Application of dermatan sulfate in promotion of cholesterol excretion

Abstract

The invention belongs to the technical field of dermatan sulfate application, and particularly relates to application of dermatan sulfate in promotion of cholesterol excretion. The cholesterol excretion promoting effect of dermatan sulfate is discussed through in-vivo and in-vitro experiments, and results show that dermatan sulfate has the effect of promoting cholesterol excretion of liver cells, not only can reduce blood fat and liver lipid and promote excretion of cholesterol to excrement, but also can inhibit expression of ASGR1, further up-regulates LXR alpha, and has the effect of promoting cholesterol excretion of liver cells. The expression of ABCA1 and ABCG5 transporters is improved to promote cholesterol excretion and reduce the in-vivo lipid level. Therefore, dermatan sulfate is a potential medicine for promoting cholesterol excretion, a new thought is provided for development of a targeted ASGR1 medicine for promoting cholesterol excretion, and a theoretical basis is provided for treatment of cardiovascular diseases such as hypercholesterolemia and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of dermatan sulfate application, and particularly relates to the application of dermatan sulfate in promoting cholesterol efflux. Background Art

[0002] Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide, and hypercholesterolemia is one of the leading causes of CVD. Hypercholesterolemia, a form of hyperlipidemia, is a major risk factor for CVD and increases the risk of cardiovascular diseases such as heart attack and stroke. Cholesterol homeostasis is achieved through a complex interplay between intestinal cholesterol absorption, plasma lipoprotein uptake, de novo biosynthesis, and cholesterol catabolism and excretion. Therefore, the development of cholesterol-lowering drugs has become a critical issue.

[0003] There are three common classes of lipid-lowering drugs used clinically. Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), lowering plasma cholesterol by reducing cholesterol biosynthesis and increasing low-density lipoprotein (LDL) uptake by upregulating the low-density lipoprotein receptor (LDLR). Ezetimibe is an intestinal cholesterol absorption inhibitor that blocks cholesterol uptake by inhibiting the endocytosis of Niemann–Pick C1 like 1 (NPC1L1). PCSK9 inhibitors increase hepatic LDL uptake by stabilizing the LDLR. However, currently, no drug lowers cholesterol by directly promoting cholesterol metabolism or excretion. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of dermatan sulfate in promoting cholesterol efflux, which has good effect and is green and safe.

[0005] The present invention provides application of dermatan sulfate in preparing a drug for promoting cholesterol efflux.

[0006] Preferably, the cholesterol efflux comprises: One or more of lowering blood lipid levels, reducing liver fat accumulation, increasing fecal lipid excretion, and regulating liver ASGR1 protein expression.

[0007] Preferably, the effective concentration of dermatan sulfate is 10-100 μM.

[0008] Preferably, the effective concentration of dermatan sulfate is 20 μM.

[0009] The present invention provides a drug for promoting cholesterol efflux, which comprises dermatan sulfate.

[0010] Beneficial effects: The present invention provides the use of dermatan sulfate in the preparation of drugs that promote cholesterol efflux. The present invention studies the effect of dermatan sulfate on cholesterol efflux from liver cells by constructing a cholesterol efflux cell model, studies the effect of dermatan sulfate on serum, liver and fecal lipids by establishing a hypercholesterolemia mouse model, and studies the cholesterol-lowering mechanism of dermatan sulfate through western-blot and qPCR experiments. The results show that dermatan sulfate has the effect of promoting cholesterol efflux from liver cells, which can reduce blood lipids and liver lipids and promote cholesterol excretion into feces; at the same time, dermatan sulfate can inhibit ASGR1, thereby upregulating LXRα, increasing the expression of ABCA1 and ABCG5 transporters to promote cholesterol efflux and reduce lipid levels in the body. Therefore, dermatan sulfate is a potential drug that promotes cholesterol efflux. The present invention provides new ideas for the development of drugs that target ASGR1 to promote cholesterol efflux, and provides a theoretical basis for the treatment of cardiovascular diseases caused by hypercholesterolemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings and tables required for use in the embodiments.

