CN104116724A - Application of FFAR2 natural ligand acetate in preparation of medicine for treating type 1 diabetes - Google Patents

Abstract

The invention relates to the technical field of medicines and in particular relates to application of FFAR2 natural ligand acetate in preparation of medicine for treating type 1 diabetes. The acetate refers to sodium acetate. Male C57BL/6 strain mice are adopted, a MLDS diabetic mouse model is established through a small dose multiple streptozotocin injection method, and a treatment effect of the FFAR2 natural ligand sodium acetate on the type 1 diabetes is observed. In the MLDS diabetic mouse model, the MLDS diabetic mice are treated through FFAR2 natural ligand sodium acetate injection, sugar metabolism states of the MLDS diabetic mouse model can be improved by sodium acetate, and the insulin secretion level is restored. The sodium acetate can be well applied to treatment of the type 1 diabetes, and test data and theoretical basis can be provided.

Description

The application of FFAR2 native ligand acetate in preparation treatment type 1 diabetes medicine

Technical field

The present invention relates to a kind of medical technical field, the particularly application of FFAR2 native ligand acetate in preparation treatment type 1 diabetes medicine.

Background technology

Type 1 diabetes is to be destroyed and cause by autoantibody because can produce the beta Cell of islet of insulin.Beta Cell of islet is unique cell that can produce insulin in body, and beta Cell of islet can be exercised the function of sugared sensor, and in sugar impression and insulin secretion, body, sugared stable state has Central Position in maintaining.Once therefore beta Cell of islet is destroyed, can cause glycemic control disorder, easily there are a series of side reactions such as ketoacidosis, as heart disease, renal failure, visually impaired etc.

Fatty acid (Fatty Acids, FAs) is the carboxylic acid with non-annularity aliphatic tail end.Fatty acid is a kind of important energy source in humans and animals body, is also a kind of signaling molecule simultaneously, has multiple physiological action.Conventionally fatty acid can be divided into short-chain fatty acid (carbochain is less than 6), medium-chain fatty acid (carbochain is between 6 to 12) and long-chain fatty acid (carbochain is no less than 12) three classes according to its aliphatic chain length of receptor.If not endogenous is synthetic, in human body in, long-chain fatty acid is usually with the esterified formation triglyceride of glycerol (Triglycerides, TG), as the energy transhipment in body and the material storing.Short-chain fatty acid usually exists with the form of dissociating.In short-chain fatty acid, the concentration of acetic acid in intestinal and blood circulation is the highest.

The short-chain fatty acid of the free states such as short-chain fat acid acceptor acetic acid is as signaling molecule, can regulate its corresponding receptor downstream signal and the expression of gene, their physiological function and the regulating and controlling effect to the Nutrition and Metabolism disease in body thereof also receive much concern for a long time.But because the specific membrane receptor of short-chain fatty acid is always undiscovered, cannot further go deep into all the time about the understanding of its molecular mechanism.In the twentieth century later stage nineties, the free fatty specific membrane receptor that research starts to find gradually and identify makes people restart to be familiar with the molecular mechanism that free fatty plays a role in Healthy People and patient body.Free-fat acid acceptor (Free Fatty Acid Receptor, FFAR) belongs to g protein coupled receptor (G-Protein-Coupled Receptor, GPCR) family.Mankind GPCR family has approximately 850 members, is the cell surface receptor superfamily with seven cross-film structures, can experience the signals such as light, abnormal smells from the patient, neurotransmitter and hormone molecule, and closely related with signal in a series of cells, physiological process.Wherein, the FFAR basis having been found that the separately length of part fatty acid chain can be divided into: long-chain fat acid acceptor, as GPR120 and GPR40 (FFAR1); Medium-chain fatty acid receptor, as GPR84; And short-chain fat acid acceptor, as GPR43 (FFAR2) and GPR41 (FFAR3).

Existing research has disclosed Adipocyte Differentiation, fat-splitting, sugared stable state, insulin sensitivity, intestinal microbial population interaction, intestinal peristalsis promoting, intestinal peptide secretion, the appetite of short-chain fat acid acceptor in energy metabolism and has all played very important effect in regulating and controlling.FFAR2 can treat for associated metabolic or the relevant disease of intestinal microbial population as a good target spot.

Type 1 diabetes is as a kind of disease that there is no method healing, along with the lasting rising of sickness rate, gradually by more researcher is paid close attention to.Reduce immunoreation and reach at present the object of so-called " treatment " by immunosuppressant, and the command range of this degree " healing " type 1 diabetes or inadequate in fact.Not definite Biological indicators are indicated the process of disease progression at present.Only can not well detect the state of development of this autoimmune disease by clinical indices such as insulin C-peptide, glycolated hemoglobin, sugar and insulin tolerances, conventionally utilize these metabolic index in the time of the terminal of research, could to show the success or not of studying, and cannot allow researcher show that clearly corresponding intervention is whether in the real control that participates in disease of duration of test.Therefore the disappearance of critical biomarker may make a lot of real effect that clinical intervention brings be hidden in noise.

