Application of sennoside A in preparation of anti-metabolic syndrome drugs and / or health care products

Abstract

The invention relates to the technical field of treatment of metabolic syndromes, in particular to application of sennoside A in preparation of anti-metabolic syndrome drugs and / or health care products. It is found that the sennoside A can improve glycolipid metabolism of metabolic syndrome model mice induced by high fat diet, inhibit obesity and improve hypercholesterolemia and increase of low-density lipoprotein; the abnormities of fasting blood glucose, fasting insulin, fasting C peptide and plasma GLP-1 of metabolic syndrome model mice can be improved, and the insulin sensitivity of OGTT, ITT and HOMA-IR can be improved; in addition, abnormal expression of WT-HFD metabolic syndrome model mouse Sirt3 can be effectively relieved, GLP-1 protein expression can be promoted, and phosphorylation of IRS-1 / PI3K / AKT insulin signal transduction pathway key molecules is promoted; meanwhile, it is found that Sirt3 is a key molecule of sennoside A for exerting the treatment effect.

Description

Technical Field

[0001] The present invention relates to the technical field of metabolic syndrome treatment, and in particular to an application of sennoside A in the preparation of anti-metabolic syndrome drugs and / or health products. Background Art

[0002] With the aging of the population and the increasing severity of overweight and obesity, metabolic syndrome (MS) has become a global disease that seriously affects human health. Currently, more than 1 billion people worldwide suffer from metabolic syndrome. From the perspective of the incidence of metabolic syndrome in different age and gender groups, men are higher than women before the age of 50, and women are higher than men after the age of 50. As people's material life has been continuously enriched in recent years, unhealthy diet and living habits have led to an increase in the incidence of metabolic syndrome year by year, and the harm caused by cardiovascular and cerebrovascular accidents has gradually emerged. Metabolic syndrome is a common cause of cardiovascular and cerebrovascular diseases, diabetes and other diseases. The risk of cardiovascular and cerebrovascular diseases in patients with metabolic syndrome is 3 times that of patients without metabolic syndrome, and the risk of death is 5 to 6 times higher than that of patients without metabolic syndrome. Metabolic syndrome also increases the incidence, prevalence and mortality of T2DM, bringing a heavy burden to patients, families and society. Metabolic syndrome and related diseases increase the total cost of medical care by 60%. The large number of patients with metabolic syndrome and the health hazards and economic expenditures caused by this disease cannot be ignored and must be given sufficient attention.

[0003] Current research suggests that the pathogenesis of MS involves metabolic stress and mitochondrial dysfunction related to obesity and insulin resistance (IR), chronic inflammation, changes in microbial flora, deoxyribonucleic acid (DNA) methylation, etc. Traditional Chinese medicine has the advantages of significant therapeutic effects, rich targets, and few side effects. It has a brighter research prospect for diseases such as metabolic syndrome that require comprehensive treatment and multi-faceted consideration.

[0004] MS has many risk factors and complex mechanisms. Current treatments mainly target the various components of MS, including changing unhealthy lifestyles, weight management, controlling blood sugar and blood pressure, and improving lipid metabolism disorders. Currently, the International Diabetes Federation still provides necessary treatment for MS based on its individual components such as hyperlipidemia and hyperglycemia, with the goal of reducing the risk of cardiovascular disease by reducing the risk of individual components. This treatment plan has the risk of patients taking too many types of medication, poor compliance, cumulative drug side effects, and increased risk of liver and kidney damage. Summary of the invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide an application of sennoside A in the preparation of anti-metabolic syndrome drugs and / or health products.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The present invention provides an application of sennoside A (also referred to as "SA") in the preparation of anti-metabolic syndrome drugs and / or health products.

[0008] In one embodiment of the present invention, the metabolic syndrome is metabolic syndrome caused by a high-fat diet.

[0009] In one embodiment of the present invention, the anti-metabolic syndrome drug and / or health product is an anti-hyperinsulinemia drug and / or health product.

[0010] In one embodiment of the present invention, the anti-metabolic syndrome drug and / or health product is an anti-low-density lipoprotein drug and / or health product.

[0011] In one embodiment of the present invention, the anti-metabolic syndrome drug and / or health product is an anti-type 2 diabetes drug and / or health product.

[0012] In one embodiment of the present invention, the anti-metabolic syndrome drug and / or health product is an anti-insulin resistance drug and / or health product.

[0013] In one embodiment of the present invention, the medicine and / or health product contains one or more pharmaceutically acceptable excipients.

[0014] In one embodiment of the present invention, the pharmaceutically acceptable excipient is selected from one or more of glycerol, butylene glycol, trehalose, betaine, allantoin, carbomer, dextrin, starch, gel cellulose, methyl cellulose, sodium carboxymethyl cellulose or polyvinyl pyrrolidone.

[0015] In one embodiment of the present invention, the dosage form of the medicine and / or health product is tablets, capsules, powders, granules, suspensions or injections.

[0016] In one embodiment of the present invention, the effective concentration of sennoside A is 30-45 mg / kg / d.

[0017] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product that inhibits the expression of cholesterol and / or low-density lipoprotein.

[0018] In one embodiment of the present invention, the drug and / or health product is a drug and / or health product that promotes the phosphorylation of IRS-1, PI3K, and AKT in the presence of Sirt3.

[0019] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product that promotes the expression of GLP-1.

[0020] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product for improving insulin sensitivity index.

[0021] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product that improves elevated fasting blood glucose and / or elevated fasting insulin and / or elevated fasting C-peptide.

[0022] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product for improving glucose tolerance and / or insulin tolerance.

[0023] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product that inhibits mitochondrial ROS activity and / or SOD activity.

[0024] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product that inhibits the expression of pro-apoptotic proteins and / or promotes the expression of anti-apoptotic proteins.

[0025] In one embodiment of the present invention, the pro-apoptotic protein is selected from one of bax and cleaved caspase-3; and the anti-apoptotic protein is Bcl-2.

