Establishment method of type II diabetes mellitus rat model

Abstract

The invention discloses a method for establishing a type II diabetes mellitus rat model in the technical field of biology, which comprises the following steps of: regulating and controlling dynamic pathology evolution, reproducing the natural disease course of type II diabetes mellitus in stages from insulin resistance-beta cell injury-multi-organ complications-flora intervention and nerve regulation, and establishing a type II diabetes mellitus rat model. The animal model is used for overcoming the defects of a traditional model in the aspect of simulating complex pathological characteristics, the animal model capable of more comprehensively reproducing the multi-dimensional pathological characteristics of the human type II diabetes mellitus is constructed by optimizing an induction strategy and integrating multi-factor intervention, and a more reliable tool is provided for diabetes mellitus mechanism research and drug development.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular is a method for establishing a type II diabetes rat model. Background Art

[0002] The pathogenesis of diabetes involves two core links: insulin resistance and pancreatic beta-cell dysfunction. Type 2 diabetes in humans is often caused by a combination of long-term energy excess (high-fat diet), obesity, genetic susceptibility, and compensatory insulin deficiency.

[0003] Long-term high-fat feeding (45-60% of energy supply comes from fat) simulates an energy-excess environment, inducing obesity and insulin resistance. While this approach closely resembles the natural course of the disease, it takes 8-16 weeks to establish a model and requires other measures to accelerate β-cell damage. Streptozotocin (STZ) selectively damages pancreatic β cells, requiring a low-dose STZ (25-35 mg / kg) combined with a high-fat diet to simultaneously trigger insulin resistance and β-cell dysfunction within 4-8 weeks, more closely resembling the pathological characteristics of human type 2 diabetes.

[0004] Existing model-building technologies primarily rely on optimized high-fat diet induction and streptozotocin-induced pancreatic beta cell destruction. While these methods can rapidly establish animal models that conform to the pathological characteristics of human type 2 diabetes, as demonstrated in patent publication CN113575516B, mismatched injection timing with the dietary induction period—for example, when the high-fat diet induction period is too short (less than four weeks), insulin resistance may not fully develop, potentially leading to atypical model pathological characteristics, manifesting as only a single pathological feature (such as hyperglycemia or insulin resistance). A high-fat diet combined with STZ alone is unlikely to mimic the chronic inflammation and multi-organ complications (such as liver damage and neuropathy) associated with type 2 diabetes. Furthermore, in studies of diabetic gastric motility disorders, rat models may not exhibit symptoms of corresponding organ complications, making subsequent research difficult.

[0005] Therefore, it is necessary to propose a method for optimizing the induction of insulin resistance and taking into account the complications of diabetes-induced rat model, such as organ damage, neuropathy and changes in gastrointestinal flora diversity, so as to establish a type 2 diabetic rat model for studying the pathology of type 2 diabetic complications. Summary of the Invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a method for establishing a type 2 diabetes rat model to address the shortcomings of traditional models in simulating complex pathological characteristics. By optimizing the induction strategy and integrating multi-factor intervention, an animal model is constructed that can more comprehensively reproduce the multidimensional pathological characteristics of human type 2 diabetes, providing a more reliable tool for diabetes mechanism research and drug development.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows: A method for establishing a type II diabetic rat model comprises the following steps:

[0008] Step 1: From week 1 to week 8, healthy 6-week-old male Sprague-Dawley rats were selected and placed in an SPF-grade animal room on a progressively high-fat free-feed diet. The fat content of the diet started at 25% in the first week and increased by 5% each week to 60% in the eighth week. Time-restricted feeding was applied on two random days each week until the end of the experiment.

[0009] Step 2: At week 9, the patients were intraperitoneally injected with increasing doses of 5 wt% STZ solution three times every five days, with a dose gradient of 15 / 20 / 25 mg / kg. The 5 wt% STZ solution was prepared by dissolving STZ powder in a citric acid buffer solution at pH 4.2 to prepare a 5 wt% STZ solution.

[0010] Step 3: From week 10 to week 18, trans fatty acids were added to the high-fat diet to induce non-alcoholic fatty liver disease for 12 weeks. The rats were confined in a custom-made restraint cylinder for 1 hour daily for 8 weeks, and rectal temperature and respiratory rate were monitored during the restraint period.

