CN112675165A - Application of ellagic acid and metabolite thereof as natural inhibitor in preparation of anti-cell apoptosis drug - Google Patents
Application of ellagic acid and metabolite thereof as natural inhibitor in preparation of anti-cell apoptosis drug Download PDFInfo
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- CN112675165A CN112675165A CN202110088079.8A CN202110088079A CN112675165A CN 112675165 A CN112675165 A CN 112675165A CN 202110088079 A CN202110088079 A CN 202110088079A CN 112675165 A CN112675165 A CN 112675165A
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- ellagic acid
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of ellagic acid and a metabolite thereof as a natural inhibitor in preparation of a medicine for resisting cell scorching. The invention finds that the high glycolipid has a promoting effect on the scorching of pancreatic cells and liver cells, and the expression levels of the scorching proteins GSDMD and GSDME in the scorching signal channel are increased, which indicates that the scorching plays a role in the high-glucose and high-lipid induced pancreatic and liver inflammatory reactions. GSDMD, GSDME mediated cell apoptosis has been implicated in diabetic pancreatic, liver injury. The invention discovers for the first time that ellagic acid and intestinal metabolite urolithin A thereof can inhibit GSDMD and GSDME mediated cell apoptosis in an in vivo experiment of a diabetes model mouse, thereby generating anti-inflammatory protection effect on cells.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of ellagic acid and a metabolite thereof as a natural inhibitor in preparation of a medicine for resisting cell scorching.
Background
Apoptosis is a newly discovered mode of programmed cell death. After the inflammasome is activated, caspase-1/4/5/11 is cut, the cut mature cysteine acts on gasdermin D (dermolide D, GSDMD), the formed GSDMD-N end forms a hole on a cell membrane through oligomerization, water inflow is generated, ion gradients inside and outside the cell membrane disappear, and the cell is swollen, osmotically dissolved and finally cracked to die.
More and more researches show that after cells are burnt and killed, the cells can form pores, and gradually expand until the cell membranes are ruptured, so that a large amount of inflammatory mediators in the cells are released, the inflammatory reaction is accelerated, and serious tissue damage is caused. Activated caspase1 in the process of focal death can convert Pro-IL-1 beta into active IL-1 beta, and can also promote GSDMD (glutathione S-methyl diphosphate) characteristic of focal death to be sheared into GSDMD-N with the effect of promoting focal death, thus completing the cell membrane perforating effect, inflammatory mediators IL-1 beta, IL-18 and cell contents are released to the outside of cells through cell membrane fissures to cause cascade inflammatory reaction, meanwhile, water molecules enter the cells through the cell membrane fissures, and the cells are swollen, ruptured and dead. Activated caspase3 also cleaves GSDME to GSDME-N to complete the process of pyro-death.
The chronic metabolic inflammatory reaction mediated by inflammatory factors plays an important role in the occurrence and development of chronic diseases, and the continuous secretion of the inflammatory factors of cells for a long time causes the cascade reaction of inflammation. Previous studies have shown that apoptosis of cells is associated with a variety of diseases, such as diabetes, atherosclerosis, diabetic nephropathy, acute lung injury, hyperuricemia, non-alcoholic fatty liver disease, psoriasis, myocardial ischemia reperfusion injury, viral hepatitis, and the like. In the case of diabetes, the inventor of the present application found that inflammatory signaling pathways are activated in diabetic mice to aggravate the development of diabetes, and mainly inflammatory factors such as TNF-alpha, CRP, IL-1 beta and the like are continuously at a slightly elevated level. Persistent hyperglycemia in diabetic patients activates NOD-like receptor protein 3(NLRP3) inflammasome, which in turn activates IL-1 β -mediated inflammatory responses upon caspase-1 activation, resulting in inflammation of the pancreas and damage to the pancreas. Therefore, the inhibition of pancreatic NLRP3/IL-1 beta signal is an important target signal for resisting islet beta cell apoptosis, resisting inflammation and protecting pancreas. However, the current clinical commonly used therapeutic drugs mainly delay diabetes by controlling blood sugar, and the long-term application of partial sulfonylurea hypoglycemic drugs even causes the exhaustion of insulin secretion, so that the research and development of natural drugs with pancreas protection effects are very necessary.
