Application of gentiopicroside in preparation of medicine for improving liver fatty degeneration and ferroptosis of type 2 diabetes mellitus

Abstract

The invention discloses application of gentiopicroside in preparation of a medicine for improving liver fatty degeneration and ferroptosis of type 2 diabetes mellitus, and belongs to the technical field of medicines. A C57BL / 6J mouse T2DM model induced by combination of high-fat feed and streptozotocin is taken as a research object, gentiopicroside is given for 8 weeks through oral administration and intragastric administration, metabolic indexes such as blood sugar, body weight, food intake and water intake of the mouse are dynamically monitored, serum and liver tissue are reserved after the mouse is killed, and the mouse can be used for preparing the gentiopicroside-gentiopicroside-gentiopicroside-gentiopicroside-gentiopicroside-gentiopicroside. Through liver pathological staining, serum and liver tissue oxidative stress index detection and PI3K / AKT / Nrf2 signal channel and ferroptosis related protein expression analysis, the pharmacological action of the gentiopicroside on T2DM is systematically evaluated, the regulation effect of the gentiopicroside on T2DM liver fatty degeneration and ferroptosis is determined for the first time, the research blank of the action mechanism of the gentiopicroside in T2DM liver complications is filled, and the application prospect of the gentiopicroside in T2DM liver complications is broadened. An experimental basis is provided for developing a novel medicine for improving T2DM liver fatty degeneration and ferroptosis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to the use of gentiopicroside in the preparation of a drug for improving liver fatty degeneration and ferroptosis in type 2 diabetes. Background Art

[0002] Ferroptosis, a new type of programmed cell death that is dependent on iron ions, is closely related to the progression of type 2 diabetes mellitus (T2DM). T2DM patients are in a state of long-term hyperglycemia and hyperlipidemia, which results in the massive generation of ROS and disordered iron metabolism, creating conditions for ferroptosis. Pancreatic β-cells are sensitive to oxidative stress. Once ferroptosis is initiated, the level of lipid peroxidation in β-cells increases dramatically, mitochondrial morphology changes, and membrane potential decreases, leading to increased β-cell apoptosis, exacerbated β-cell dysfunction, decreased insulin secretion, and more severe imbalance in blood glucose regulation. Moreover, after ferroptosis occurs in cells such as adipose tissue and muscle, insulin sensitivity decreases, insulin signaling is blocked, and the body's ability to absorb and utilize glucose continues to decline. This shows that ferroptosis is a key node in the onset and progression of T2DM. Among the many pathways that regulate oxidative stress and ferroptosis, the PI3K / AKT / Nrf2 signaling pathway plays an important role in maintaining redox balance and inhibiting ferroptosis.

