JP7353246B2 - Nucleic acids and their use for the treatment or improvement of male metabolic syndrome - Google Patents

Description

The present invention relates to nucleic acids, in particular for causing apoptosis of originally mature Leydig cells in a male mammal, for regenerating Leydig stem cells in said male mammal to mature Leydig cells, and for increasing testosterone content in said male mammal. and for the treatment, amelioration or prevention of metabolic syndrome in male mammals, such as obesity and/or type 2 diabetes.

Metabolic syndrome refers to a pathological condition in which the human body has a metabolic disorder of substances such as proteins, fats, and carbohydrates. Metabolic syndrome is not just one type of disease, but a series of complex metabolic disorder syndromes such as abdominal fat accumulation, high triglycerides, high cholesterol, high blood pressure, hyperglycemia, etc. The central part of which is obesity and insulin. It is resistance.

The World Health Organization has already officially defined obesity as a BMI >30 and considers it a disease in its own right. Obesity is also a risk factor for type 2 diabetes, cardiovascular disease, hypertension, stroke, and multiple types of tumors.

The results of many clinical studies have shown that extracorporeal testosterone supplementation (testosterone replacement therapy) can effectively improve the sexual function of obese or diabetic patients, can effectively reduce the content of body fat, and can also reduce serum testosterone. It has been shown that increasing the content can significantly reduce fasting blood sugar levels and insulin resistance. However, long-term extracorporeal testosterone supplementation tends to cause symptoms such as abnormal liver function, hyperlipidemia, decreased bone density, and emotional instability. This is related to the fact that extracorporeal hormone replacement does not match the human body's rhythmic demand for androgens and that long-term use disrupts the balance of hormone metabolism in the body.

Leydig cells are cells that have the function of synthesizing and secreting androgens according to physiological rhythms regulated by the hypothalamus-pituitary-gonadal axis, and are the most major source of androgens in the male body. There are two different types of Leydig cells in the development of a male individual: fetal Leydig cells (Fetal LC, FLC) and mature Leydig cells. The latter is formed in the male adolescent testis, and in the process of differentiation and development, it is divided into Leydig stem cells (Stem LC, SLC), Leydig progenitor cells (Progenitor LC, PLC), immature Leydig cells (Immature LC, ILC), and mature Leydig cells. It is divided into four stages of cells (Adult LC, ALC). Once ALCs are formed, their number does not change significantly in healthy animals even when males enter a state of senescence, but their hormone synthesis function gradually declines with increasing age. Recently, after using ethane-1,2-dimethanesulfonic acid to induce apoptosis of mature Leydig cells in the testes, Leydig stem cells, which were initially dormant, became able to proliferate and eventually differentiate into new mature Leydig cells. There is research that has revealed this. These regenerative cells secrete androgens according to physiological rhythms, similarly regulated by the hypothalamus-pituitary-gonadal axis. Regenerated Leydig cells similarly pass through four stages in the proliferation and differentiation process: Leydig stem cells, Leydig progenitor cells, immature Leydig cells, and mature Leydig cells. More interestingly, activation, proliferation and differentiation of embryonic Leydig cells can be achieved in the testis of both young and old male animals. However, ethane 1,2-dimethanesulfonic acid has low safety as a drug, and has visceral toxicity such as hepatotoxicity, which greatly limits its clinical use.

One aspect of the present invention is that the sequence is SEQ ID No. 1 is provided.

In one specific embodiment, the nucleic acid is deoxyribonucleic acid and/or ribonucleic acid. For the sake of brevity, the present invention uses the ribonucleic acid form, ie SEQ ID No. Although only SEQ ID No. 1 is shown, when the nucleic acid of the present invention is a deoxyribonucleic acid, for example, SEQ ID No. It will be understood that u in 1 is changed to t. Specifically, the sequence in the case of ribonucleic acid is gguaccagaa gagagguuuu cuggcucu guuucacauc uuaauuaccc ucccacaccc aaggcuugca ggagagcaag, and if "u" in this sequence is replaced with "t" Deoxyribonucleic acid.

In one specific embodiment, the sequence of said nucleic acid is SEQ ID No. 1, and the truncated sequence has the same function as the original sequence.

In one specific embodiment, the sequence of said nucleic acid is SEQ ID No. It has 80% or more identity with the sequence shown in 1 and has the same function as the original sequence.