[0026] In one embodiment of the present invention, the medicine and / or health product is a medicine and / or health product that improves the loss of colon mucosal contents and / or epithelial cell shedding and / or mucosal gland atrophy.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention found that SA can improve the glucose and lipid metabolism of metabolic syndrome model mice induced by a high-fat diet, inhibit obesity, and improve hypercholesterolemia and elevated low-density lipoprotein.

[0029] (2) The present invention found that SA can improve fasting blood glucose, fasting insulin, fasting C-peptide, and plasma GLP-1 abnormalities in metabolic syndrome model mice, and improve OGTT, ITT, and HOMA-IR insulin sensitivity. However, the above therapeutic effects of SA were significantly inhibited in SIRT3 KO mice.

[0030] (3) The present invention found that SA can effectively alleviate the abnormal expression of Sirt3 in WT-HFD metabolic syndrome model mice, promote the expression of GLP-1 protein, and promote the phosphorylation of key molecules in the IRS-1 / PI3K / AKT insulin signaling pathway. However, the above therapeutic effects of SA were significantly inhibited in SIRT3 KO mice.

[0031] (4) The present invention compares the experimental results of Sirt3-KO mice and WT mice, indicating that Sirt3 is the key molecule for sennoside A to exert the above therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figures are the test results of mouse body weight and blood lipids; A. Dynamic weight monitoring of mice; B. Cholesterol (mmol / L); C. Low-density lipoprotein (mmol / L); D. Triglyceride (mmol / L); n=8 / group, data are expressed as mean±SD, *P<0.05, ns means not significant.

[0033] Figure 2 The figure is the test result of fasting blood glucose of mice; the data are expressed as mean±SD, ****P<0.0001, *P<0.05.

[0034] Figure 3 The figure is the test result of fasting insulin in mice; the data are expressed as mean±SD, ****P<0.0001, *P<0.05.

[0035] Figure 4 The figure is the detection result of fasting C-peptide in mice; the data are expressed as mean±SD, *P<0.05, ns means not significant.

[0036] Figure 5 The results of the test for the mouse insulin sensitivity index HOMA-IR are shown in Figure 1. The data are expressed as mean±SD, ****P<0.0001, *P<0.05, and ns means no significance. (n=8)

[0037] Figure 6 This is a graph showing the results of a glucose tolerance test in mice.

[0038] Figure 7 The figure shows the area under the glucose curve of the glucose tolerance test in mice; the data are expressed as mean±SD, ****P<0.0001, *P<0.01.

[0039] Figure 8This is a graph showing the results of the mouse insulin tolerance test; the data are expressed as mean±SD, ****P<0.0001, **P<0.01.

[0040] Fig. 9 The figure is the detection result of mouse plasma GLP-1; the data are expressed as mean±SD, *P<0.01, ns means not significant.

[0041] Fig.10 This is the result of H&E staining of mouse colon tissue;

[0042] Fig.11 The results of Sirt3 and GLP-1 mRNA expression levels are shown in Figure 2; A: GLP-1 mRNA expression, B: Sirt3 mRNA expression; each group was normalized using WT-CON as the standard; data are expressed as mean±SD, ****P<0.0001; n means not significant.

[0043] Fig.12 The results of the expression levels of Sirt3, GLP-1, and IRS-1 / PI3K / AKT insulin signaling pathway proteins in mouse colon; relative expression of AP-IRS1 / IRS1; relative expression of BP-PI3K / PI3K; relative expression of CP-AKT / AKT; D. Sirt3 protein expression; E. GLP-1 protein expression. All groups were normalized with WT-CON as the standard; data are expressed as mean±SD, **P<0.01, ****P<0.0001, ns is not significant.

[0044] Fig.13 SA improves the body weight, fasting blood glucose and insulin levels of HFD mice; A. Body weight; B. Fasting blood glucose; C. Fasting plasma insulin; D. HOMA-IR index. Data are expressed as mean±SD (n=6). **P<0.01; **P<0.05vs NCD. #P<0.05vs HFD.

[0045] Fig.14 This is a comparison chart of the body shapes of mice in each group after the experiment;

[0046] Fig.15SA improved glucose homeostasis and circulating GLP-1 levels in HFD mice. A. OGTT blood glucose (2 g / kg body weight); B. OGTT glucose area under the curve; C. Fasting plasma GLP-1. Data are expressed as mean±SD (n=6). ***P<0.001. ##P&<0.01, ns=not significant vs HFD. OGTT and body weight data were analyzed by two-way analysis of variance (two-way ANOVA). Fasting blood glucose, fasting insulin, HOMA-IR, blood glucose area under the curve (AUC) and plasma GLP-1 levels were analyzed by Student's t test.

[0047] Fig.16 SA regulates colonic mitochondrial ROS level and SOD activity in HFD mice. A. Mitochondrial ROS level in colonic tissue; B. Total SOD activity in mouse colonic tissue; C. MnSOD activity in mouse colon; D. MnSOD / total SOD activity ratio. Data are expressed as mean±SD (n=6). ****P<0.0001; ***P<0.001; *P<0.05vs NCD. ###P<0.001; ##P<0.01vs HFD.

[0048] Fig.17 SA regulates the expression of SIRT3 protein, acetylated MnSOD (acetyl-K68, -K122) and Bcl-2 / Bax / Cleaved caspase-3 mitochondrial apoptosis signaling pathway proteins in the colon of HFD mice. The expression of each protein relative to β-actin. Data are expressed as mean±SD (n=6). ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05vs NCD. ###P<0.001; ##P<0.01; #P<0.05; ns=not significant vs HFD.

[0049] Fig.18 This is the result of HE staining of mouse colon. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0051] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0052] Example 1

[0053] This example provides an analysis of the effect of sennoside A in treating metabolic syndrome.

