Application of 9-cis-retinoic acid in preparation of products for treating, preventing or improving obesity

Abstract

The invention relates to application of 9-cis-retinoic acid in preparation of products for treating, preventing or improving obesity, and belongs to the technical field of biological medicine. Experimental evaluation of obese mice induced by high-fat diet proves that the 9-cis-retinoic acid can reduce blood fat and improve the glycometabolism function, and lipid accumulation of the liver is improved mainly by increasing expression of main regulatory protein PPAR-alpha oxidized by liver beta, so that the 9-cis-retinoic acid can be used for preparing medicines for treating obesity. The traditional Chinese medicine composition can be used for treating, preventing or improving metabolic symptoms caused by high fat diet, and a new strategy is provided for treating obesity and related metabolic syndromes. Besides, 9-cis-retinoic acid is a natural active metabolite of vitamin A, does not show obvious toxic reaction in animal experiments, and has better biocompatibility.

Description

Technical Field

[0001] The present invention relates to the use of 9-cis retinoic acid in the preparation of products for treating, preventing or improving obesity, and belongs to the technical field of biomedicine. Background Art

[0002] A long-term high-fat diet is likely to lead to obesity, which in turn causes metabolic disorders, including insulin resistance, lipid metabolism disorders, oxidative stress, inflammatory responses, and gut microbiota dysbiosis, etc. Excessive fat will accumulate in the liver, leading to obesity and metabolic syndrome (Lian C Y, Zhai Z Z, Li Z F, et al. High fat diet-triggered non-alcoholic fatty liver disease: A review of proposed mechanisms[J]. Chemico-biological interactions, 2020, 330:109199.). Hepatic steatosis is generally considered a benign disease, but it can progress to non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and even more severe liver diseases such as cirrhosis and liver cancer (Ferramosca A, Di Giacomo M, Zara V. Antioxidant dietary approach in treatment of fatty liver: New insights and updates[J]. World journal of gastroenterology, 2017, 23(23):4146; Friedman S L, Neuschwander-Tetri B A, Rinella M, et al. Mechanisms of NAFLD development and therapeutic strategies[J]. Nature medicine, 2018, 24(7):908-922; Armstrong M J, Adams L A, Canbay A, et al. Extrahepatic complications of nonalcoholic fatty liver disease[J]. Hepatology, 2014, 59(3):1174-1197.). The pathogenesis of obesity caused by a high-fat diet is complex and involves multiple factors. Among them, insulin resistance is the key driving factor, which leads to a large amount of free fatty acids entering the liver by enhancing lipolysis, triggering fatty deposition in hepatocytes (Petersen M C, Shulman G I. Mechanisms of insulin action and insulin resistance[J]. Physiological reviews, 2018.).The treatment goals for obesity caused by a high-fat diet include inducing weight loss, anti-hyperglycemia, controlling the progression of inflammation and fibrosis, metabolic enzyme inhibitors, and energy metabolism regulation, etc. (Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesisto treatment concepts in humans[J]. Molecular Metabolism, 2021, 50: 101122.). Lifestyle and dietary interventions are the foundation. Improving insulin sensitivity through a healthy diet, weight management, and exercise can help reverse early fatty liver disease. Drug treatment is still being explored. Existing obesity treatment drugs, such as orlistat (which inhibits fat absorption) or GLP-1 receptor agonists (which delay gastric emptying), mostly target single metabolic links and have limited effects on complications such as liver damage and insulin resistance associated with obesity. Therefore, it is of great significance to study a new type of drug for alleviating obesity caused by a high-fat diet.

[0003] Previous studies have shown that all-trans retinoic acid (ATRA) can inhibit lipid deposition through RAR signaling. However, it mainly binds to RARs and has a very low affinity for RXRs. Therefore, its indirect regulation of lipid metabolism is weak and its impact on lipid metabolism is limited. At the same time, ATRA is more widely distributed, but has a single action target, which may increase the risk of side effects in non-target organs (such as skin and bones). Moreover, its regulation of lipid metabolism is only theoretical research without corresponding valid data for support. Its structure is shown below. 9-cis-retinoic Acid (9-cRA) is an isomer of ATRA (structure shown below), that is, the 9th position of ATRA becomes cis, which is substantially different from the structure of ATRA. And 9-cis-retinoic acid is one of the important active derivatives produced by the metabolism of vitamin A (retinol) and is an important factor in cell survival, differentiation, and death (Rhee E J, Plutzky J. Retinoid metabolism and diabetes mellitus[J]. Diabetes&metabolism journal, 2012, 36(3):167.). The cis configuration of 9-cis-retinoic acid enables it to activate both nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) simultaneously. These nuclear receptors can regulate the expression of a series of genes related to cell differentiation, proliferation, apoptosis, and metabolism by binding to the retinoic acid response elements (RAREs) of specific genes (Yang H, Su M, Liu M, et al. Hepatic retinaldehyde deficiency is involved in diabetes deterioration by enhancing PCK1-and G6PC-mediated gluconeogenesis[J]. Acta Pharmaceutica Sinica B, 2023, 13(9):3728-3743.). Currently, there is no report on the prevention and treatment effects of 9-cis-retinoic acid on chronic metabolic diseases such as obesity. Therefore, 9-cis-retinoic acid is expected to become a promising candidate for the treatment, prevention, or improvement of obesity, which is of great significance for broadening the treatment methods for obesity caused by high-fat diet.

