Application of triple functional components in birch juice in relieving hyperuricemia

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of triple active ingredients in birch juice in relieving hyperuricemia. According to the invention, firstly, the influence of birch juice on the weight, blood uric acid, blood urea nitrogen, serum creatinine and visceral organ coefficient of a hyperuricemia mouse is evaluated through animal experiments; in order to reveal the potential mechanism of the active ingredients of the birch juice for relieving the hyperuricemia, the invention further explores the ability of three main active ingredients of betulin, betulinic acid and kaempferol in the birch juice for regulating and controlling the high-grade conformation of nucleic acid in a hyperuricemia-related gene promoter region; the molecular mechanism of the triple active components in the birch juice for relieving hyperuricemia is speculated and explained from the gene level. In conclusion, the invention provides a brand-new theoretical basis and mechanism elaboration of a gene level for relieving metabolic diseases by natural functional small molecules.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of three functional components in birch sap in alleviating hyperuricemia. Background Art

[0002] Birch sap is a liquid directly collected from the trunk of Betula platyphylla, and is known as the "plant mineral water". According to research and determination, birch sap contains more than 60 kinds of nutrients required by the human body, such as various vitamins, minerals, amino acids, fatty acids, etc., which helps to promote human metabolism, and thus has been favored by more and more consumers in recent years. In addition, the natural, mild characteristics and anti-inflammatory and bactericidal effects of birch sap have been developed for use in health products, pharmaceuticals, cosmetics and other fields.

[0003] The three main active components of birch sap, betulinol, betulinic acid and kaempferol, have natural anti-inflammatory, analgesic and urate-dissolving effects, which help to relieve the symptoms of hyperuricemia and promote the metabolism and excretion of uric acid. Research has confirmed that the three active components play an important role in the expression regulation of the key pathogenic genes of hyperuricemia, Uric Acid Transporter 1 (URAT1) and glucose transporter 9 (GLUT9). URAT1 is mainly responsible for the reabsorption of uric acid, while GLUT9 is involved in the transport of uric acid. When both are overexpressed, it will lead to the accumulation of uric acid in the body, thus causing hyperuricemia. However, the active components of birch sap may regulate gene expression through different pathways: on the one hand, the active components may directly act on the gene regulatory region and inhibit its transcription and translation processes; on the other hand, it may indirectly regulate gene expression by affecting the intracellular signal transduction pathway. Therefore, further clarifying the gene expression regulation pathway of the active components helps to relieve the symptoms of hyperuricemia patients and reduce the frequency of symptom attacks.

[0004] The present invention first evaluated the effects of birch sap on the body weight, blood uric acid, blood urea nitrogen, serum creatinine and organ coefficients of hyperuricemic mice through animal experiments. In order to reveal the potential mechanism of the active components of birch sap in alleviating hyperuricemia, the present invention respectively selected the promoter sequences of URAT1 and GLUT9 genes with the potential to form G-quadruplexes, and further explored the ability of the three main active components in birch sap, betulinol, betulinic acid and kaempferol, to regulate the nucleic acid higher-order conformation of the promoter regions of hyperuricemia-related genes, and speculated and explained the molecular mechanism of the three active components in birch sap in alleviating hyperuricemia at the gene level. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention aims to propose an application of the functional components of birch sap in alleviating hyperuricemia.

[0006] To achieve the above object, on the one hand, the present invention provides the application of the DNA sequence shown in any one of SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.9 as a binding target on the URAT1 promoter in the regulation of URAT1 gene expression by betulin, betulinic acid, and kaempferol.

[0007] On the other hand, the present invention provides the application of the DNA sequence shown in any one of SEQ ID NO.14 or SEQ ID NO.15 as a binding target on the GLUT9 promoter in the regulation of GLUT9 gene expression by betulin and betulinic acid.

[0008] On the other hand, the present invention provides the application of the DNA sequence shown in SEQ ID NO.4 as a binding target of betulin in the regulation of URAT1 gene expression by betulin.

[0009] On the other hand, the present invention provides the application of the DNA sequence shown in SEQ ID NO.15 as a binding target of betulin in the regulation of GLUT9 gene expression by betulin.

[0010] On the other hand, the present invention provides the application of the DNA sequence shown in SEQ ID NO.9 as a binding target of betulinic acid in the regulation of URAT1 gene expression by betulinic acid.

[0011] On the other hand, the present invention provides the application of the DNA sequence shown in SEQ ID NO.14 as a binding target of betulinic acid in the regulation of GLUT9 gene expression by betulinic acid.

