Application of a selenium-rich polypeptide in inhibiting NLRP3 inflammasome-related inflammation and regulating cholesterol transport to protect against abnormal lipid metabolism

Abstract

The present invention belongs to the field of active peptides, and specifically relates to an application of a selenium-rich polypeptide for inhibiting NLRP3 inflammasome-related inflammation and regulating cholesterol transport to protect against abnormal lipid metabolism. The selenium-rich polypeptide is prepared by chelating the Adi-Apo fusion protein shown in SEQ ID NO: 3 with sodium selenite (Na2SeO3); the selenium peptide chelate has the effect of significantly reducing the concentration of TC / TG in serum, high antioxidant and high anti-inflammatory activity to reduce liver lipid deposition, inflammation levels and the proportion of atherosclerotic plaque area, and can be used to treat and / or prevent metabolic-related fatty liver disease, coronary atherosclerosis and other lipid metabolism abnormalities.

Description

Technical Field

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

[0002] Adiponectin is an adipokine secreted by adipose tissue that has multiple biological functions, including anti-inflammation, reducing lipid deposition, anti-atherosclerosis, and regulating insulin sensitivity. Studies have shown that adiponectin plays an important metabolic regulatory role by interacting with its receptors AdipoR1 and AdipoR2, improving insulin sensitivity, promoting fatty acid oxidation, and inhibiting hepatic gluconeogenesis. In addition, adiponectin has also been found to have significant anti-inflammatory effects, inhibiting endogenous inflammatory responses, and reducing the risk of metabolic-related fatty liver disease, atherosclerosis, and cardiovascular disease. However, decreased adiponectin levels are closely related to the occurrence of diseases such as obesity, metabolic-related fatty liver disease, diabetes, and metabolic syndrome. Therefore, enhancing the biological function and level of adiponectin is considered to be an important direction for regulating abnormal lipid metabolism 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 transport of cholesterol. ApoA-I promotes the transport of cholesterol from peripheral tissues to the liver by binding to ATP-binding transporter A1 (ABCA1), and then is metabolized or excreted, thereby reducing the total cholesterol level in liver tissue and blood. Studies have found that the level of ApoA-I is negatively correlated with the risk of (lipid metabolism disorders and cardiovascular diseases), and the deficiency or dysfunction of ApoA-I is closely related to the occurrence of metabolic-related fatty liver disease and atherosclerosis. ApoA-I not only has the function of reverse cholesterol transport, but also can reduce damage to hepatocytes and vascular endothelium through anti-inflammatory effects, slowing down the progression of fatty liver and arteriosclerosis. Therefore, increasing the level of ApoA-I or enhancing its function is considered to be an important strategy to slow down lipid metabolism disorders such as fatty liver and atherosclerosis.

[0004] The NLRP3 inflammasome is an important intracellular immune regulatory structure. Its activation can trigger a series of inflammatory responses, which are particularly closely related to a variety of metabolic diseases such as metabolic-related fatty liver disease, atherosclerosis, and obesity. Recent studies have shown that selenium-rich peptides have significant anti-inflammatory effects and can reduce the inflammatory response caused by them by regulating the activation of the NLRP3 inflammasome. Selenium-rich peptides can effectively inhibit the activation of the NLRP3 inflammasome by regulating oxidative stress and anti-inflammatory pathways, thereby alleviating chronic low-grade inflammatory responses and helping to restore abnormal lipid metabolism. However, the effectiveness of selenium-rich peptides in clinical applications is currently limited. How to regulate abnormal lipid metabolism while inhibiting the NLRP3 inflammasome remains an important research direction.