GSDMDM is the last co-effector protein downstream of inflammatory body activation, a key conserved gate protein involved in inflammation, is cleaved by caspases at the junction of the N-terminal domain (GSDMDM-NT) and the self-inhibitory C-terminal domain (GSDMDM-CT), and, when activated by caspase, gasdermin D (GSDMD) is cut in half, releasing the active fragment, known as gasdermin-D-NT. GSDMDM-NT can combine with acidic phospholipid on the inner surface of plasma membrane and perforate the membrane to release inflammatory cytokines such as IL-1 beta and IL-18, and induce apoptosis. The discovery of GSDMD as the last common step in the activation of inflammasome to cause apoptosis and inflammatory cytokine release provides a new approach to the targeted treatment of inflammatory diseases such as sepsis. Therefore, the development of an inhibitor of GSDMD is of great interest because it may inhibit the inflammatory response triggered by any stimulus, which is co-induced by activation of all the inflammasome pathways, unlike NLRP3 inhibitors which inhibit only one specific inflammasome. In addition, blocking GSDMDM-mediated cell membrane perforation may inhibit the release of a variety of inflammatory molecules associated with apoptosis, unlike IL-1 β or IL-18 inhibitors which act on only one downstream pathway. GSDMD is a gatekeeper of the pathway leading to apoptosis and inflammatory cytokine extravasation.
gasdermin E (dermatan E, GSDME) is a newly discovered protein, and the research on the function and the structure of the protein promotes the development of targeted drugs, so that various diseases related to cell apoptosis can be treated. GSDME is mainly expressed in tissues such as brain, cochlea, heart, kidney, placenta, etc. Caspase-3 associated with apoptosis can specifically cleave GSDME under the activation of chemotherapeutic drugs, tumor necrosis factor, virus infection and the like, causing cell apoptosis in GSDME-highly expressed cells, and caspase-3 activation induces cell apoptosis and secondary necrosis in GSDME-deficient cells. GSDME-associated deafness usually develops before 15 years of age and is manifested as progressive hearing loss, and its main mechanism is that deletion of exon 8 of the GSDME-encoding gene causes "gain of function" frame shift mutation, resulting in truncated C-terminal domain of the GSDME protein, which cannot effectively inhibit pore-forming activity of N-terminal domain, eventually causing apoptosis, but the reason why cochlear capillaries are particularly sensitive to GSDME gene mutation is not clear at present.
In conclusion, GSDMD and GSDME are important factors causing cell apoptosis, and if a compound capable of inhibiting the expression of the GSDMD and the GSDME can be found, the GSDMD and the GSDME can become an attractive drug target to prevent the cell apoptosis when needed so as to prevent various inflammatory chronic diseases. However, no natural drug has been reported as a GSDMD, GSDME inhibitor so far.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of ellagic acid and metabolites thereof as natural inhibitors in preparation of anti-cell apoptosis drugs.
The invention aims to provide application of ellagic acid and a metabolite thereof as a natural inhibitor in preparing a medicine for resisting cell scorching, wherein the metabolite is a metabolite urolithin A of ellagic acid in intestinal tracts.
Preferably, the application of the ellagic acid and the metabolites thereof as natural inhibitors in the preparation of anti-apoptosis drugs is to use one or the combination of two of the ellagic acid and the urolithin A in the preparation of anti-apoptosis drugs, or use one or the combination of two of the ellagic acid and the urolithin A in the preparation of anti-apoptosis drugs. .
Preferably, the ellagic acid and its metabolites are used as natural inhibitors in the preparation of anti-apoptosis drugs, wherein the cells are pancreatic cells or liver cells.
Preferably, the application of the ellagic acid and metabolites thereof as natural inhibitors in preparing drugs for resisting cell apoptosis caused by GSDME, GSDME-N, GSDMD and GSDMDM-N, and the apoptosis caused by clear-caspase 1 and clear-caspase 3 is provided.
Preferably, the application of the ellagic acid and the metabolites thereof as natural inhibitors in preparing drugs for resisting cell apoptosis is used, and the ellagic acid or the urolithin A is used for preparing expression inhibitors of GSDME, GSDME-N, GSDMD and GSDMDM-N.
Preferably, the application of the ellagic acid and the metabolites thereof as natural inhibitors in preparing anti-apoptosis drugs is to prepare an expression inhibitor of LC3II/I, p62, the urolithin A is used for preparing an expression promoter of LC3II/I, the urolithin A is used for preparing an expression inhibitor of p62, and the ellagic acid or the urolithin A is used for preparing expression promoters of ATG5 and Parkin.
Preferably, the application of the ellagic acid and the metabolite thereof as natural inhibitors in preparing anti-cell apoptosis medicines, and the ellagic acid or the urolithin A are used for preparing mitochondrion autophagy regulators so as to protect pancreatic or liver cells and reduce the cell damage caused by the diabetic environment.
Preferably, the application of the ellagic acid and the metabolites thereof as natural inhibitors in preparing anti-cell apoptosis drugs, and the ellagic acid or the urolithin A are used for preparing expression inhibitors of clear-caspase 1 and clear-caspase 3.
Preferably, the application of the ellagic acid and the metabolite thereof as natural inhibitors in preparing anti-cell apoptosis medicines is used for preparing TXNIP, NLRP3 and IL-1 beta expression inhibitors.
