Application of target protein in improving cognitive hypofunction of type 2 diabetes mellitus

Abstract

The invention discloses application of a target protein in improving cognitive hypofunction of type 2 diabetes mellitus, and belongs to the field of biological medicine. Protein expression of the MTMR12 is determined to be remarkably reduced in hippocampus of a T2DM mouse with cognitive impairment through proteomics sequencing, and the cognitive impairment of the mouse is improved through brain stereotactic injection of MTMR12 overexpression lentivirus. Under the condition that the total swimming distance and the swimming speed are not obviously different, the cognitive function of mice in a single MTMR12 overexpression group is not obviously changed compared with that of mice in a control group, but the escape latency of T2DM mice injected with MTMR12 overexpression lentivirus groups is about 46% shorter than that of the T2DM mice, the number of times of platform crossing is about 2.8 times larger than that of the T2DM mice, and the staying time in a target quadrant is increased by 1.3 times.

Description

TECHNICAL FIELD

[0001] The application relates to an application of a target protein in improving cognitive dysfunction in type 2 diabetes, and belongs to the field of biological medicine. BACKGROUND

[0002] Diabetes mellitus (DM) is a systemic metabolic disease characterized by insulin resistance and hyperglycemia. According to the data of International Diabetes Federation (IDF) in 2021, the number of DM patients is increasing, which will bring a serious burden to social development. Type 2 diabetes mellitus (T2DM) is closely related to various complications, and cognitive dysfunction (CD) is one of the common complications of the nervous system in T2DM patients. Studies have shown that the decline in cognitive ability, cognitive impairment or dementia in T2DM patients is related to the influence of long-term hyperglycemia.

[0003] T2DM cognitive dysfunction is insidious in onset and its progression is related to the control of blood glucose level. There is currently no effective treatment drug for delaying and reversing the progression of cognitive impairment disease, so in the treatment of type 2 diabetes cognitive dysfunction, the current guidelines usually recommend a combination of lifestyle interventions and methods such as antidiabetic drug therapy. Although hypoglycemic drugs have a protective effect on cognitive function, they have greater side effects and reduce the sensitivity of target organs to drugs, so drug treatment targeting T2DM cognitive dysfunction-related targets may become an important means of treatment and prevention. SUMMARY

[0004] To solve the above problems, the application determines that the expression of Myotubularin Related Protein 12 (MTMR12) in the hippocampal tissues of T2DM mice and Control mice has a significant difference through proteomics sequencing of the hippocampal tissues of the T2DM mice and the Control mice, and the expression amount of MTMR12 in the T2DM group is significantly reduced. In the later stage, it is found that the cognitive function of the mice is improved through brain stereotactic injection of MTMR12 overexpression lentivirus into the hippocampal tissues of the mice. On this basis, the patent uses MTMR12 as a target protein to prevent and treat cognitive dysfunction in type 2 diabetes.

[0005] The application provides a MTMR12 protein as a prevention and / or treatment target in the preparation of a diabetes cognitive dysfunction drug action target.

[0006] The sequence of the MTMR12 protein is shown in SEQ ID NO. 3:

[0007] MLGKGGVGGGGGTKAPKPSFVSYVRPEEIHTDEKEVTEKEVTLHLLPGEQLLCEASTVLKYVQEDSCQRGVYGRLVCTDFKISFLGDEDSALDNGGEAQFKNKIIGVNDVPLHCVDQIYGVFDEKKKPLFGQLKKYPEKLVIHCKDLRVLHFCLRYTKEEEVKRIVSGIIHHTQSPKLLKRLFLFSYAAAVHGTATDSRNCTVMFDTPKDWCWELERTKGSVKYRTVSVNEGYRVSDRLPAYFVVPTPLPEDDVRRFQGHGIPIWCWSCHNGSALLKMSALPKEQDDGALQVQKSFLDGIYKTIHRPPYEMVKTEDLSSNFLSLQEIQSAYCKFKQLFLIDNSSEFWDTDIKWFSLLESSSWLDIIRRCLKKAIEITECLEAQNVNVLLLEENASDLCCLLSSLVQVMMDAHCRTRTGFQSLIQKEWVMGGHSFLDRCNHLHQSDKEEVPIFLLFLDCVWQLVHQYPPAFEFTETYLTVLSDSLYIPIFSTFFFNSPHQKDTNMGRENLDAQSKPLTLLTVWDWSVQFEPKAQTLLRNPLYVEKPKLDRGQQKGSRFKHQRQLSLPLTQSKSSPKRGFFREETDHLIKNLLGKRISKLINSSDDLQDSSREFYDSWHSKPTDYHGLLLPHIEGPEIKVWAQRYLRWIPEAQILGGGRVATMGKLLEMMEEVQSLQEKIEARHHRQEAVHAEAPGLLRNSVRLSSLFPFALLQRHSAKPVLPTSGWKALGGEDDLAKREDEFVDLGDV