[0012] On the other hand, the present invention provides the application of the DNA sequence shown in SEQ ID NO.2 as a binding target of kaempferol in the regulation of URAT1 gene expression by kaempferol.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. The present invention discloses for the first time multiple G-quadruplex-forming potential sequences located in the promoter regions of hyperuricemia-related genes.

[0015] 2. The present invention firstly proposes that three active ingredients in birch sap, namely betulin, betulinic acid, and kaempferol, can regulate gene expression by acting on the G-quadruplex-forming potential sequences in the promoter regions of URAT1 or GLUT9.

[0016] 3. The sequences with G-quadruplex-forming potential in the promoter regions of URAT1 and GLUT9 proposed by the present invention can be used as potential regulatory targets for functional natural small molecules, thereby regulating the expression of disease-related genes, and having great application prospects in the field of screening food-grade natural products and corresponding small molecule drugs.

[0017] 4. The present invention evaluated the effects of orally administered birch sap on the body weight, blood uric acid, blood urea nitrogen, serum creatinine and organ coefficients of hyperuricemic mice, and demonstrated the alleviating effect of orally administered birch sap on hyperuricemia at the animal level, providing new ideas for the treatment of metabolic diseases and drug development. Description of the Drawings

[0018] Figure 1 Shows the changes in body weight of mice in each group (Note: ****, ***, ** represent P < 0.0001, P < 0.001, P < 0.01 respectively).

[0019] Figure 2 Shows the body weight gain of mice in each group (Note: *, ns represent P < 0.05, P > 0.05 respectively).

[0020] Figure 3 Shows the effects of each experimental group on the blood uric acid of hyperuricemic mice (Note: ****, **, ns represent P < 0.0001, P < 0.01, P > 0.05 respectively).

[0021] Figure 4 Shows the effects of each experimental group on the blood urea nitrogen and serum creatinine of hyperuricemic mice (Note: ****, ***, **, *, ns represent P < 0.0001, P < 0.001, P < 0.01, P < 0.05, P > 0.05 respectively). A shows the effect on blood urea nitrogen; B shows the effect on serum creatinine.

[0022] Figure 5 Shows the effects of each experimental group on the organ coefficients of hyperuricemic mice (Note: ns represents P > 0.05). A shows the effect on the kidney; B shows the effect on the liver; C shows the effect on epididymal fat.

[0023] Figure 6 Shows the changes in the fluorescence intensity of the binding of betulinol to the sequences. A shows the changes in the fluorescence intensity of the binding of betulinol to URAT1 1263 (SEQ ID NO.4); B shows the changes in the fluorescence intensity of the binding of betulinol to GLUT9 1625 (SEQ ID NO.15).

[0024] Figure 7 Shows the changes in the fluorescence intensity of the binding of betulinic acid to the sequences. A shows the changes in the fluorescence intensity of the binding of betulinic acid to URAT1 1497 (SEQ ID NO.9); B shows the changes in the fluorescence intensity of the binding of betulinic acid to GLUT9 972 (SEQ ID NO.14).

[0025] Figure 8 Shows the changes in the fluorescence intensity of the binding of kaempferol to the sequence URAT1 1221 (SEQ ID NO.2).

[0026] Figure 9 The circular dichroism spectrum of URAT1 1263 (SEQ ID NO.4) bound to betulinol.

[0027] Figure 10 The circular dichroism spectrum of GLUT9 1625 (SEQ ID NO.15) bound to betulinol.

[0028] Figure 11 The circular dichroism spectrum of URAT1 1497 (SEQ ID NO.9) bound to betulinic acid.

[0029] Figure 12 The circular dichroism spectrum of GLUT9 972 (SEQ ID NO.14) bound to betulinic acid.

[0030] Figure 13 The circular dichroism spectrum of URAT1 1221 (SEQ ID NO.2) bound to kaempferol. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0032] Example 1. Birch sap relieves hyperuricemia in mice

[0033] Male Kunming mice (25 ± 2.0 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Breeding conditions: temperature 22 ± 2 °C, humidity 50 ± 10%, with a continuous 12 h / 12 h light-dark cycle. Before the experiment, the mice were first acclimated to free drinking water and food for 1 week. This protocol was approved by the Experimental Animal Welfare and Animal Experiment Ethics Review Committee of China Agricultural University. All animals were humanely treated in accordance with the "Guide for the Care and Use of Laboratory Animals" of the Ministry of Science and Technology of the People's Republic of China, and the animal protocol was approved by the Ethics Committee of China Agricultural University.

[0034] The mice were randomly divided into 4 groups (7 mice in each group) and were fed adaptively for 7 days before the experiment.