[0003] The natural product gentiopicroside (GPS) is a cyclic ether terpene glycoside compound found widely in Gentianaceae plants such as Gentiana striata, Gentiana scabra, Gentiana triflora, and Gentiana scabra. Since its discovery, research has shown that it possesses numerous pharmacological effects, including anti-inflammatory and analgesic, hepatoprotective and choleretic, antidepressant, anti-tumor, anti-diabetic, and anti-osteoporotic properties. Notably, with regard to diabetes and its complications, GPS significantly improves peripheral neuropathy in STZ-induced diabetic rats, potentially by improving nerve blood flow and regulating dyslipidemia. GPS also significantly improves diabetic retinopathy and diabetic nephropathy by inhibiting inflammation and oxidative stress. GPS activates the PI3K / AKT signaling pathway by targeting progesterone and lipid Q3 receptor (PAQR3), significantly ameliorating glucose and lipid metabolism disorders in mice with high-fat diet and streptozotocin-induced type 2 diabetes. Furthermore, in a palmitic acid-induced insulin resistance model in L02 hepatocytes, GPS maintains intracellular glucose homeostasis by inhibiting gluconeogenesis. In a high-fat diet-induced ApoE mouse model, GPS significantly improved atherosclerosis by regulating intestinal flora and fecal metabolites. However, whether GPS can improve hepatic steatosis and hepatic ferroptosis in T2DM mice remains unclear. Therefore, this study aimed to clarify the effects of GPS on hepatic steatosis and hepatic ferroptosis in T2DM mice. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of gentiopicroside in the preparation of a drug for improving liver steatosis and ferroptosis in type 2 diabetes. The present invention uses a T2DM model formed by C57BL / 6J mice induced by a high-fat diet (HFD) combined with streptozotocin (STZ) as the object, and orally administers gentiopicroside by gavage for 8 weeks. During the experiment, the blood glucose, body weight, food intake, and water intake of the mice are measured. When the mice are killed, serum, liver, etc. are collected, and the liver is pathologically stained, oxidative stress-related indicators in serum and liver are measured, and PI3K / AKT / Nrf2 signaling pathways and ferroptosis-related proteins in the liver are detected. The pharmacological effects of GPS on type 2 diabetes are comprehensively evaluated, and the effects of GPS on liver steatosis and ferroptosis in T2DM are investigated. It is clear that GPS can effectively improve liver steatosis and ferroptosis in type 2 diabetes, and its development provides an experimental basis for a new drug to improve liver steatosis and ferroptosis in T2DM.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] Application of gentiopicroside in the preparation of drugs for improving liver steatosis and ferroptosis in type 2 diabetes.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] The present invention uses a T2DM model of C57BL / 6J mice induced by a high-fat diet combined with streptozotocin as the research object, administers gentiopicroside by oral gavage for 8 weeks, and dynamically monitors metabolic indicators such as blood glucose, body weight, food intake, and water intake of the mice. After the mice are killed, serum and liver tissue are collected. Liver pathological staining (HE, Oil Red, PAS staining), determination of lipid metabolism-related indicators in serum (TG, TC, LDL-C, HDL-C), detection of oxidative stress indicators in serum and liver tissue (SOD, MDA, GSH, etc.), expression analysis of PI3K / AKT / Nrf2 signaling pathway and ferroptosis-related proteins (TFR1, FTH1, GPX4, etc.) are performed to systematically evaluate the pharmacological effects of gentiopicroside on T2DM. This is the first time to clarify that gentiopicroside has a regulatory effect on hepatic steatosis and ferroptosis in T2DM, filling the research gap in the mechanism of action of gentiopicroside in T2DM liver complications, and providing an experimental basis for the development of new drugs for improving hepatic steatosis and ferroptosis in T2DM. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 :GPS reduces blood glucose in T2DM mice;

[0010] Figure 2 :GPS improves oral glucose tolerance and insulin resistance in T2DM mice;

[0011] Figure 3:GPS improves liver pathological morphological abnormalities and fatty degeneration in T2DM mice;

[0012] Figure 4 :GPS activates the PI3K / AKT / Nrf2 signaling pathway in the liver of T2DM mice;

[0013] Figure 5 : GPS alleviates ferroptosis in the liver of T2DM mice. DETAILED DESCRIPTION

[0014] 1. Test drug.

[0015] Name: Gentiopicroside, purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0016] Solvent: 0.5% sodium carboxymethyl cellulose, distilled water;

[0017] Preparation method: According to experimental needs, 0.5% sodium carboxymethyl cellulose GPS is used to prepare suspensions of different concentrations.

[0018] 2. Animals.

[0019] 1) Normal mice: Male C57BL / 6J mice, weighing 20-22 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0020] 2) Construction of type 2 diabetes model, grouping and drug administration.

[0021] After one week of adaptive feeding in the animal house, purchased C57BL / 6J mice were randomly divided into a high-fat diet group and a standard diet group. After 10 weeks of feeding, mice in the HFD group fasted for 8 hours and then received an intraperitoneal injection of streptozotocin (35 mg / kg) for 5 consecutive days. Mice in the standard diet group received an intraperitoneal injection of the corresponding volume of citrate buffer (pH = 4.4). Following STZ injection, mice were fed for another week and then fasted for 6 hours. Fasting blood glucose (FBG) was measured by tail tip blood sampling. A FBG ≥ 200 mg / dL was considered a successful type 2 diabetes model.

[0022] Subsequently, T2DM mice were randomly divided into a T2DM model group (Mod), a low-dose GPS group (25 mg / kg, GPS-L), and a high-dose GPS group (50 mg / kg, GPS-H) based on body weight, FBG, and random blood glucose (RBG). Mice fed a normal diet served as a control group (Nor). Subsequently, the control and model groups were gavaged with 0.5% sodium carboxymethylcellulose (CMC-Na) of the corresponding volume for 8 weeks. After 8 weeks of administration, the mice were sacrificed and their livers and serum were collected. Body weight, RBG, FBG, food intake, and water intake of the mice were measured weekly during the experiment.