[0054] (1.1) Experimental animals

[0055] 8-week-old male C57BL / 6N SIRT3 knockout mice were constructed with the assistance of Saiye (Suzhou) Biotechnology Co., Ltd., and a complete knockout mouse model of Sirt3 gene was established by using CRISPR / cas-mediated genome engineering technology. All mice were identified as SIRT3 KO homozygotes (- / -) by PCR genotyping. At the same time, 8-week-old male C57BL / 6N wild-type mice (WT) were also provided by Saiye (Suzhou) Biotechnology Co., Ltd., license number SCXK (Su) 2018-0003. All mice were raised in the Experimental Animal Center of the Affiliated Municipal Hospital of Traditional Chinese Medicine of Shanghai University of Traditional Chinese Medicine, under temperature (22±2℃), humidity (60±5%), and 12h dark / light cycle conditions.

[0056] (1.2) Animal model establishment and grouping method

[0057] C57BL / 6N WT mice (hereinafter referred to as "WT mice") and C57BL / 6N SIRT3 KO mice (hereinafter referred to as "KO mice") were divided into two groups each.

[0058] The mice in the control group (CON group) were fed with experimental mouse maintenance diet (n=8+8, 13.5% of calories came from fat). The mice in the model group (n=20+20) were fed with a high-fat diet with 60% fat energy supply for 8 weeks. After the mice in the model group were fed with a high-fat diet for 8 weeks, 30 mg / kg streptozotocin (STZ) was intraperitoneally injected under fasting conditions. After the injection, sufficient drinking water and feed were provided. Five days later, the mice underwent fasting blood glucose (FBG) testing. When FBG was greater than 7.8 mmol / L, it was considered that the T2DM model was successfully established (n=16+16). The mice with successfully established T2DM model were randomly divided into two groups: ① HFD group (HFD, n=8+8); ② SA administration group (SA, n=8+8). Thereafter, the mice in the control group continued to be fed with maintenance diet, and the HFD group and SA group were fed with a high-fat diet.

[0059] Among them, the experimental mouse maintenance feed (101009, SPF grade, 5.2% fat, 61.0% carbohydrates, 18.8% protein) and high-fat feed (XTHF60, purified SPF grade, 60% fat, 20% carbohydrates, 20% protein) were purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. and sterilized by irradiation; SA was administered by gavage at a dose of 35 mg / kg / d for 8 weeks.

[0060] The methods for establishing the animal model, the screening of successful modeling animals, and the drug administration intervention measures are shown in Table 1:

[0061] Table 1 Summary of drug administration interventions

[0062]

[0063] (1.3) Experimental methods

[0064] 1.3.1) Body weight, blood sugar, OGTT, ITT test

[0065] After the SA treatment started, the body weight of mice in each group was recorded every 2 weeks, and each mouse was weighed 3 times, and the average value was taken and the decimal point was taken as the body weight value. The mice were fasted overnight (12h) and the fasting blood glucose level was measured after the tail vein blood was collected. After 8 weeks of SA treatment, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed.

[0066] Among them, the OGTT experiment was conducted after the mice were fasted for 12 hours. The mice were gavaged with 2g / kg of glucose, and the blood glucose levels at 0 minutes, 15 minutes, 30 minutes, 60 minutes and 120 minutes were measured by Roche One Touch blood glucose meter from the tail vein.

[0067] On the day of the ITT experiment, the subjects were fasted for 4 hours, and Novolin 30R insulin injection was prepared into 0.1U / ml solution and injected intraperitoneally at a dose of 0.1ml / kg. The blood glucose levels were measured and recorded at 0min, 15min, 30min, 60min, and 120min after injection.

[0068] 1.3.2) Fasting GLP-1, insulin, and C-peptide ELISA tests

[0069] After the mice rested for 1 week, they were anesthetized by intraperitoneal injection of pentobarbital (35 mg / kg). Blood was then collected by removing the eyeballs to detect fasting GLP-1 and insulin. Heparin anticoagulant tubes containing ethylenediaminetetraacetic acid (EDTA) and dipeptidyl peptidase 4 inhibitor (Vildagliptin) (1 mM) were prepared before the experiment. The mouse blood was collected in the anticoagulant tubes, and the plasma was collected after centrifugation at 3000g at 4°C for 10 min and stored in a -80°C refrigerator. The levels of GLP-1, insulin, and C-peptide in plasma were detected using a double antibody sandwich ELISA kit. The ELISA experiment was carried out step by step according to the instructions provided by the ELISA kit manufacturer, and then the absorbance of the enzyme-linked immunosorbent assay system was measured at a wavelength of 450nm using a BioTek Synergy 2 microplate reader.

[0070] 1.3.3) Calculation of insulin sensitivity index HOMA-IR

[0071] The insulin sensitivity index HOMA-IR was calculated based on fasting blood glucose and fasting insulin using the formula HOMA-IR = [fasting insulin (mIU / L) × fasting blood glucose (mmol / L) / 22.5].

[0072] 1.3.4) Tissue sample collection

[0073] After blood collection, mice were killed by cervical dislocation and colon tissue was obtained. Some colon tissues were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator; some colon tissues were fixed in 4% paraformaldehyde solution and subsequently subjected to HE staining experiments. The animal experiment process was approved by the Experimental Animal Ethics Committee of the Shanghai University of Traditional Chinese Medicine Affiliated Hospital of Traditional Chinese Medicine.

[0074] 1.3.5) HE staining to observe the colon structure of mice

[0075] The colon tissue was quickly sliced ​​on ice, fixed with paraformaldehyde, and then dehydrated with gradient ethanol, embedded in epoxy resin and ultrathin sectioned to prepare electron microscopic samples. The ultrastructure of mouse colon mitochondria was observed using a biological transmission microscope and stained with hematoxylin and eosin.