[0011] Step 4: From week 19 to week 22, prepare drinking water containing broad-spectrum antibiotics and administer the drug by gavage for 7 consecutive days. During this period, fecal samples were collected daily for 16S rRNA sequencing and the residual index of bacterial diversity was detected. A mixed suspension of Akkermansia muciniphila and butyrate-producing bacteria in a ratio of 1:1 was prepared and 1 mL of the bacterial solution was administered using a ball-tipped gavage needle at 9:00 a.m. daily for 4 weeks. At week 19, the rats were anesthetized and fixed in a stereotaxic apparatus. A midline abdominal incision was made to expose the gastric cardia. A bipolar platinum-iridium alloy electrode was placed around the main trunk of the vagus nerve. The wire was led to the back of the neck through a subcutaneous tunnel and connected to an external pulse generator. Intermittent electrical stimulation was used for 6 hours per day, synchronized with the feeding period, for 4 weeks.

[0012] Furthermore, tail vein blood was collected at the end of each step to detect HbA1c, FFA and IL-6 indicators, and feces were collected at fixed time points every week and frozen at -80°C for metagenomic and metabolomic testing. At the end of the experiment, pancreas, liver and hippocampal tissues were obtained for multi-omics analysis.

[0013] Furthermore, the feed in step 1 is a gradient fat feed prepared by weight percentage, the fat sources of which are lard (60%), soybean oil (30%) and cholesterol (10%), and 15% sucrose is added to simulate human refined sugar intake.

[0014] Furthermore, the time-restricted feeding in step one is to compress the daily eating time to 4 hours, and only drinking water is provided during the rest of the time. During the time-restricted feeding intervention, fasting body weight is measured weekly, tail vein blood is collected to detect serum free fatty acids (FFA) and HOMA-IR index, and the dynamic curve of insulin resistance is calculated.

[0015] Furthermore, in step three, 2% hydrogenated vegetable oil is added to the high-fat diet to induce non-alcoholic fatty liver disease, and liver ultrasound elastography is performed every 4 weeks to detect the degree of fatty degeneration.

[0016] Furthermore, at the end of the 18th week in step 3, the rat livers were subjected to histopathological examination.

[0017] Furthermore, at the end of the 18th week in step three, the rats were subjected to behavioral tests, including an open field test and an elevated plus maze test.

[0018] Furthermore, in step 4, the weight ratio of the broad-spectrum antibiotic drinking water formula is 1 part vancomycin, 2 parts neomycin and 2 parts metronidazole, the final concentration of vancomycin is 0.5 g / L, it is stored in the dark and replaced daily.

[0019] Further, in step 4, the mixed suspension is diluted to a concentration of 10 8 CFU / mL.

[0020] Furthermore, in step 4, the intermittent electrical stimulation parameters were 50 Hz, 0.5 ms pulse width, and 30 s on / 90 s off cycle. After the stimulation, brain tissue sections were obtained and the density of c-Fos-positive cells in the paraventricular nucleus of the hypothalamus was detected by immunofluorescence.

[0021] The basic approach works by progressively increasing the body's fat content from 25% to 60% to induce insulin resistance. This simulates the energy-excess environment associated with a long-term high-fat diet in humans, promoting adipose tissue expansion and ectopic lipid deposition, activating inflammatory responses (such as elevated IL-6) and oxidative stress, and gradually inducing systemic insulin resistance. Time-restricted feeding compresses the daily eating window to four hours, further exacerbating metabolic stress and accelerating the decline in insulin sensitivity by disrupting circadian rhythms and feeding behavior.

[0022] Controllable β-cell dysfunction: Low-dose STZ (15-25 mg / kg) selectively damages pancreatic β-cells while avoiding excessive damage that could deviate from the type 2 diabetes phenotype. A progressive injection strategy simulates the progressive decline in β-cell function from the pre-diabetes stage to the onset of diabetes.