Preferably, the ellagic acid and the metabolite thereof are used as natural inhibitors to prepare drugs for resisting cell apoptosis, and the ellagic acid or the urolithin A are used for preparing drugs for reducing blood sugar, drugs for resisting pancreatitis or drugs for resisting hepatitis.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention finds that the high glycolipid has promotion effect on the scorching of pancreatic cells and liver cells, and the expression levels of the scorching proteins GSDMD and GSDME in the scorching signal channel are increased. The mechanism of the method is that the high-sugar lipid promotes the increase of the expression of key molecules GSDMD and GSDME in pancreas and liver, which indicates that the scorching plays a role in the inflammation reaction of pancreas and liver induced by high-sugar and high-fat. GSDMD, GSDME mediated cell apoptosis has been implicated in diabetic pancreatic, liver injury.
2. Ellagic Acid (EA) and an intestinal bacteria metabolite urolithin A (Urothin A, Uroa) thereof are natural polyphenol compounds, and the invention discovers that the Ellagic Acid (EA) and the intestinal bacteria metabolite urolithin A can inhibit cell apoptosis mediated by GSDMD and GSDME for the first time in an in vivo experiment of a diabetes model mouse, so that an anti-inflammatory protection effect is generated on cells.
Drawings
FIG. 1 is a hematoxylin-eosin staining pattern of example 1 of the present invention;
FIGS. 1a-d are Normal, Model, EA, and UroA sets, respectively;
FIG. 2 is a western blot detection chart of GSDMD, GSDMDM-N, GSDME-N of example 1;
lanes 1-4 are UroA, Normal, Model and EA panels, respectively;
FIG. 3 is a western blot assay of LC3II/I, ATG5, Parkin, ATG7, PINK1, p62 of example 1 of the present invention;
lanes 1-4 of FIG. 3A are UroA, Normal, Model and EA panels, respectively; lanes 1-5 of FIG. 3B are UroA, Model 1, Normal, Model 2 and EA panels, respectively;
FIG. 4 is a diagram showing liver tissues of a mouse after an experiment according to example 2 of the present invention;
FIGS. 4a-d are Normal, Model, EA, and UroA sets, respectively;
FIG. 5 is a diagram of the pathological morphological structure of liver tissue of a mouse observed by hematoxylin-eosin staining according to example 2 of the present invention;
FIGS. 5a-d are Normal, Model, EA, and UroA sets, respectively;
FIG. 6 is a western blot assay of a cell apoptosis-related protein according to example 2 of the present invention;
lanes 1-4 are UroA, Normal, Model and EA panels, respectively;
FIG. 7 shows the results of the measurement of the relative expression level of proteins involved in apoptosis in cells in example 2 of the present invention;
FIG. 8 is a western blot assay of apoptosis-related proteins of example 2 of the present invention;
a is caspase1, clear-caspase 1, B is clear-caspase 3; lanes 1-4 of FIG. 8A are UroA, Normal, Model and EA panels, respectively; lanes 1-5 of FIG. 8B are UroA, Model 1, Normal, Model 2 and EA panels, respectively;
FIG. 9 shows the results of measuring the relative expression level of apoptosis-related protein in example 2 of the present invention
FIG. 10 is a graph of TXINIP (A), NLRP3 and IL-1 beta (B) western blot;
lanes 1-4 of FIG. 10A are UroA, Normal, Model and EA panels, respectively; lanes 1-5 of FIG. 10B are UroA, Model 1, Normal, Model 2 and EA panels, respectively;
FIG. 11 shows the results of measuring the relative expression amounts of TXNIP, NLRP3 and IL-1. beta.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention to be implemented, the present invention will be further described with reference to the following specific embodiments and accompanying drawings.
The invention provides application of ellagic acid and metabolites thereof as natural inhibitors in preparation of anti-apoptosis drugs, which comprises the following embodiments.
Example 1 use of ellagic acid and its metabolites as natural inhibitors for the protection of pancreatic cell inflammation
1.1 Experimental animals
SPF grade 10 week old c57BL/6 male mice, with a body mass of 24. + -.2 g, were purchased from the animal center of Xinjiang medical university (animal license number: SYXK (New) 2016-0002).
1.2 materials of the experiment
The common feed is purchased from the animal experiment center of Xinjiang medical university; high fat diet purchased from south-bound telofix diet ltd; STZ was purchased from Sigma bio ltd; EA purchased from Shanghai Michelin Biochemical technology Limited (purity not less than 98%); UroA is purchased from Shanghai Yangyi BioNcGmbH (purity is more than or equal to 98%); glucagon (GC) ELISA test kits, C-reactive protein (CRP) ELISA test kits, all available from Sibobestbio Biotech, Inc., Shanghai; NLRP3, cleaved cysteine aspartic protease 3(cleaved-caspase3), IL-1 β antibody, caspase1/cleaved-caspase1, PINK1, Parkin, ATG7, ATG5, LC3II/I, p62, goat anti-rabbit IgG-HRP from Shenyang Wanleibo Biol Ltd, goat anti-mouse IgG-HRP from Hangzhou Hua mountain Biotechnology Ltd. GSDMD is available from CST Biotechnology, Inc., and GSDME is available from abcam Biotechnology, Inc.