[0076] 1.3.6) Western blot analysis of protein expression

[0077] 1) Sample protein extraction: Take fresh frozen mouse colon tissue, remove surrounding fat, wash with ice-cold 1×PBS 3 times, cut the colon tissue into small pieces with scissors, put in 2 steel beads, add 200μL WB lysis buffer, set 60HZ, 60s tissue crusher to crush. Then put it in a pre-cooled centrifuge at 4 degrees, 12000rpm / min, centrifuge for 15min, and take the supernatant for testing.

[0078] 2) Protein quantification: Follow the BCA protein quantification instructions, detect the OD value at 562nm on a microplate reader, calculate the sample protein concentration based on the standard curve, and configure the protein system so that the sample protein concentration reaches 2-3μg / μL.

[0079] 3) Protein denaturation: Add 5× loading buffer to the mixed protein sample according to each ratio, heat in a 100°C metal bath for 10 min to denature the protein, and then the sample to be tested is obtained.

[0080] 4) Electrophoresis: Prepare separation gel and concentration gel according to the instructions. Load 20 μg of sample protein to be tested into each well, run electrophoresis at 80V to the lower separation gel, then adjust the voltage to 160V until the loading buffer runs to the bottom of the gel.

[0081] 5) Transfer: Make a "sandwich" of the activated PVDF membrane and gel and put it into the transfer box. Keep the transfer tank in an ice bath and transfer the membrane at a constant current of 400 mA for 60 minutes.

[0082] 6) Blocking: After transfer, wash the membrane with TBST for 5 minutes and place it in fast and efficient blocking solution for 15 minutes.

[0083] 7) Primary antibody incubation: Take out the blocked membrane, add primary antibodies respectively, and place in a shaker at 4°C refrigerator overnight.

[0084] 8) Secondary antibody incubation: Wash the membrane with washing buffer for 10 minutes × 3 times; add HRP-labeled secondary antibody of the corresponding species, incubate at room temperature for 1 hour, and repeat the washing step.

[0085] 9) Development: Take out the PVDF membrane containing the sample protein, add an appropriate amount of ECL chemiluminescent detection agent, and use a developer to obtain the image.

[0086] 1.3.7) qt-PCR analysis of changes in mRNA of related factors

[0087] 1) Total RNA extraction: Take 15-20 mg of colon tissue, add 600 μl of lysis buffer, and homogenize with a micro-electric homogenizer. Centrifuge at 14000×g for 2 minutes, transfer the supernatant to a new centrifuge tube, and then extract total RNA according to the instructions of the centrifugal column RNA extraction kit.

[0088] 2) Reverse transcription:

[0089] The reaction system is configured as shown in Table 2:

[0090] Table 2 Reaction system

[0091]

[0092]

[0093] Mix the reaction system and then centrifuge to precipitate the liquid. Reverse transcription conditions: incubate at 42℃ for 10min, incubate at 80℃ for 10min, and 4℃ forever. The cDNA of the reaction product is stored in a -20℃ refrigerator.

[0094] 3) Real-time quantitative PCR:

[0095] The reaction system shown in Table 3 was configured, wherein at least three replicate wells were made for each gene.

[0096] Table 3 Reaction system

[0097]

[0098] The reaction procedure is as follows:

[0099] a. Pre-denaturation: 95℃ for 2min;

[0100] b. Denaturation: 95℃ for 15sec;

[0101] c. Annealing / extension: 60℃ for 15-30 seconds;

[0102] d. Repeat steps b and c for a total of 40 cycles;

[0103] e. Melting curve analysis (optional): 95℃15sec, 60℃15sec, 95℃15sec.

[0104] 4) Result processing: The relative expression of the target gene was calculated using the 2-ΔΔCt method. ΔΔCt = (Ct value of target gene in sample A - Ct value of internal reference gene in sample A) - (Ct value of target gene in sample B - Ct value of internal reference gene in sample B).

[0105] Among them, sample A and sample B refer to different control group samples in the same group.

[0106] 5) The detection primer sequences are shown in Table 4:

[0107] Table 4 Primer sequence summary

[0108]

[0109]

[0110] 1.3.8) Statistical analysis

[0111] The data obtained in the experiment were expressed as mean ± SD and analyzed using Graphpad 9.2 statistical software. One-way ANOVA was used to compare samples among multiple groups, and the rest were analyzed using Student's T test. P < 0.05 was considered statistically significant.

[0112] 1.4) Experimental results

[0113] 1.4.1) Mouse body weight and blood lipid detection

[0114] After the T2DM mice were successfully modeled, they were treated with SA by intragastric administration. The body weight of the mice was monitored every 2 weeks thereafter. Figure 1As shown in A and Table 5, the mice in the WT-CON and KO-CON groups were normal in appearance, responsive, and had moderately dry and loose feces. The mice in the WT-HFD and KO-HFD groups were obese, with significantly higher body weight than the corresponding two CON groups, slow in movement, dry in fur, and green in feces with moderately dry and loose feces. The mice in the WT-SA and KO-SA groups were obese in appearance. Although they still ate a high-fat diet after SA gavage treatment, their weight was controlled, their reactions were responsive, their fur was slightly dry, and their feces were loose. At the same time, Figure 1 B. Figure 1 C. Figure 1 D and Table 6, the cholesterol, triglyceride, and low-density lipoprotein indexes of mice in the WT-KFD group and KO-HFD group were significantly higher than those of the corresponding WT-CON group and KO-CON group. SA can significantly improve hypercholesterolemia and elevated low-density lipoprotein in mice, but has no significant therapeutic effect on abnormal triglycerides in mice. There was no significant difference in the therapeutic effect of SA between the KO-SA group and the WT-SA group.

[0115] Table 5 Mouse weight monitoring

[0116]

[0117] Table 6 Blood lipid detection in mice

[0118]

[0119]

[0120] 1.4.2) Fasting blood glucose in mice

[0121] After 8 weeks of SA treatment, the fasting blood glucose of mice in each group was tested. Figure 2 As shown in Table 7, the blood glucose levels of mice in the WT-HFD group and the KO-HFD group were significantly higher than those of the corresponding WT-CON group and the KO-CON group. The blood glucose levels of mice in the WT-SA group were significantly lower than those in the WT-HFD group, indicating that SA gavage treatment can improve the elevated fasting blood glucose levels in T2DM mice. At the same time, the therapeutic effect of SA on the fasting blood glucose levels of HFD mice in the KO group was significantly inhibited. Although there was still improvement, the improvement effect was far less significant than that of SA in the WT group.