[0004] Summary of the Invention

[0005] The object of the present invention is to provide a new application of 9-cis-retinoic acid, specifically providing the application of 9-cis-retinoic acid in the preparation of products for the treatment, prevention, or improvement of obesity.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] Use of 9-cis retinoic acid in the preparation of a product for treating, preventing or improving obesity.

[0008] Furthermore, the obesity is obesity caused by a high-fat diet.

[0009] Furthermore, the obesity caused by a high-fat diet refers to weight gain, insulin resistance and fatty liver metabolic disorder caused by a high-fat diet.

[0010] Preferably, the 9-cis retinoic acid can reduce the weight gain caused by a high-fat diet.

[0011] Preferably, the 9-cis retinoic acid can reduce the contents of total cholesterol, triglyceride and low-density lipoprotein in the serum.

[0012] Preferably, the 9-cis retinoic acid can improve the abnormal glucose metabolism in obesity caused by a high-fat diet.

[0013] More preferably, the 9-cis retinoic acid can improve the abnormal glucose metabolism in obesity caused by a high-fat diet, including that the 9-cis retinoic acid can improve glucose tolerance, insulin resistance and hyperglycemia.

[0014] Preferably, the 9-cis retinoic acid can reduce the contents of aspartate aminotransferase and alanine aminotransferase in the serum.

[0015] Preferably, the 9-cis retinoic acid can improve lipid accumulation in the liver by up-regulating the expression of PPAR-α in hepatocytes.

[0016] Preferably, the effective dosage of the 9-cis retinoic acid is 0.01 - 10 μg / g body weight / day (μg / g BW / day).

[0017] Furthermore, the 0.01 - 10 μg / g BW / day means that the effective dosage per 1 g body weight per day is 0.01 - 10 μg.

[0018] In the above technical solution, the product for treating, preventing or improving obesity is a drug or a health food.

[0019] Furthermore, when the product is a drug, it includes the above-mentioned 9-cis retinoic acid and other pharmaceutically acceptable excipients.

[0020] Even further, when the product is a drug, its pharmaceutical dosage form is powder, granule, tablet, capsule, oral liquid, powder, suspension or film.

[0021] Further, when the product is a health food, it includes the above-mentioned 9-cis retinoic acid and other food-acceptable excipients.

[0022] The present invention compares the improvement effects of 9-cis retinoic acid and all-trans retinoic acid on obesity caused by high-fat diet, and confirms that 9-cis retinoic acid has a more obvious improvement effect on obesity caused by high-fat diet than all-trans retinoic acid, and higher safety and biocompatibility. 9-cis retinoic acid can reduce the body weight and blood lipid levels of obesity caused by high-fat diet, and improve liver fat vacuoles and insulin tolerance. 9-cis retinoic acid has a very high affinity for RXR, and PPAR-α mainly exists in the liver. Therefore, 9-cis fatty acid has a stronger accumulation ability in the liver, and thus acts more directly on the hepatocyte lipid metabolism pathway.

[0023] Beneficial effects of the present invention: The present invention first proposes a new application of 9-cis retinoic acid in the treatment, prevention or improvement of obesity. By activating the PPAR-α receptor and regulating lipid metabolism, glucose metabolism and liver function at the same time, it achieves multi-target effects, making it more comprehensive in the treatment of obesity and related metabolic syndromes, and the treatment effect is significantly better than all-trans retinoic acid. In addition, 9-cis retinoic acid is a natural active metabolite of vitamin A and does not show significant toxic reactions in animal experiments, with better biocompatibility and safety. Therefore, 9-cis retinoic acid provides a new strategy for the treatment of obesity and related metabolic syndromes. Brief Description of the Drawings

[0024] Figure 1 It is a flow chart of the experiment of 9-cRA and ATRA intervening in obese mice caused by high-fat diet in Example 1.