[0023] 3. Materials: Gentiopicroside (lot number J04HS1.84065, purity ≥97%, Shanghai Yuanye Biotechnology Co., Ltd.); STZ (Sigma); high-fat diet (cat. no. SYHF60-1, Shuyu Biotechnology (Shanghai) Co., Ltd.). The high-fat diet consisted of 40.7% standard feed, 27% lard, 8% sucrose, 10.8% whole milk powder, and 13.5% casein, with 20% energy provided by protein, 60% by fat, and 20% by carbohydrates. Low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglyceride (TG) assays were performed by Beijing Zhongsheng Beikong Biotechnology Co., Ltd. Reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), MDA, and total antioxidant capacity (T-AOC) kits were purchased from Nanjing Jiancheng Bioengineering Institute; monoclonal antibodies to transferrin receptor 1 (TFR1), ferritin heavy chain 1 (FTH1), acyl-CoA synthetase long-chain family member 4 (ACSL4), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) were purchased from CST (USA); monoclonal antibody AK T, p-AKT, PI3K, p-PI3K, Nrf2, NADPH quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), glutamylcysteine ​​ligase catalytic subunit (GCLC), and β-actin were provided by Wuhan Sevier Biotechnology Co., Ltd.; CCK8 kit, BCA protein kit, RIPA lysis buffer, protease and phosphatase inhibitors, chemiluminescence reagent, horseradish peroxidase-conjugated rabbit secondary antibody, and mouse secondary antibody were provided by Beijing Pulilai Gene Technology Co., Ltd.

[0024] 4. Determination method.

[0025] 1) Measurement of Basic Metabolic Parameters in Mice: Body weight, random blood glucose (RBG), fasting blood glucose (FBG), and food intake were measured weekly during the experiment. After the final dose, blood was collected from the venous plexus of the inner canthus, and then the mice were sacrificed. Whole blood was allowed to stand at room temperature for 2 hours and then centrifuged at 6000 rpm for 10 minutes to collect serum.

[0026] 2) Superoxide dismutase (SOD) assay: Oxygen free radicals oxidize hydroxylamine to form nitrite, which, under the action of a color developer, produces a purple-red color. When SOD is present in the sample being tested, it specifically inhibits superoxide anion free radicals, reducing the formation of nitrite. SOD activity is measured colorimetrically at 550nm. SOD plays a crucial role in the body's oxidative and antioxidant balance. This enzyme scavenges superoxide anion free radicals to protect cells from damage, and its activity indirectly reflects the body's ability to scavenge oxygen free radicals.

[0027] 3) Malondialdehyde (MDA) assay: MDA in lipid peroxidation degradation products can be condensed with thiobarbituric acid to form a red product with a maximum absorption peak at 532 nm. The amount of MDA tested can reflect the degree of lipid peroxidation in the body, and indirectly reflect the severity of free radical attack on the body cells.

[0028] 4) Reduced glutathione (GSH) assay: GSH can react with dithio-dinitrobenzoic acid to form a yellow compound, and the GSH content can be determined by colorimetric method at 405 nm. GSH is the most important non-enzymatic antioxidant in the body, which can scavenge O 2- , H2O2, LOOH, so the amount of GSH is an important factor to measure the antioxidant capacity of the body.

[0029] 5) Determination of lipid metabolism related indicators in mouse serum: After the mice were sacrificed after the orbital blood was taken, the whole blood was allowed to stand for 2 h, and then centrifuged at 4℃, 6000r·min -1 for 10 min, and the supernatant was aspirated. Then the lipid related indicators in the serum were determined according to the instructions: TG, HDL-C, TC, LDL-C.

[0030] 6) Mouse glucose tolerance test (OGTT) and insulin tolerance test (ITT) experiment: OGTT and ITT experiments were performed at 6 and 7 weeks of administration, respectively. The blood glucose of the mice was measured at 0 min before fasting. For OGTT experiment, the mice were given oral gavage of glucose (2g / kg) after fasting for 10h, and for ITT experiment, the mice were subcutaneously injected with insulin (0.75U / kg) after fasting for 6h. Then the blood was taken from the tail tip at 30min, 60min, 90min and 120min after the administration of glucose and insulin, and the blood glucose at each time point was measured. According to the blood glucose value at each time point, the blood glucose time curve was drawn, and the area under the curve (AUC) of each group was calculated.

[0031] 7) Mouse liver histological staining analysis: A part of the liver of the mice was fixed in neutral paraformaldehyde, and after 24h it was embedded in paraffin and prepared into a section with a thickness of 5μm. Then the skeletal muscle and pancreas were subjected to liver hematoxylin-eosin (HE) staining, oil red staining and glycogen staining (PAS) according to the instructions of the kit. Finally, all the sections were observed under a microscope and analyzed by taking pictures.