[0122] Table 7 Fasting blood glucose in mice

[0123]

[0124] 1.4.3) Fasting insulin, fasting C-peptide and insulin sensitivity index HOMA-IR in mice

[0125] After 8 weeks of SA treatment, fasting insulin and fasting C-peptide were detected in each group of mice. Figure 3 As shown in Table 8, the fasting insulin of mice in the WT-HFD group and the KO-HFD group was significantly higher than that of the corresponding WT-CON group and the KO-CON group, indicating that HFD mice had obvious abnormal insulin secretion, and long-term excessive insulin secretion was an important factor in inducing insulin resistance. The fasting insulin of mice in the WT-SA group was significantly lower than that of the WT-HFD group, indicating that SA gavage treatment can improve the abnormal insulin secretion of HFD mice. At the same time, the therapeutic effect of SA on abnormal insulin secretion in T2DM mice in the KO group was also inhibited.

[0126] C-peptide is also secreted by pancreatic β cells and has a common precursor with insulin. Testing the level of C-peptide can indirectly reflect the level of insulin secretion. Figure 4 As shown in Table 8 , the fasting C-peptide levels of mice in each group were detected, and the results showed that the therapeutic effect of SA on abnormal C-peptide secretion in HFD mice was also inhibited by SIRT3 knockout.

[0127] The insulin sensitivity index HOMA-IR is an indicator used to evaluate an individual's insulin resistance level. Figure 5 As shown in the results, SA could effectively improve the insulin resistance level of HFD mice, while SIRT3-KO significantly reduced the therapeutic effect of SA.

[0128] Table 8 Fasting insulin, fasting C-peptide and HOMA-IR in mice

[0129]

[0130]

[0131] 1.4.4) OGTT and ITT assays in mice

[0132] The glucose tolerance test (OGTT) is a glucose load test that can evaluate the body's ability to regulate blood sugar. Figure 6As shown in Table 9, the blood glucose levels of mice in the WT-CON and KO-CON groups dropped rapidly after oral administration of glucose, and the blood glucose levels were close to the fasting state at 120 minutes after taking the glucose. The fasting blood glucose levels of mice in the WT-HFD and KO-HFD groups were significantly increased, and oral glucose tolerance was significantly impaired, as shown by the significantly higher blood glucose levels at 15 minutes, 30 minutes, 60 minutes, and 120 minutes after oral glucose than those in the normal diet group. The fasting blood glucose levels of mice in the WT-SA administration group were lower than those in the model group, and the blood glucose levels at 15 minutes, 30 minutes, 60 minutes, and 120 minutes after the glucose tolerance test were lower than those in the WT-HFD group, indicating that SA can improve OGTT. However, there was no significant improvement in OGTT in the KO-SA group compared with the KO-HFD group, indicating that Sirt3-KO antagonized the therapeutic effect of SA. At the same time, the area under the OGTT glucose curve (Area undercurve, AUC) also proved this point (such as Figure 7 shown).

[0133] Table 9 Mouse OGTT test

[0134]

[0135] The insulin tolerance test (ITT) is a test of blood sugar changes and insulin sensitivity by intraperitoneal injection of insulin. The insulin resistance state with reduced insulin sensitivity is a typical symptom of type 2 diabetes. Figure 8 As shown in Table 10, the ITT test of mice in the WT-HFD group and the KO-HFD group showed that the insulin sensitivity was significantly impaired. After 8 weeks of SA gavage treatment, the ITT tolerance test of mice in the WT-SA group was significantly improved, indicating that SA can significantly improve the insulin sensitivity of HFD mice. However, the therapeutic effect of SA on mice in the KO group was significantly inhibited.

[0136] Table 10 Mouse ITT test

[0137]

[0138] 1.4.5) Mouse plasma GLP-1 detection

[0139] Glucagon-like peptide-1 (GLP-1) can inhibit the secretion of glucagon and delay gastric emptying, reducing food intake through central appetite suppression, thereby achieving the effect of lowering blood sugar. Fig. 9 As shown in the figure, after 8 weeks of SA treatment, ELISA was used to detect GLP-1 in mouse plasma, and the results showed that SA could improve the abnormal secretion of GLP-1 in HFD mice and promote the secretion of GLP-1. However, the therapeutic effect of SA on mice in the KO-CON, KO-HFD, and KO-SA groups was significantly inhibited.

[0140] 1.4.6) HE staining to observe the effect of sennoside A on colon tissue pathological morphology

[0141] like Fig.10 From the HE staining photos of mouse colon, we can see that compared with the WT-CON and KO-CON mice, the WT-HFD and KO-HFD mice showed morphological changes such as loss of colon mucosal contents, epithelial cell shedding, and mucosal gland atrophy; the colon epithelium of the WT-SA group was relatively smooth and complete, the glands were arranged more neatly, and the number of atrophic glands was significantly reduced; while the treatment effect of the KO-SA group was not obvious compared with the KO-HFD group.

[0142] 1.4.7) qtPCR detection of Sirt3 and GLP-1 mRNA expression levels in mouse colon

[0143] like Fig.11 As shown, SA could significantly improve the decreased mRNA expression of Sirt3 and GLP-1 molecules in HFD mice, while the therapeutic effect of SA on the abnormal expression of GLP-1 mRNA in Sirt3-KO mice was significantly inhibited.