[0035] The experimental method is as follows:

[0036] (1) Mice in the normal control group were gavaged with 0.5 mL of normal saline for 7 consecutive days, and gavaged with 0.5% carboxymethyl cellulose sodium (CMC-Na) from day 8 to day 21.

[0037] (2) The mice in the model group were intragastrically administered with 0.5 mL of normal saline continuously for 7 days, and potassium oxonate (PO) (250 mg / kg) was given from the 8th to the 21st day.

[0038] (3) The mice in the positive control group were intraperitoneally injected with allopurinol 5 mg / kg every day for 7 days, and from the 8th day to the end, PO (250 mg / kg) and allopurinol 5 mg / kg were given at an interval of one hour.

[0039] (4) The mice in the birch sap group were intragastrically administered with birch sap 10 mg / kg continuously for 7 days, and from the 8th day to the end, PO (250 mg / kg) and allopurinol 5 mg / kg were given at an interval of one hour.

[0040] On the last day, food was withheld 12 hours before drug administration, and water was available ad libitum. All drug administrations were based on the body weight measured before each administration. One hour after the last drug administration, the mice were anesthetized and sacrificed, and peripheral blood of the ophthalmic vein was collected. The blood coagulated at room temperature for about 1 hour and was centrifuged at 3000 rpm for 10 minutes to obtain serum. The serum samples were stored at -20 °C. The levels of serum uric acid (SUA), creatinine (Cr), and blood urea nitrogen (BUN) were measured by the phosphotungstic acid method. The liver was excised, rinsed with ice-cold saline, and liver tissue sections were stored at -80 °C.

[0041] During the whole experiment, the mice in each group were in a relatively stable state, without showing listlessness, decreased activity, fighting, etc., and there were no deaths. Figure 1 and Figure 2 It can be seen that the weight gain rate of the model group was faster than that of the blank control group. Both the birch sap group and the positive control group could reduce the effect of potassium oxonate on body weight, and there was no significant difference in body weight between the birch sap group and the blank control group. Compared with the first weighing, at the last weighing, the body weight of the model group was significantly increased compared with the blank group (P < 0.0001); the positive control group had an inhibitory effect on weight gain compared with the model group but not significantly, and the birch sap treatment group significantly inhibited the increase in body weight of mice compared with the model group (P < 0.001). This indicates that birch sap can relieve the increase in body weight of mice caused by hyperuricemia to a certain extent.

[0042] The serum uric acid (SUA) content of hyperuricemic mice in different treatment groups was further measured. Figure 3It can be seen that, compared with the blank control group, the SUA in the model group was significantly increased (P<0.0001), indicating successful modeling. The birch sap group significantly decreased the SUA content in mice compared with the model group (P<0.01), and there was no significant difference in its SUA value compared with the blank group (P>0.05). The positive control group had a more significant effect on reducing SUA compared with the model group (P<0.0001), and also significantly reduced SUA compared with the blank group (P<0.0001). The above experimental results prove that oral administration of birch sap can effectively reduce the SUA content in hyperuricemic mice.

[0043] Furthermore, the contents of serum urea nitrogen (SUN) and serum creatinine (SCr) in hyperuricemic mice in different treatment groups were explored ( Figure 4 ). The SUN in the model group was increased compared with the blank group, but the difference was not statistically significant (P>0.05). There was a downward trend in SUN in the positive control group and the birch sap group compared with the model group, but the difference in the positive control group compared with the model group was not statistically significant (P>0.05), and the difference in the birch sap group compared with the model group was significant (P<0.05). Both the birch sap group and the positive control group significantly reduced the SUN in hyperuricemic mice compared with the blank control group (P<0.05)( Figure 4 A). For the SCr content, the SCr in the model group was significantly increased in mice compared with the blank group (P<0.0001). The positive control group significantly reduced the SCr compared with the model group and the blank group (P<0.0001 and P<0.001). The birch sap group also significantly reduced the SCr compared with the model group and the blank group (P<0.001 and P<0.01)( Figure 4 B). This indicates that birch sap can alleviate the increase in SUN and SCr contents caused by hyperuricemia to a certain extent.

[0044] In addition, as Figure 5 can be seen, there was no significant difference in the kidney, liver and epididymal fat indexes among the groups (P>0.05).

[0045] Example 2. Mining of G-quadruplex-forming potential sequences in the promoter regions of URAT1 and GLUT9

[0046] URAT1 is a recombinant protein on the cell membrane and belongs to a subfamily of the organic anion transporter (OAT) family. It plays an important role in regulating uric acid. The functional structure of the URAT1 protein is quite complex, and it plays a very important role in uric acid metabolism.