[0025] Figure 2 It is a comparison chart of body weights of 9-cRA and ATRA intervening in obese mice caused by high-fat diet in Example 1 after 3 weeks.

[0026] Figure 3 It is a comparison chart of serum total cholesterol contents of 9-cRA and ATRA intervening in obese mice caused by high-fat diet in Example 1 after 3 weeks.

[0027] Figure 4 It is a comparison chart of serum triglyceride contents of 9-cRA and ATRA intervening in obese mice caused by high-fat diet in Example 1 after 3 weeks.

[0028] Figure 5 It is a comparison chart of serum high-density lipoprotein cholesterol contents of 9-cRA and ATRA intervening in obese mice caused by high-fat diet in Example 1 after 3 weeks.

[0029] Figure 6Comparison chart of serum low-density lipoprotein cholesterol content in obese mice induced by high-fat diet after 9-cRA and ATRA intervention for 3 weeks in Example 1.

[0030] Figure 7 Comparison chart of fasting blood glucose content in obese mice induced by high-fat diet after 9-cRA and ATRA intervention for 3 weeks in Example 1.

[0031] Figure 8 Comparison chart of glucose tolerance test in obese mice induced by high-fat diet after 9-cRA and ATRA intervention for 3 weeks in Example 1.

[0032] Figure 9 Comparison chart of insulin tolerance test in obese mice induced by high-fat diet after 9-cRA and ATRA intervention for 3 weeks in Example 1.

[0033] Figure 10 Comparison chart of gross, HE pathological sections and liver index of liver tissue in obese mice induced by high-fat diet after 9-cRA intervention for 3 weeks in Example 1.

[0034] Figure 11 Comparison chart of serum alanine aminotransferase in obese mice induced by high-fat diet after 9-cRA and ATRA intervention for 3 weeks in Example 1.

[0035] Figure 12 Comparison chart of serum aspartate aminotransferase in obese mice induced by high-fat diet after 9-cRA and ATRA intervention for 3 weeks in Example 1.

[0036] Figure 13 Comparison chart of serum malondialdehyde in obese mice induced by high-fat diet after 9-cRA and ATRA intervention for 3 weeks in Example 1.

[0037] Figure 14 Comparison chart of mRNA expression of ppar-α in liver tissue of obese mice induced by high-fat diet after 9-cRA intervention for 3 weeks in Example 1.

[0038] Figure 15 Comparison chart of oil red O pathological sections of liver tissue and relative area of quantified liver lipid droplets in obese mice induced by high-fat diet after 9-cRA intervention for 3 weeks in Example 1.

[0039] Figure 16 Comparison chart of PPAR-α immunohistochemistry experiment and its quantified positive comparison in liver tissue of obese mice induced by high-fat diet after 9-cRA intervention for 3 weeks in Example 1.

[0040] Figure 17It is the comparison diagram of HE pathological sections of epididymal white adipose tissue, the comparison diagram of epididymal white adipose index, and the comparison diagram of relative sizes of quantified adipocytes in obese mice caused by high-fat diet after 9-cRA intervention for 3 weeks in Example 1. Detailed implementation mode

[0041] The following non-limiting examples can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0042] In the following examples, the test methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0043] Example 1

[0044] Study on the improvement effect of 9-cRA on obesity induced by high-fat diet:

[0045] Construction of high-fat diet-induced obesity model: Male C57BL / 6J mice were used as experimental animals, raised at room temperature, and fed freely; after 1 week of adaptive feeding, the mice were modeled for 7 weeks. During modeling, the mice were fed a high-fat diet - H10060 (the fat energy ratio was 60%, purchased from Beijing Huafukang Biotechnology Co., Ltd.).

[0046] The obese model mice caused by high-fat diet were randomly divided into 6 groups, with 6 mice in each group. The experimental conditions of each group are shown in Table 1:

[0047] Table 1. Experimental conditions of each group of mice

[0048]

[0049] After the modeling of each experimental group of mice was completed, 9-cRA (purchased from Shandong Xiya Chemical Co., Ltd.) and ATRA (purchased from Shandong Xiya Chemical Co., Ltd.) were respectively dissolved in DMSO, diluted with normal saline, and intervened by tail vein injection according to the experimental conditions in Table 1 for 3 weeks ( Figure 1 ).