[0012] Figure 1 The effect of the DS provided by the present invention on cholesterol efflux from Huh-7 cells; Figure 2 Effects of the DS provided by the present invention on cholesterol efflux in ASGR1-overexpressing HEK 293T cells; Figure 3 The effect of the DS provided by the present invention on the expression of ASGR1 signaling pathway proteins; Figure 4 The antagonistic effect of the AMPK and LXRα inhibitors provided by the present invention on DS-induced cholesterol efflux; Figure 5 The DS provided by the present invention accelerates the degradation of ASGR1 protein and its binding ability with ASGR1 protein; Figure 6 To verify the high-fat mouse model provided by the present invention; Figure 7 The effects of the DS provided by the present invention on the food intake and body weight of mice during the entire administration cycle; Figure 8 The effects of the DS provided by the present invention on subcutaneous and visceral fat in mice; Figure 9 The effect of the DS provided by the present invention on the blood lipid level of mice; Figure 10 The effects of the DS provided by the present invention on mouse liver morphology (H&E) and liver lipids; Figure 11 The effect of the DS provided by the present invention on the lipid level in mouse feces; Figure 12 Mouse jejunum sections (H&E staining) provided by the present invention; Figure 13 The effect of the DS provided by the present invention on the expression levels of ASGR1 protein and its downstream signaling pathway proteins in mouse liver; Figure 14 The present invention provides an effect of the DS on the mRNA levels of ASGR1 and its downstream pathway genes in mouse liver. DETAILED DESCRIPTION

[0013] In the present invention, unless otherwise specified, the equipment, raw materials and methods used are conventional.

[0014] The dermatan sulfate described in the present invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a product number of D303943 and a CAS number of 54328-33-5.

[0015] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] The experimental methods and raw materials are described as follows: Experimental cells: The human hepatocellular carcinoma cell line Huh-7 (catalog number: TCHu182) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in complete medium containing 10% high-quality fetal bovine serum (FBS) and 90% DMEM.

[0017] HEK-293T cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in complete medium containing 10% high-quality fetal bovine serum (FBS) and 90% DMEM.

[0018] Experimental animals: Male C57BL / 6J mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., housed in cages of five. They were maintained in a room with a temperature of 20–24°C and a humidity of 50%. The animal room was equipped with a KG316TA timer to maintain a 12 / 12 h light / dark cycle, and mice had free access to food and water. The protocol adhered to the guidelines and regulations of the Laboratory Animal Care and Use Committee of Qingdao University and the ethical standards of international associations.

[0019] Statistical processing All data are expressed as mean ± standard deviation (SD). t The data were statistically analyzed using 1-way ANOVA, 2-way ANOVA, and other methods. n represents different batches of cells or different mice. p When the p-value is less than 0.05, there is a statistically significant difference.

[0020] Example 1 The cholesterol efflux experiment process is as follows: (1) Inoculation of cells: Huh-7 cells in the logarithmic growth phase were taken and counted, and the number of cells was 0.2×10 6 / mL was inoculated into a 96-well plate and incubated in a cell culture incubator for 24 h.

[0021] (2) Staining and first administration: Discard the culture medium and add 100 μL PBS to each well for washing to remove non-adherent cells and dead cells. The cells were divided into solvent control group, administration group and positive control group. Among them, the solvent control group, also known as the blank control group, was added with the same amount of DMSO as the positive control group and 50 μL Bodipy-Cholesterol (0.0625 mM); Drug-treated groups: dermatan sulfate test drug groups (dermatan sulfate concentrations were 0.3, 1, 3, 10, 30, 50, and 100 µM), with 50 µL of test drug and 50 µL of Bodipy-Cholesterol (0.0625 mM) per well; Positive control group: 50 μL GW3965 (1 μM) and 50 μL Bodipy-Cholesterol (0.0625 mM); The solvent control group, drug-treated group and positive control group were placed in a cell culture incubator and incubated for 24 h.