[0144] 1.4.8) Western Blot detection of Sirt3, GLP-1, IRS-1 / PI3K / AKT insulin signaling pathway protein expression levels in mouse colon

[0145] The above results prove that SA can effectively improve the insulin sensitivity of HFD mice and improve the impaired GLP-1 secretion of HFD mice, and this therapeutic effect is closely related to the SIRT3 gene. Next, we detected the expression levels of Sirt3, GLP-1, and IRS-1 / PI3K / AKT insulin signaling pathway proteins in mice ( Fig.12 A left figure is a schematic diagram of protein bands). Fig.12 As shown in (AE), SA gavage treatment can effectively alleviate the abnormal expression of Sirt3 in HFD diabetic model mice, promote the expression of GLP-1 protein, and promote the phosphorylation of key molecules in the IRS-1 / PI3K / AKT insulin signaling pathway. At the same time, it can be seen that the three groups of Sirt3-KO mice have no sirt3 protein expression ( Fig.12 D), while the above therapeutic effects of SA were inhibited in KO mice ( Fig.12 AC and Fig.12 E).

[0146] In summary, the present example found that SA can improve the glucose and lipid metabolism of high-fat diet (HFD) type II diabetes model mice, inhibit obesity, and improve hypercholesterolemia and elevated low-density lipoprotein; SA can improve fasting blood glucose, fasting insulin, fasting C-peptide, and plasma GLP-1 abnormalities in HFD diabetes model mice, and improve OGTT, ITT, and HOMA-IR insulin sensitivity; and the above therapeutic effects of SA are significantly inhibited in SIRT3 KO mice; SA can effectively alleviate the abnormal expression of Sirt3 in WT-HFD diabetes model mice, promote GLP-1 protein expression, and promote the phosphorylation of key molecules in the IRS-1 / PI3K / AKT insulin signaling pathway, and the above therapeutic effects of SA are significantly inhibited in KO group mice; by comparing the experimental results of KO group mice and WT group mice, it is shown that Sirt3 is the key molecule for sennoside A to exert the above therapeutic effects.

[0147] Example 2

[0148] This example provides a discussion on the mechanism of sennoside A protecting enteroendocrine L cells in the treatment of T2DM based on SIRT3.

[0149] (2.1) Experimental animals

[0150] 8-week-old male C57BL / 6J mice (SPF grade) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. Mice were raised at the Shanghai University of Traditional Chinese Medicine Laboratory Animal Center under the conditions of temperature (22±2°C), humidity (60±5%), and 12h dark / light cycle. The establishment method of the animal model, the screening of successfully modeled animals, and the drug intervention measures are shown in Table 11.

[0151] (A) Animal grouping method:

[0152] (A1) Mice in the control group (Normal Chow's diet, NCD group) were fed with normal chow (n=6, 13.5% of calories came from fat).

[0153] (A2) HFD mice (n=20) were fed a high-fat diet (60% of calories from fat) for 12 weeks.

[0154] (A3) Fasting blood glucose (FBG) was tested in HFD mice, and a T2DM model was successfully established when FBG was greater than 7.8 mmol / L (n=12).

[0155] (A4) HFD-induced T2DM mice were randomly divided into 2 groups:

[0156] ①HFD group (HFD, n = 6)

[0157] ②SA administration group (SA, n=6).

[0158] Based on the results of previous studies on the effects of different oral doses of SA on improving insulin resistance in mice, SA was administered by oral gavage at a dose of 30 mg / kg / d for 8 weeks.

[0159] Table 11 Establishment and grouping of animal models

[0160]

[0161] (B) Sample collection:

[0162] At the end of SA treatment, mice were anesthetized by intraperitoneal injection of pentobarbital (35 mg / kg). Blood was then collected by orbital bleeding, and the mice were killed by cervical dislocation to obtain colon tissue. Part of the colon tissue was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. Part of the colon tissue was fixed in 4% paraformaldehyde solution and subsequently stained with H&E. Part of the fresh colon tissue was placed on ice for subsequent mitochondrial extraction and ROS and SOD activity detection. The animal experiment process was approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM201009001).

[0163] (2.2) Experimental methods

[0164] 2.2.1) Weight measurement, fasting blood glucose test and OGTT test

[0165] During SA treatment, the weekly weight changes of mice in each group were recorded, and each mouse was weighed 3 times, and the average value was taken and the decimal point was taken as the weight value. After the mice fasted overnight (12h), the tail vein blood was taken to measure the fasting blood glucose level. The oral glucose tolerance test (OGTT) was performed after the mice fasted for 12 hours. The mice were gavaged with 2g / kg of glucose, and the tail vein blood was taken to measure the blood glucose level at 0 minutes, 15 minutes, 30 minutes, 60 minutes and 120 minutes using Roche One Touch blood glucose meter.

[0166] 2.2.2) Calculation of fasting insulin and insulin sensitivity index HOMA-IR

[0167] Blood samples were collected from mice after fasting for 8 hours to detect fasting insulin. The blood of mice was collected in EP tubes containing ethylenediaminetetraacetic acid (EDTA) and dipeptidylpeptidase-4 inhibitor (DPP-4I) (1 mM), and plasma was collected after centrifugation at 3000g at 4°C for 10 minutes and stored in a -80°C refrigerator.

[0168] The plasma insulin level was detected using a double antibody sandwich ELISA kit according to the instructions provided by the reagent manufacturer, and then the absorbance of the enzyme-linked immunosorbent assay system was measured at a wavelength of 450 nm using a BioTek Synergy 2 microplate reader.

[0169] The insulin sensitivity index HOMA-IR was calculated based on fasting blood glucose and fasting insulin using the formula HOMA-IR = [fasting insulin (mIU / L) × fasting blood glucose (mmol / L) / 22.5].

[0170] 2.2.3) Plasma GLP-1 detection

[0171] After fasting for 8 hours, the mice were bled by orbital sampling into EP tubes containing EDTA and DPP-4I (1 mM), and the plasma was collected after centrifugation at 3000g for 10 minutes at 4°C and stored at -80°C. The level of GLP-1 in plasma was detected using a double antibody sandwich ELISA kit. The experimental process followed the instructions provided by the reagent manufacturer, and then the absorbance of the enzyme-linked immunosorbent assay system was measured at a wavelength of 450 nm using a BioTek Synergy 2 microplate reader.