A second aspect of the present invention provides a drug comprising the nucleic acid according to any one of the first aspects of the present invention and a pharmaceutically acceptable carrier. The carrier may preferably be a common injection carrier in the field, and may also include diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, adsorption carriers, and surfactants. Other carriers common in the art such as agents or lubricants may also be used.

The third aspect of the present invention is that the nucleic acid according to any one of the first aspects of the present invention or the drug according to the second aspect of the present invention causes apoptosis of originally mature Leydig cells in a male mammal, Leydig stem cells of at least one of regenerating (ie, activated and developing) into mature Leydig cells and increasing testosterone content in said male mammal.

In one specific embodiment, the third aspect of the present invention provides that the nucleic acid according to any one of the present invention causes apoptosis of originally mature Leydig cells in a male mammal, Use is provided for at least one of regenerating Leydig stem cells into mature Leydig cells and increasing testosterone content in said male mammal. In this case, the third aspect of the present invention provides that the nucleic acid according to any one of the first aspects of the present invention causes apoptosis in the original mature Leydig cells in the male mammal, and converts the Leydig stem cells in the male mammal into mature Leydig cells. and increasing the testosterone content in said male mammal.

In one specific embodiment, the male mammal is a human male. Generally, the male mammal using the nucleic acid is an adult male mammal, so in the case of humans, it is an adult male.

A fourth aspect of the present invention provides the use of the nucleic acid according to any one of the first aspects of the present invention for producing a drug for treating, ameliorating, or preventing a disease caused by testosterone deficiency in male mammals.

In one specific embodiment, the male mammal is preferably an adult male.

In one specific embodiment, the disease due to testosterone deficiency includes at least one of male sexual dysfunction, impaired spermatogenesis, impaired sperm maturation, obesity, diabetes, osteoporosis, and cognitive decline.

In one specific embodiment, said diabetes is type 2 diabetes.

The nucleic acid sequence in the present invention can be synthesized by a company with nucleic acid synthesis capabilities (for example, Nanjing Jinsirui Biological Technology Co., Ltd.).

The nucleic acids of the present invention can be used by subcutaneous injection into the scrotum. For cells, the amount used is 200 nanomoles per liter (nM/L), and for rats or mice, the injection volume is 30 μL of miR-LC solution (20 micromoles) per testis on one side. When converted from amount can be detected.

The present invention provides for causing the proliferation and differentiation of Leydig stem cells located in the testis of a male mammal by causing apoptosis of the original mature Leydig cells in the testis of the male mammal to regenerate new mature Leydig cells; For the first time, a nucleic acid capable of inducing an increase in the content of testosterone in the testis has been discovered, and this nucleic acid is characterized by strong specificity as it has no activity to induce apoptosis in other somatic cells within the testis. Since the prior art shows that increasing testosterone content can improve the decreased gonadal function in male mammals, the nucleic acids of the present invention can improve sexual function and sperm formation and maturation disorders in male mammals. . Furthermore, from the research results of the prior art and the present invention, it has been confirmed that regeneration of mature Leydig cells and/or improvement of testosterone content can improve obesity or type 2 diabetes. It can be used to improve, treat or prevent obesity or type 2 diabetes in mammals, such as lowering body fat content, increasing the sensitivity of pancreatic islet cells, reducing insulin resistance and lowering blood sugar concentrations.