[0022] (3) Second administration and induction of cholesterol efflux: The culture medium was discarded and 100 μL of PBS was added to each well for washing. Then, 50 μL of DMSO (solvent control group), various concentrations of DS (drug administration group), and GW3965 (positive control group) were added to each well and incubated in a cell culture incubator for 6 h to induce HDL cholesterol efflux.

[0023] (4) Collect 80 μL of extracellular fluid and intracellular fluid into a black 96-well plate to evenly distribute the liquid, and measure the fluorescence intensity using a Flexstation 3 multifunctional microplate reader. Figure 1 (exist Figure 1Figure A shows the effect of DS on Huh-7 cell viability determined by resazurin conversion assay (n=4); Figure B shows the dose-effect curve of DS in promoting cholesterol efflux, and the half effective concentration EC was calculated. 50 =6.725 μM (n=4), all the above data are expressed as mean ± standard deviation, **** p <0.0001, ns indicates no statistical difference (one-way ANOVA) The results of this experiment showed that DS had no effect on the viability of Huh-7 cells within the range of 1-100 μM ( Figure 1 A in the figure); Using GW3965 as a positive control, it can be seen that DS began to exhibit cholesterol efflux activity in Huh-7 cells starting from 10 μM, and the activity was concentration-dependent. The half effective concentration (EC 50 ) is 6.725 µM ( Figure 1 B in ).

[0024] Example 2 Effects of Dermatan Sulfate DS on ASGR1-Overexpressing HEK-293T Cells Dermatan sulfate DS promotes cholesterol efflux in Huh-7 cells. Since ASGR1 is highly expressed in Huh-7 liver cells and plays a regulatory role in cholesterol efflux, an ASGR1-overexpressing HEK-293T cell model was constructed to study the mechanism by which DS promotes cholesterol efflux. The results are shown in Figure 2 (exist Figure 2 Figure A shows the effect of DS on HEK-293T cell viability determined by resazurin conversion assay (n=4); Figure B shows the construction of ASGR1 overexpression HEK293T cell model (n=3); Figure C shows the effect of DS on cholesterol efflux in ASGR1 overexpression HEK-293T cells (n=4); All data are expressed as mean ± SD, * p <0.05,*** p <0.001, **** p <0.0001, ns indicates no statistical difference (one-way ANOVA).

[0025] Combine Figure 2 Figure A shows that the safe dose of DS for HEK-293T cell growth was determined by resazurin conversion assay. The study showed that DS at a concentration of 1-100 μM had no obvious toxic effect on HEK-293T cell growth.

[0026] HEK-293T cells were transfected to overexpress ASGR1, and western-blot experiments were performed to verify whether the modeling was successful. Figure 2 Figure B in .

[0027] Combine Figure 2 The results in Figure B show that the expression level of ASGR1 protein in transfected cells was significantly increased, and the overexpression model was successfully established.

[0028] The effect of safe concentration of DS on the cholesterol efflux capacity of ASGR1 overexpression model was tested. The results are shown in Figure 2 Figure C in .

[0029] Combine Figure 2 As shown in Figure C, compared with the control group, the cholesterol efflux of HEK-293T cells overexpressing ASGR1 was reduced. However, 0.3-100 μM DS could reverse the phenomenon of reduced cholesterol efflux caused by ASGR1 overexpression. The half effective concentration (EC) of DS to promote cholesterol efflux in HEK-293T cells overexpressing ASGR1 was calculated. 50 is 7.309 µM.