[0005] Currently, most treatment strategies for dyslipidemia and chronic inflammation focus on single-target therapies. However, due to the complexity of the diseases, single targets often fail to provide ideal therapeutic effects. Therefore, fusion proteins combining the adiponectin and apoA-I proteins have emerged as a new research direction. This fusion protein not only enhances the lipid metabolism and anti-inflammatory functions of adiponectin, but also leverages the role of apoA-I in reverse cholesterol transport, reducing hepatic lipid deposition, and combating atherosclerosis. Furthermore, the fusion protein may have the potential to inhibit activation of the NLRP3 inflammasome, further alleviating metabolic disorders triggered by inflammation. By combining multiple mechanisms of action, this technology not only improves dyslipidemia but also effectively suppresses the associated chronic inflammation, making it a promising therapeutic strategy. Summary of the Invention

[0006] In order to address the deficiencies in the existing technology for the treatment of lipid metabolism disorders and inflammatory response-related diseases such as metabolism-related fatty liver disease and atherosclerosis, the present invention provides a selenium-rich polypeptide that can effectively inhibit the activation of NLRP3 inflammasomes, regulate cholesterol transport, and regulate lipid metabolism disorders.

[0007] Specifically, the selenium-rich polypeptide is prepared by a chelation reaction between an Adi-Apo fusion protein and inorganic selenium, wherein the Adi-Apo fusion protein is a polypeptide selected from SEQ ID NO: 3, and the inorganic selenium is sodium selenite (Na2SeO3). This seleno-peptide chelate not only significantly reduces the concentrations of total cholesterol (TC) and triglycerides (TG) in serum, but also has highly effective 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 metabolic-related fatty liver disease, coronary atherosclerosis and other lipid metabolism disorders.

[0008] In certain embodiments, the selenium-rich polypeptide chelates sodium selenite and Adi-Apo fusion protein to form a chelate having excellent biological activity in vivo, capable of dually regulating cholesterol transport and inflammatory response, thereby promoting the recovery of abnormal lipid metabolism.

[0009] The present invention also provides a preparation method, which comprises chelating the Adi-Apo fusion protein shown in SEQ ID NO: 3 with sodium selenite (Na2SeO3). This method is simple, feasible, and has good application prospects.

[0010] Finally, the present invention provides applications of the selenium-rich polypeptide, including its use in the preparation of a medicament for treating and / or preventing metabolic-related fatty liver disease, coronary atherosclerosis, and other lipid metabolism disorders. By applying the selenium-rich polypeptide to related medicaments, it is possible to effectively improve lipid metabolism disorders, reduce inflammatory responses, and slow the progression of lipid metabolism disorders such as fatty liver and atherosclerosis, thereby playing a positive protective role in the body's health.

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

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

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

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

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

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

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

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

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

[0020] Figure 7 Detection of the proportion of atherosclerotic plaque area by Adi-ApoA fusion protein and its selenium chelate. DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

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

[0023] According to NCBI record Adiponectin precursor [Homo sapiens] (NCBI Reference Sequence: NP_001171271.1), the mature peptide sequence does not include the signal peptide:

[0024] GHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIR FTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN(SEQ ID NO:1).

[0025] According to NCBI, the mature peptide sequence of ApoA-I [Homo sapiens] (GenBank: CAA30377.1) excluding the signal peptide is as follows:

[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 Co., Ltd., and the constructed plasmid (pET-28a(+)-Adi-ApoA) was transformed into the Escherichia coli BL21 (DE3) strain. The plasmid was introduced into Escherichia coli using a chemical transformation method. The competent Escherichia coli was mixed with the recombinant plasmid, heat-shocked (42°C, 45 seconds) after an ice bath, and then placed in an ice bath for 5 minutes. Successfully transformed Escherichia coli colonies were selected using ampicillin. Positive clones were selected and cultured on a small scale. The plasmid was extracted using a plasmid extraction kit for verification. The screened positive clones were inoculated into 5 mL of LB medium containing antibiotics and cultured in a shaking incubator at 37°C overnight (16 to 18 hours). The next day, the overnight culture was inoculated into 1 L of fresh LB medium and cultured at 37°C until OD600 reached 0.6-0.8; when the cell growth reached the logarithmic phase, IPTG (concentration of 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). Cells were lysed using an ultrasonic disruptor (on ice). After cell lysis, 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 approximately 54.31 kDa, which was in line with expectations, and the protein had a high purity and could be used for subsequent biological activity experiments.