[0008] In an embodiment, the cognitive dysfunction of type 2 diabetes is a dysfunction of the nervous system caused by type 2 diabetes, and early symptoms include impaired language and visual memory, executive function, motor function, and information processing speed, and as the disease progresses, later symptoms include mental and behavioral abnormalities, delirium, and the like, and eventually there is a probability of developing Alzheimer's disease or dementia.

[0009] In an embodiment, the prepared drug is in any pharmaceutically acceptable dosage form.

[0010] In an embodiment, the prepared dosage form comprises: tablets, inhalation preparations, oral liquids, injections, external preparations.

[0011] In an embodiment, the prepared medicine comprises a pharmaceutically acceptable excipient.

[0012] In an embodiment, the prepared medicine excipient comprises one or more of probiotics, lactose, maltose, microcrystalline cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, starch, sucrose, glucose, aspartame, water, glycerol, whey protein powder, chitooligosaccharide.

[0013] In an embodiment, the medicine needs to have the following functions:

[0014] (1) acting on the target protein: MTMR12, increasing the expression of the target protein;

[0015] (2) improving cognitive dysfunction or slowing the progression of cognitive decline.

[0016] In an embodiment, the viral vector comprises an adeno-associated viral vector, a lentiviral vector, an adenoviral vector, and a retroviral vector.

[0017] In an embodiment, the medicine comprises an MTMR12 overexpression lentiviral vector, and the lentiviral vector is constructed using primers comprising nucleotide sequences as shown in SEQ ID NO. 1-2.

[0018] In an embodiment, the dosage form of the medicine is any one of suspension, granules, capsules, powders, tablets, emulsions, dripping pills, injections, suppositories, enemas, aerosols, patches, or drops.

[0019] In an embodiment, the administration route of the medicine is oral administration, sublingual administration, rectal administration, skin mucosa administration, inhalation administration, or injection administration.

[0020] The present application also provides a use of an MTMR12 protein activator in the preparation of a medicine for preventing and / or treating diabetic cognitive dysfunction.

[0021] In an embodiment, the MTMR12 protein activator comprises any viral vector, plasmid, or small molecule compound that can promote the expression of MTMR12 protein.

[0022] In an embodiment, the MTMR12 protein activator is an MTMR12 overexpression lentiviral vector, and the lentiviral vector is constructed using primers comprising nucleotide sequences as shown in SEQ ID NO. 1-2.

[0023] SEQ ID NO. 1: gcgaattcgaagtatacctcgagGCCACCatgctggggaaaggggga;

[0024] SEQ ID NO. 2: gcgatcgcagatccttggatcctcacacgtcccctaggtccac.

[0025] In an embodiment, the medicine comprises a pharmaceutically acceptable excipient.

[0026] In an embodiment, the medicine comprises a pharmaceutically acceptable dosage form.

[0027] In an embodiment, the dosage form of the medicine is any one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a dripping pill, an injection, a suppository, an enema, an aerosol, a patch, or a drop.

[0028] In an embodiment, the administration route of the medicine is oral administration, sublingual administration, rectal administration, skin mucosal administration, inhalation administration, or injection administration.

[0029] The present application also provides a use of the MTMR12 protein in screening a medicine for preventing or treating diabetic cognitive dysfunction.