1.3 animal model preparation
c57BL/6 mice were acclimatized to normal diet for 1 week before use in the experiment, with illumination intensity of 200lx from 9 am to 21 pm daily. The feeding environment is controlled at 22-24 deg.C and relative humidity of 30-40%, and is kept in a relatively stable clean environment. Food and water are freely available. All animals were cared for according to the convention for animal protection, and the study was approved by the ethical committee of the university of medical, Xinjiang.
Mice were randomly divided into 4 groups (10 mice per group) by body weight, specifically, a Normal control group (Normal group), a diabetes Model group (Model group), an ellagic acid group (EA group) and a urolithin a group (UroA group), the Normal control group was fed with a Normal diet, and the Model group, the EA group and the UroA group were fed with a high fat diet. After high-fat feeding for 6 weeks, 2d of continuous intraperitoneal injection of STZ (85mg/kg/d) after fasting without water prohibition for 12 hours induces diabetes, and the same volume of citric acid-sodium citrate buffer solution is intraperitoneally injected in a normal group. After 2 days, cutting the tail and taking blood to measure the blood sugar, and successfully molding by taking fasting blood sugar >11.1 mmol/L.
1.4 animal administration
After each group (Model group, EA group and UroA group) is fed with high-fat feed for 6 weeks, 2d of intraperitoneal injection of STZ is continuously carried out, after the molding is successful, distilled water is fed into the Normal group and the Model group by intragastric gavage at the weight of 0.1mL/10g, EA and UroA are respectively fed into the EA group and the UroA group at the weight dose of 50mg/kg, and the intervention time of each group is 8 weeks.
1.5 Biochemical index of plasma
At the end of the experiment, all mice were fasted for 6h without water deprivation, weighed and fasting blood glucose measured. Collecting blood from the fundus vein2Centrifuging at 3000r/min for 10min in an EDTA coated anticoagulant tube to separate bloodAnd (5) subpackaging the slurry, and freezing and storing at-80 ℃ for later use. For the detection of Glucagon (GC), C-reactive protein (CRP). See tables 1-2 for results.
TABLE 1 GC and fasting blood glucose assay results
In Table 1, P is compared with Normal group<0.01; compared with a Model group##P<0.01. The results in Table 1 show that fasting plasma glucose and glucagon GC are significantly increased in Model group mice compared to Normal group mice (P<0.01); EA or UroA intervention mice had significantly lower fasting plasma glucose and plasma GC (P) compared to Model group mice<0.01). Therefore, EA and UroA can improve the pancreatic hormone secretion disorder of diabetes and have the treatment effect on diabetes.
TABLE 2 CRP assay results
In Table 2, P is compared with Normal group<0.05，**P<0.01; compared with a Model group#P<0.05，##P<0.01. Compared with Normal group mice, the plasma C-reactive protein CRP content of Model group mice is remarkably increased (P)<0.01); the plasma CRP levels were significantly reduced in EA or UroA-intervened mice (P) compared to Model group mice<0.01). In the state of type 2 diabetes, a large amount of inflammatory factors are generated in islet tissues, so that an inflammatory cascade reaction occurs, the islet tissues can directly or indirectly act on pancreas and damage pancreatic cells, the death of the pancreatic cells is promoted, the secretion function of the pancreas is disordered, and hyperglycemia is caused. CRP is protein which rises sharply in blood plasma when an organism is infected or tissues are damaged, and the results in Table 2 suggest that EA and UroA can reduce the secretion of proinflammatory factors, further reduce the tissue damage caused by inflammatory reaction, and the effects of EA and UroA are equivalent.
1.6 staining with hematoxylin-eosin to observe pathological changes
After the administration, the mouse pancreas tissue is soaked in 4% (v/v) paraformaldehyde for 24h, and after the fixation is completed, dehydration, paraffin embedding, slicing (thickness 3-5 μm), and then the mouse pancreas tissue is placed in an oven at 60 ℃ overnight. Soaking paraffin sections in xylene for 20min × 2 times, soaking in anhydrous alcohol for 10min × 2 times, sequentially soaking in 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5min, and washing with distilled water. And placing the rehydrated pancreatic tissue slices in Harris hematoxylin staining solution for staining for 10min, lightly washing with tap water, differentiating with 1% by volume of hydrochloric acid ethanol for several seconds, lightly washing with tap water, turning blue with 0.5% by volume of ammonia water, and washing with tap water again. Hematoxylin-stained pancreatic tissue sections were immersed in eosin stain for approximately 3min, xylene 5min x 2 times, neutral gum mounted, coverslipped, viewed under a microscope and photographed. Results referring to FIG. 1 and Table 3, FIGS. 1a-d are for the Normal group, Model group, EA group, and UroA group, respectively.
