CN112999232A - Application of nitric oxide spin-trapping nano-targeted antioxidant in preparation of drugs for preventing diabetes - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of medicine and pharmacology, and particularly relates to an application of a nitric oxide spin trapping nano-targeting antioxidant capable of targeting liver mitochondria in preparation of a medicament for preventing diabetes. The nano-targeted antioxidant provided by the invention can obviously inhibit the increase of glucose tolerance of a diabetic mouse and the increase of gluconeogenesis capability of the diabetic mouse, and can prevent the occurrence and development of diabetes by up-regulating the expression of liver antioxidant protein, increasing the generation of liver mitochondria of the mouse and improving the energy metabolism function of the mitochondria. Therefore, the nano antioxidant can be used for preparing medicines or health products for preventing diabetes.
Description
Technical Field
The invention belongs to the technical field of medicine and pharmacology, and particularly relates to an application of a nitric oxide spin trapping nano-targeting antioxidant capable of targeting liver mitochondria in preparation of a medicament for preventing diabetes.
Background
Type 2 diabetes (T2 DM) is a global disease burden, and is considered one of the major challenges in human health in the 21 st century, and is a serious threat to human health and life. In 2017, the 8 th edition of global diabetes distribution data published by the International Diabetes Federation (IDF) showed that there are currently 4.25 million adults (20-79 years) worldwide with diabetes at a prevalence of about 8.8%. Diabetes mellitus is the leading cause of visual loss, amputation and end-stage renal disease, and is also an important risk factor for atherosclerosis, which in turn induces the occurrence of cardiovascular disease, ultimately leading to death. These striking statistics are in sharp contrast to the few effective treatment strategies to date for this multifactorial disease.
The islets of langerhans, liver and muscle are key organs involved in the homeostasis of blood sugar in the body. Two factors that contribute to hyperglycemia are increased hepatic glucose production (release of glucose) and decreased glucose consumption by peripheral tissues (mainly adipose tissue and muscle). An imbalance in hepatic glucose release and peripheral tissue glucose uptake can lead to persistent hyperglycemia, a major factor in the development of type one and type two diabetes. The glucose produced by the liver accounts for about 90% of the endogenous glucose production and is critical to the glucose homeostasis of the whole body. Therefore, hepatic glucose metabolism disorder in patients with T2DM is an attractive therapeutic target. Oxidative stress is a major cause of the development and progression of diabetes and its complications, and therefore, reducing hepatic oxidative stress can alleviate insulin resistance, thereby preventing the occurrence of diabetes. At present, some antioxidants have been reported to have good application prospects in diabetes animal models, but the clinical application effect is not very ideal. Therefore, the research and development of more effective medicines capable of preventing the occurrence and development of diabetes are particularly important, and the market value and significance are very wide.
Disclosure of Invention
The invention aims to provide application of a nitric oxide spin trapping nano-targeting antioxidant capable of targeting liver mitochondria in preparation of a medicament for preventing and treating diabetes.
The nitric oxide spin-trapping nano-targeted antioxidant can obviously inhibit the increase of glucose tolerance of a diabetic mouse and the increase of gluconeogenesis capability of the diabetic mouse, and can prevent the occurrence and development of diabetes by up-regulating the expression of liver antioxidant protein, increasing the generation of liver mitochondria of the mouse and improving the energy metabolism function of the mitochondria. Experiments show that:
the nano-targeted antioxidant can obviously inhibit the glucose tolerance increase of diabetic mice;
the nano-targeted antioxidant can inhibit gluconeogenesis increase of diabetic mice;
the nano-targeted antioxidant can up-regulate the expression of the antioxidant protein of the liver;
the nano-targeting antioxidant can increase mouse liver mitochondrion generation, activate mitochondrion generation related protein PGC-1 alpha and increase expression of mitochondrion generation upstream regulatory protein Nrf 1;
the nano-targeted antioxidant can improve energy metabolism of mitochondria.
Therefore, the nano-targeted antioxidant can be used for preparing medicines for preventing and treating diabetes and also can be used for preparing health-care products for preventing and treating diabetes.