[0030] The present application also provides a medicine capable of preventing or treating diabetic cognitive dysfunction, the medicine comprising an MTMR12 protein activator.

[0031] In an embodiment, the MTMR12 protein activator comprises any virus vector, plasmid, or small molecule compound capable of promoting expression of the MTMR12 protein.

[0032] In an embodiment, the MTMR12 protein activator is an MTMR12 overexpression lentivirus vector, and the lentivirus vector is constructed using primers comprising nucleotide sequences as shown in SEQ ID NO. 1 and 2.

[0033] SEQ ID NO. 1: gcgaattcgaagtatacctcgagGCCACCatgctggggaaaggggga;

[0034] SEQ ID NO. 2: gcgatcgcagatccttggatcctcacacgtcccctaggtccac.

[0035] In an embodiment, the medicine comprises a pharmaceutically acceptable excipient.

[0036] In an embodiment, the drug comprises a pharmaceutically acceptable dosage form.

[0037] In an embodiment, the dosage form of the drug is any one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a dripping pill, an injection, a suppository, an enema, an aerosol, a patch, or a drop.

[0038] In an embodiment, the administration route of the drug is oral administration, sublingual administration, rectal administration, skin mucosa administration, inhalation administration, or injection administration.

[0039] Advantages

[0040] (1) The present application determines that the expression of MTMR12 protein is significantly down-regulated in the hippocampal tissue of T2DM mice with cognitive dysfunction through proteomic sequencing, and the cognitive dysfunction of the mice is improved by stereotactic injection of MTMR12 overexpression lentivirus. The expression amounts of PSD95, BDNF, and Synaspinl proteins are all significantly increased in the hippocampus of the mice injected with the MTMR12 overexpression lentivirus. Among them, the expression amount of PSD95 protein is up-regulated by about 1.6 times, the expression amount of BDNF is up-regulated by about 1.8 times, and the expression amount of Synaspinl protein is up-regulated by about 1.6 times. On this basis, the present application uses MTMR12 as a new target protein to prevent and / or treat diabetic cognitive dysfunction.

[0041] (2) The target protein MTMR12 in the present application can alleviate the cognitive dysfunction induced by type 2 diabetes. It can be observed through the water maze behavior experiment that the learning and memory ability of the T2DM mice injected with the MTMR12 overexpression lentivirus is obviously improved. In the case that there is no significant difference in the total swimming distance and swimming speed, the cognitive function of the mice in the MTMR12 overexpression group alone has no obvious change compared with the control group of mice, but the escape latency of the T2DM mice injected with the MTMR12 overexpression lentivirus is about 46% shorter than that of the T2DM mice, the number of times of crossing the platform is about 2.8 times more, and the time of staying in the target quadrant is increased by 1.3 times. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The blood glucose value changes of the T2DM mice and the control group of mice 1-8 weeks after injection of STZ;

[0043] Figure 2Behavioral experiments on the water maze were conducted on T2DM mice and control mice 8 weeks after STZ injection. A represents the latency curves for finding the hidden platform during the experimental training period from day 1 to day 5; B represents the swimming routes of the two groups of mice within 1 minute after the platform was removed on day 6; C represents the number of times the two groups of mice crossed the location of the glass platform within 1 minute on day 6; D represents the time spent in the target quadrant within 1 minute for the two groups of mice; and E represents the swimming latency of the two groups of mice on day 6.

[0044] Figure 3 This image shows differentially expressed proteins and pathway enrichment maps between the control group and type 2 diabetic (T2DM) mice in proteomics sequencing analysis; where A is a clustering heatmap and B is a GO enrichment analysis of the differentially expressed proteins.

[0045] Figure 4 To screen and validate differentially expressed proteins, the study included: A) qPCR validation of differentially expressed genes, selecting 15 differentially expressed proteins from 45 downregulated proteins, and examining their mRNA levels in Control and HG group cells; BC Western blotting to detect MTMR12 expression in Control and HG group cells, examining MTMR12 protein levels at the cellular level; DE Western blotting to detect MTMR12 protein expression in the hippocampus of Control and T2DM group mice, examining MTMR12 protein levels at the animal level; and F qPCR detection of MTMR12 mRNA levels in the hippocampus of Control and T2DM group mice, examining MTMR12 mRNA levels at the animal level.