Figure 1 is an analysis of miR-LC inducing apoptosis of mature rat Leydig cells. Figure 1A is a morphological observation; in the figure, the blue fluorescence (on the right) is the cell nucleus, the green fluorescence (on the left) is the activated Caspase-3 enzyme in the cell, and Figure 1B is the data statistics. In the figure, *** indicates p<0.001, normal rat Leydig cells treated with ethane 1,2-dimethanesulfonic acid group vs. normal control group, normal rat Leydig cells treated with miR-LC vs. This is a control group of normal rat Leydig cells. Figure 2 is serum testosterone content analysis of rats on day 7 after miR-LC treatment. In the figure, *** is p<0.001, normal control group vs. type 2 diabetes model group, n=10, ### is p<0.001, normal control group vs. normal model miR-LC treatment group, n=10, $$$ p<0.001, type 2 diabetes model group vs type 2 diabetes model miR-LC treated group, n=10. n represents the number of rats. Figure 3 is serum testosterone content analysis of rats on day 56 after miR-LC treatment. In the figure, ** indicates p<0.01, type 2 diabetes model group vs type 2 diabetes model miR-LC treated group, n=10, and # indicates p<0.05, normal model miR-LC treated group. vs type 2 diabetes model miR-LC treated group, n=10, n represents the number of rats. FIG. 4 is a morphological analysis of mature Leydig cells in type 2 diabetes model rat testis after miR-LC treatment. In the diagram, the three diagrams in the first row exhibit blue-violet fluorescence, the three diagrams in the second row exhibit green fluorescence, and the three diagrams in the third row correspond to those in the first and second rows. This is a superimposed diagram of the following figures. Figure 5 is an analysis of rat fasting blood glucose content at different stages of miR-LC treatment. Figure 5A is the day of miR-LC treatment (day 0), Figure 5B is the 21st day after miR-LC treatment, and Figure 5C is the 35th day after miR-LC treatment. D in FIG. 5 is the 56th day after miR-LC treatment. *p<0.05, **p<0.01, type 2 diabetes model group vs type 2 diabetes model miR-LC treated group, n=10, n represents the number of animals. Figure 6 is an analysis of insulin content in rats at different stages of miR-LC treatment. Figure 6A is the 7th day after miR-LC treatment, Figure 6B is the 21st day after miR-LC treatment, and Figure 6C is the 35th day after miR-LC treatment. , FIG. 6D is the 56th day after miR-LC treatment. *: p<0.05, normal control group vs. normal model miR-LC treated group, n=10; #: p<0.05, type 2 diabetes model group vs. type 2 diabetes model miR-LC treated group , n=10, where n represents the number of animals. Figure 7 is a morphological analysis of rat testis tissue on day 21 after miR-LC treatment, with a magnification of 200x. Figure 8 is an analysis of mouse serum testosterone content at different stages of miR-LC treatment. Among them, for the normal control group and the obesity model group, only the results on the 56th day after treatment are shown. * p<0.05, normal control group vs. obese model group, n=10, n represents the number of animals. Figure 9 is an analysis of mouse weight change trends during different stages of miR-LC treatment. Among them, only the results on the 56th day after treatment are shown for the normal control group and obesity model group. *** p<0.001, normal control group vs. obese model group, n=10, n represents the number of animals.

Hereinafter, the above content of the present invention will be explained in more detail with reference to preferred examples, but this is not intended to limit the present invention.

Unless otherwise specified, all reagents used in the examples of the present invention can be purchased commercially.

miR-LC (SEQ ID No. 1) short chain ribonucleic acid was synthesized by Nanjing Jinsirui Biological Technology Co., Ltd., China.

Example 1
Short-chain ribonucleic acid miR-LC can induce apoptosis in rat mature Leydig cells Based on the knowledge of the prior art, the mechanism by which mature Leydig cells undergo apoptosis is mainly to activate Caspase-3 enzyme in the cells to induce subsequent nuclear activation. The NucView 488 dye can bind to the activated Caspase-3 enzyme, making it useful for fluorescence microscopy observation or enzyme labeling equipment (Fluoroskan, Dice) in detecting the apoptotic status of mature Leydig cells. This can be achieved by measuring the fluorescence value by Mokufei Shier Technology (China) Co., Ltd.).