In the invention, the nitrogen oxide spin trapping Nano antioxidant capable of targeting the liver is preferably Nano-mitoPBN and Nano-PBN, and the structural formulas are respectively as follows:
（Arroyo, Carmen M., and Masahiro Kohno. "Difficulties encountered in the detection of nitric oxide (NO) by spin trapping techniques. A cautionary note." Free radical research communications 14.2 (1991): 145-155.）。
compared with the prior art, the invention has the following beneficial technical effects:
the invention discloses application of a nitric oxide spin trapping type nano antioxidant capable of targeting liver mitochondria in developing a medicament for preventing diabetes, the nano antioxidant can obviously inhibit the increase of glucose tolerance of a diabetic mouse and the increase of gluconeogenesis capability of the diabetic mouse, and the occurrence and the development of diabetes are prevented by up-regulating the expression of liver antioxidant protein, increasing the generation of mouse liver mitochondria, activating mitochondria to generate related protein PGC-1 alpha, increasing the expression of mitochondria to generate upstream regulatory protein Nrf1 and improving the energy metabolism function of mitochondria, so that the nano antioxidant has a good application prospect in the prevention of the occurrence and the development of diabetes.
Drawings
Fig. 1 is two nitric oxide spin-trapping nano-antioxidants that can target the liver: the Nano-MitoPBN and the Nano-PBN can be absorbed by the liver and promote the cell viability of the liver cells under the STZ stress; wherein, A and B are liver absorption curves of Nano-MitoPBN and Nano-PBN, the abscissa is time, and the ordinate is the concentration of the drug contained in the unit protein; c is two nitric oxide spin-trapping nano antioxidants targeting the liver, and can promote the cell viability of the liver cells under the stress of STZ.
FIG. 2 shows that the nano antioxidant can obviously inhibit the increase of the glucose tolerance of diabetic mice. Wherein: the abscissa is time and the ordinate is glucose concentration.
FIG. 3 is a graph of the increase in gluconeogenesis inhibitory ability of nano-antioxidants in diabetic mice. Wherein: the abscissa is time and the ordinate is glucose concentration. Wherein: (A) is a graph; (B) is a statistical chart.
FIG. 4 shows that the nano antioxidant can up-regulate the expression of antioxidant protein of liver. Wherein, (A) is a Western blot result graph; (B) the Western blot statistical chart is shown, the abscissa represents the protein name, and the ordinate represents the relative protein expression level content.
FIG. 5 shows that the nano antioxidant can increase mouse liver mitochondrion generation and activate the expression of mitochondrion generation related protein. Wherein: (A) the copy number of mitochondria, the abscissa of the gene name and the ordinate of the relative copy number of mitochondria; (B) is a Western blot result graph; (C) the Western blot statistical chart is shown, the abscissa represents the protein name, and the ordinate represents the relative protein expression level content.
Fig. 6 shows that the nano antioxidant can improve energy metabolism of mitochondria. Wherein: the abscissa is time and the ordinate is oxygen consumption rate.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
Experimental Material
Glucometers were purchased from rotz; grx2, Trx2, and Prx3 antibodies were purchased from protein technology companies (china); antibodies p-PGC-1 α, PGC-1 α and Nrf1 were purchased from Cell Signaling Technology, Inc. (USA).
Diabetic mouse model establishment
KM male mice (25-30 g in weight) were purchased from Jiejie animals (Shanghai, China). Mice were divided into 4 groups: control group (Ctl), diabetes (Db), diabetes + control intervention (Db + V) and diabetes + nano antioxidant intervention (Db + NM), 8-10 per group. The diabetic groups were fasted for 12 h, i.e., the first day was intraperitoneally injected with 0.1M citric acid zotocin (STZ) solution (pH 4.5) at a dose of 80 mg/kg, the second day was intraperitoneally injected with 0.1M citric acid zotocin solution (pH 4.5) at a dose of 70 mg/kg, and high fat-fed mice (60% calories from fat) were followed by intraperitoneally injected with Nano-antioxidant (Nano-MitoPBN or Nano-PBN) at a dose of 2.5 mg/kg daily, and the mice were sacrificed three weeks after the injection. Tissues and plasma were collected and stored at-80 ℃ for further analysis.