[0046] Figure 5 To conduct behavioral experiments on the water maze in T2DM mice after stereotactic injection into the brain and injection of a lentivirus overexpressing MTMR12 into the hippocampus.

[0047] Figure 6 Western blot analysis was performed to verify the role of PSD95, Synapsin1, BDNF, and cognitive function-related proteins.

[0048] Figure 7 A schematic diagram illustrating the construction of the T2DM mouse model.

[0049] Figure 8 A schematic diagram illustrating the construction of a T2DM mouse model overexpressing MTMR12. Detailed Implementation

[0050] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0051] The raw materials used in the following examples: streptozotocin (STZ) was purchased from Sigma-Aldrich; HMC3 cells were purchased from Wuhan Boster Biological Engineering Co., Ltd.; Mice were purchased from 40 male 6-8 week old C57BL / 6 mice purchased from Spafar (Beijing) Biotechnology Co., Ltd.; MTMR12 overexpression lentivirus was purchased from Shanghai Jiman Biotechnology Co., Ltd.; High-fat feed formula: Wuxi Fanpo Biotechnology Co., Ltd. (21.8 kJ / g, 60% fat); basal feed was purchased from Wuxi Fanpo Biotechnology Co., Ltd. (14.7 kJ / g, 13% fat)

[0052] The method of hippocampal brain stereotactic injection involved in the following examples is as follows:

[0053] For hippocampal brain stereotactic injection in mice, the anesthetized mouse was fixed on the brain stereotactic instrument, the head hair was removed, the scalp was cut longitudinally, the skull was fully exposed, the periosteum was wiped off, the Bregma point (fontanel point) was marked as zero, and the injection coordinates of the hippocampal tissue were determined and marked according to the anatomical atlas ( Figure 8 ):

[0054] X = 1.5 mm (left and right);

[0055] Y = 2.0 mm (anterior and posterior);

[0056] Z = 2.0 mm (depth);

[0057] A dental drill was used to slowly drill a hole at the marked position, a microsyringe needle was slowly inserted to the predetermined depth using a microsyringe pump, and each hippocampus was injected at a speed of 0.2 μL / min. After the injection was completed, the needle was slowly pulled out, and the scalp of the head was sutured with absorbable suture. Behavioral testing was performed after 6 weeks.

[0058] The method for detecting protein concentration involved in the following examples

[0059] The hippocampal tissue was weighed and lysed on ice for 30 min, and the supernatant was obtained by centrifugation. The protein concentration was determined using a BCA kit, the OD value was measured at 567 nm, and the concentration was calculated by a standard curve. After uniforming the protein concentration, protein buffer was added to prepare the sample for electrophoresis. According to the molecular weight of the target protein, an SDS-PAGE gel was prepared, and an electrophoresis device was connected. 20 μg of protein and 3 μL of protein marker were added to each sample well, and the electrophoresis time was set. After electrophoresis, the protein was transferred from the gel to the PVDF membrane and blocked in 5% skim milk for 1 h; the required primary antibody was added, and the incubation was performed overnight on a shaking bed at 4°C; after recovering the primary antibody, the secondary antibody was added, and the membrane was placed in a gel imager for exposure and development to detect the target band. Finally, the gray value of the target band was analyzed, and the protein expression level was evaluated by comparing with the internal reference band.

[0060] HMC3 cells and hippocampal tissues were lysed with TRIZOL, chloroform was added, after shaking, centrifugation and standing, the supernatant was taken, an equal amount of isopropanol was added to precipitate the sediment, and the sediment was washed with 75% ethanol to obtain relatively pure RNA. An appropriate amount of enzyme-free water was added to dissolve the sediment, and the 260 / 280 absorbance was measured to dilute the concentration to 300-500. According to the different concentrations of each group, 3 μg of RNA was taken and diluted to 12 μL, 5x buffer 3 μL was added, mixed, incubated at 42°C for 2 min, 4x buffer 5 μL was added, mixed, and RT-PCR was performed to obtain cDNA. 2 μL of cDNA was taken and mixed with 5 μL of mix, 0.4 μL of forward and reverse primers, and 2.6 μL of water to perform RT-PCR detection in a 96-well plate. The primer sequences are shown in Table 2. The relative expression of the target gene was calculated by 2-ΔΔCt method and normalized to β-actin. All experimental steps were performed according to the manufacturer's instructions.