Adult Leydig cells from normal 3 month old rats are isolated and purified according to conventional methods. Briefly, normal 3-month-old rats without any defects are killed by carbon dioxide asphyxiation, and then the testes are removed. A digestive fluid was prepared (prepared by adding 16 mg of collagenase D and 1 mg of deoxyribonuclease to 100 mL of ultrapure water), and the testes were left in the digestive fluid in a constant temperature water bath shaker at 37°C. and processed for 20 minutes. The convoluted tubules were then filtered using a 200 mesh cell screen to obtain a Leydig cell suspension. The suspension was placed in a cell separation solution (Percoll discontinuous density gradient solution, density gradient 1.068 to 1.088 g/mL), and the cells were collected by density gradient centrifugation (24500 r/min, 40 min). After diluting with phosphate buffer (PBS, pH=7.4) and washing twice (250 r/min, 5 min), the cells were counted and left in a 24-well cell culture plate (cells per well). 500,000 cells) and cultured overnight at 34° C. (14 to 16 hours) using Dulbecco Eagle's minimum essential medium (DMEM). Thereafter, the cells were divided into three groups: a normal control group (negative control), an ethane 1,2-dimethanesulfonic acid treated group (positive control), and a miR-LC treated group. For the normal control group, a blank liposome reagent (HiPerFect Transfection Reagent, Qiagen, product number: 301704, USA) was added to the serum-free DMEM medium at a volume ratio of 1:24 (blank liposome reagent: serum-free DMEM medium), The mixture was mixed uniformly to obtain a blank liposome medium. For the ethane 1,2-dimethane sulfonic acid treatment group, ethane 1,2-dimethane sulfonic acid (prepared by dissolving it in dimethyl sulfoxide) at a final concentration of 75 milligrams per liter (mg/L) was prepared in serum-free DMEM medium. ) was added to obtain an ethane 1,2-dimethanesulfonic acid medium. For the miR-LC treatment group, first, a blank liposome reagent (same origin as above) was added to the serum-free DMEM medium at a volume ratio of 1:24 (blank liposome reagent: serum-free DMEM medium) and mixed uniformly. A blank liposome medium was obtained, and then miR-LC was added into the blank liposome medium to reach a final concentration of miR-LC of 200 nanomoles/liter (nM/L), and incubated for 10 min at 25°C. Then, liposome coating was performed to obtain a liposome medium coated with miR-LC. Next, blank liposome medium, ethane 1,2-dimethane sulfonic acid medium, and miR-LC-coated liposome medium (200 μL each) were added to a 96-well culture plate (1 x 10 cells/well) in which mature Leydig cells were cultured. ) was added dropwise, and cultured at 34°C for 24 hours. Each group was then added to NucView 488 dye (Biotium, Product No. 30029, Hayward, Canada) at a final concentration of 5 micromol/liter (·M/L), incubated at 25°C for 30 min, and then phosphorized. After washing the cells twice with acid buffer, the cell nuclei labeled with blue fluorescence and the activated Caspase-3 enzyme in the cells labeled with green fluorescence (Figure 1-A) were detected using a fluorescence microscope. I was able to observe it. Furthermore, we used a fluorescence microscope to count and analyze cells expressing green fluorescence, and found that among the normal control group, only 14.5 ± 1.3% of cells expressed green fluorescence; Among the miR-LC-treated group of normal rat Leydig cells, 71.8 ± 6.6% of cells expressed green fluorescence, and among the ethane-1,2-dimethanesulfonic acid-treated group of normal rat Leydig cells, This shows that 87.4±4.5% of cells express green fluorescence (Figure 1-B). From this, compared to the normal control group, the ethane-1,2-dimethanesulfonic acid treatment group of normal rat Leydig cells could significantly promote the occurrence of apoptosis in mature Leydig cells (P<0.001 (normal rat Leydig cells treated with ethane 1,2-dimethanesulfonic acid group vs. normal control group); similarly, normal rat Leydig cells treated with miR-LC also significantly promoted the occurrence of apoptosis in mature Leydig cells. (P<0.001 miR-LC treated group of normal rat Leydig cells vs. normal control group), demonstrating that miR-LC has the ability to induce apoptosis of mature Leydig cells.

Example 2
Construction of Type 2 Diabetic Rat Model In order to observe whether miR-LC has the effect of regulating rat serum androgen content, the present invention first constructed a type 2 diabetic rat model.

Three-month-old adult rats were purchased and raised in an environment with a constant temperature of 24±2°C, relative humidity of 50% to 70%, and a regular day/night cycle of L:D=12:12. The rats were divided into two groups, the first group received an intraperitoneal injection of streptozotocin at a dosage of 35 milligrams per kilogram of body weight (mg/kg) (streptozotocin was administered in citrate buffer in an ice bath, shaded and dry environment). (dissolved in 0.1 mmol/liter (mM/L), pH=4.4) and completed use within 5 min), and for the second group as a normal control group, equal proportions of citrate buffer 0. 1 mmol/liter (mM/L), pH=4.4). After 4 days, blood was collected from the rat's tail, and the fasting blood glucose concentration of each rat injected with streptozotocin was measured using a blood glucose meter. Rats whose blood glucose concentration reaches 11 mmol/liter (mM/L) or higher are considered successful model construction. For individual rats whose blood sugar concentration did not reach the target concentration, streptozotocin was additionally injected intraperitoneally at a dose of 15 mg/kg, and 4 days later, the blood sugar level was detected again and the blood sugar content was 11 mmol/liter. (mM/L) or more.