[0030] Example 3 Dermatan sulfate promotes cholesterol efflux via the ASGR1-LXRα-ABCA1 / ABCG5 signaling pathway (1) Verification of the regulation of key target proteins of ASGR1 According to EC 50 =7.309 μM, and 20 μM DS was selected for mechanism study. By western-blot experiments, the changes in the expression levels of ASGR1 protein and cholesterol efflux-related proteins (LXRα, ABCA1, and ABCG5) in wild-type and ASGR1-overexpressing HEK-293T cells were determined. The results are shown in Figure 3 (exist Figure 3 Figure A shows the expression level of ASGR1 protein (n=6); Figure B shows the expression level of LXRα protein (n=5); Figure C shows the expression level of ABCA1 protein (n=5); Figure D shows the expression level of ABCG5 protein (n=5); All data are expressed as mean ± SD, * p <0.05,*** p <0.001, **** p <0.0001, ns indicates no statistical difference (one-way ANOVA).

[0031] Combine Figure 3 It can be seen that 20 μM DS can inhibit the expression of ASGR1 in the cell model, upregulate LXRα, and then upregulate the expression of cholesterol transporters ABCA1 and ABCG5.

[0032] (2) The mechanism by which ASGR1 inhibits the regulation of cholesterol efflux through the AMPK-LXRα cascade pathway Inhibition of ASGR1 can activate AMPK. Activated AMPK increases the level of LXRα protein, thereby transcriptionally upregulating the expression of cholesterol transporters ABCA1 and ABCG5, ultimately promoting cholesterol efflux. To verify this pathway, intervention experiments were performed using the specific AMPK inhibitor Compound C (CC) and the LXRα antagonist GSK2033. Huh-7 liver cells were divided into six groups: blank control group (DMSO), AMPK inhibitor CC group (10 µM), LXRα antagonist GSK2033 group (10 µM), DS administration group (20 µM), CC and DS combination group, and GSK2033 and DS combination group. After 24 hours of intervention, cholesterol efflux activity was detected, and the results are shown in Table 1. Figure 4 Figure A (in Figure 4 Figure A shows the antagonistic effect of AMPK and LXRα inhibitors on DS-induced cholesterol efflux. All data are expressed as mean ± standard deviation, n = 4, * p <0.05,** p <0.01 (one-way ANOVA)).

[0033] Combine Figure 4 As shown in Figure A, both CC and GSK2033 significantly antagonized the cholesterol efflux-promoting effect of DS.

[0034] Huh-7 liver cells were divided into six groups: blank control group (DMSO), inhibitor CC group (10 μM), AMPK inhibitor CC group (10 μM), LXRα antagonist GSK2033 group (20 μM), CC combined with DS group, and GSK2033 combined with DS group. After 24 h of treatment, the cells were collected and the expression levels of related proteins were detected by western blotting. The results are shown in Figure 5. Figure 4 BD diagram (in Figure 4 Figure B shows the expression level of LXRα protein (n=6); Figure C shows the expression level of ABCA1 protein (n=5); Figure D shows the expression level of ABCG5 protein (n=5); all data are expressed as mean ± standard deviation, ** p <0.01,*** p <0.001, **** p <0.0001 (one-way ANOVA).

[0035] Combine Figure 4As shown in the BD diagram, the AMPK inhibitor CC and the LXRα antagonist GSK2033 significantly inhibited the upregulation of LXRα, ABCA1, and ABCG5 expression by DS, respectively. This confirms that DS activates the AMPK-LXRα signaling axis by inhibiting ASGR1, which in turn transcriptionally upregulates ABCA1 / ABCG5 expression, ultimately promoting cholesterol efflux. This multi-target synergistic mechanism suggests that DS has the potential to be developed as a cholesterol-lowering therapeutic targeting the ASGR1-LXRα-ABCA1 / G5 pathway.

[0036] Example 4 Dermatan sulfate promotes ASGR1 protein degradation by direct binding To investigate how DS inhibits ASGR1 protein levels, a protein degradation experiment was performed to investigate the changes in the degradation rate of ASGR1 protein before and after DS treatment. Cycloheximide (CHX) can inhibit protein synthesis and is commonly used to study protein degradation. The cells were divided into a CHX (50 µg / mL) group (denoted as Control) and a CHX (50 µg / mL) + DS (20 µM) co-treatment group (denoted as DS). Huh-7 cells were collected at 0 h, 1 h, 3 h, 6 h, 9 h, and 12 h after treatment to investigate the degradation rate of ASGR1 protein. The results are shown in Table 1. Figure 5 (exist Figure 5 Figure A shows the representative band of ASGR1 protein; Figure B shows the quantitative analysis of ASGR1 protein expression (n=4); Figure C shows the surface plasmon resonance results. All data are expressed as mean ± standard deviation, n=4, ** p <0.01,*** p <0.001 (one-way ANOVA)).