[0061] The primers involved in the following examples are shown in the following table:

[0062] Table 1: Primer sequences

[0063]

[0064]

[0065] The Morris water maze (MWM) experiment involved in the following examples is as follows:

[0066] The Morris water maze (MWM) experiment includes two aspects, namely spatial acquisition training and spatial search training. On the first day at 8:00, the mice were moved into the water maze room to adapt to the surrounding environment in advance, reducing the influence caused by environmental mutation. The water maze is a circular pool with a radius of 120 cm and a depth of 60 cm. The pool is divided into four quadrants, and different colored and shaped cards such as red pentagrams and yellow squares are pasted on the pool walls of each quadrant. A 40 cm high cylindrical organic glass platform is placed in the middle of the fourth quadrant. In order to avoid the mice seeing the platform and to easily separate the mice from the water, milk powder is poured into the water, the water temperature is maintained at 21-25°C, and the liquid level is maintained at 1-2 cm above the platform.

[0067] The MWM experiment lasted for 6 days. On the first day at 8:00, the mice were moved into the water maze room to adapt to the surrounding environment in advance, reducing the influence caused by environmental mutation. The water maze is a circular pool with a radius of 120 cm and a depth of 60 cm. The pool is divided into four quadrants, and different colored and shaped cards such as red pentagrams and yellow squares are pasted on the pool walls of each quadrant. A 40 cm high cylindrical organic glass platform is placed in the middle of the fourth quadrant. In order to avoid the mice seeing the platform and to easily separate the mice from the water, milk powder is poured into the water, the water temperature is maintained at 21-25°C, and the liquid level is maintained at 1-2 cm above the platform.

[0068] On the last day, the platform was removed, and the mice were fixed to enter the water from the opposite quadrant of the original platform location, and timing was performed for 60 s. The latency, the number of crossings of the target platform, the residence time in the target quadrant, the total path of swimming, and the swimming speed were recorded.

[0069] Example 1: Construction of T2DM mice

[0070] The model construction is shown in Figure 7 .

[0071] After adaptive feeding for 0-7 days, C57BL / 6J mice were randomly divided into a control Control group and a model T2DM group; the specific steps are as follows:

[0072] (1) Days 7-35: Feeding with high-fat feed or normal feed

[0073] The Control group was maintained on a normal feed (normal feed) diet (14.7 kJ / g, 13% energy as fat) throughout the experiment for 4 weeks.

[0074] The T2DM group was given high-fat feed (21.8 J, 60% fat, D12492) for 4 weeks.

[0075] (2) Days 36-45

[0076] The T2DM group (model group): After 4 weeks of feeding, on days 36-40, after 12 h of daily fasting without water deprivation, STZ was injected intraperitoneally at 50 mg / kg. The STZ was dissolved in a citric acid-trisodium citrate buffer and stored in the dark under refrigeration. The injection was completed within 30 min after preparation, and the injection was performed for 5 consecutive days. After intraperitoneal injection of the mice, high-fat feed was given for days 40-44, and the mice were fasted without water deprivation for 10 h on day 45. The fasting blood glucose (FBG) was measured.

[0077] The Control group (blank group): After 4 weeks of feeding, on days 36-40, the mice were injected with the same dose of citric acid-trisodium citrate buffer as the model group after 12 h of daily fasting without water deprivation. The injection was performed for 5 consecutive days. After intraperitoneal injection of the mice, the mice were fed with normal feed for days 40-44, and the mice were fasted without water deprivation for 10 h on day 45. The fasting blood glucose (FBG) was measured. The results are shown in Table 2.