[0023] Simultaneously simulating multiple organ complications, the addition of hydrogenated vegetable oils (trans fatty acids) interferes with hepatocyte lipid metabolism, inducing non-alcoholic fatty liver disease and reproducing the pathological features of diabetic liver damage. High-frequency stimulation of the vagus nerve directly interferes with gastric motility regulation, simulating diabetic autonomic neuropathy. Restraint stress activates the HPA axis, simulating the amplifying effect of psychological stress on blood sugar fluctuations. After broad-spectrum antibiotics eliminate the preexisting bacterial flora, specific probiotics are implanted to simulate diabetes-related bacterial imbalance and metabolic remodeling after intervention.

[0024] Dynamic pathological evolution regulation, from insulin resistance (8 weeks) - β-cell damage (9 weeks) - multiple organ complications (10-18 weeks) - microbiome intervention and neural regulation (19-22 weeks), reproduces the natural course of type 2 diabetes in stages, supports mechanism research and drug intervention effect evaluation at different stages of diabetes.

[0025] The beneficial effects of the basic program are: 1. Traditional models focus on a single pathological indicator (such as hyperglycemia), while this method uses multi-factor intervention to simultaneously reproduce core pathological characteristics such as insulin resistance, β-cell dysfunction, fatty liver, neuropathy and dysbiosis, and each indicator maintains a dynamic balance in the experiment to prevent the model from deviating from the typical type 2 diabetes phenotype.

[0026] 2. Through the simultaneous collection of tail vein blood, feces and tissue samples, it supports multi-dimensional analysis of metabolome, metagenome, transcriptome and proteome, providing systematic data for analyzing the molecular mechanism of diabetes.

[0027] 3. The microbiota modulation and neural stimulation phase (weeks 19-22) can independently evaluate the therapeutic potential of drugs for specific complications (such as gastroparesis) or test the improvement effect of microbiota intervention on metabolic abnormalities.

[0028] 4. This model can simulate the multi-target treatment needs of diabetic patients (e.g., hypoglycemic drugs + liver-protecting drugs + neuroprotective agents + probiotics), providing an experimental platform for the development of comprehensive treatment plans. The implantation of nerve stimulation electrodes supports precise intervention research for diabetic autonomic neuropathy.

[0029] 5. Compared with genetically engineered models (such as ZDF rats), this method achieves pathological simulation through environmental intervention and drug injection, which is lower in cost and avoids the ethical controversy caused by genetic manipulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the method for establishing a type 2 diabetes rat model in an embodiment of the present invention.

[0031] Figure 2 Schematic diagram of the process of inducing non-alcoholic fatty liver disease in an embodiment of the present invention.

[0032] Figure 3 Schematic diagram of the neuropathy induction step in an embodiment of the present invention.

[0033] Figure 4 Schematic diagram of the intestinal flora disturbance and colonization steps in an embodiment of the present invention.

[0034] Figure 5 Schematic diagram of the vagus nerve electrical stimulation step in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The following is further described in detail through specific implementation methods:

[0036] Example 1

[0037] Basically as attached Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 A method for establishing a type II diabetic rat model is shown, comprising the following steps:

[0038] Step 1: From week 1 to week 8, healthy 6-week-old male SD rats were selected and subjected to a progressive high-fat free-feeding diet in an SPF animal room environment. The feed was a gradient fat feed prepared by weight percentage, and the fat sources were lard (60%), soybean oil (30%) and cholesterol (10%). 15% sucrose was added to simulate human refined sugar intake. The fat proportion in the feed started from 25% in the first week and increased by 5% each week to 60% in the eighth week. Time-restricted feeding was performed on two random days each week until the end of the experiment. Time-restricted feeding compressed the daily eating time to 4 hours, and only drinking water was provided for the rest of the time. During the time-restricted feeding intervention period, fasting body weight was measured weekly, and tail vein blood was collected to detect serum free fatty acids (FFA) and HOMA-IR index, and the dynamic curve of insulin resistance was calculated.

[0039] Step 2: At week 9, the patients were intraperitoneally injected with increasing doses of 5 wt% STZ solution three times every five days, with a dose gradient of 15 / 20 / 25 mg / kg. The 5 wt% STZ solution was prepared by dissolving STZ powder in a citric acid buffer solution at pH 4.2 to prepare a 5 wt% STZ solution.