[0037] Combine Figure 5 As shown by AB, starting from 6 h, the CHX and DS combined group significantly reduced ASGR1 protein expression compared with the CHX group, suggesting that DS can significantly promote ASGR1 protein degradation and inhibit ASGR1 protein.

[0038] From the structural level analysis, combined Figure 5 Surface plasmon resonance (SPR) binding studies confirmed a high-affinity interaction between DS and the ASGR1 extracellular carbohydrate recognition domain. Real-time binding kinetic data showed rapid binding (binding rate constant Ka = 3.08 × 10 2 M⁻ 1 s -1 ) and slowly dissociated (dissociation rate constant Kd = 0.023 s -1) characteristics, and the equilibrium dissociation constant (KD) was calculated to be 74.1 μM, which is consistent with the characteristics of biologically significant ligand-receptor interactions.

[0039] Example 5 Establishment of a hypercholesterolemia mouse model A high-fat diet was used to induce a hypercholesterolemia mouse model to evaluate the lipid-lowering activity of DS. The model was established by feeding the high-fat diet for 14 weeks. Mice were fasted for 12 hours before lipid testing. Blood was collected from the eye sockets and serum lipid levels were measured using an in vitro enzyme-linked immunosorbent assay (ELISA). The results are shown in the table. Figure 6 (exist Figure 6 Figure A shows the total cholesterol (TC) content in serum; Figure B shows the triglyceride (TG) content in serum; all data are expressed as mean ± SD (n = 8 mice), **** p <0.0001 (Student's t test)).

[0040] Combine Figure 6 The HFD group (high-fat diet) showed significantly higher plasma TC and TG levels than the ND group (normal diet), increasing by 77% and 91%, respectively. These results indicate that the hypercholesterolemia mouse model was successfully established.

[0041] Example 6 Effects of Dermatan Sulfate on Hypercholesterolemia Mouse Model After the hypercholesterolemia mouse model was successfully established, the high-fat diet group mice were randomly divided into a model control group (HFD-Control), a positive drug atorvastatin group (HFD-Atorvastatin, 10 mg / kg), and a DS administration group. The DS administration group was divided into low-dose (HFD-DS 5 mg / kg), medium-dose (HFD-DS 15 mg / kg), and high-dose (HFD-DS 50 mg / kg) groups and administered the corresponding drugs by gavage. The control group was given the same volume of CMC-Na solution for 10 weeks. The body weight and food intake of the mice were recorded 1-2 times a week at fixed times. Results are shown in the table. Figure 7 (exist Figure 7 Figure A shows the food intake of mice in each group after oral gavage; Figure B shows the weight changes of mice in each group, n=12); the changes in organ indexes of mice in each treatment group are shown in Table 1.

[0042] Table 1 Mouse organ index

[0043] Combine Figure 7As shown in Table 1, food intake remained similar among the groups after oral administration. This indicates that DS has no effect on food intake, suggesting that its lipid-lowering effects are achieved by affecting lipid metabolism rather than reducing total lipid intake. While the ND-Control and HFD-Control groups reached a plateau in body weight around 17 weeks, the atorvastatin and DS-treated groups experienced some weight loss starting at 16 weeks (two weeks after oral administration), but this did not differ significantly from the control group, consistent with expected results. Organ indices are important parameters for toxicological evaluation, effectively reflecting changes in the physiological status of specific organs in experimental animals and serving as a crucial basis for assessing the effects of long-term drug exposure on experimental organs. The results show that there were no significant differences in organ indices among the groups after DS administration. This suggests that long-term administration of DS has no significant toxic side effects and does not affect any organs. Combined with changes in body weight and food intake, the DS dosage is within the safe range.