[0078] Table 2: Blood glucose values (unit: mmol / L) of the Control group and the T2DM group

[0079]

[0080] The results show that the fasting blood glucose (FBG) is determined, and the model is successful when the FBG is greater than or equal to 11.1 mmol / L for two times. The FBG of the mice in the Control group is less than or equal to 7 mmol / L.

[0081] (3) Day 46 to day 106

[0082] The mice in the T2DM group and the Control group are continuously fed with the respective feed for 8 weeks (day 46 to day 106);

[0083] At the same time, on day 101, the mice are subjected to the MWM experiment for 6 days. The results are shown in the following table and figure: Figure 2

[0084] Table 3: The escape latency (unit: s) of the Control group and the T2DM group during the spatial collection training experiment (day 1-5)

[0085]

[0086] The average number of times of crossing the platform for the Control group and the T2DM group is 3 times and 1 time, respectively;

[0087] The average time of staying in the target quadrant for the Control group and the T2DM group is 20.13 s and 6.92 s, respectively;

[0088] The average escape latency on the sixth day for the Control group and the T2DM group is 12.75 s and 30.93 s, respectively.

[0089] Example 2: Determination of MTMR12 protein

[0090] The animal experiment is the same as in Example 1. After the experiment is completed, the mice are sacrificed and subjected to protein analysis. The specific steps are as follows:

[0091] (1) Hippocampal proteomics analysis of T2DM cognitive dysfunction mice

[0092] The hippocampal tissues of the two groups of mice are rapidly frozen in liquid nitrogen after being sacrificed, and subjected to proteomics sequencing analysis to screen for differential proteins.

[0093] The results are shown in the following table and figure: Figure 3

[0094] Figure 3 A is a clustering heat map showing that there are 23 up-regulated proteins and 45 down-regulated proteins in the T2DM (DN) group compared with the Control group mice; Figure 1 ​​B. GO enrichment analysis of differential proteins showed that the down-regulated differential proteins were involved in the regulation of pathways related to memory and neural inflammation.

[0095] (2) Screening of differential proteins

[0096] As shown in Figure 4 :

[0097] Figure 4 To screen differential proteins, Figure 4 A. Through literature research, 15 differential proteins were selected from 45 down-regulated proteins, and mRNA level test was performed on the cells of Control group and HG group; the specific steps are as follows:

[0098] The frozen HMC3 cells were thawed and recovered, and were cultured in a 60mm culture dish with MEM medium. The logarithmic growth phase cells were trypsinized, passaged, and inoculated in a 6-well plate. The Ctrl group and the HG group (35mmol / L glucose was added) were set up, each group had 6 replicates, and incubated for 48h. The cells of each group were collected, and the expression levels of 15 differential genes in the cells were detected by RT-PCR.

[0099] The results showed that Thoc2, Mtmr12 and Tgfbrap1 were the most significant differences; Figure 4 B- Figure 4 C. The MTMR12 protein level test was performed at the cellular level.

[0100] In the present application, MTMR12 was selected for subsequent experiments;

[0101] Figure 4 D- Figure 4 E. The MTMR12 protein level test was performed at the animal level. Figure 4 F. The MTMR12 mRNA level test was performed at the animal level, and the results were consistent, that is, the mRNA and protein levels of MTMR12 were significantly reduced under high glucose conditions.

[0102] Example 3: Overexpression of MTMR12 can improve cognitive dysfunction of T2DM mice

[0103] The specific steps are as follows:

[0104] 1. Preparation of experimental reagents

[0105] The lentiviral vector for injection was stored at -80℃ before injection, and was taken out to room temperature for thawing before injection, and then injected.

[0106] Construction of MTMR12 OE (lentivirus overexpressing MTMR12) for injection: The specific construction was entrusted to a company (Shanghai Jiman Company) for construction.