[0040] Step 3: From weeks 10 to 18, 2% hydrogenated vegetable oil was added to the high-fat diet to induce non-alcoholic fatty liver disease for 12 weeks. Ultrasound elastography of the liver was performed every 4 weeks to detect the degree of fatty degeneration. At the end of this step, the rats' livers were histopathologically examined. Rats were confined in a custom restraint cylinder for 1 hour daily for 8 weeks. Rectal temperature and respiratory rate were monitored during the restraint period. At the end of this step, the rats underwent behavioral testing, including open field test and elevated plus maze test.

[0041] Step 4: From week 19 to week 22, prepare broad-spectrum antibiotic drinking water. The weight ratio of the broad-spectrum antibiotic drinking water formula is 1 part vancomycin, 2 parts neomycin and 2 parts metronidazole. The final concentration of vancomycin is 0.5 g / L. Store in the dark and change it every day. Gavage for 7 consecutive days. During this period, fecal samples are collected every day for 16S rRNA sequencing and the residual index of bacterial diversity is tested. A mixed suspension of Akkermansia muciniphila and butyric acid-producing bacteria in a ratio of 1:1 is prepared, and the mixed suspension is diluted to a concentration of 10 8 CFU / mL, 1 mL of bacterial solution was administered using a ball-tipped gavage needle at 9:00 am every day for 4 weeks. At week 19, the rats were anesthetized and fixed in a stereotaxic apparatus. A midline abdominal incision was made to expose the gastric cardia. A bipolar platinum-iridium alloy electrode was placed around the vagus nerve trunk, and the wire was led to the back of the neck through a subcutaneous tunnel and connected to an external pulse generator. Intermittent electrical stimulation was used for 6 hours every day, synchronized with the feeding period, for 4 weeks. The intermittent electrical stimulation parameters were 50 Hz, 0.5 ms pulse width, and 30 s on / 90 s off cycle. After the stimulation, brain tissue sections were obtained, and the density of c-Fos-positive cells in the paraventricular nucleus of the hypothalamus was detected by immunofluorescence.

[0042] The specific experimental process is as follows:

[0043] 1. Experimental Materials and Animal Preparation

[0044] 1. Experimental animals: 120 healthy 6-week-old male SD rats, weighing 180-200 g, were divided into an experimental group (n=100) and a control group (n=20) and housed in an SPF environment (temperature 22±1°C, humidity 50%, 12 h light / 12 h dark cycle).

[0045] 2. Main reagents: streptozotocin (STZ), citric acid buffer, hydrogenated vegetable oil, vancomycin, neomycin, metronidazole; Akkermansia muciniphila (ATCC BAA-835), butyric acid-producing bacteria (Clostridium butyricum, CGMCC 1.5202).

[0046] 3. Experimental equipment: ultrasonic elastography, open field experimental system and two-photon microscope.

[0047] 2. Experimental Process

[0048] 1. Feed preparation and feeding

[0049] Prepare a gradient fat diet with the following fat composition: lard 60% (containing saturated fatty acids), soybean oil 30% (containing polyunsaturated fatty acids), and cholesterol 10%. Adjust the fat ratio: initially increase the fat ratio by 25% and increase it by 5% each week (25% in the first week to 60% in the eighth week). Keep sucrose at a fixed ratio of 15%.

[0050] Time-restricted feeding intervention: Randomly select 2 days per week (such as Tuesday and Friday), limit the daily feeding time to 8:00-12:00, and only provide sterilized drinking water at other times.

[0051] 2. Metabolic monitoring

[0052] Fasting body weight: measured at 8:00 am every Monday (electronic balance with an accuracy of 0.1 g). Tail vein blood sampling: after the restricted feeding period ends every Friday, 200 μL of blood is collected for testing after fasting for 12 hours. Serum free fatty acids (FFA): enzyme colorimetric method, fasting insulin (FINS): ELISA method

[0053] HOMA-IR calculation: HOMA-IR = (fasting blood glucose mmol / L × FINS μU / mL) / 22.5

[0054] 3. Preparation of STZ solution, citric acid buffer: 0.1M citric acid + 0.1M sodium citrate, pH 4.2, pre-cooled at 4℃,

[0055] STZ dissolution: Prepare immediately before use by dissolving STZ powder in pre-cooled buffer to prepare a 5 wt% solution (operate on ice in the dark and complete injection within 15 minutes)

[0056] 4. Fractional injection schedule: injection time points: 9th week, day 1 (15 mg / kg), day 6 (20 mg / kg), day 11 (25 mg / kg), fast for 6 hours before injection (free access to water), intraperitoneal injection of STZ solution, supplemented with 5% glucose water within 2 hours after injection (to prevent hypoglycemic shock).