The first group of rats that reached the above blood sugar concentration index and the corresponding normal control group (second group) were kept for 4 weeks, and then changes in their fasting blood sugar content were monitored. As a result of monitoring, the fasting blood glucose content of the rats in the first group continued to increase, and the average fasting blood glucose content was as high as 14.15 ± 3.10 mmol/liter (mM/L), but compared to the normal control group animals. The fasting blood glucose level was 4.86±0.40 mmol/liter (mM/L). These results showed that the type 2 diabetic rat model was successfully constructed.

Example 3
The serum androgen content of normal control group and type 2 diabetic rats can be reduced by subcutaneously injecting miR-LC into the scrotum.Normal control group (subcutaneously injecting 30 μL of saline per testis on one side) , normal model miR-LC treated group (scrotal subcutaneous injection), type 2 diabetes model group (scrotal subcutaneous injection of 30 μL of saline per testis on one side), type 2 diabetes model miR-LC treated group (scrotal subcutaneous injection) (subcutaneous injection into the capsule) Four groups were established. Ten rats per group were housed normally. The method for preparing and using the miR-LC solution is to add a blank liposome reagent to ultrapure water at a volume ratio of 1:24 (blank liposome reagent: ultrapure water) and mix uniformly to obtain a blank liposome solution. Then, miR-LC was added to the blank liposome solution until the final concentration of miR-LC reached 20 micromol/liter (M/L), and the solution was left standing at 25°C for 10 min to coat the liposomes. A liposome suspension coated with miR-LC (ie, miR-LC solution) was obtained. 60 μL of miR-LC solution was taken for testis-scrotal subcutaneous injection (30 μL per testis on one side). On the seventh day after treatment, blood was collected from the tail vein, and serum testosterone content was measured by radioenzyme-linked immunoassay, and the results are shown in FIG.

From the experimental results, the average serum testosterone content of rats in the normal control group was 1.82±0.39 nanograms/milliliter (ng/mL), and the average serum testosterone content of rats in the type 2 diabetes model group was 0.67±0. It was found to be 15 nanograms per milliliter (ng/mL). On the 7th day after miR-LC treatment of normal control group rats and type 2 diabetes model rats, the average serum testosterone content of both groups of rats decreased below the detection threshold.

Since more than 95% of the testosterone in the male body is synthesized and secreted by mature Leydig cells, the above results indicate that the serum testosterone of the type 2 diabetes model group rats is clearly lower than that of the normal control group rats, indicating that miR-LC treatment is capable of inducing the occurrence of apoptosis in the original mature Leydig cells in the testis of normal control group rats, and inducing the occurrence of apoptosis in the original mature Leydig cells in the testis of type 2 diabetes model group rats; It was also found to cause a drastic drop in serum testosterone content in rats.

Example 4
Regeneration of Leydig cells improves serum testosterone content in type 2 diabetic rats. Normal control group (subcutaneous injection of physiological saline into the scrotum), normal model miR-LC treated group (subcutaneous injection into the scrotum), type 2 diabetes model group ( Four groups were established: a type 2 diabetes model miR-LC treatment group (subcutaneous injection into the scrotum) and a type 2 diabetes model miR-LC treatment group (subcutaneous injection into the scrotum). There were 10 rats per group. It was treated in the same manner as in Example 3.

On the 56th day after injection, blood was collected from the tail vein, and serum testosterone content was detected by radioisotope enzyme-linked immunoassay, and the results are shown in FIG. The serum testosterone content of the type 2 diabetes model group rats was clearly lower than that of the normal control group, and the serum testosterone levels of the normal control group and type 2 diabetes model group rats were 2.12±0.48 and 0.56±0. It was 27 nanograms per milliliter (ng/mL). On day 56 after miR-LC treatment of normal control rats, their serum testosterone content had recovered to the normal level before miR-LC treatment and was 2.61 ± 0.22 nanograms per milliliter (ng/mL). ), and on the 56th day after miR-LC treatment of type 2 diabetes model group rats, their serum testosterone content reached 1.73±0.36 nanograms/milliliter (ng/mL). The serum testosterone in the miR-LC-treated type 2 diabetes model group rats is still obviously lower than the normal control group and the normal model miR-LC treatment group, but compared to the type 2 diabetes model group, the serum testosterone content is extremely low. Significant improvement. These results indicate that the ability of the regenerated mature Leydig cells to synthesize testosterone corresponds to that of the original healthy mature Leydig cells, and that after miR-LC treatment, the ability to synthesize testosterone in the testis of type 2 diabetic model rats The mature Leydig cells have a stronger androgen synthesis ability than the original mature Leydig cells, so it was found that they can improve the serum testosterone level in the body and improve the condition of type 2 diabetes.