[0107] 2. Successfully constructed a model specifically overexpressing MTMR12

[0108] The mouse T2DM model and the blank group model were respectively constructed according to the method of Example 1, wherein the number of mice n = 16 in the Control group and the number of mice n = 20 in the T2DM group, the Control group was fed with ordinary maintenance feed, and the T2DM group was continued to be fed with HFD; after adaptive feeding for 0-7 days, the C57BL / 6J mice were randomly divided into: a control Control group and a model T2DM group;

[0109] The specific steps are as follows Figure 8 ) :

[0110] (1) 7-35 days: feeding with high-fat feed or ordinary feed

[0111] The Control group was maintained on a normal feed (ordinary feed) diet (14.7 kJ / g, 13% energy as fat) throughout the experiment and was fed for 4 weeks.

[0112] The T2DM group was given high-fat feed (21.8J, 60% fat, D12492) and was fed for 4 weeks.

[0113] (2) 36-45 days:

[0114] The T2DM group (model group): after 4 weeks of feeding, from day 36 to day 40, after 12 hours of daily fasting without water, STZ 50 mg / kg was injected intraperitoneally, STZ was dissolved in citric acid-trisodium citrate buffer and stored in the dark under refrigeration, and was injected within 30 minutes after preparation, for 5 consecutive days; after intraperitoneal injection of the mice, from day 40 to day 44, the mice were fed with high-fat feed, and on day 45, the mice were fasted without water for 10 hours, and the fasting blood glucose (FBG) of the mice was measured;

[0115] The Control group (blank group): after 4 weeks of feeding, from day 36 to day 40, the mice were fasted without water for 12 hours daily, and the mice were injected with the same dose of citric acid-trisodium citrate buffer as the model group, for 5 consecutive days; after intraperitoneal injection of the mice, from day 40 to day 44, the mice were fed with ordinary feed, and on day 45, the mice were fasted without water for 10 hours, and the fasting blood glucose (FBG) of the mice was measured;

[0116] (3) 46-101 days

[0117] The T2DM group (model group) and the Control group (blank group) mice continue to be fed with respective feed (day 46-day 101) ;

[0118] (4) On day 101, the Control group and the T2DM group mice are randomly divided into 2 groups, and the specific grouping is as follows:

[0119] Blank group-1: ND+vector;

[0120] Blank group-2: ND+MTMR12 OE;

[0121] Model group: T2DM+vector;

[0122] Treatment group: T2DM+MTMR12 OE.

[0123] Among them:

[0124] ND+vector group (blank group-1): 8, injecting control lentiviral vector vector into the hippocampus of each mouse in the blank group, 2 μL in one side of the hippocampus, and 4 μL in both sides;

[0125] ND+MTMR12 OE group (blank group-2): 8, injecting MTMR12 overexpression lentivirus into the hippocampus of each mouse in the blank group, 2 μL in one side of the hippocampus, and 4 μL in both sides;

[0126] T2DM+vector group (model group): 10, injecting control lentiviral vector vector into the hippocampus of each mouse in the model group, 2 μL in one side of the hippocampus, and 4 μL in both sides;

[0127] T2DM+MTMR12 OE group (treatment group): 10, injecting MTMR12 overexpression lentivirus into the hippocampus of each mouse in the model group, 2 μL in one side of the hippocampus, and 4 μL in both sides.

[0128] According to the above grouping, the mice hippocampus is injected by brain stereotactic positioning.

[0129] Day 101-148:

[0130] On day 101, after injecting the mice, each group is fed with respective feed (blank group: normal feed, model group and treatment group: high-fat feed) (day 101-148); and behavioral detection is performed on day 143-148.

[0131] 3. Experimental results

[0132] The results show that:

[0133] (1) During the spatial acquisition training experiment (days 1-5), the escape latency of the MTMR12 OE group mice was significantly shorter than that of the T2DM group mice, Figure 5 The data after the experiment on day 5 are shown in the following table:

[0134] Table 4: Escape latency of mice (unit: s)

[0135]

[0136] The same results were obtained in the spatial search experiment after removing the transparent platform on day 6 Figure 5 ).

[0137] (2) Other behavioral test results:

[0138] The average number of times that the mice in the ND+vector, ND+MTMR12 OE, T2DM+vector, and T2DM+MTMR12 OE groups crossed the platform was 3.8, 3.2, 1, and 3 times, respectively; the number of times that the mice in the MTMR12 OE group crossed the platform increased relative to the T2DM group mice.