[0057] 5. Blood glucose monitoring and dose adjustment. Blood glucose testing: test fasting blood glucose on the 3rd and 5th days after injection. Dose adjustment: if fasting blood glucose is >16.7mmol / L, suspend subsequent injections; if <11.1mmol / L, add an additional 5mg / kg dose.

[0058] 6. Induction of non-alcoholic fatty liver disease: Diet adjustment: Add 2% hydrogenated vegetable oil (trans fatty acids ≥ 30%) to a 60% fat diet. Liver assessment: Ultrasound elastography: Rats were anesthetized every 4 weeks, and hepatic steatosis was assessed using FibroScan (CAP values ≥ 280 dB / m were considered fatty liver). Histopathology: At the endpoint, the right lobe of the liver was obtained, fixed with 4% paraformaldehyde, paraffin-embedded, and stained with hematoxylin and eosin (NAS score: 0-8). Apoptosis assay: Western blot analysis was performed to measure the BCL-2 / BAX protein ratio in liver tissue (BCL-2 antibody: Abcam ab32124; BAX antibody: Abcam ab32503).

[0059] 7. Neuropathy Induction and Behavioral Testing. Chronic restraint stress: Rats were restrained daily between 2:00 PM and 3:00 PM using a transparent polycarbonate restraint tube (5 cm diameter, 20 cm length). Monitoring parameters included rectal temperature (terminated when >39°C) and respiratory rate (terminated when >120 breaths / minute). Behavioral assessments: Open field test: An 80 × 80 cm square apparatus was used to record time spent in the central area, number of upright positions, and total distance traveled over a 10-minute period (analyzed by the EthoVision system). Elevated plus maze: The ratio of time spent in the open to closed arms (<30% was considered anxiety-indicating).

[0060] 8. Intestinal flora disturbance, broad-spectrum antibiotic drinking water preparation: Vancomycin 0.5g / L + Neomycin 1g / L + Metronidazole 1g / L, store in dark, continuous gavage for 7 days, daily gavage volume 1mL / 100g body weight. Probiotic colonization: Bacterial solution preparation: Akkermansia muciniphila and butyrate-producing bacteria are mixed and diluted to 10 8 CFU / mL (suspended in PBS under anaerobic conditions), 1 mL of bacterial solution was gavaged at 9:00 every day for 4 weeks.

[0061] 9. Vagus nerve electrical stimulation, surgical implantation: Anesthetize the rat (isoflurane inhalation anesthesia), make a midline abdominal incision to expose the gastric cardia, encircle the vagus nerve trunk with a bipolar platinum-iridium electrode, and lead the wire through a subcutaneous tunnel to the back of the neck. Suture the incision and apply antibiotic ointment.

[0062] Electrical stimulation parameters: Stimulation period was synchronized with food intake every day (8:00-12:00 + 14:00-16:00), parameter settings: 50 Hz, 0.5 ms pulse width, 30 s on / 90 s off cycle (pulse generator: AM Systems 2100).

[0063] Neuronal activity verification: Brain tissue sections were taken and immunofluorescence staining for c-Fos protein (antibody: Abcam ab190289). Positive cell count: ≥50 cells / mm in the paraventricular nucleus of the hypothalamus 2For effective activation.