[0044] Example 7 Effects of Dermatan Sulfate on Subcutaneous and Visceral Fat in Hypercholesterolemia Mice The mice were scanned and imaged using in vivo small animal CT imaging technology 10 weeks after administration. The results are shown in Figure 8 Figure A in Figure 8 In Figure A, the first row of images represent Micro-CT abdominal cross-sectional images of mice, where white represents bones or food in the gastrointestinal tract, dark gray represents fat, and light gray represents internal organs; the second row of images in Figure A represent Micro-CT modeling images of mice, where yellow represents subcutaneous fat and green represents visceral fat).

[0045] Depend on Figure 8 As shown in Figure A, subcutaneous and visceral fat content was significantly increased in mice fed a high-fat diet (HFD) compared to the ND-control group, indicating that a high-fat diet can cause subcutaneous and visceral fat accumulation in mice. Administration of dermatan sulfate significantly alleviated this fat accumulation in a dose-dependent manner. Modeling and rendering revealed a significant increase in visceral fat volume in the HFD-control group, as reflected in the abdominal cross-section image, and a corresponding decrease after administration.

[0046] The scan data is used to quantify the fat volume by building a 3D model. Figure 8 BC in (in Figure 8 Figure B shows the relative volume of subcutaneous fat (mm 3 / g); Figure C shows the relative volume of visceral fat (mm 3 / g). ATOR is Atorvastatin. Data are expressed as mean ± standard deviation, n = 3, * p<0.05,** p <0.01, ns indicates no statistical difference (one-way AVOVA).

[0047] Combine Figure 8 As shown in the BC graph, the subcutaneous and visceral fat contents of mice fed a high-fat diet were 2.12 and 4.21 times higher than those of the control diet group, respectively. After administration, compared with the HFD-control group, the subcutaneous and visceral fat contents of mice in the atorvastatin group and the medium- and high-dose DS groups were significantly reduced, with subcutaneous fat decreasing by 48.66%, 46.04%, and 52.62%, respectively, and visceral fat decreasing by 51.19%, 58.83%, and 69.37%, respectively. This indicates that DS can significantly improve the deposition of subcutaneous and visceral fat in mice with high cholesterol.

[0048] Example 8 Effect of Dermatan Sulfate on Serum Lipid Metabolism Profile in Hypercholesterolemia Mice The lipid content in the serum of mice in the above treatment groups was detected using a lipid content determination kit. The results are shown in Figure 9 (exist Figure 9 Panel A shows serum TC levels (mM); Panel B shows serum TG levels (mM); Panel C shows serum HDL-C levels (mM); and Panel D shows serum LDL-C levels (mM). ATOR stands for atorvastatin. Data are expressed as mean ± SD, n = 8. * p <0.05,** p <0.01,*** p <0.001, **** p <0.0001, ns indicates no statistical difference (one-way ANOVA).

[0049] Combine Figure 9 The data showed that the serum TC, TG, HDL-C and LDL-C of mice in the HFD-Control group were 1.23, 1.36, 0.89 and 1.27 times that of the ND-Control group, respectively. After 10 weeks of oral administration of dermatan sulfate, the serum TC of mice in the positive control atorvastatin group decreased by 30.53%, and the serum TC of mice in the dermatan sulfate 50 mg / kg group decreased by 28.94% compared with the HFD-Control group ( Figure 9 Compared with the HFD-Control group, the serum TG of mice in the atorvastatin group decreased by 40.94%, and the serum TG of mice in the dermatan sulfate 5, 15 and 50 mg / kg groups decreased by 15.94%, 18.63% and 30.18%, respectively ( Figure 9Compared with the HFD-Control group, the serum HDL-C level of mice in the dermatan sulfate 50 mg / kg group increased by 53.59% ( Figure 9 Compared with the HFD-Control group, the serum LDL-C level of mice in the atorvastatin group decreased by 36.54%, and the serum LDL-C level of mice in the dermatan sulfate 50 mg / kg group decreased by 24.97% ( Figure 9 D).