[0172] 2.2.4) ROS detection

[0173] Colon mitochondria were isolated according to the instructions of the tissue mitochondria isolation kit: After the mice were killed, fresh colon tissue was taken and placed on ice. A small piece of colon was cut, and about 100 mg of colon tissue was weighed in a 1.5 ml EP tube, and washed with pre-cooled PBS on ice. The tissue was placed in an EP tube on ice and cut into small tissue fragments with scissors. 10 volumes of pre-cooled PBS were added and ice bathed for 3 minutes. 600g centrifuged for 10 to 20 seconds to precipitate the tissue sample and discard the supernatant. 8 volumes of pre-cooled trypsin digestion solution prepared in the kit were added and ice bathed for 20 minutes. 600g centrifuged for 10 to 20 seconds to precipitate the tissue sample and discard the supernatant. 2 volumes of mitochondrial isolation reagent prepared in the kit were added to resuspend the tissue to wash away residual trypsin. 600g centrifuged for 10 to 20 seconds to precipitate the tissue sample and discard the supernatant. 8 volumes of pre-cooled mitochondrial isolation reagent were added and homogenized with a glass homogenizer on an ice bath for 20 to 30 times. Centrifuge the homogenate at 600g and 4℃ for 5 minutes. Transfer the supernatant to another EP tube and centrifuge at 11,000g and 4℃ for 10 minutes. Remove the supernatant and the precipitate is the isolated mitochondria. Resuspension of mitochondrial samples: Resuspend mitochondria with the mitochondrial storage solution in the mitochondrial isolation kit to obtain a mitochondrial suspension; determine the protein concentration of the mitochondrial suspension sample by the BCA method, and adjust the protein concentration of each sample to 3mg / ml.

[0174] DCFH-DA probe loading (Cat. No. S0033, Bio-Technology): Dilute the DCFH-DA dye probe with the mitochondrial storage solution in the mitochondrial isolation kit at a ratio of 1:1000 to a final concentration of 10mM. Loading probe: Take 1 volume of mitochondrial suspension sample, add an equal volume of diluted DCFH-DA, and incubate in a cell culture incubator at 37°C for 20 minutes. Invert and mix every 3-5 minutes to allow the probe to fully contact the mitochondria. Wash the mitochondria three times with mitochondrial storage solution to fully remove the DCFH-DA that has not entered the mitochondria. Centrifuge at 600g, 4°C for 3 minutes, and take the precipitate to obtain the mitochondria loaded with the probe. Detection of relative fluorescence intensity: Resuspend the mitochondrial sample loaded with the probe in one volume of mitochondrial storage solution, and add 0.1ml of mitochondrial suspension sample to each well of the 96-well plate, that is, the mitochondrial protein content added to each well is 300μg. The fluorescence intensity of each sample was measured using a BioTek Synergy 2 microplate reader set to an excitation wavelength of 485 nm and an emission wavelength of 528 nm according to the manufacturer's instructions. The results of ROS content were expressed as relative fluorescence intensity.

[0175] 2.2.5) SOD activity detection

[0176] The total SOD and MnSOD activities of mouse colon tissue were detected using the CuZn / Mn-SOD activity detection kit (WST-8 method) (Cat. No. S0103, Bio-Technology Co., Ltd.):

[0177] The total SOD and MnSOD activities of mouse colon tissue were detected using the CuZn / Mn-SOD activity detection kit (WST-8 method) (Cat. No. S0103, Bio-Technology Co., Ltd.):

[0178] (A) Sample preparation: Take an appropriate amount of colon tissue sample, add PBS precooled at 4°C or in an ice bath, cut it into small pieces with scissors at 4°C or in an ice bath, and homogenize it with a glass homogenizer. Centrifuge the homogenate at 4°C and take the supernatant as the sample to be tested. Determine the protein concentration of each sample by the BCA method. After determining the SOD activity units of each sample, the protein concentration of the tissue homogenate is used for homogenization.

[0179] Inhibition of Cu / Zn-SOD: Mix Cu / Zn-SOD inhibitor A and appropriately diluted samples in a centrifuge tube at a volume ratio of 1:24 and incubate at 37°C for 1 hour; add 20μl of Cu / Zn-SOD inhibitor B to 780μl ultrapure water and mix well, i.e., dilute 40 times. Then mix the 40-fold diluted Cu / Zn-SOD inhibitor B and the above mixture at a volume ratio of 1:25 and incubate at 37°C for another 15 minutes. Then measure the SOD activity.

[0180] (B) Sample determination and calculation of SOD activity units: The SOD content in the sample was determined according to the manufacturer's instructions of the kit and a blank control was set up, and the absorbance was measured at 450 nm.

[0181] Calculation of SOD activity in samples: ① Calculate the inhibition percentage: Inhibition percentage = (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100%. ② When the inhibition percentage in the xanthine oxidase coupling reaction system is 50%, the SOD enzyme activity in the reaction system is defined as one enzyme activity unit. ③ The calculation formula for SOD enzyme activity: SOD enzyme activity unit in the sample to be tested = inhibition percentage / (1 - inhibition percentage) IU.

[0182] (C) According to the protein concentration and dilution factor of the sample, the SOD activity unit was converted into IU / mg protein.

[0183] 2.2.6) Detection of acetylated MnSOD

[0184] Western blot detection of acetylated MnSOD (acetyl-K68, -K122) levels:

[0185] The colon tissue was lysed in ice-cold Western and IP lysis buffer. Protein samples were separated on 12% SDS-PAGE gel and transferred to a 0.22 μm PVDF membrane. Blocked with 5% bovine serum albumin (BSA) solution and incubated with primary antibody at 4°C overnight. Wash the PVDF membrane with TBST solution for 5 min × 4 times. Incubate with fluorescent dye-labeled secondary antibody (anti-mouse IgG, #5470; anti-rabbit IgG, #5151, Cell Signaling Technology) for 1 hour.