[0064] 3. Experimental Results

[0065] At the end of the eighth week, the HOMA-IR index of the experimental group rats increased 3.2 times compared to the control group (P < 0.01), and the serum FFA level increased by 180%. At the end of the ninth week, the proportion of fasting blood glucose in the experimental group greater than 11.1 mmol / L reached 92%, and the mortality rate was less than 8%. The results are shown in Tables 1 and 2 below:

[0066] Table 1. Results of insulin resistance induction

[0067]

[0068] P<0.001

[0069] Table 2. STZ pulse administration experimental results

[0070]

[0071] At week 18, 40 rats were randomly selected from the 92 surviving experimental group rats as the intermediate experimental group, and 10 rats were randomly selected from the control group as the intermediate control group. Behavioral tests were first performed and then histopathological studies were performed on the livers of the rats in the intermediate experimental group and the intermediate control group.

[0072] The intermediate experimental group's central open field residence time was shortened to 35% of that of the intermediate control group (P < 0.05), and the elevated plus maze open arm entry rate was < 25%. HE staining showed that the proportion of fat vacuoles in hepatocytes of rats in the intermediate experimental group was > 60%, and the NAS score was ≥ 5 points. The BCL-2 / BAX ratio decreased to 0.3 ± 0.1. The results are shown in Table 3 below:

[0073] Table 3. Liver pathology and behavioral assessment results

[0074]

[0075] P<0.01

[0076] During the process of intestinal flora perturbation and recolonization, the Shannon index of the experimental group decreased by 72% after antibiotic intervention, and probiotic colonization restored butyrate concentration to 80% of the control group. After the vagus nerve activation experiment, the density of c-Fos-positive cells in the paraventricular nucleus of the hypothalamus in the experimental group reached 68±12 cells / mm 2 , which was significantly higher than that of the control group (P < 0.01). The results are shown in Table 4 below:

[0077] Table 4. Results of intestinal flora and gut-brain axis regulation

[0078]

[0079] P<0.001

[0080] Finally, the achievement rates of diabetes and various complications were calculated, and the pathological correlation was analyzed using statistics. The results are shown in Table 5 below:

[0081] Table 5. Correlation between the success rate of the comprehensive model and pathology

[0082]

[0083] The above results show that this method can be used to comprehensively model diabetes and its complications, can be used to study the pathological effects caused by different diabetic complications, and can serve as a reliable tool for diabetes mechanism research and drug development.

[0084] Example 2

[0085] The difference from the above embodiment is that, as shown in the attached Figure 1 As shown: At the end of each step, tail vein blood was collected to detect HbA1c, FFA and IL-6 indicators. Feces were collected at fixed time points every week and frozen at -80℃ for metagenomic and metabolomic analysis. At the end of the experiment, pancreas, liver and hippocampus tissues were obtained for multi-omics analysis.

[0086] The specific implementation process is as follows: HbA1c increased from 6.8% at week 8 to 9.6% at week 18, with simultaneous increases in FFA and IL-6, confirming the development of insulin resistance and inflammation. A high-fat diet led to a 72% decrease in butyrate, which returned to 93% of baseline after probiotic intervention (18.9 vs 20.3 μmol / g). PDX1 downregulation and abnormal proinsulin indicate impaired pancreatic β-cell function, AMPK inhibition, and 4.5-fold TAG accumulation indicate impaired liver lipid metabolism. Activation of the NF-κB pathway and inhibition of BDNF methylation indicate hippocampal neuroinflammation. The results are shown in Tables 6, 7, and 8 below:

[0087] Table 6. Results of tail vein blood test at each stage

[0088]

[0089]

[0090] P<0.001

[0091] Table 7. Stool test results at key time points

[0092]

[0093] *: P < 0.05 compared with baseline; **: P < 0.01 compared with baseline; #: P < 0.05 compared with week 18 (recovery after probiotic intervention).

[0094] Table 8. Results of key biomarker detection in pancreas, liver, and hippocampus tissues

[0095]

[0096] This model system integrates multi-dimensional pathological characteristics of metabolism, microorganisms, and nerves, providing a highly simulated platform for type 2 diabetes mechanism research and drug development.