[0050] Example 9: Dermatan Sulfate Alleviates Fat Accumulation in the Liver of Hypercholesterolemia Mice The morphology and histology of the livers of mice in the above treatment groups were evaluated, and the results are shown in Figure 10 A (in Figure 10 In Figure A, the first row of pictures shows the representative liver morphology of mice in each group; the second row of pictures shows the representative H&E-stained liver sections of mice in each group).

[0051] Combine Figure 10 As shown in Figure A, the livers of mice in the ND-Control group were reddish-brown with a uniform color distribution, free of blood streaks, and smooth in surface. In contrast, the livers of mice in the HFD-Control group were mildly edematous, soft and easily eroded, and yellowish-brown in color, suggesting a degree of fatty liver lesions in the HFD-Control group. Treatment with atorvastatin and dermatan sulfate effectively alleviated these liver lesions. At a dose of 50 mg / kg, the livers had become distinctly reddish-brown with a uniform color and a smooth surface. H&E staining revealed that the liver cells in the ND-Control group were densely packed, uniform in size, with uniform cytoplasm and clear, centrally located nuclei. Compared with the ND-Control group, the livers of the HFD-Control group showed more disorganized and unevenly sized cells, and prominent cytoplasmic vacuoles, indicating fatty liver degeneration. Treatment with atorvastatin and dermatan sulfate reduced cytoplasmic vacuoles, which gradually decreased with increasing doses, alleviating fatty liver degeneration. The above phenomena indicate that dermatan sulfate can effectively alleviate fatty lesions in hypercholesterolemia mice.

[0052] The lipid content in the liver of mice was measured after 10 weeks of oral gavage treatment. Figure 10 BD ( Figure 10 B represents the liver TC level (mM); Figure 10 C represents liver TG level (mM); Figure 10 D represents the level of total bile acid TBA in the liver (mM). Data are expressed as mean ± standard deviation (n=6-8). *p <0.05, **p <0.01, ***p<0.001, ****p <0.0001, ns indicates no statistical difference (one-way ANOVA).

[0053] The results showed that the liver TC, TG and TBA of mice in the HFD-Control group were 1.60, 3.67 and 1.74 times that of the ND-Control group, respectively. After 10 weeks of oral administration of dermatan sulfate, the liver TC of mice in the positive control atorvastatin group decreased by 29.38%, and the serum TC of mice in the dermatan sulfate 50 mg / kg group decreased by 35.82% compared with the HFD-Control group ( Figure 10 B); Compared with the HFD-Control group, the serum TG of mice in the atorvastatin group decreased by 55.82%, and the serum TG of mice in the sulfated skin 50 mg / kg group decreased by 70.61% ( Figure 10 Compared with the HFD-Control group, the serum TBA level of mice in the atorvastatin group decreased by 15.18%, and the serum TBA level of mice in the dermatan sulfate 50 mg / kg group decreased by 52.51% ( Figure 10 The quantitative results of the liver lipid profile were consistent with the results of liver morphology and H&E staining.

[0054] Example 10 Dermatan sulfate can promote lipid excretion and repair small intestinal villi damage in mice fed a high-fat diet Ten weeks after oral administration, fresh feces of mice in each treatment group were tested using a lipid determination kit. The results are shown in Table 2. Figure 11 (exist Figure 11 Figure A shows the level of TC in feces (mM); Figure B shows the level of TG in feces (mM). Data are expressed as mean ± standard deviation, n = 8, * p <0.05,** p <0.01,*** p <0.001, **** p <0.0001, ns indicates no statistical difference (one-way ANOVA).