[0032] 8) Western blot analysis of mouse liver protein: Livers from four mice were randomly selected from each group. 50 mg of tissue was weighed and added to 500 μL of RIPA lysis buffer containing protease and phosphatase inhibitors. The tissue was minced with surgical scissors and thoroughly homogenized using a mechanical homogenizer. Lysis was performed on ice for 40 min and centrifuged at 4°C (12,000 rpm) for 10 min. The supernatant was diluted 10-fold and the protein concentration was determined using the BCA assay. After adjusting the protein concentration to the same level, 5× loading buffer was added and denatured by boiling. After electrophoresis on a 10% polyacrylamide gel, the membrane was transferred to a PVDF membrane, blocked at room temperature for 2 h, and incubated with the primary antibody overnight at 4°C. The membrane was washed three times and incubated with the secondary antibody for 2 h at room temperature. After development, grayscale values ​​were calculated using Image Lab.

[0033] 5. Experimental process and experimental data.

[0034] (1) GPS lowers blood glucose in T2DM mice.

[0035] During the experiment, the metabolic indicators of mice were measured. Figure 1 【(A) Body weight, (B) Random blood glucose, (C) Fasting blood glucose, (D) Glycated hemoglobin level, (E) Food intake, (F) Water intake. The results are expressed as mean ± standard error, n = 10. Compared with the Nor group, # P<0.05, ## P < 0.01; *P < 0.05, **P < 0.01 compared with the Mod group.] During the experiment, the weight of mice in the Nor group gradually increased. Before STZ injection, the weight of mice on a HFD was significantly higher than that of mice in the Nor group. After STZ injection, the weight of mice in the HFD group decreased significantly. After grouping, the weight of mice on a Mod group was significantly lower than that of the Nor group. Compared with the Mod group, there was no significant change in the weight of mice in the high- and low-dose GPS groups. During the administration period, RBG and FBG values ​​of mice in the Mod group were significantly higher than those in the Nor group. High-dose GPS significantly reduced RBG and FBG in T2DM mice starting from the second week of administration, while low-dose GPS showed a trend toward lower RBG and FBG. HbA1c measurements further demonstrated that high-dose GPS significantly reduced blood glucose in T2DM mice. Food and water monitoring results showed that food and water intake in Mod mice increased significantly compared with the Nor group, while GPS reduced food and water intake in T2DM mice in a dose-dependent manner.

[0036] The above results indicate that GPS significantly improves blood glucose disorders in T2DM mice.

[0037] (2) GPS improves oral glucose tolerance and insulin resistance in T2DM mice.

[0038] To further investigate the effect of GPS on blood glucose in T2DM mice, OGTT and ITT experiments were performed during the experiment. Figure 2 【(A) Mouse OGTT test, (B) Area under the curve of OGTT test, (C) Mouse ITT test, (D) Area under the curve of ITT test. The results are expressed as mean ± standard error, n = 10. Compared with the Nor group, # P<0.05, ## P < 0.01; *P < 0.05, **P < 0.01 compared with the Mod group.] After glucose loading or insulin injection, blood glucose levels and AUC in the model group increased significantly at all time points compared with the Nor group, indicating impaired glucose tolerance and insulin resistance in T2DM mice. GPS treatment, however, showed a dose-dependent decrease in blood glucose levels and AUC at 30, 60, 90, and 120 minutes after glucose loading or insulin injection compared with the model group.

[0039] The above results show that GPS significantly improves oral glucose tolerance and insulin resistance in T2DM mice.

[0040] (3) GPS improves lipid metabolism disorders in T2DM mice.

[0041] To clarify the effect of GPS on serum lipid levels in T2DM mice, serum lipid-related indicators were measured, and the results are shown in Table 1. As shown in Table 1, compared with the normal control group, the serum TG, TC, and LDL-C levels of the model group mice were significantly increased, and the HDL-C level was significantly decreased, indicating that T2DM mice had dyslipidemia; low-dose GPS significantly reduced the serum TC level in T2DM mice, with a trend of reducing serum TG and LDL-C levels and increasing HDL-C levels; high-dose GPS significantly reduced the serum TG, TC, and LDL-C levels in T2DM mice and significantly increased the serum HDL-C level.

[0042] Table 1. GPS improves lipid metabolism disorders in T2DM mice ( n=10)

[0043]

[0044] Note: Compared with the normal group, # P<0.01, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0045] (4) GPS improves liver pathological morphological abnormalities and fatty degeneration in T2DM mice.