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

[0032] Dissolve the purified Adi-ApoA fusion protein in PBS buffer at a protein concentration of 8 mg / mL. The pH of the buffer is set to 7.4 to maintain protein stability. Take an appropriate amount of sodium selenite (Na2SeO3) and dissolve it in deionized water to a concentration of 2 mM. Ensure that the pH is 7.0, which helps ensure good solubility and reactivity of sodium selenite. Slowly add the sodium selenite solution to the Adi-ApoA fusion protein solution and stir. Ensure that gentle stirring is maintained throughout the process to ensure that the sodium selenite is evenly distributed and reacts with the protein. Set the reaction temperature to 20-25°C and the reaction time to 24 hours. After the reaction is complete, transfer the reaction system to an 800Da dialysis bag, place it in a container containing buffer, and dialyze to remove excess sodium selenite. The dialysis fluid is deionized water and is replaced every 4 hours. The dialyzed sample was freeze-dried to obtain Adi-ApoA fusion protein selenium chelate powder, which was stored at -20°C.

[0033] The selenium content is determined using the 3,3′-diaminobenzidine colorimetric method: the chelate sample is mixed with a 3,3′-diaminobenzidine (DAB) solution and reacted in an acidic environment. Under acidic conditions, selenium reacts with DAB to form a dark blue complex that has a distinct absorption peak at a specific wavelength. By measuring the absorbance of this absorption peak, the selenium content in the sample can be calculated based on the standard curve. The selenium chelation rate is calculated according to formula (1):

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

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

[0036] Potential analysis:

[0037] The potential changes of Adi-ApoA fusion protein and its selenium chelate were measured by Nano-2sZEN3600 nanoparticle size potentiometry to evaluate whether the chelation reaction was successful. 1.0 mg of Adi-ApoA fusion protein and Adi-ApoA fusion protein selenium chelate were dissolved in 1 mL of deionized water at a concentration of 1.0 mg / mL. After reacting at 37°C for 10 minutes, the potential was measured using a potentiometry instrument. Each sample was measured 3 times and the average value was taken to compare the potential difference. If the potential change is significant, it indicates that selenium has successfully chelated with the Adi-ApoA fusion protein; if the change is small, the chelation reaction is not complete, 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-Apo A 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 the potential value of the selenium chelate was significantly different from that of 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 the Disease Model of Abnormal Lipid Metabolism

[0040] This study aimed to evaluate the efficacy of Adi-ApoA fusion protein and its selenium chelate on the prevention and treatment of lipid abnormalities in New Zealand white rabbits. Eight-week-old male New Zealand white rabbits were randomly divided into four groups: a normal control group (normal diet), a model group (high-fat diet), a fusion protein group (high-fat diet combined with Adi-ApoA fusion protein, intraperitoneal injection of 200 mg / kg daily), a selenium chelate group (high-fat diet combined with Adi-ApoA fusion protein selenium chelate, intraperitoneal injection of 200 mg / kg daily), and a positive control group (high-fat diet combined with atorvastatin, intraperitoneal injection of 4 mg / kg daily). The model, fusion protein, selenium chelate, and positive control groups were fed a high-fat diet (containing 2% cholesterol, 12% soybean oil, and 8% sucrose) for 8 weeks to establish a lipid abnormality model.

[0041] At the end of the 8th week of the experiment, blood samples were collected from the ear vein of each group of rabbits. TC and TG in serum were determined using a fully automatic biochemical analyzer. According to the operating procedures, serum samples were extracted and quantitatively analyzed using relevant kits. Each group of samples was measured three times to ensure the reliability of the data, and statistical analysis was performed to compare the differences between the groups. Figure 3 .

[0042] Figure 3 The results showed that the serum TC (P<0.01) and TG (P<0.01) levels of the rabbits in the model group were higher than those in the blank control group, indicating that the modeling of rabbits with abnormal lipid metabolism was successful; compared with the model group, the serum TC and TG levels of the fusion protein group, selenium chelate group and positive control group were significantly reduced (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 8th week of the experiment, blood samples were collected from the ear vein of each group of rabbits, and the serum GPX4 and GSH indicators were detected by enzyme-linked immunosorbent assay (ELISA) kit to evaluate the level of antioxidant stress. The measurement was repeated 3 times for each group of samples to ensure the reliability of the data. Statistical analysis was performed to compare the differences between the groups. Figure 4 .