[0055] Combine Figure 11 As can be seen, the TC and TG levels in the HFD group were 5.98-fold and 1.71-fold higher than those in the ND group, respectively. Compared with the HFD-Control group, TC excretion increased by 49.82%, 49.56%, and 59.14% in the atorvastatin, 15 mg / kg, and 50 mg / kg dermatan sulfate groups, respectively. TG levels increased by 21.89% and 30.51% in the atorvastatin and 50 mg / kg dermatan sulfate groups, respectively. This suggests that DS can produce a long-lasting and stable lipid-lowering effect by promoting fecal lipid excretion.

[0056] Based on the positive effect of DS on cholesterol excretion, H&E staining sections were performed on the jejunum after DS treatment in each treatment group. Figure 12 .

[0057] Combine Figure 12 Dermatan sulfate repaired intestinal villi damage in mice fed a high-fat diet. Specifically, the villi in the jejunum of mice fed a standard diet were neatly arranged, structurally intact, and relatively slender. In the high-fat diet control group, the villi shortened and thickened, with necrotic ends and flabby hypertrophy. Treatment with atorvastatin and DS ameliorated the high-fat diet-induced villi damage. In the 50mg / kg DS group, the villi regained their slender shape, with damage to the villi ends repaired and the villi becoming tightly packed.

[0058] Example 11 Dermatan Sulfate Regulates the Liver ASGR1-LXRα-ABCA1 / ABCG5 Signaling Pathway Western-blot experiments were used to study the effect of dermatan sulfate on the expression of various proteins in the ASGR1 pathway. The results are shown in Figure 13 (exist Figure 13 Figure A shows the protein representative bands; Figure B shows the quantitative expression level of ASGR1 protein; Figure C shows the quantitative expression level of LXRα protein; Figure D shows the expression level of ABCA1 protein; Figure E shows the quantitative expression level of ABCG5 protein. All data are expressed as mean ± standard deviation, n = 6, * p <0.05,** p <0.01,*** p <0.001, **** p <0.0001, ns indicates no statistical difference (one-way ANOVA).

[0059] Combine Figure 13 The results showed that the 50 mg / kg dose of dermatan sulfate could reduce the expression of ASGR1 protein in the mouse liver by 53.73%, and significantly upregulated the levels of LXRα, ABCA1 and ABCG5 proteins.

[0060] Quantitative PCR was used to study the effect of dermatan sulfate on the transcription of downstream target genes in the ASGR1 pathway. Figure 14 (exist Figure 14 Figure A shows the mRNA level of ASGR1; Figure B shows the mRNA level of LXRα; Figure C shows the mRNA level of ABCA1; Figure D shows the mRNA level of ABCG5. All data are expressed as mean ± standard deviation, n = 6, * p <0.05,** p <0.01,**p <0.01, **** p <0.0001, ns indicates no statistical difference (one-way ANOVA).

[0061] Combine Figure 14 Results showed that quantitative PCR analysis revealed no changes in ASGR1 mRNA levels, confirming that dermatan sulfate's inhibitory effect on ASGR1 occurs at the post-translational level. Following ASGR1 protein inhibition by dermatan sulfate, LXRα mRNA levels increased, thereby enhancing transcriptional activation of the cholesterol transporters ABCA1 and ABCG5 at the mRNA level. This suggests that dermatan sulfate regulates lipid levels at multiple levels in mouse liver via the ASGR1-LXRα-ABCA1 / ABCG5 signaling pathway.

[0062] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of dermatan sulfate in the preparation of drugs that promote cholesterol efflux.

2. The use according to claim 1, characterized in that The cholesterol efflux includes: One or more of lowering blood lipid levels, reducing liver fat accumulation, increasing fecal lipid excretion and regulating liver ASGR1 protein expression.

3. The use according to claim 1, characterized in that The effective concentration of dermatan sulfate is 10-100 μM.

4. The use according to claim 3, characterized in that The effective concentration of dermatan sulfate is 20 µM.

5. A drug for promoting cholesterol efflux, characterized in that: The drug includes dermatan sulfate.

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