As can be seen from FIG. 1, the islet cells of the Normal group of mice are Normal in morphology, uniform in size, large in islet number and cells in the islets, full in cytoplasm, and clear in islet boundary. Compared with a Normal group, the Model group mice have disorganized islet cell morphology and structure, extremely small islet quantity, severe atrophy of the islets, disordered shape, loose cytoplasm, fuzzy islet boundaries, irregular shape and extremely few cells in the islets; some had mild punctate necrosis and edema. Compared with the Model group, the mouse islets of the EA group and the UroA group are smaller, but the shape and the structure are basically normal, the boundaries of the islets and the exocrine part are clearer, the number of the islet cells is increased compared with that of the diabetes Model group, and the islet area is increased. In the UroA group, the islets were smaller and the number of islets was smaller than in the EA group. HE staining results suggest a better EA effect.
TABLE 3 summary of EA and UroA improvement of pathological morphological structure of pancreatic tissue in diabetic mice
Note: a greater number of "+" indicates a more pronounced necrotic edema.
1.7 detection of mouse pancreas-related protein expression quantity by western blot method
After the administration, the pancreatic tissue of the mouse is cut into pieces, a proper amount of protein buffer solution is added, protein is extracted, the protein quantification is carried out, and the protein concentration is adjusted. Each group takes 20 mu L of sample volume, contains 40 mu g of protein sample, and is mixed with the sample buffer solution in equal volume; then, 10% SDS-PAGE is used for electrophoretic separation, the obtained product is transferred to a PVDF membrane and is sealed for 1 hour by containing 5% skimmed milk powder; then, the primary antibodies are incubated overnight at 4 ℃ (in this experiment, the primary antibodies used are GSDMD, GSDMD-N, GSDME and GSDME-N respectively, the dilution ratio is GSDMD, GSDMD-N, GSDME and GSDME-N is 1: 1000, then, the secondary antibodies lgG-HRP (1: 5000) are incubated for 45min at 37 ℃, ECL luminescence detection is carried out after TBST rinsing, beta-actin is used as internal reference protein, the gray value of the protein bands is analyzed by Image J software, the experiment is repeated at least 3 times, and the average value is obtained, and the results are shown in the following tables 4-6 and FIGS. 2-3.
TABLE 4 Effect of EA/UroA on mouse pancreatic pyroptosis protein expression
In Table 4, P is compared with Normal group<0.05，**P<0.01; compared with a Model group#P<0.05，##P<0.01; compared with EA group&P<0.05，&&P<0.01. Table 4 results show that the pancreatic tissue of Model group mice has significantly increased expression of GSDMD, GSDMD-N, GSDME, and GSDME-N proteins (P) compared to that of Normal group mice (P)<0.01); EA and UroA intervention mice showed significant reduction of GSDMD, GSDMD-N, GSDME and GSDME-N protein expression in pancreatic tissue (P) compared to Model group mice<0.01); compared with the EA group, the protein expression of the GSDME and the GSDME-N in the pancreatic tissues of the mice intervened by the UroA is increased, the GSDMD is reduced, and the GSDMDM-N is not different, which indicates that the EA inhibits the GSDME and the GSDME-N more obviously, while the function of the UroA in inhibiting the GSDMD is stronger, and the UroA in inhibiting the GSD is inhibitedThe two functions in the MD-N aspect are similar, and no difference is found. FIG. 2 is a western blot analysis of GSDMD, GSDMDM-N, GSDME-N, and lanes 1-4 are the UroA group, the Normal group, the Model group, and the EA group, respectively.
The research of the embodiment finds that pancreatic cells have the phenomenon of apoptosis in the diabetic high glycolipid state, and the expression levels of the apoptosis protein GSDMD and GSDME in the apoptosis signal pathway are increased. The mechanism of the method is that the high-sugar lipid promotes the key molecule GSDMD of the burn-out in the pancreas, the GSDME expression is increased, and the burn-out is suggested to play a role in the pancreatic inflammatory reaction induced by the high-sugar lipid. GSDMD, GSDME-mediated apoptosis of cells is involved in diabetic pancreatic injury. After EA or UroA stem prognosis, the process of pancreatic caspase1/GSDMD mediated apoptosis and caspase3/GSDME mediated apoptosis-to-focalism is obviously inhibited, and further, the pancreatic inflammation caused by the apoptosis of the diabetic mouse cells is inhibited.