[0047] GLUT9 is a glucose facilitative transporter present on the basolateral membrane of proximal tubule epithelial cells and is a novel high-capacity, low-affinity uric acid transporter, which plays an important role in maintaining the uric acid level in the body.

[0048] Intervention and regulation of the promoter region can affect the expression of downstream related genes. Therefore, to study whether there are G-quadruplex elements in the promoter regions of hyperuricemia-related genes URAT1 and GLUT9, the G-quadruplex prediction website G4 Hunter (https: / / bioinformatics.ibp.cz / # / analyse / quadruplex) was used to predict and analyze the G-quadruplex structures of the promoter sequences before the transcription start sites (TSS) of URAT1 and GLUT9, and 15 nucleic acid sequences with scores greater than 0.9 were obtained, as shown in SEQ ID NO.1 - 15, indicating that these 15 sequences may have the potential to form G-quadruplex higher-order conformations (Table 1).

[0049] Table 1 Summary of nucleic acid sequences

[0050]

[0051]

[0052] Example 3. Verification of the interaction between nucleic acid sequences and the active ingredients of birch sap by fluorescence experiment

[0053] Betulinol and betulinic acid are the signature triterpenoid compounds in birch sap, which have anti-inflammatory, analgesic, and urate-dissolving effects, and help reduce the deposition of urate in joints and relieve joint pain and inflammation caused by hyperuricemia.

[0054] Kaempferol is a flavonoid compound with a relatively high content in birch sap and has good anti-inflammatory activity. By activating and inhibiting the NLRP3 inflammasome and inhibiting the expression of inflammatory factors, etc., it effectively regulates and reduces the inflammatory response and tissue damage caused by hyperuricemia.

[0055] The study used fluorescence allosteric analysis to verify the conformational regulation ability of the three active ingredients in birch sap, betulinol, betulinic acid, and kaempferol, respectively, on the folded G-quadruplex of 15 nucleic acid sequences (SEQ ID NO.1 - 15). This method first induces the nucleic acid sequences to fold into G-quadruplex structures and enhances the fluorescence signal by binding to thioflavin T (ThT); if the active ingredient is then added and the fluorescence signal decreases, it indicates that the active ingredient has interacted with the G-quadruplex, affecting its structural stability and leading to the disintegration of the structure, and has the potential to regulate gene expression.

[0056] The specific experimental operations are as follows: Centrifuge the EP tube containing the freeze-dried nucleic acid sequence at 10,000 - 12,000 r / min for 10 - 15 min, add ultrapure water to dilute it into a nucleic acid mother solution with a concentration of 100 μmol / L and mix well. Subsequently, heat it in a PCR instrument at 95 °C for 10 min for denaturation, and then cool it down to 4 °C in a gradient for standby. Dilute the nucleic acid sequence to 1 μmol / L with 20 mmol / L Tris-K + buffer solution (pH 7.4, containing 30 mmol / L KCl), and mix it well with 5 μmol / L ThT solution. Subsequently, add 2 μmol / L active substance (betulin, betulinic acid, and kaempferol) solutions respectively, vortex and mix well. After incubating at room temperature for 5 min, collect the fluorescence signal using a fluorescence microplate reader at an excitation wavelength of 442 nm and an emission wavelength of 490 nm. In the control sample, the active substance solution is replaced with the same volume of Tris-K + buffer solution.

[0057] As Figures 6 - 8 shown, after betulin binds to the G-quadruplexes formed by URAT1 1263 (SEQ ID NO.4) and GLUT9 1625 (SEQ ID NO.15) respectively, the ThT fluorescence intensity decreases by 6.75% and 12.45%. After betulinic acid binds to the G-quadruplexes formed by URAT1 1497 (SEQ ID NO.9) and GLUT9 972 (SEQ ID NO.14) respectively, the ThT fluorescence significantly decreases by 8.36% and 23.18%. After kaempferol binds to the G-quadruplex formed by URAT1 1221 (SEQ ID NO.2), the ThT fluorescence decreases by 3.13%. It indicates that betulin, betulinic acid, and kaempferol can interact with the G-quadruplexes formed by the corresponding nucleic acid sequences respectively, induce conformational changes, release some embedded ThT, and lead to a decrease in the fluorescence intensity of the system.

[0058] In summary, through the fluorescence allosteric analysis method, it is verified that the selected nucleic acid sequence has the potential to form G-quadruplexes. At the same time, the three main active substances in birch sap, betulin, betulinic acid, and kaempferol, have the ability to regulate the higher-order conformation of specific sequences in the promoter region of key pathogenic genes for hyperuricemia.