[0139] The average escape latency time of the mice in the ND+vector, ND+MTMR12 OE, T2DM+vector, and T2DM+MTMR12 OE groups on day 6 was 11.50 s, 6.87 s, 40.77 s, and 18.80 s, respectively; the latency was shortened.

[0140] The average time that the mice in the ND+vector, ND+MTMR12 OE, T2DM+vector, and T2DM+MTMR12 OE groups spent in the target quadrant was 14.23 s, 14.30 s, 8.78 s, and 11.33 s, respectively; the time spent in the target quadrant was prolonged.

[0141] (3) Protein samples were prepared from the hippocampal tissues of the mice for WB experiments to detect the expression of three proteins, PSD95, Synapsin1, and BDNF Figure 6 ), and the results are shown in the following table:

[0142] Table 5: Expression of different proteins in mice in different groups

[0143]

[0144] The results show that MTMR12 OE can upregulate the expression of these three proteins in the hippocampus of T2DM mice. Moreover, the overexpression efficiency of MTMR12 is confirmed in the WB.

[0145] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. Application of MTMR12 protein as a preventive and / or therapeutic target in the preparation of drugs for the treatment of cognitive impairment in diabetes.

2. The application according to claim 1, characterized in that, The drug can promote the expression of MTMR12 protein; Preferably, the drug contains an agonist of the MTMR12 protein, as well as any viral vector, plasmid, or small molecule compound that can promote the expression of the MTMR12 protein. Preferably, the viral vector includes adeno-associated virus vector, lentiviral vector, adenovirus vector, and retroviral vector; Preferably, the drug comprises an MTMR12 overexpression lentiviral vector, the lentiviral vector being constructed using primers containing nucleotide sequences as shown in SEQ ID NO. 1-2.

3. The application according to claim 2, characterized in that, The dosage form of the drug is any one of suspension, granules, capsules, powders, tablets, emulsions, pills, injections, suppositories, enemas, aerosols, patches, or drops; Preferably, the route of administration of the drug is oral, sublingual, rectal, transdermal or mucosal, inhalation, or injection.

4. Application of MTMR12 protein activator in the preparation of drugs for the prevention and / or treatment of cognitive impairment in diabetes.

5. The application according to claim 4, characterized in that, The MTMR12 protein activator includes any viral vector, plasmid, or small molecule compound that can promote MTMR12 protein expression; Preferably, the MTMR12 protein activator is an MTMR12 overexpression lentiviral vector, and the lentiviral vector is constructed using primers containing nucleotide sequences as shown in SEQ ID NO. 1-2; Preferably, the drug comprises pharmaceutically acceptable excipients; Preferably, the drug comprises a pharmaceutically acceptable dosage form.

6. The application according to claim 5, characterized in that, The dosage form of the drug is any one of suspension, granules, capsules, powders, tablets, emulsions, pills, injections, suppositories, enemas, aerosols, patches, or drops; Preferably, the route of administration of the drug is oral, sublingual, rectal, transdermal or mucosal, inhalation, or injection.

7. Application of MTMR12 protein in screening drugs for the prevention or treatment of cognitive impairment in diabetes.

8. A drug capable of preventing or treating diabetic cognitive impairment, characterized in that, The drug contains an MTMR12 protein activator; Preferably, the MTMR12 protein activator includes any viral vector, plasmid, or small molecule compound that can promote MTMR12 protein expression; Preferably, the MTMR12 protein activator is an MTMR12 overexpression lentiviral vector, and the lentiviral vector is constructed using primers containing nucleotide sequences as shown in SEQ ID NO. 1-2.

9. The drug according to claim 7 or 8, characterized in that, The drug includes pharmaceutically acceptable excipients; Preferably, the drug comprises a pharmaceutically acceptable dosage form.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug is any one of suspension, granules, capsules, powders, tablets, emulsions, pellets, injections, suppositories, enemas, aerosols, patches, or drops; preferably, the route of administration of the drug is oral administration, sublingual administration, rectal administration, skin and mucous membrane administration, inhalation administration, or injection administration.

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