[0050] The body size and weight of each group were measured to study the effects of 9-cRA and ATRA on the body weight of obese mice caused by high-fat diet. The results are as Figure 2 shown. The data are expressed as mean ± standard deviation, n = 6 in each group, * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01 vs. HFD. Figure 2It can be seen that after 3 weeks of intervention, the weight loss in the AL group (27.00±0.8198 g) and the AH group (26.87±2.115 g) was more significant than that in the TAL group (29.33±1.442 g) and the TAH group (27.95±1.319 g) (p>0.05 or p<0.05). At the same dose, the weight loss effect in the AL group was 2.664 times that in the TAL group; the weight loss effect in the AH group was 1.324 times that in the TAH group. It can be seen that 9-cRA can effectively reduce the body weight of mice (p<0.01).

[0051] The sera of the mice in the above groups were measured for TC, TG, HDL, and LDL to explore the effects of 9-cRA and ATRA on the blood lipid levels in the obese mouse model induced by high-fat diet. The results of the contents of TG, TC, HDL-c, and LDL-c in the sera of the mice are as Figures 3 - 6 shown, and the data are expressed as mean±standard deviation, with n = 6 in each group, * p<0.05, ** p<0.01, *** p<0.001 vs. HFD. It can be Figures 3 - 6 seen that after 3 weeks of intervention, the TG levels in the mice in the AL group (0.5008±0.08275 mmol / L) and the AH group (0.4786±0.06246 mmol / L) decreased significantly, showing significant differences compared with the HFD group (p<0.01), and were more significant than those in the TAL group (0.6017±0.04482 mmol / L) and the TAH group (0.5880±0.06569 mmol / L) (p<0.05). At the same dose, the reduction effect in the AL group was 1.780 times that in the TAL group; the reduction effect in the AH group was 1.764 times that in the TAH group. The TC levels in the mice in the AL group (2.783±0.1454 mmol / L) and the AH group (2.446±0.3817 mmol / L) also decreased significantly, showing significant differences compared with the HFD group (p<0.01), and were more significant than those in the TAL group (3.349±0.4275 mmol / L) and the TAH group (3.314±0.1948 mmol / L) (p<0.05). At the same dose, the reduction effect in the AL group was 2.101 times that in the TAL group; the reduction effect in the AH group was 2.581 times that in the TAH group. In addition, in the cholesterol measurement results, the HDL and LDL levels in the AL group and the AH group also decreased significantly, and the results were significantly equivalent to those in the TAL group and the TAH group.

[0052] The above groups of mice were subjected to an oral glucose tolerance test (GTT), an intraperitoneal insulin tolerance test (ITT), and a fasting blood glucose experiment to explore the effects of 9-cRA and ATRA on glucose metabolism in the obese mouse model induced by high-fat diet. The results are asFigures 7 - 9 As shown, the data are expressed as mean ± standard deviation, with n = 6 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD. From Figures 7 - 9 It can be seen that after 3 weeks of intervention, the blood glucose levels of mice in the AL group (7.500 ± 0.9899 mmol / L) and the AH group (6.983 ± 0.4579 mmol / L) were significantly decreased compared with those in the HFD group (9.100 ± 1.099 mmol / L), showing significant differences (p < 0.05), and were significantly equivalent to those in the TAL group (7.733 ± 0.4457 mmol / L) (p < 0.05) and the TAH group (7.417 ± 0.4401 mmol / L) (p > 0.05). At the same dose, the reduction effect of the AL group was 1.170 times that of the TAL group; the reduction effect of the AH group was 1.258 times that of the TAH group. In the GTT and ITT experiments, the average blood glucose of mice in the HFD group was above that of each group within 0 - 120 min; compared with the HFD group, the areas under the curve (AUC) of AL and AH groups of mice decreased, showing significant differences (p < 0.01), which were more significant than those of the TAL group and the TAH group (p > 0.05). At the same dose, in the AUC statistical results of GTT and ITT, the reduction effects of the AL group were 4.011 times and 2.534 times that of the TAL group respectively; the reduction effect of the AH group was 2.733 times that of the TAH group.