[0059] Example 4. Verification of the interaction between nucleic acid sequences and the active components of birch sap using circular dichroism spectroscopy

[0060] Furthermore, use circular dichroism spectroscopy to verify the ability of the three active components in birch sap, betulin, betulinic acid, and kaempferol, to interact with the above-mentioned specific nucleic acid sequences respectively.

[0061] The specific operations are as follows: 20 μmol / L betulin, betulinic acid, and kaempferol were respectively mixed with 10 μmol / L specific nucleic acid sequences in a pH 7.4 Tris-HCL buffer solution to prepare a test solution with a total volume of 200 μL. Using a JASCO J-1500 circular dichroism spectrometer, at 20 °C, scanning was performed at a rate of 1 nm / s, and the scanning range was 220 - 320 nm. Before sample determination, the background signal of the pH 7.4 Tris-HCL buffer solution was scanned separately and automatically subtracted during subsequent sample determination.

[0062] It can be seen from Figures 9 - 13 that the nucleic acid sequences all showed negative peaks around 240 nm and positive peaks around 260 nm, which are typical circular dichroism peaks of G-quadruplexes, indicating that the alternative sequences all have G-quadruplex structures.

[0063] Figures 9 - 11 In , the bindings of betulin to URAT1 1263 (SEQ ID NO.4) and GLUT9 1625 (SEQ ID NO.15), and the binding of betulinic acid to URAT1 1497 (SEQ ID NO.9) all showed an increase in ellipticity. On the contrary, Figures 12 - 13 in , the bindings of betulinic acid to GLUT9 972 (SEQ ID NO.14) and kaempferol to URAT1 1221 (SEQ ID NO.2) both showed a significant decrease in ellipticity. This is because the intensity of the circular dichroism characteristic peak is related to the number of DNA helical bases. Betulin and betulinic acid are embedded in the higher-order conformations formed by the folding of the corresponding nucleic acid sequences, changing the number of bases in the initially formed conformations, thereby causing changes in the intensity of the circular dichroism characteristic peaks, resulting in a downward or upward shift of the negative peak at 245 nm and the positive peak at 265 nm. In addition, Figures 10 - 11 in , after the bindings of betulin to GLUT9 1625 (SEQ ID NO.15) and betulinic acid to URAT1 1497 (SEQ ID NO.9), a slight red shift of some characteristic peaks occurred, indicating that the active molecules had a certain impact on the order and compactness of the G-quadruplex structures formed by the nucleic acid sequences.

[0064] In summary, based on the changes in the positions and intensities of the characteristic peaks of circular dichroism, it is shown that the three active components in birch sap, betulin, betulinic acid, and kaempferol, cause changes in the higher-order conformations of nucleic acids through interactions with specific nucleic acid sequences.

[0065] In summary, combining the results of fluorescence experiments and circular dichroism experiments, it was revealed that the three active components in birch sap, betulin, betulinic acid, and kaempferol, all interacted with specific nucleic acid sequences in the promoter region of the key pathogenic genes for hyperuricemia. By causing changes in the higher-order nucleic acid conformations such as G-quadruplexes, the expression of genes GLUT9 and URAT1 was regulated, thus affecting the reabsorption of uric acid to a certain extent, achieving the effect of reducing serum uric acid levels and decreasing the risk of developing hyperuricemia.

Claims

1. Use of the DNA sequence shown in any one of SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.9 as a binding target on the URAT1 promoter in the regulation of URAT1 gene expression by betulinol, betulinic acid, or kaempferol.

2. Use of the DNA sequence shown in any one of SEQ ID NO.14 or SEQ ID NO.15 as a binding target on the GLUT9 promoter in the regulation of GLUT9 gene expression by betulinol or betulinic acid.

3. Use of the DNA sequence shown in SEQ ID NO.4 as a binding target of betulinol in the regulation of URAT1 gene expression by betulinol.

4. Use of the DNA sequence shown in SEQ ID NO.15 as a binding target of betulinol in the regulation of GLUT9 gene expression by betulinol.

5. Use of the DNA sequence shown in SEQ ID NO.9 as a binding target of betulinic acid in the regulation of URAT1 gene expression by betulinic acid.

6. Use of the DNA sequence shown in SEQ ID NO.14 as a binding target of betulinic acid in the regulation of GLUT9 gene expression by betulinic acid.

7. Use of the DNA sequence shown in SEQ ID NO.2 as a binding target of kaempferol in the regulation of URAT1 gene expression by kaempferol.

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