Example 5
The number of Leydig cells can be increased by miR-LC treatment of type 2 diabetic rats. CYP11A1 is one of the key enzymes in the androgen synthesis pathway in Leydig cells, and at the same time, it is a specific marker protein of Leydig cells in the testis. It is also one of the. Therefore, by analyzing the fluorescence intensity of the enzyme in cells, changes in the number of Leydig cells and the amount of enzyme expression within the cells can be reflected.

There were three groups: a normal control group (subcutaneous injection of physiological saline into the scrotum), a type 2 diabetes model group (subcutaneous injection of physiological saline into the scrotum), and a type 2 diabetes model miR-LC treatment group (subcutaneous injection into the scrotum). Established. There were 10 rats per group. It was treated in the same manner as in Example 3.

On day 56 post-injection, rat testes were collected and frozen sectioned. Detection was performed by immunofluorescence. The specific method is to section the tissue in a thermostatic freezing microtome, then leave it at room temperature to equilibrate for 15 minutes, fix it using a 4% paraformaldehyde solution for 15 minutes, and then cut it in phosphate buffer (PBS). was washed three times (5 min each time). Block the slices with blocking solution (PBS containing 10% goat serum) for 1 hour at room temperature, remove the blocking solution using a pipette, and immediately insert 1 milliliter (mL) of Rabbit Anti-Rat CYP11A1 (Abcam). , product number: ab175408, Shanghai) (1 μL of antibody was diluted in 999 μL of PBS solution) and incubated overnight at 4°C. After the slices were left at room temperature to equilibrate for 30 min, the slices were washed 3 times with PBS and added with 1 milliliter (mL) of Goat Anti-Rabbit secondary antibody (Abcam, product number: ab150077, Shanghai) solution (2 μL). Antibodies were diluted in 998 μL of PBS solution) and incubated for 1 hour at room temperature, after which the slices were washed 5 times with PBS (5 min each time). The cells were stained with DAPI staining solution for 5 minutes and washed three times with PBS (5 minutes each time). After sealing the slices using a cover glass, they were observed using a confocal microscope and photographs were taken. The results are shown in Figure 4.

In Figure 4, immunofluorescence detection analysis for CYP11A1 protein revealed that the green fluorescence intensity of mature Leydig cells in the type 2 diabetes model group was clearly reduced compared to the normal group, and type 2 diabetes was found to be associated with the number of mature Leydig cells in the rat testes. It is shown that this caused a reduction in On day 56 after miR-LC treatment of type 2 diabetes model, the green fluorescence intensity of Leydig cells in type 2 diabetes rat testis was lower than that of the normal control group; The fluorescence intensity was obviously improved, indicating that the number of Leydig cells was partially recovered by regeneration after miR-LC treatment of the type 2 diabetes model.

Example 6
Improving blood sugar levels and insulin resistance in type 2 diabetic rats through the regeneration process of Leydig cells Normal control group (subcutaneous injection of saline in the scrotum), normal model miR-LC treatment group (subcutaneous injection in the scrotum), type 2 diabetes model Four groups were established: a group treated with saline (subcutaneous injection into the scrotum) and a type 2 diabetes model miR-LC treatment group (subcutaneous injection into the scrotum). There were 10 rats per group. It was treated in the same manner as in Example 3.

