours. After the reaction is completed, transfer the reaction system to an 800 Da dialysis bag, place it in a container containing buffer solution, and perform dialysis treatment to remove excess sodium selenite. The dialysis time is 24 hours. The dialysis solution is deionized water and the dialysis solution is changed every 4 hours. The dialyzed sample is obtained as a powder of Adi-ApoA fusion protein selenium chelate by freeze-drying and stored at -20 °C.

[0033] The selenium content was determined by the 3,3′-diaminobenzidine colorimetric method: Mix the chelate sample with 3,3′-diaminobenzidine (DAB) solution and react in an acidic environment. Selenium reacts with DAB under acidic conditions to form a dark blue complex, which has an obvious absorption peak at a specific wavelength. By measuring the absorbance of this absorption peak, the selenium content in the sample can be calculated according to the standard curve. Calculate the selenium chelation rate according to formula (1):

[0034] Selenium chelation rate (%) = (A1 / A2) × 100% (1)

[0035] Where: A1 is the total amount of selenium in the Adi-ApoA fusion protein selenium chelate in g; A2 is the total amount of selenium in the reaction system in g; The results showed that the selenium chelation rate was 78 + 0.52%.

[0036] Potential analysis:

[0037] The potential changes of the Adi-ApoA fusion protein and its selenium chelate were measured by a Nano-2sZEN3600 nanoparticle size potentiometer to evaluate whether the chelation reaction was successful. 1.0 mg of the Adi-ApoA fusion protein and the Adi-ApoA fusion protein selenium chelate were respectively dissolved in 1 mL of deionized water with a concentration of 1.0 mg / mL. After reacting at 37 °C for 10 minutes, the potential was measured using a potentiometer. Each sample was measured 3 times and the average value was taken to compare the potential differences. If the potential changes significantly, it indicates that selenium has successfully chelated with the Adi-ApoA fusion protein; if the change is small, the chelation reaction is not completed, see Figure 2 .

[0038] Figure 2The results showed that the zeta potential value of the Adi-ApoA fusion protein selenium chelate was higher than that of the Adi-ApoA fusion protein, indicating that after chelation, the negative charge on the surface of the fusion protein was significantly reduced. That is, the potential measurement results showed that there was a significant difference in the potential value between the selenium chelate and the Adi-ApoA fusion protein solution, indicating that selenium was successfully chelated with the Adi-ApoA fusion protein.

[0039] Example 3 Study on the effects of Adi-ApoA fusion protein and its selenium chelate on dyslipidemia models

[0040] This experiment aimed to evaluate the preventive and therapeutic effects of Adi-ApoA fusion protein and its selenium chelate on dyslipidemia model New Zealand white rabbits. Male New Zealand white rabbits at 8 weeks of age were randomly divided into four groups: normal control group (normal diet), model group (high-fat diet), fusion protein group (high-fat diet + Adi-ApoA fusion protein, intraperitoneally injected with Adi-ApoA fusion protein at 200 mg / kg per day), selenium chelate group (high-fat diet + Adi-ApoA fusion protein selenium chelate, intraperitoneally injected with Adi-ApoA fusion protein selenium chelate at 200 mg / kg per day), and positive control group (high-fat diet + atorvastatin, intraperitoneally injected with atorvastatin at 4 mg / kg per day). The model group, fusion protein group, selenium chelate group, and positive control group were fed with a high-fat diet (containing 2% cholesterol, 12% soybean oil, and 8% sucrose) for 8 weeks to establish a dyslipidemia model.

[0041] At the end of the 8-week experiment, ear marginal vein blood samples were taken from the rabbits in each group. A fully automatic biochemical analyzer was used to measure TC and TG in the serum. According to the operating procedures, serum samples were extracted and quantitative analysis was performed using relevant kits. Each group of samples was measured 3 times to ensure the reliability of the data, and statistical analysis was carried out to compare the differences between the groups, as shown in Figure 3 .

[0042] Figure 3 The results showed that TC (P<0.01) and TG (P<0.01) in the serum of rabbits in the model group were increased compared with the blank control group, indicating that the dyslipidemia rabbit model was successfully established; compared with the model group, the serum TC and TG levels in the fusion protein group, selenium chelate group, and positive control group were significantly decreased (P<0.01), and there were significant differences; at the same time, the lipid-lowering effect of the selenium chelate group was better than that of the fusion protein and atorvastatin.

[0043] At the end of the 8-week experiment, ear marginal vein blood samples were taken from the rabbits in each group, and ELISA kits were used to detect serum GPX4 and GSH indicators to evaluate the antioxidant stress level. Each group of samples was measured 3 times to ensure the reliability of the data, and statistical analysis was carried out to compare the differences between the groups, as shown in Figure 4 .

[0044] Figure 4 The results showed that compared with the blank control group, the levels of GPX4 (P<0.05) and GSH (P<0.05) in the serum of rabbits in the model group decreased, indicating that the rabbit model of abnormal lipid metabolism was successfully established; compared with the model group, the levels of serum GPX4 and GSH in the selenium chelate group were significantly increased, with significant differences (P<0.01), indicating that the selenium chelate of Adi-ApoA fusion protein had obvious antioxidant effects and could indirectly inhibit the inflammasome through the antioxidant pathway.