TABLE 5 results of relative expression level detection of autophagy-related proteins ATG7, ATG5 and LC3II/I
TABLE 6 detection results of relative expression of autophagy-related proteins p62, PINK1 and Parkin
In tables 5-6, P compared to Normal group<0.05，**P<0.01; ratio to Model group#P<0.05，##P<0.01; compared with EA group&P<0.05，&&P<0.01. The expression of autophagy-related protein in pancreatic tissues is determined by a Western Blot method, and compared with Normal mice, the expression of LC3II/I, P62 protein in pancreatic tissues of Model mice is obviously increased (P)<0.01), significant reduction in ATG5 and Parkin protein expression (P)<0.01), the expression of PINK1 and ATG7 proteins is obviously not different (P)>0.01); compared with Model group mice, the expression of P62 protein in pancreatic tissues of mice with EA or UroA intervention is remarkably reduced (P)<0.01), the expression of ATG5 protein is obviously increasedHigh (P)<0.01); significant reduction of LC3 II/protein I EA group (P)<0.01), the UroA group was significantly elevated (P)<0.01), no significant difference in ATG7 expression was observed; compared with the EA group, the expression of LC3II/I, P62 and Parkin proteins in pancreatic tissues of the mice is obviously increased by UroA intervention (P)<0.01), the expression of ATG5 protein is increased (P)<0.05), the expression of PINK1 and ATG7 has no obvious difference.
FIG. 3 is a western blot assay of LC3II/I, ATG5, Parkin, ATG7, PINK1, p62 of example 1 of the present invention; lanes 1-4 are UroA, Normal, Model and EA panels, respectively; lanes 1-5 of FIG. 3B are UroA, Model 1, Normal, Model 2 and EA sets, respectively (two Model set lanes are made herein for ease of comparing the bands).
Unlike "apoptosis," autophagy "is a self-digestion process that degrades proteins and organelles under cellular stress, and can maintain cellular homeostasis, facilitating cell survival. ATG5 and ATG7 as autophagy-related proteins can participate in the formation of autophagosomes, and PINKL/Parkin is a main mode for controlling the autophagy of mitochondria by removing damage and redundant mitochondria through large autophagy
The results show that LC3II/I and p62 expressions are obviously increased and ATG5 and Parkin are obviously reduced in pancreatic tissues of diabetes model mice, which indicates that autophagosome in pancreatic of the diabetes model mice generates more and degrades less, and then autophagy flow disorder occurs; after EA stem cell, LC3II/I, p62 protein is obviously reduced, and ATG5 is obviously increased, which indicates that EA enables pancreatic autophagosome to generate less and degrade more, and improves autophagy flow disorder; interestingly, after UroA intervention, LC3II/I, ATG5 and Parkin were significantly elevated and p62 expression was significantly reduced, UroA cleared pancreatic autophagy flow. No significant difference was observed between groups, ATG7 and PINK 1. In conclusion, the results suggest that PINK1/Parkin mediated mitophagy plays a role in resisting high fat combined with STZ induced pancreatic apoptosis, and the autophagy regulation and control effect provides a potential treatment strategy for diabetic pancreatic injury.
In conclusion, the study of this example shows that EA and the metabolite UroA thereof have protective effects on high fat combined STZ-induced pancreatic injury of type 2 diabetic mice, and the mechanism of EA and the metabolite UroA thereof is related to inhibition of apoptosis and inflammatory signal pathways and regulation of pancreatic mitochondrial autophagy process. The research preliminarily reveals the benefit of the plant polyphenol ellagic acid and the metabolite urolithin A thereof on the protection of the pancreas of the diabetes mellitus, and is worthy of further research and development.
Example 2 use of ellagic acid and its metabolites as natural inhibitors for the protection of liver cell inflammation
1. Materials and methods
Experimental animals, experimental materials, preparation of animal models, administration of animals, and observation of pathological morphological structures of liver tissues of mice by hematoxylin-eosin staining are respectively referred to 1.1, 1.2, 1.3, 1.4, and 1.6 of example 1.
After the animal administration experiment was completed, liver tissues of the mice were taken and photographed for appearance as shown in FIG. 4, and FIGS. 4a to d are Normal group, Model group, EA group and UroA group, respectively. The results show that the liver of the Normal group mouse is fresh and pink; model group mice showed white ischemia of liver, high fat content; the liver color of the mice in the EA group is obviously improved and is pink; the appearance of the liver of the Uroa group mice is obviously improved, and the mice are pink.
2. Results of the experiment
(1) EA and UroA for improving pathological morphological structure of liver tissue of diabetic mouse
Hematoxylin-eosin staining observation of pathological morphological structure of mouse liver tissue is shown in FIG. 5, and FIGS. 5a-d are Normal group, Model group, EA group and UroA group, respectively. The liver lobule structure of Normal mice is Normal, and no fat change and edema are shown under an optical microscope; the liver lobular structural disorder, severe edema and severe steatosis of liver cells of Model group mice have fat drops with different sizes and punctate necrosis; the liver lobule structure of the mice in the EA group is normal, slight edema, slight steatosis and slight punctate necrosis; the liver lobules of the Uroa group mice were normal in structure, without edema, slight steatosis, slight punctate necrosis.