Tail vein blood was collected on the day after the injection and at four stages on the 21st, 35th, and 56th day after the injection, and the fasting blood glucose content of the animal was detected using a glucometer, and the results were recorded. Shown in Figure 5. The results showed that the fasting blood glucose content of normal control group rats and normal model miR-LC treated group rats remained stable throughout the treatment process (four stages), and the average fasting blood glucose content of normal control group rats Blood sugar content was maintained at an average of about 4.35 mmol/liter (mM/L), and the average fasting blood glucose content of normal model miR-LC treated group rats was about 4.18 mmol/liter (mM/L). The results revealed that the reduction and recovery of serum testosterone content by miR-LC treatment did not clearly change the fasting blood glucose content in normal rats. The results showed that in type 2 diabetes model rats, their fasting blood glucose concentration was maintained at a much higher level, with an average concentration of 22.5 mmol/liter (mM/L) in the four stages, and miR- It was further revealed that after LC treatment, the fasting blood glucose content of type 2 diabetes model miR-LC treated group rats tended to gradually decrease over time (i.e., during the regeneration process of Leydig cells). . On the 35th day after miR-LC treatment, its fasting blood glucose content was 18.61 ± 2.322 mmol/liter (mM/L), and on the 56th day after miR-LC treatment, its mean fasting The blood sugar content was 14.06±3.7 mmol/liter (mM/L), which was 38% lower than the fasting blood sugar content of the type 2 diabetes model group rats. These data revealed that the serum testosterone level improved by regenerated Leydig cells can improve (reduce) the hyperglycemia level in the serum of type 2 diabetic rats, and at the same time, the mature Leydig cells formed by regeneration and development revealed that It has also been shown that these cells have a stronger androgen synthesis ability than the original Leydig cells.

Tail vein blood was collected on the 7th, 21st, 35th, and 56th days after injection, and the insulin content in the animal blood was measured using an insulin content enzyme-linked immunodetection kit. The results are shown in FIG. The results showed that the insulin content in the rats' bodies in the type 2 diabetes model group remained at a high level throughout the experimental period, exceeding 20 mmol/liter (mM/L), approximately 1.5 times that of the normal control group. This indicates that insulin resistance phenomenon exists in type 2 diabetes model rats. Serum insulin content in rats increased 7 days after normal model miR-LC treatment, but recovered to a level comparable to the normal control group after 21 days. These results indicate that serum testosterone content has a regulatory effect on insulin synthesis. In the early period after miR-LC treatment, the serum insulin content of type 2 diabetes model rats gradually decreases as the Leydig cells regenerate and develop, but on day 56, the serum insulin content decreases to 15.13±. It was 2.8 mmol/liter (mM/L), and no obvious difference was seen from the normal control group. These results revealed that the phenomenon of insulin resistance in type 2 diabetes model rats can be effectively resolved by regeneration and growth of Leydig cells.

Example 7
Effect of miR-LC on normal model rat testis tissue To observe whether miR-LC had an effect on cells in the rat testis (e.g. Sertoli cells, pericytes, etc.) and the structure of convoluted seminiferous tubules. Two groups were established: a normal control group (subcutaneous injection of physiological saline into the scrotum) and a normal model miR-LC treatment group (subcutaneous injection into the scrotum). There were 10 rats per group. It was treated in the same manner as in Example 3.

On the 21st day after injection, testicular tissues were taken for pathological analysis. Briefly, testicular tissue was taken, placed in a 10% formalin solution, and fixed for 24 hours, then placed in an embedding cassette, and the tissue was washed with running water for 30 min to remove the fixative. The tissue was sequentially dehydrated using alcohol and xylene solutions, and then embedded in melted wax. After the embedded tissue blocks were cured, they were sectioned with a microtome. The slices were dried in an oven at 60°C for 120 min, then dewaxed with xylene solution for 30 min, and then dewaxed with absolute ethanol, followed by 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and distilled water. Each sample was dewaxed for 5 minutes, and all the liquid was absorbed using absorbent paper. After dewaxing, the slices were impregnated with hematoxylin staining solution for 15 minutes, and then washed with ultrapure water for 25 minutes. The slices were then impregnated with 0.5% eosin stain for 2-5 min, and the eosin stain was washed away with running water. The slices were dehydrated for 5 min in the order of 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and absolute ethanol, then further dehydrated in xylene solution for 15 to 30 min, and finally, the slices were dried. , and sealed the slices with neutral rubber. The results of microscopic observation are shown in FIG. The experimental results show that, compared to cells in the normal control group rat testes, the convoluted seminiferous tubule structure in the miR-LC-treated normal model rat testis is completely preserved, and both Sertoli cells and pericytes are It was found that there were no obvious lesions and the density of Leydig cells was clearly lower than that of normal control group rats.This result, combined with the results of Example 3, indicates that the original mature Leydig cells were induced and apoptosis occurred. revealed. In addition, in FIG. 7, there are a small amount of Leydig cells in Leydig, and these cells should be regenerated Leydig cells. The above results revealed that, other than Leydig cells, miR-LC treatment did not cause obvious damage to the testicular seminiferous tubule structure and other somatic cells within the testis.