[0186] Signals were detected using the LI-COR Odyssey CLx imaging system. Protein immunoblots were quantified using ImageJ v2.1.0 software.

[0187] 2.2.7) Detection of SIRT3 and mitochondria-mediated apoptosis signaling pathway

[0188] Western blot was used to detect the expression levels of Bcl-2 / Bax / Cleaved caspase-3 signaling pathway proteins. The primary antibodies used were as described in step 1.3.6) of Example 1, and the rest of the methods were the same as those in step 2.3.3) of this Example.

[0189] 2.2.8) Histochemical analysis

[0190] After the mice were killed, 1 cm colon segments were taken and fixed in 4% paraformaldehyde solution for 48 hours. The fixed tissue blocks were routinely embedded in paraffin. Dehydrated in gradients of 50%, 70%, 80%, 90%, 95% and anhydrous ethanol, transparentized with xylene, immersed in wax, embedded, and sliced ​​at 5 μm. The slices were dried, stained with hematoxylin for 5 minutes, washed with tap water for 5 minutes, differentiated with hydrochloric acid and ethanol for 30 seconds, and washed with tap water 3 times, each time for 5 minutes. Eosin staining for 2 minutes. The slices were dehydrated, transparent, and sealed with neutral resin, and observed and photographed under a Zeiss AxioLab 5 optical microscope.

[0191] (2.3) Results

[0192] 2.3.1) In this example, the body weight, fasting blood glucose (FBG) and fasting insulin of mice in the Chow's diet control group (NCD, n=6), high-fat diet control group (HFD, n=6) and SA-administered high-fat diet group (SA, n=6) were measured.

[0193] During the SA treatment period, the mice in each group were weighed weekly. Compared with the HFD control group, the SA group significantly reduced the weight gain of mice ( Fig.13 A, Fig.14 ). Comparison of FBG between the HFD control group and the NCD group ( Fig.13 B) and fasting insulin values ​​( Fig.13 C), the results showed that hyperglycemia and insulin resistance (IR) symptoms were established in the model. SA improved fasting blood glucose ( Fig.13 B) and lower fasting insulin ( Fig.13 C), thereby improving the insulin sensitivity index of HOMA-IR ( Fig.13 D).

[0194] 2.3.2) OGTT showed that SA stabilized postprandial hyperglycemia ( Fig.15 A) and improve the glucose area under the curve (AUC) ( Fig.15 B). GLP-1 inhibits the secretion of glucagon and can delay gastric emptying, reducing food intake through central appetite suppression, thereby achieving the effect of lowering blood sugar. It has the effect of significantly reducing weight and increasing postprandial insulin secretion. ELISA results show that the level of circulating GLP-1 has been restored to a certain extent after 8 weeks of SA administration ( Fig.15 C).

[0195] 2.3.3) Mitochondria are the main source of intracellular ROS. This example detected the ROS level and SOD activity of mitochondria isolated from the colon of each group (n=6) of mice. Fig.16A) and total SOD activity ( Fig.16 B) increased, MnSOD activity ( Fig.16 C) decreased, and the ratio of MnSOD / total SOD activity also decreased ( Fig.16 D). SA suppressed ROS levels compared with HFD ( Fig.16 A) and total SOD activity ( Fig.16 B) increased, increasing the activity of Mn-SOD ( Fig.16 C), increased the ratio of MnSOD / total SOD activity.

[0196] 2.3.4) Western blot results showed that SIRT3 decreased in the colon of HFD mice, and SA treatment could partially correct this phenomenon. The levels of MnSOD (K68-Acetyl) and MnSOD (K122-Acetyl) in the colon of HFD mice were significantly increased, while the increase in the SA group was limited ( Fig.17 ).

[0197] The levels of pro-apoptotic Bax and cleaved Caspase-3 were increased, and the level of anti-apoptotic Bcl-2 was decreased in the HFD group ( Fig.17 ); SA treatment weakened the expression of this apoptosis signaling pathway protein ( Fig.17 ).

[0198] 2.3.5) Activation of Caspase-3 is involved in the apoptotic cell clearance mechanism of pathological intestinal epithelial shedding. To prove the activation of Caspase-3 in the colon, the colons of mice in each group were examined histologically, and it was observed that SA improved the morphological changes of colon mucosal content loss, epithelial cell shedding, and mucosal gland atrophy in HFD mice ( Fig.18 ).

[0199] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of sennoside A in the preparation of anti-metabolic syndrome drugs and / or health products.

2. The use according to claim 1, characterized in that: The metabolic syndrome is a metabolic syndrome caused by a high-fat diet.

3. The use according to claim 1, characterized in that: The anti-metabolic syndrome drug and / or health product is a drug and / or health product for resisting hyperinsulinemia.

4. The use according to claim 1, characterized in that: Anti-metabolic syndrome drugs and / or health products are anti-low-density lipoprotein drugs and / or health products.

5. The use according to claim 1, characterized in that: Anti-metabolic syndrome drugs and / or health products are drugs and / or health products against type 2 diabetes.

6. The use according to claim 1, characterized in that: Anti-metabolic syndrome drugs and / or health products are drugs and / or health products against insulin resistance.

7. The use according to claim 1, characterized in that: The medicine and / or health product contains one or more pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipient is selected from one or more of glycerol, butylene glycol, trehalose, betaine, allantoin, carbomer, dextrin, starch, gel cellulose, methyl cellulose, sodium carboxymethyl cellulose or polyvinyl pyrrolidone.

9. The use according to claim 1, characterized in that: The dosage form of the medicine and / or health product is tablet, capsule, powder, granule, suspension or injection.

10. The use according to claim 1, characterized in that: The effective concentration of the sennoside A is 30-45 mg / kg.

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