Summary of the invention

The object of this invention is to provide the effect of FFAR2 native ligand acetate in treatment type 1 diabetes, provide theoretical foundation and experimental data for treating clinically type 1 diabetes.

The present invention relates to the application of FFAR2 native ligand acetate in preparation treatment type 1 diabetes medicine.Preferably, described acetate is sodium acetate.

The clinical resource of this research and utilization Shanghai Ruijin Hospital department of endocrinology; collect the normal healthy people of onset type 1 diabetes people and age-sex's coupling; made the variation of mrna expression spectrum of its PBMC; analysis result shows: onset type 1 diabetes patient FFAR2 in PBMC mrna expression amount with the horizontal positive correlation of patient's insulin C-peptide; and FFAR2 high expressed group has lower HbA1c level, illustrate that FFAR2 high expressed plays a protective role to type 1 diabetes patient.

Male C57BL/6 Strains of Mouse for this experiment, low dose repeatedly streptozotocin injection (MLDS) builds diabetic mice model, observes the effect of FFAR2 native ligand sodium acetate to type 1 diabetes.Sodium acetate, as the activator of FFAR2, is because acetate is the native ligand of FFAR2 on the one hand, on the other hand because acetic acid is compared the activation FFAR2 that other short-chain fatty acid can be more special.In MLDS diabetic mice model, FFAR2 native ligand sodium acetate can improve mice carbohydrate metabolism state, recovers insulin secretion level, for treatment type 1 diabetes provides foundation.

Beneficial effect of the present invention is:

1, the present invention has verified that FFAR2 mrna expression level in onset type 1 diabetes patient's PERIPHERAL BLOOD MONONUCLEAR CELL PBMC raises.Mrna expression amount is with the horizontal positive correlation of patient's insulin C-peptide in PBMC for onset type 1 diabetes patient FFAR2, and FFAR2 high expressed group has lower HbA1c level, illustrates that FFAR2 high expressed plays a protective role to type 1 diabetes patient.

2. the present invention is in MLDS diabetic mice model, and FFAR2 native ligand sodium acetate injection treatment MLDS diabetic mice, has verified that sodium acetate can improve MLDS diabetic mice carbohydrate metabolism state, recovers insulin secretion level.For the sodium acetate application in treatment type 1 diabetes better, test data and theoretical foundation are provided.

Brief description of the drawings

Fig. 1 is the random blood sugar test comparison figure of MLDS diabetic mice after sodium acetate injection.

Fig. 2 is the blood glucose insulin level test comparison figure of MLDS diabetic mice after sodium acetate injection.

Fig. 3 is the table sugar tolerance test comparison figure of MLDS diabetic mice after sodium acetate injection.

Fig. 4 is that the IL6 gene expression of MLDS diabetic mice after sodium acetate injection detects comparison diagram.

Detailed description of the invention

Below in conjunction with embodiment, the invention will be further described:

Embodiment

1, the structure of MLDS diabetic mice model

It is male C57BL/6 Strains of Mouse that this institute adopts mouse model, and experiment is 6-8 age in week while beginning.At specific pathogen free microorganism (Specific Pathogen Free, SPF) under rank environment, raise, room temperature maintains 20-25 DEG C and gives 12 hour daytime that rule is certain, 24 hour daily cycle at 12 hour night, and the common Mus grain of the customization that gives to eat at any time and the sterilized water drunk at any time.

Adopt repeatedly streptozotocin injection (multiple-low-dose streptozocin, MLDS) structure diabetic mice model of low dose.Experiment mice is given lumbar injection streptozotocin (streptozocin, STZ), injects STZ, injected dose continuous 5 day every day: 50mg/kg body weight.Mice STZ configures in 0.05mmol/L citrate buffer (pH4.5).

2, the research of FFAR2 native ligand sodium acetate.

The sodium acetate processing of MLDS diabetic mice model, detects FFAR2 native ligand sodium acetate diabetes is occurred to for the impact developing.

Experiment grouping: whether whether experiment be divided into injection sodium acetate (adding FFAR2 part) according to injection MLDS (making diabetes model): Vehicle+PBS group, Vehicle+ sodium acetate group, MLDS+PBS group, MLDS+ sodium acetate group, four groups altogether.

The sodium acetate medication of sodium acetate injection group: inject latter two hours at STZ while injection two groups of mices of injection sodium acetate continuous three days, STZ before STZ injection, and inject sodium acetate rear continuous 8 week every day in STZ injection, per injection dosage is: 500mg/kg body weight.