[0053] The livers of mice in each group above were observed and weighed, and the liver tissues were sectioned and stained with hematoxylin - eosin (HE staining), and AST, ALT, and MDA in the serum were detected to explore the effects of 9 - cRA and ATRA interventions on the livers of obese mouse models induced by high - fat diet. The results are as Figures 10 - 13 shown, the data are expressed as mean ± standard deviation, with n = 6 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD. From Figure 10 It can be seen that there were no obvious differences in the gross photographs of mouse livers observed by the naked eye. The results of liver indices showed that there were no significant differences between each 9 - cRA intervention group and the HFD group, but there was a tendency of relative reduction; after HE staining of the sectioned mouse livers, it was found that the liver sections of mice in the CON group showed clear hepatic lobule structures, normal hepatocyte morphology, neatly and closely arranged, large and round cell nuclei, and no abnormalities were seen; no obvious abnormalities were seen in the HFD group and the 9 - cRA intervention groups. From Figures 11 - 13It can be seen that the levels of ALT, AST, and MDA in the sera of the AL and AH groups of mice decreased significantly, showing significant differences (p < 0.01), and were more significant than those in the TAL and TAH groups (p > 0.05).

[0054] Immunohistochemistry experiments, Oil Red O staining, and immunoblotting experiments were performed on the livers of the mice in the above groups to explore the effects of 9-cRA intervention on liver lipid metabolism and the mRNA expression levels of ppar -α and the protein expression of PPAR-α in the livers of obese mouse models caused by high-fat diet. The results are as Figures 14 - 16 shown. The data are expressed as mean ± standard deviation, with n = 6 in each group, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 vs . HFD. It can be Figure 14 seen that after homogenizing the mouse livers and performing qPCR experiments to detect the ppar mRNA expression of ppar -α, the results showed that after 9-cRA intervention, the mRNA expression of Figure 15 -α in the mouse livers increased significantly, showing significant differences (p < 0.01); it can be Figure 16 seen that after slicing the mouse livers and performing Oil Red O staining, the results showed that there were obvious lipid droplets in the livers of the HFD group mice, showing uniform distribution (the lipid droplets were red). After 9-cRA intervention, the distribution of red lipid droplets in the mouse livers decreased significantly, showing significant differences (p < 0.001); it can be

[0055] seen that after slicing the mouse livers and performing immunohistochemistry experiments, the results showed that PPAR-α in the liver cell nuclei of the mouse liver sections was dark brown. After 9-cRA intervention, the expression levels of PPAR-α in the livers of the AL and AH groups were both increased compared with the HFD group, showing significant differences (p < 0.001). Figure 17 shown. The data are expressed as mean ± standard deviation, with n = 6 in each group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD. It can be Figure 17It can be seen that the results of the fat index showed that there was no significant difference between each intervention group and the HFD group (p > 0.05), but there was a tendency of relative decrease; after HE staining of the epididymal white fat sections of mice, it was found that the fat sections of the CON group showed a cell structure with uniform size, arranged neatly and tightly, and a relatively large number of cells could be observed within a certain visible field of view; the fat sections of the HFD group showed numerous relatively large fat cells. After intervention with 9-cRA, the size of the fat cells in mice decreased significantly, and there were significant differences compared with the HFD group (p < 0.001).

[0056] In summary, compared with ATRA, 9-cRA has better effects on reducing blood lipids, improving glucose metabolism function, and improving hepatic lipid accumulation. 9-cRA mainly improves hepatic lipid accumulation by increasing the expression of the main regulatory protein PPAR-α for hepatic β-oxidation, and can be used for the treatment, prevention or improvement of metabolic symptoms caused by a high-fat diet, which has extremely important clinical significance.

Claims

Use of 9-cis retinoic acid in the preparation of a product for treating, preventing or improving obesity.

2. The application according to claim 1, wherein: The obesity is obesity caused by a high-fat diet.

3. The obesity caused by a high-fat diet according to claim 2, characterized in that: The obesity caused by a high-fat diet refers to weight gain, insulin resistance and liver lipid metabolism disorder caused by a high-fat diet.

4. The application according to claim 1 or 2, characterized in that: The 9-cis retinoic acid can reduce the weight gain caused by a high-fat diet.

5. The application according to claim 1 or 2, characterized in that: The 9-cis retinoic acid can reduce the contents of total cholesterol, triglyceride and low-density lipoprotein in the serum of obesity caused by a high-fat diet.

6. The application according to claim 1 or 2, characterized in that: The 9-cis retinoic acid can improve the abnormal glucose metabolism of obesity caused by a high-fat diet.

7. The application according to claim 1 or 2, characterized in that: The 9-cis retinoic acid can reduce the contents of aspartate aminotransferase and alanine aminotransferase in the serum of obesity caused by a high-fat diet.

8. The application according to claim 1 or 2, characterized in that: The 9-cis retinoic acid can improve the lipid accumulation in the liver by upregulating the expression of PPAR-α in hepatocytes.

9. The application according to claim 1, wherein: The effective dosage of the 9-cis retinoic acid is 0.01 - 10 μg / g body weight per day.

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