[0097] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0098] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for establishing a type II diabetic rat model, characterized by: The following steps are involved: Step 1: From week 1 to week 8, healthy 6-week-old male Sprague-Dawley rats were selected and placed in an SPF-grade animal room on a progressively high-fat free-feed diet. The fat content of the diet started at 25% in the first week and increased by 5% each week to 60% in the eighth week. Time-restricted feeding was applied on two random days each week until the end of the experiment. Step 2: At week 9, the patients were intraperitoneally injected with increasing doses of 5 wt% STZ solution three times every five days, with a dose gradient of 15 / 20 / 25 mg / kg. The 5 wt% STZ solution was prepared by dissolving STZ powder in a citric acid buffer solution at pH 4.2 to prepare a 5 wt% STZ solution. Step 3: From week 10 to week 18, trans fatty acids were added to the high-fat diet to induce non-alcoholic fatty liver disease for 12 weeks. The rats were confined in a custom-made restraint cylinder for 1 hour daily for 8 weeks, and rectal temperature and respiratory rate were monitored during the restraint period. Step 4: From week 19 to week 22, a broad-spectrum antibiotic-containing drinking water was prepared and administered orally for 7 consecutive days. During this period, fecal samples were collected daily for 16S rRNA sequencing and residual bacterial diversity index testing. A 1:1 mixed suspension of Akkermansia muciniphila and butyrate-producing bacteria was prepared and 1 mL of the bacterial suspension was administered orally at 9:00 a.m. daily for 4 weeks using a ball-tipped gavage needle. At week 19, the rats were anesthetized and fixed in a stereotaxic apparatus. A midline abdominal incision was made to expose the gastric cardia. A bipolar platinum-iridium alloy electrode was placed around the vagus nerve trunk. The wire was led to the back of the neck through a subcutaneous tunnel and connected to an external pulse generator. Intermittent electrical stimulation was applied for 6 hours per day, synchronized with the feeding period, for 4 weeks.

2. The method for establishing a type II diabetic rat model according to claim 1, wherein: At the end of each step, tail vein blood was collected to detect HbA1c, FFA, and IL-6 indicators. Feces were collected at fixed time points each week and frozen at -80°C for metagenomic and metabolomic analysis. At the end of the experiment, pancreas, liver, and hippocampal tissues were collected for multi-omics analysis.

3. The method for establishing a type II diabetic rat model according to claim 2, wherein: The feed in step 1 is a gradient fat feed prepared by weight percentage, the fat sources of which are lard (60%), soybean oil (30%) and cholesterol (10%), and 15% sucrose is added to simulate human refined sugar intake.

4. The method for establishing a type II diabetes rat model according to claim 3, wherein: The time-restricted feeding in step one is to compress the daily eating time to 4 hours, and only provide drinking water during the rest of the time. During the time-restricted feeding intervention, fasting body weight is measured weekly, tail vein blood is collected to detect serum free fatty acids (FFA) and HOMA-IR index, and the dynamic curve of insulin resistance is calculated.

5. The method for establishing a type II diabetic rat model according to claim 4, wherein: In step three, 2% hydrogenated vegetable oil was added to the high-fat diet to induce non-alcoholic fatty liver disease, and liver ultrasound elastography was performed every 4 weeks to detect the degree of fatty degeneration.

6. The method for establishing a type II diabetic rat model according to claim 5, wherein: In step three, at the end of week 18, the rat livers were subjected to histopathological examination.

7. The method for establishing a type II diabetic rat model according to claim 6, wherein: In step three, at the end of week 18, the rats were subjected to behavioral tests, including open field test and elevated plus maze test.

8. The method for establishing a type II diabetes rat model according to claim 7, wherein: In step 4, the weight ratio of the broad-spectrum antibiotic drinking water formula is 1 part vancomycin, 2 parts neomycin and 2 parts metronidazole, with a final concentration of vancomycin of 0.5 g / L. Store in the dark and change daily.

9. The method for establishing a type II diabetes rat model according to claim 8, wherein: In step 4, the mixed suspension is diluted to a concentration of 10 8 CFU / mL.

10. The method for establishing a type II diabetes rat model according to claim 9, characterized in that: In step 4, the intermittent electrical stimulation parameters were 50 Hz, 0.5 ms pulse width, and 30 s on / 90 s off cycle. After the stimulation, brain tissue sections were obtained and the density of c-Fos-positive cells in the paraventricular nucleus of the hypothalamus was detected by immunofluorescence.

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