(2) Effect of EA/UroA on relative expression levels of T2DM mouse liver tissue apoptosis key proteins GSDME, GSDME-N, GSDMD and GSDMDM-N
FIG. 6 is a western blot assay of GSDME, GSDME-N, GSDMD and GSDMDM-N, and FIG. 7 is a relative expression level measurement result of GSDME, GSDME-N, GSDMD and GSDMDM-NComparison with Normal group,. P<0.05，**P<0.01; in comparison with the Model set,#P<0.05；##P<0.01; in comparison to the group of EAs,△P<0.05；△△P<0.01. the results showed that the expression of GSDME, GSDME-N, GSDMD and GSDMDM-N protein was significantly increased in liver tissue of Model group mice compared to that of Normal group mice (P<0.01); EA and UroA intervention in mouse liver tissue GSDME, GSDME-N, GSDMD and GSDMDM-N protein expression is reduced (P) compared with Model group mice<0.05)。
(3) Effect of EA/UroA on relative expression level of apoptosis-related protein in liver tissue of T2DM mouse
FIG. 8 is a western blot analysis of an apoptosis-related protein map, with caspase1, clear-caspase 1 in FIG. 8A, and clear-caspase 3 in FIG. 8B, and with UroA, Normal, Model, and EA panels in lanes 1-4 of FIG. 8A, respectively; lanes 1-5 of FIG. 8B are UroA, Model 1, Normal, Model 2 and EA sets (two Model set lanes are shown herein for ease of comparing bands), respectively, FIG. 9 shows the relative expression measurements of clear-caspase 1 and clear-caspase 3, as compared to the Normal set, P<0.05，**P<0.01; in comparison with the Model set,#P<0.05；##P<0.01; in comparison to the group of EAs,△P<0.05；△△P<0.01. compared with Normal group mice, the expression of clear-caspase 1 and clear-caspase 3 proteins in liver tissues of Model group mice is remarkably increased (P)<0.01); EA and UroA intervention mice liver tissue clear-caspase 1 and clear-caspase 3 protein expression is reduced (P) compared with diabetes model group mice<0.01，P<0.05)。
(4) Effect of EA/UroA on relative expression levels of T2DM mouse liver tissue inflammation-associated proteins TXNIP, NLRP3 and IL-1 beta
FIG. 10 is a western blot detection chart showing lanes 1 to 4 in FIG. 10A, which are UroA group, Normal group, Model group and EA group, respectively; lanes 1-5 of FIG. 10B are UroA, Model 1, Normal, Model 2 and EA sets (two Model set lanes are shown for convenience of comparing bands), respectively, and the relative expression level of TXNIP, NLRP3 and IL-1 is shown in FIG. 11, compared to the Normal set<0.05，**P<0.01; in comparison with the Model set,#P<0.05；##P<0.01; in comparison to the group of EAs,△P<0.05；△△P<0.01。
compared with Normal group mice, the liver tissues of Model group mice have increased TXNIP, NLRP3 and IL-1 beta protein expression (P < 0.01); compared with Model group mice, the expression of TXNIP and IL-1 beta protein in liver tissues of EA and UroA intervention mice is obviously reduced (P <0.05), and the TXNIP inhibition of the liver tissues of the diabetes mice is more obvious (P <0.01) by the UroA intervention mice.
It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. The application of ellagic acid and its metabolite as natural inhibitor in preparing medicine for resisting cell scorching is characterized in that the metabolite is metabolite urolithin A of ellagic acid in intestinal tract.
2. Use of ellagic acid and its metabolites as natural inhibitors in the manufacture of a medicament against apoptosis of cells according to claim 1, characterized in that one or a combination of both of said ellagic acid and said urolithin a is used in the manufacture of a medicament against apoptosis of cells, or a combination of one or both of said ellagic acid and said urolithin a is used in the manufacture of a medicament against apoptosis of cells.
3. Use of ellagic acid and its metabolites as natural inhibitors in the preparation of a medicament against apoptosis of cells according to claim 2, wherein said cells are pancreatic or liver cells.
4. The use of ellagic acid and metabolites thereof as natural inhibitors for the preparation of a medicament against apoptosis of cells according to claim 3, wherein said apoptosis is caused by GSDME, GSDME-N, GSDMD and GSDMDM-N, and said apoptosis is caused by clear-caspase 1 and clear-caspase 3.