[0044] Figure 4 The results showed that the GPX4 (P<0.05) and GSH (P<0.05) levels in the serum of the rabbits in the model group were decreased compared with those in the blank control group, indicating that the modeling of abnormal lipid metabolism in rabbits was successful; compared with the model group, the serum GPX4 and GSH levels in the selenium chelate group were significantly increased, and there was a significant difference (P<0.01), indicating that the Adi-ApoA fusion protein selenium chelate has a significant antioxidant effect, which can indirectly inhibit inflammasomes through antioxidant pathways.

[0045] At the end of the 8th week of the experiment, blood samples were collected from the ear vein of each group of rabbits, and the serum TNF-β and IL-1β indicators were detected by enzyme-linked immunosorbent assay (ELISA) kit to evaluate the level of inflammatory factor inhibition. The measurement was repeated 3 times for each group of samples to ensure the reliability of the data. Statistical analysis was performed to compare the differences between the groups. Figure 5 .

[0046] Figure 5 The results showed that the levels of TNF-β (P<0.05) and IL-1β (P<0.05) in the serum of the rabbits in the model group were increased compared with those in the blank control group, indicating that the modeling of rabbits with abnormal lipid metabolism was successful; compared with the model group, the levels of serum TNF-β and IL-1β in the selenium chelate group were significantly reduced, and there was a significant difference (P<0.01), indicating that the Adi-ApoA fusion protein selenium chelate has a significant inhibitory effect on the level of inflammatory factors, which directly inhibits inflammasomes.

[0047] Assessment of Fat Deposition in Liver Cells: At the end of the 8th week of the experiment, liver tissue samples were collected from each group and immediately fixed in 4% paraformaldehyde for 4 hours. After fixation, the liver tissue was washed with PBS buffer. The fixed liver tissue was sliced into 4-6 μm sections. The sections were stained with 0.5% Oil Red O in isopropyl alcohol for 30 minutes. After staining, the sections were gently washed with PBS buffer to remove excess dye. The sections were dehydrated by sequentially soaking in 70%, 80%, 90%, and 100% ethanol solutions for 10 minutes each. The sections were then cleared with xylene for 10 minutes. The sections were mounted with mounting adhesive and allowed to dry before microscopic examination. The stained sections were observed using a light microscope and images were captured for subsequent analysis. Representative areas were selected for observation to ensure clear visualization of the distribution of fat droplets and hepatocyte structure. Oil Red O staining of liver tissue reveals fat droplets within hepatocytes as red areas. An increase in fat deposition typically manifests as more red areas, while a decrease in fat deposition after treatment manifests as a decrease in red areas. Quantitative analysis of liver fat deposition can assess changes in lipid metabolism.

[0048] The liver sections of each group were stained with Oil Red O, observed and photographed using a microscope, and then the fat deposition area was quantified using image analysis software. Fat Deposition Area: The area of the Oil Red O-stained area in the liver section was calculated using image analysis software; Liver Tissue Area: The total area of the liver tissue area measured by image analysis software. Fat Deposition Ratio: The ratio of the fat deposition area to the total liver tissue area, expressed as a percentage, see 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 effect of the selenium chelate group in reducing fat deposition was significantly better than that of the fusion protein group and the positive control group.

[0050] Detection of atherosclerotic plaque area: At the end of the 8th week of the experiment, arterial specimens of each group of rabbits were taken and fixed, and then stained with Oil Red O to clearly show the plaque area. Then, a microscope was used to observe and take high-definition images, and quantitative analysis was performed 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, in order to improve the accuracy of the data, attention should be paid to the consistency of sample processing and the uniformity of the staining effect during the detection process. Figure 7 .

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

[0052] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection 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; the Adi-Apo fusion protein is selected from the polypeptide shown in SEQ ID NO: 3; and the inorganic selenium is selected from sodium selenite (Na2SeO3).

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

3. Use of the selenium-rich polypeptide according to any one of claims 1-2 in the preparation of a drug for treating metabolic-related fatty liver disease and coronary atherosclerosis.

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