[0045] At the end of the 8-week experiment, ear marginal vein blood samples were taken from rabbits in each group, and the serum TNF-β and IL-1β indexes were detected by an enzyme-linked immunosorbent assay (ELISA) kit to evaluate their inhibitory effects on the levels of inflammatory factors. Each sample was measured 3 times to ensure the reliability of the data, and statistical analysis was performed to compare the differences between groups, as shown in Figure 5 .

[0046] Figure 5 The results showed that compared with the blank control group, the levels of TNF-β (P<0.05) and IL-1β (P<0.05) in the serum of rabbits in the model group increased, indicating that the rabbit model of abnormal lipid metabolism was successfully established; compared with the model group, the levels of serum TNF-β and IL-1β in the selenium chelate group were significantly decreased, with significant differences (P<0.01), indicating that the selenium chelate of Adi-ApoA fusion protein had obvious inhibitory effects on the levels of inflammatory factors and directly inhibited the inflammasome.

[0047] Detection of the proportion of fat deposition in liver cells: At the end of the 8-week experiment, liver tissue samples of rabbits in each group were taken and immediately fixed in 4% paraformaldehyde solution for 4 hours. After fixation, the liver tissue was washed with PBS buffer. The fixed liver tissue was sectioned into a thickness of 4 - 6 μm. The sections were immersed in 0.5% Oil Red O solution for staining for 30 minutes. The Oil Red O solution was Oil Red O dissolved in isopropanol. After staining, the sections were gently washed with PBS buffer to remove the excess dye. The sections were successively immersed in 70%, 80%, 90%, and 100% ethanol solutions for dehydration, with each immersion for 10 minutes. Then, the sections were cleared with xylene for 10 minutes. The tissue sections were mounted with mounting medium and observed under a microscope after drying. The stained tissue sections were observed using an optical microscope, and images were taken for subsequent analysis. Representative areas were selected for observation to ensure that the distribution of fat droplets and the hepatocyte structure could be clearly shown. Through Oil Red O staining of liver tissue, the fat droplets in hepatocytes were seen as red areas. An increase in the proportion of fat deposition was usually manifested as more red-stained areas. Conversely, a decrease in fat deposition after treatment was manifested as a reduction in the red area. By quantitatively analyzing the degree of fat deposition in the liver, the changes in lipid metabolism could be evaluated.

[0048] Oil Red O staining was performed on the liver sections of each group, observed and photographed using a microscope, and then the fat deposition area was quantitatively analyzed using image analysis software. Fat Deposition Area: The area of the Oil Red O-stained region in the liver section was calculated using image analysis software; Liver Tissue Area: The total area of the liver tissue region measured by the image analysis software. Fat Deposition Ratio: The ratio of the fat deposition area to the total area of the liver tissue, expressed as a percentage, as shown in Figure 6 .

[0049] Figure 6 The results showed that compared with the model group, the selenium chelate group could significantly reduce the fat content in liver cells (P < 0.01), and the selenium chelate group was significantly superior to the fusion protein group and the positive control group in reducing fat deposition.

[0050] Detection of atherosclerotic plaque area: At the end of the 8-week experiment, arterial specimens of rabbits in each group were taken and fixed, and then stained with Oil Red O stain to clearly show the plaque area. Subsequently, a microscope was used for observation and high-definition images were taken, and quantitative analysis was performed in combination with image analysis software to calculate the percentage of plaque area. This method can accurately reflect the degree of arteriosclerosis and provide a reliable basis for clinical diagnosis and research. At the same time, to improve the accuracy of data, attention should be paid to the consistency of sample processing and the uniformity of staining effect during the detection process, see Figure 7 .

[0051] Figure 7 The results showed that compared with the model group, the selenium chelate group could significantly reduce the proportion of atherosclerotic plaque area (P<0.01), and the selenium chelate group was significantly superior to the fusion protein group and the positive control group in reducing atherosclerotic plaques.

[0052] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A selenium-rich polypeptide that inhibits NLRP3 inflammasome-related inflammation and regulates cholesterol transport to protect against abnormal lipid metabolism, characterized in that: The selenium-rich polypeptide is prepared by chelating Adi-Apo fusion protein with inorganic selenium.

2. The selenium-enriched polypeptide according to claim 1, characterized in that The Adi-Apo fusion protein is selected from the polypeptide shown in SEQ ID NO:

3.

3. The selenium-enriched polypeptide according to claim 1 or 2, characterized in that The inorganic selenium is selected from sodium selenite (Na2SeO3).

4. A method for preparing a selenopeptide chelate, characterized in that: The Adi-Apo fusion protein as shown in SEQ ID NO: 3 is subjected to a chelation reaction with selenium, wherein sodium selenite (Na2SeO3) is used as a selenium source in the chelation reaction.

5. Use of the selenium-rich polypeptide according to any one of claims 1 to 3 in the preparation of drugs for treating and / or preventing abnormal lipid metabolism diseases such as metabolism-related fatty liver disease and coronary atherosclerosis.

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