[0046] The liver is an important organ that regulates blood sugar homeostasis and lipid metabolism. It can be damaged by various causes. The most common damage in T2DM is liver fat accumulation. To investigate whether GPS has an effect on liver fat accumulation in T2DM mice, liver tissues were stained with HE, PAS, and oil red. Figure 3 【(A) HE, PAS and Oil Red staining of mouse liver (n=5), (B) TG content in liver (n=10), (C) TC content in liver (n=6), (D) Statistical results of Oil Red staining of liver (n=6). The results are expressed as mean ± standard error, n=6. Compared with the Nor group, # P<0.05, ## P < 0.01; compared with the Mod group, *P < 0.05, **P < 0.01], compared with the Nor group, the hepatocytes of the Mod group were significantly swollen and vacuolated, with numerous round fat droplets of varying sizes filling the cytoplasm. Inflammatory cell infiltration was also observed. GPS administration for 8 weeks inhibited hepatic lipid accumulation to varying degrees and also improved hepatocyte morphology. Oil red staining also showed that both high and low doses of GPS significantly reduced lipid accumulation in the livers of T2DM mice. Glycogen staining results indicated that GPS increased glycogen synthesis in the livers of T2DM mice.

[0047] To further verify that GPS can indeed improve hepatic fatty degeneration, the TG and TC levels in the liver were measured. The results showed that the TG and TC levels in the liver of Mod mice were significantly higher than those in the normal control group, while high-dose GPS significantly reduced the TG and TC levels in the liver, and low-dose GPS had a trend of reducing TG and TC in the liver.

[0048] The above results indicate that GPS inhibits hepatic fatty degeneration, suggesting that its improvement of T2DM symptoms may be related to inhibiting hepatic fat accumulation and thereby increasing the liver's sensitivity to insulin.

[0049] (5) GPS activates the PI3K / AKT / Nrf2 signaling pathway in the liver of T2DM mice.

[0050] The expression of proteins related to the PI3K / AKT / Nrf2 signaling pathway in the liver was determined. Figure 4 (A) Representative Western blot images of liver proteins p-PI3K and p-AKT, (B) Grayscale analysis bar graphs of liver proteins p-PI3K and p-AKT, (C) Representative Western blot images of liver proteins Nrf2, Keap1, HO-1, and NQO1, (D) Grayscale analysis bar graphs of liver proteins Nrf2, Keap1, HO-1, and NQO1. The results are expressed as mean ± standard error, n = 10. Compared with the Nor group, #P<0.05, ## P<0.01; compared with the Mod group, *P<0.05, **P<0.01], compared with the Nor group, the expression of p-PI3K, p-AKT, Nrf2, HO-1, and NQO1 proteins in the liver of the Mod group was significantly decreased, while the expression of Keap1 protein was significantly increased. After 8 weeks of GPS treatment, the expression of p-PI3K, p-AKT, Nrf2, HO-1, and NQO1 proteins in the liver of T2DM mice was significantly increased, while the expression of Keap1 was decreased.

[0051] The above results indicate that GPS may activate the PI3K / AKT / Nrf2 signaling pathway in the liver of T2DM mice and improve liver function abnormalities.

[0052] (6) GPS alleviates the occurrence of ferroptosis in the liver of T2DM mice.

[0053] Further determination of liver iron death related indicators showed that Figure 5 (A) GSH content in liver (n=10), (B) SOD activity in liver (n=10), (C) MDA content in liver (n=10), (D) 4-HNE content in liver (n=10), (E) Representative Western blot images of ferroptosis-related proteins in liver, (F) Relative optical density histogram of protein expression of Nrf2, GPX4, FTH-1, SLC7A11 and TFR-1 in liver (n=6). The results are expressed as mean ± standard error. Compared with the Nor group, # P<0.05, ## P < 0.01; compared with the Mod group, *P < 0.05, **P < 0.01] As shown in the results, compared with the Nor group, the GSH content and SOD activity in the liver of the Mod group were significantly decreased, the MDA and 4-HNE contents were significantly increased, the expression of the liver proteins Nrf2, GPX4, FTH-1, and SLC7A11 was significantly decreased, while the expression of the protein TFR-1 was significantly increased, indicating that ferroptosis occurred significantly in the liver of T2DM mice. After 8 weeks of GPS treatment, the GSH content and SOD activity in the liver of T2DM mice were significantly increased, the MDA and 4-HNE contents were significantly decreased, the expression of the liver proteins Nrf2, GPX4, FTH-1, and SLC7A11 was significantly increased, while the expression of the protein TFR-1 was significantly decreased.

[0054] The above results indicate that GPS can alleviate liver ferroptosis in T2DM mice.

Claims

1. Application of gentiopicroside in the preparation of drugs to improve liver fatty degeneration and ferroptosis in type 2 diabetes.

Images