Experimental methods
(1) Oral Glucose Tolerance Test (OGTT)
After 1 g/kg of glucose is injected into the abdominal cavity of the fasting mouse, measuring the blood sugar by a glucometer at 15 min, 30 min, 60 min and 120 min respectively;
(2) pyruvic acid tolerance test (PPT)
After fasting for 15 h, measuring the blood sugar by a glucometer after injecting pyruvic acid 1-2 g/kg into the abdominal cavity for 15 min, 30 min, 60 min and 120 min respectively;
（3）Western blot
carrying out gel running by using 10% acrylamide gel, wherein the loading amount of protein is 10 mu g, carrying out transfer printing by using a PVDF membrane, then sealing, incubating a primary antibody, carrying out overnight at 4 ℃, cleaning the primary antibody, incubating a secondary antibody, carrying out 1 h at room temperature, cleaning the secondary antibody, and carrying out chemiluminescence;
(4) mitochondrial copy number detection
1) Total DNA extraction from liver tissue
Extracting total DNA of the liver tissue by using the kit, wherein the operation steps are carried out according to the instruction;
2) real-time fluorescent quantitative PCR (Real-time PCR)
SYBR Green method is used for carrying out the reaction, the reaction system comprises 1 mu L of cDNA, 5 mu L of 2 XSYBR Premix Ex Taq II, 0.5 mu L of upstream and downstream primer mixed liquor (10 mu M), and sterile water is added to 10 mu L; reaction conditions were as described, 95 ℃ melting for 10 min, 40 cycles of PCR (each cycle comprised 95 ℃ 30 s, 55 ℃ 30 s, 72 ℃ 20 s), and finally melting curves were observed (95 ℃ 15 s, 60 ℃ 15 s, 95 ℃ 15 s); 18S rRNA was used as an internal control, and the primer sequences used in the experiment were:
16S rRNA
Forward: 5’- CCGCAAGGGAAAGATGAAAGAC -3’（SEQ. ID. NO 1）
Reverse: 5’- TCGTTTGGTTTCGGGGTTTC -3’ （SEQ. ID. NO 2）
ATP6
Forward: 5′-TAGCCATACACAACACTAAAGGACGA -3’（SEQ. ID. NO 3）
Reverse: 5′-GGGCATTTTTAATCTTAGAGCGAAA -3’（SEQ. ID. NO 4）
18S rRNA
Forward: 5’- GTAACCCGTTGAACCCCATT -3’（SEQ. ID. NO 5）
Reverse: 5’- CCATCCAATCGGTAGTAGCG -3’（SEQ. ID. NO 6）
(5) statistical analysis
Results are expressed as Mean ± SD and data analysis using One Way-ANOVA analysis method with statistically significant meaning of p <0.05, p <0.01, p < 0.001.
Conclusion of the experiment
(1) Two nitric oxide spin trapping nano antioxidants capable of targeting the liver: Nano-mitoPBN and Nano-PBN can be absorbed by liver and promote the cell viability of liver cells under STZ stress
As shown in fig. 1 (a and B), both drugs are absorbed by the liver, especially Nano-MitoPBN, which is present in the liver at a higher concentration than Nano-PBN; as shown in FIG. 1 (C), the hepatocyte L02 is under oxidative stress and has reduced cell activity under the stimulation of STZ with concentration of 1mmol/L, while different concentrations (5. mu. mol/L and 20. mu. mol/L) of the nitric oxide spin-trapping nano antioxidant targeting the liver can promote the cell activity of the hepatocyte;
(2) the PBN-like nano-targeted antioxidant can obviously inhibit the glucose tolerance increase of diabetic mice
Fig. 2 shows that after the diabetic mouse takes glucose orally for 30 min, the blood glucose concentration reaches the highest value, which is 20-30 mmol/L, and then continuously decreases, at 30 min, 60 min and 120 min, the blood glucose of the diabetic mouse is obviously increased compared with a control group, compared with the diabetic mouse, the blood glucose of the diabetic mouse after continuous intraperitoneal injection of a control buffer solution has no obvious change, and the increase of the glucose tolerance of the diabetic mouse can be obviously inhibited after three weeks of intraperitoneal continuous injection of a nano antioxidant;
(3) PBN-like nano-targeted antioxidant for inhibiting diabetic mouse gluconeogenesis increase
3A-3B show that after pyruvic acid is injected into the abdominal cavity of a diabetic mouse for 30 min, the blood sugar concentration reaches the highest value and is 15-20 mmol/L, then the blood sugar concentration continuously decreases, the blood sugar of the diabetic mouse is obviously increased compared with a control group at 30 min, 60 min and 120 min, the blood sugar of the diabetic mouse after the control buffer is continuously injected into the abdominal cavity has no obvious change compared with the diabetic mouse, and the nano antioxidant can obviously inhibit the increase of the gluconeogenesis ability of the diabetic mouse after the nano antioxidant is continuously injected into the abdominal cavity for three weeks;
(4) PBN-like nano-targeted antioxidant capable of up-regulating expression of liver antioxidant protein
All proteins Prx2, Trx3 and Grx2 are anti-oxidation related proteins, and fig. 4 shows that in a diabetes model group, the expressions of the three proteins are all rapidly reduced, and after three weeks of continuous intraperitoneal injection of the nano antioxidant, the expressions of the three anti-oxidation proteins in the liver are all obviously up-regulated, which indicates that the nano antioxidant indeed has a very good anti-oxidation function, and plays a role in preventing diabetes by regulating the redox balance of the liver;