Example 8
Construction of obese mouse model 60-day-old C57BL/6J mice were purchased and raised in an environment with an environmental temperature of 24 ± 2°C, relative humidity of 50% to 70%, and L:D = 12:12 with regular day and night conditions. The mice were divided into two groups, and the first group was fed ad libitum with high-fat feed (60% high-fat feed, product number: D12492, Nanjing Model Animal Research Institute) every day. After being kept for 60 consecutive days, the mice were weighed, and mice weighing 30 g or more were used as obesity models. The second group, which served as a normal model group, was fed normal food ad libitum every day.

Example 9
Effect of miR-LC treatment on serum testosterone content of obese model mice Normal control group (subcutaneous injection of physiological saline into the scrotum), normal model miR-LC treatment group (subcutaneous injection into the scrotum), obese model group (normal saline injection) Four groups were established: an obese model miR-LC treatment group (subcutaneous injection into the scrotum) and an obesity model miR-LC treatment group (subcutaneous injection into the scrotum). There were 10 mice in each group and they were housed normally on normal chow. The same treatment as in Example 3 was performed.

Tail vein blood was collected on the 7th, 21st, and 56th days after treatment, and the serum testosterone content was measured by radioenzyme-linked immunoassay, and the results are shown in FIG. 8. The results showed that the testosterone content in the bodies of mice in the obese model group was clearly lower than that in the bodies of mice in the normal control group (p<0.05), and on the 56th day after feeding, the serum testosterone content in the mice in the obese model group was remained clearly unchanged, and the serum testosterone content of the obese model miR-LC treated group mice decreased below the detection threshold on day 7, but as the Leydig cells regenerated, the serum testosterone content of the obese model miR-LC treated group mice decreased. The serum testosterone content gradually recovered, and on the 56th day, it was found that the content was no different from the serum testosterone content of normal control group mice.

Example 10
Effect of miR-LC treatment on the body weight of obese model mice. Four groups were established: a subcutaneous injection into the scrotum) and an obesity model miR-LC treatment group (subcutaneous injection into the scrotum). There were 10 mice per group and fed normally with normal chow. The same treatment as in Example 3 was performed.

The animals were weighed on the 7th and 56th day after feeding, and the results are shown in FIG. The results showed that the average body weight of normal model group animals was 21.64 g, the average body weight of obese model group animals was 32.68 g, and after miR-LC injection treatment, the body weight of obese model miR-LC treated group mice was It was found that the weight decreased from an average of 31.56 g on the 7th day to an average of 24.30 g on the 56th day. These results indicate that miR-LC treatment has ameliorating and therapeutic effects on obesity.

SEQ ID NO: 1 represents a short ribonucleic acid.

Claims (8)

The array is SEQ ID No. 1. A nucleic acid characterized by the following. The nucleic acid according to claim 1, wherein the nucleic acid is deoxyribonucleic acid and/or ribonucleic acid. at least one of causing apoptosis of originally mature Leydig cells in a male mammal, regenerating Leydig stem cells in said male mammal to mature Leydig cells, and increasing testosterone content in said male mammal. The nucleic acid according to claim 1 or 2, which is used for manufacturing a drug for . The nucleic acid according to claim 1 or 2, which is used for producing a drug for treating, ameliorating, or preventing a disease caused by testosterone deficiency in male mammals. The nucleic acid according to claim 4, wherein the disease caused by testosterone deficiency includes at least one of male sexual dysfunction, spermatogenesis disorder, sperm maturation disorder, obesity, diabetes, osteoporosis, and cognitive decline. . 6. The nucleic acid according to claim 5, wherein the diabetes is type 2 diabetes. The nucleic acid according to any one of claims 3 to 6, wherein the male mammal is an adult male. A drug comprising the nucleic acid according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.