During modeling and processing, observe continuously the drinking-water feed situation of mice every day, and detect weekly body weight change.Adopt blood glucose meter and supporting blood sugar test paper monitoring mouse blood sugar.Abdominal cavity carbohydrate tolerance experiment (intraperitoneal glucose tolerance test, IPGTT) is carried out after STZ injection finishes 4 weeks, and mice is given glucose dosage and is: 2g/kg body weight.Experiment is in mice fasting after 10-14 hour, the morning 8-10 point start to carry out.Plasma insulin level uses ELISA (enzyme-linked immunosorbent assay) test kit (R & D Systems) to detect, and gets the blood time and be after STZ injection the 8th week, and mice is by fasting after 12 hours.

Experimental result: after MLDS modeling, the phenomenon of obvious polydipsia polyphagia has appearred in mice, and polydipsia polyphagia is also one of typical phenotype of diabetes, and the success of modeling has been described from the side.And in MLDS diabetic mice group, the injection of additional sodium acetate, although can see diet situation and the solvent injection group no significant difference to sodium acetate not; But amount of drinking water has significantly reduced, sodium acetate injection group is compared with solvent injection group, has improved the phenotype that diabetic mice drinking-water increases.

This research simultaneously also continues to have detected the random blood sugar state of each group of mice, and as shown in Figure 1, non-MLDS diabetic mice does not all have the difference of blood glucose to result in the situation that having or not sodium acetate, and random blood sugar fluctuation is also not obvious; But after MLDS modeling, STZ injection finishes second day, be the 10th day shown in Fig. 1, MLDS modeling group mice has occurred that obvious blood glucose rises, more than random blood sugar rises to 13mmol/L, this is also the most typical and direct evidences of diabetes, illustrates that MLDS has caused beta Cell of islet destruction, mouse blood sugar is regulated disorderly, diabetes model modeling success.And simultaneously in lasting observation, the blood glucose situation of sodium acetate administration group is obviously better than group of solvents (P<0.01), illustrates that the part acetate of FFAR2 can improve the phenotype of diabetes blood sugar increasing.

Observing on the basis of random blood sugar improvement, be easy to associate whether variation has also occurred insulin level.So this research is measured the insulin level between experimental group, as shown in Figure 2, can see, MLDS diabetic model group is compared with control treatment mice, and insulin level significantly declines, and fall 49.8% further illustrates modeling success; And acetate injection is compared with PBS solvent injection, mouse islets element secretion level is significantly replied, and ratio reaches 37.5%.Prove that FFAR2 part sodium acetate has certain protective effect to MLDS diabetic mice really, can improve its insulin secretion state.

This research, also at 4 weeks of sodium acetate processing, has further been carried out lumbar injection glucose tolerance experiment IPGTT, detects sodium acetate and processes the carbohydrate metabolism ability of rear MLDS mice.As shown in Figure 3 (as, dotted line is expressed as MLDS injected in mice sodium acetate, solid line represents MLDS injected in mice PBS solvent), consistent with expection, after sodium acetate is processed, mouse glucose tolerance is significantly improved.Illustrate that the sugared stable state that FFAR2 part sodium acetate can improve mice to a certain extent maintains ability.

In islets of langerhans, the apoptotic process of β cell is usually accompanied by immunoreation.Because spleen is the place that in a large amount of blood, immunocyte is assembled, therefore the inflammatory conditions of spleen has reflected the inflammatory conditions of mice whole body, for this this research has detected the mrna expression of the indicator cell line factor IL6 of inflammatory reaction in mouse spleen.Result is as shown in Figure 4: diabetic model group is compared with control treatment mice, and the mRNA level of IL6 significantly raises.And the remarkable decline compared with model group of acetate processed group plasma insulin level.FFAR2 part acetate can improve the inflammatory conditions of insulin mice to a certain extent.

3, FFAR2 high expressed in type 1 diabetes human peripheral blood single nucleus cell (PBMC)

The clinical resource of this research and utilization Shanghai Ruijin Hospital department of endocrinology, collect the normal healthy people of onset type 1 diabetes people and age-sex's coupling, the mRNA level of getting 10 routine normal persons and 10 routine type 1 diabetes patients' PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) Free Fat acid acceptor FFAR2 compares, result shows, the mRNA level of type 1 diabetes patient's PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) Free Fat acid acceptor FFAR2 significantly raises, and is 4.9 times (p<0.05) of normal group.This research further utilizes the method for quantitative fluorescent PCR, by same cDNA sample proofing chip result, and other two member FFAR1 and FFAR3 of simultaneous verification FFAR2 family, testing result is: FFAR2 does not have significant difference with family member FFAR1 and FFAR3 between onset type 1 diabetes example and normal control.In onset type 1 diabetes (T1D) patient's PBMC, the mrna expression of FFAR2 is raised nearly 8 times.

Claims (2)

1. The application of 1.FFAR2 native ligand acetate in preparation treatment type 1 diabetes medicine.
2. 2. application claimed in claim 1, is characterized in that: described acetate is sodium acetate.