(5) the PBN-like nano-targeting antioxidant can increase mouse liver mitochondrion generation, can activate mitochondrion generation related protein PGC-1 alpha and increase expression of mitochondrion generation upstream regulatory protein Nrf1
The number of mitochondria has certain influence on the function of the mitochondria, generally, the reduction of the number of the mitochondria can reduce the function of the mitochondria, PGC-1 alpha molecule is taken as a transcription factor to regulate the generation of the mitochondria, and the transcription factor A is mainly used for jointly transcribing mitochondrial constituent protein and mtTFA with nuclear respiratory factor 1 and factor 2 (Nrf 1 and Nrf 2), and the replication and the transcription of the mitochondrial DNA are controlled by the mitochondrial transcription factor A, so that the transcription of the nuclear DNA is combined with the transcription of the mitochondrial DNA; FIGS. 5A-5C show that the number of mitochondria in the liver of diabetic mice was reduced, and that three weeks of continuous intraperitoneal injection of nano-antioxidants significantly increased mitochondrial generation and activated PGC-1 α and upregulated Nrf1 expression;
(6) PBN-like nano-targeting antioxidant capable of improving energy metabolism of mitochondria
Mitochondria generate ATP through a series of redox reactions to provide energy for cells, and the energy metabolism function of the mitochondria is important for maintaining the normal function of the mitochondria; by detecting the energy metabolism function of mitochondria through a hippocampal energy meter, as can be seen from figure 6, the maximum respiration value and ATP production can be obviously increased by injecting the nano antioxidant into the abdominal cavity compared with a diabetic mouse model.
The experimental results prove that the liver-targeting nano antioxidant can effectively inhibit the increase of glucose tolerance of a diabetic mouse, inhibit the increase of gluconeogenesis capability of the diabetic mouse, up-regulate the expression of liver antioxidant protein, increase the generation of liver mitochondria of the mouse, activate mitochondria to generate related protein PGC-1 alpha, increase the expression of mitochondria to generate upstream regulatory protein Nrf1 and improve the energy metabolism function of mitochondria; therefore, the nano antioxidant can regulate the glucose metabolism balance by regulating the redox balance in the liver, so as to prevent the occurrence and development of diabetes, has good application prospect, and provides a new basis for developing new diabetes prevention food and medicines.
The embodiments given above are preferable examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition and replacement made by the technical characteristics of the technical scheme of the invention by a person skilled in the art belong to the protection scope of the invention.
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Claims (7)
1. An application of a nitric oxide spin-trapping nano-targeting antioxidant capable of targeting liver mitochondria in the preparation of drugs or health products for preventing diabetes.
2. The use of claim 1, wherein the Nano-targeted antioxidant is selected from the group consisting of Nano-MitoPBN, Nano-PBN.
3. The use of claim 1 or 2, wherein the medicament is a medicament for inhibiting an increase in glucose tolerance in a diabetic mouse.
4. The use of claim 1 or 2, wherein the medicament is a medicament for inhibiting an increase in gluconeogenic ability in diabetic mice.
5. The use of claim 1 or 2, wherein the medicament is a medicament that upregulates expression of a hepatic antioxidant protein.
6. The use of claim 1 or 2, wherein the medicament is a medicament for increasing mouse liver mitochondrial production, activating mitochondrial production associated protein PGC-1 α, increasing expression of mitochondrial production upstream regulatory protein Nrf 1.
7. The use of claim 1 or 2, wherein the medicament is a medicament for increasing mitochondrial energy metabolism.
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| US20030017148A1 (en) * | 2000-05-11 | 2003-01-23 | Oklahoma Medical Research Foundation | Prevention of insulin-dependent diabetes, complications thereof, or allograft rejection by inhibition of cyclooxygenase-2 activity |
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| CN110151700A (en) * | 2019-06-06 | 2019-08-23 | 复旦大学 | Nano-MitoPBN is preparing the application in anti-oxidant and treatment diabetes medicament |
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| US20030017148A1 (en) * | 2000-05-11 | 2003-01-23 | Oklahoma Medical Research Foundation | Prevention of insulin-dependent diabetes, complications thereof, or allograft rejection by inhibition of cyclooxygenase-2 activity |
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