5. Use of ellagic acid and its metabolites as natural inhibitors in the preparation of drugs against apoptosis of cells according to claim 4, characterized in that said ellagic acid or said urolithin A is used for the preparation of inhibitors of expression of GSDME, GSDME-N, GSDMD and GSDMDM-N.
6. Use of ellagic acid and its metabolites as natural inhibitors in the preparation of drugs against apoptosis of cells, according to claim 4, characterized in that said ellagic acid is used for the preparation of expression inhibitors of LC3II/I, p62, said urolithin A is used for the preparation of expression promoters of LC3II/I, said urolithin A is used for the preparation of expression inhibitors of p62, said ellagic acid or said urolithin A is used for the preparation of expression promoters of ATG5 and Parkin.
7. Use of ellagic acid and its metabolites as natural inhibitors in the preparation of anti-apoptosis drugs according to claim 6, wherein said ellagic acid or said urolithin A is used to prepare modulators of mitophagy to protect pancreatic or liver cells and to alleviate the above-mentioned cellular damage caused by diabetic conditions.
8. The use of ellagic acid and its metabolites as natural inhibitors in the preparation of anti-apoptosis drugs according to claim 4, wherein said ellagic acid or said urolithin A is used to prepare expression inhibitors of clear-caspase 1 and clear-caspase 3.
9. Use of ellagic acid and its metabolites as natural inhibitors in the preparation of a medicament against apoptosis of cells according to claim 4, characterized in that said ellagic acid or said urolithin A is used in the preparation of an inhibitor of the expression of TXNIP, NLRP3 and IL-1 β.
10. Use of ellagic acid and its metabolites as natural inhibitors in the preparation of a medicament against apoptosis of cells, according to claim 3, characterized in that said ellagic acid or said urolithin A is used in the preparation of hypoglycemic, anti-pancreatitis or anti-hepatitis drugs.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115128283A (en) * | 2022-07-20 | 2022-09-30 | 郑州大学第一附属医院 | Marker for severe acute pancreatitis and application thereof |
| CN115819548A (en) * | 2021-11-16 | 2023-03-21 | 北京美德泰康生物科技有限公司 | Marker and method for detecting inflammation-related diseases |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103442594A (en) * | 2010-12-23 | 2013-12-11 | 阿马曾提斯公司 | Compositions and methods for improving mitochondrial function and treating neurodenegenerative diseases and cognitive disorders |
-
2021
- 2021-01-22 CN CN202110088079.8A patent/CN112675165A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103442594A (en) * | 2010-12-23 | 2013-12-11 | 阿马曾提斯公司 | Compositions and methods for improving mitochondrial function and treating neurodenegenerative diseases and cognitive disorders |
Non-Patent Citations (9)
| Title |
|---|
| ANTONIO J. AMOR等: "Ellagic Acid as a Tool to Limit the Diabetes Burden Updated Evidence", 《ANTIOXIDANTS》 * |
| BAHETIBIEKE TUOHETAERBAIKE等: "Pancreas protective effects of Urolithin A on type 2 diabetic mice induced by high fat and streptozotocin via regulating autophagy and AKT/mTOR signaling pathway", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
| 古丽海夏•哈勒玛合拜等: "鞣花酸对糖尿病小鼠胰腺炎的改善作用", 《中成药》 * |
| 巴合提别克·托合塔尔拜克: "尿石素A对糖尿病小鼠胰腺的作用及其机制研究", 《中国优秀博硕士学位论文全文数据库（硕士） 医药卫生科技辑》 * |
| 杨柳等: "尿石素A对2型糖尿病小鼠脂肪组织胰岛素抵抗的影响", 《中成药》 * |
| 田亚丽: "鞣花酸及其代谢物对糖尿病小鼠肝脏胰岛素抵抗的影响及机制", 《中国优秀博硕士学位论文全文数据库（硕士） 医药卫生科技辑》 * |
| 田亚丽等: "尿石素A激活自噬抑制含NLR家族Pyrin域蛋白3炎症信号改善糖尿病小鼠胰腺损伤研究", 《中国临床药理学杂志》 * |
| 田亚丽等: "尿石素A激活自噬改善糖尿病小鼠肝脏胰岛素抵抗研究", 《中草药》 * |
| 田亚丽等: "鞣花酸促进自噬性清除改善2型糖尿病小鼠肝脏胰岛素信号通路", 《时珍国医国药》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115819548A (en) * | 2021-11-16 | 2023-03-21 | 北京美德泰康生物科技有限公司 | Marker and method for detecting inflammation-related diseases |
| CN115819548B (en) * | 2021-11-16 | 2023-09-01 | 北京美德泰康生物科技有限公司 | Marker and method for detecting inflammation-related diseases |
| CN115128283A (en) * | 2022-07-20 | 2022-09-30 | 郑州大学第一附属医院 | Marker for severe acute pancreatitis and application thereof |
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