Application of androsterone and its combination with fibroblast growth factor in the treatment of hyperglycemia

Abstract

The present invention relates to the use of androsterone and a composition containing the androsterone in preparing a medicine for preventing and / or treating diabetes. The composition containing the androsterone also contains a fibroblast growth factor.

Description

Technical Field

[0001] The present invention relates to the field of hyperglycemia treatment, and in particular to the use of androsterone and its combined use with fibroblast growth factor in the prevention and / or treatment of hyperglycemia. Background Art

[0002] Leydig cells (LC) are cells that synthesize and secrete androgens according to physiological rhythms and are regulated by the hypothalamus-pituitary-gonad axis. They are the main source of androgens in males. There are two different types of Leydig cells in male individual development: embryonic Leydig stem cells (Fetal LC, FLC) and adult Leydig cells. The latter are formed in the testes of male puberty and are divided into four stages during differentiation and development: Leydig stem cells (Stem LC, SLC), Leydig progenitor cells (Progenitor LC, PLC), immature Leydig cells (Immature LC, ILC) and mature Leydig cells (Adult LC, ALC).

[0003] The expression timing of different steroidogenic enzymes in Leydig cells is different, which leads to different androgen end products synthesized by Leydig cells at different developmental stages: Leydig stem cells cannot synthesize androgens, Leydig progenitor cells mainly synthesize androstenone, immature Leydig cells mainly synthesize dihydrotestosterone, and mature Leydig cells mainly synthesize testosterone.

[0004] The development and differentiation of Leydig cells are generally limited to embryogenesis and early puberty. Once ALC is formed, its number in the healthy population will not change significantly even when males enter the aging state, but the hormone synthesis function will gradually decline with age. However, the original mature Leydig cells in the testicles can be specifically induced to apoptosis (for example, using the compound ethane dimethane sulfonate (EDS) to induce), and then the dormant Leydig stem cells are activated, proliferating and eventually differentiating into new mature Leydig cells. These regenerated Leydig cells will also go through four stages: Leydig stem cells, Leydig progenitor cells, immature testicular cells and mature testicular cells, and the androgen end products synthesized by the regenerated cells in each stage are the same as those of the original corresponding type of Leydig cells. For example, in rats, EDS induced apoptosis of pre-existing ALCs, and the apoptotic process lasted for 4 days. Meanwhile, SLCs began to proliferate and differentiate from the 1st to the 7th day of EDS induction. From the 7th to the 28th day, the regenerated cells were mainly in the PLC stage. From the 35th to the 56th day, the regenerated cells were mainly in the ILC stage. After the 56th day, the regenerated cells differentiated into the ALC stage.

[0005] However, the functions of androgens remain to be explored. Summary of the invention

[0006] One aspect of the present invention provides the use of androsterone in the preparation of a medicament for preventing and / or treating hyperglycemia (i.e., a condition in which the blood sugar level is higher than the normal level).

[0007] In one embodiment, the factor causing the hyperglycemia is at least one of prediabetes, diabetes, obesity and aging. For example, aging or obesity can cause mild hyperglycemia.

[0008] In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0009] In a specific embodiment, the androsterone is used to prevent and / or treat hyperglycemia in males (or expressed as men).

[0010] The second aspect of the present invention provides a combined drug comprising fibroblast growth factor and androsterone.

[0011] In a specific embodiment, the fibroblast growth factor is at least one of fibroblast growth factor 21, fibroblast growth factor 19 and fibroblast growth factor 23.

[0012] In a specific embodiment, the mass ratio of the fibroblast growth factor to the androsterone is 1:(1-800).

[0013] In a specific embodiment, the mass ratio of the fibroblast growth factor to the androsterone is 1:(8-100).

[0014] The third aspect of the present invention provides a cell that simultaneously expresses a first factor and a second factor, wherein the first factor is a fibroblast growth factor, and the second factor is a steroidogenic factor-1 (also known as NR5A1) and / or a liver receptor homolog factor-1 (Liver Receptor Homolog-1, LRH-1; also known as NR5A2), and the expression of the second factor enables the cell to synthesize and secrete androsterone. It is well known to those skilled in the art that the expression of steroidogenic factor-1 can activate key enzymes in the androsterone synthesis pathway, and thus, the expression of steroidogenic factor-1 can induce the cell to differentiate into Leydig cells with the function of synthesizing androsterone. It is also well known to those skilled in the art that LRH-1 can also induce the cell to differentiate into Leydig cells, and thus, the expression of LRH-1 can also enable the cell to synthesize androsterone.

[0015] In a specific embodiment, the fibroblast growth factor is at least one of fibroblast growth factor 21, fibroblast growth factor 19 and fibroblast growth factor 23.

[0016] In a specific embodiment, the cell is a transdifferentiated cell obtained by activating the expression of the first factor and the expression of the second factor therein.

[0017] In a specific embodiment, the transdifferentiated cell is obtained by transferring the CRISPR / dCas9 activation gene expression system for activating the expression of the first factor and the expression of the second factor into the first recipient cell, wherein:

[0018] The CRISPR / dCas9 activated gene expression system includes at least one dCas9 expression cassette, at least one guide RNA (guild RNA, gRNA) expression cassette in the promoter region of the first factor, and at least one guide RNA ((guild RNA, gRNA)) expression cassette in the promoter region of the second factor. For example, the CRISPR / dCas9 activated gene expression system can be on the same plasmid, that is, a dCas9 expression cassette, at least one guide RNA (guild RNA, gRNA) expression cassette in the promoter region of the first factor, and at least one guide RNA ((guild RNA, gRNA)) expression cassette in the promoter region of the second factor on one plasmid; or the CRISPR / dCas9 activated gene expression system can also be distributed on two or three plasmids, for example, three expression cassettes are located on three plasmids.

[0019] The recombinant vector of the CRISPR / dCas9 activated gene expression system can be realized using commercial vectors, such as PB-TRE-dCas9-VPR (Addgene; Catalog No.: 63800) or PB-SAM (Addgene; Catalog No.: 102559). In addition, PB-TRE-dCas9-VPR and pX330A-1x4 can be used together, or PB-SAM and pX330A-1x4 can be used together, wherein pX330A-1x4 needs to be simply modified to remove the Cas9 gene therein.

[0020] In a specific embodiment, the cell is a transdifferentiated cell in which the expression of the first factor and the expression of the second factor are activated and regulated by a first signaling pathway regulator.

[0021] In a specific embodiment, the cell is the transdifferentiated cell obtained by transferring the CRISPR / dCas9 activated gene expression system into the first recipient cell and regulating it using the first signaling pathway regulator.

[0022] In a specific embodiment, the cell is an engineered cell obtained by transforming the expression vector carrying the first factor expression cassette and the second factor expression cassette into the second recipient cell and regulating with the second signaling pathway regulatory factor.

[0023] In a specific embodiment, the first signaling pathway regulator and the second signaling pathway regulator independently include at least one of insulin, hedgehog pathway agonist SAG, cyclic adenosine monophosphate, transforming growth factor β signaling pathway inhibitor LY2109761, fibroblast growth factor 2, luteinizing hormone and rosiglitazone.

[0024] In a specific embodiment, the regulation of the first signaling pathway regulatory factor and the regulation of the second signaling pathway regulatory factor are independently divided into two stages, wherein the signaling pathway regulatory factors in the first stage include insulin, hedgehog pathway agonist SAG, cyclic adenosine monophosphate, transforming growth factor β signaling pathway inhibitor LY2109761, fibroblast growth factor 2 and luteinizing hormone, and the signaling pathway regulatory factors in the second stage include insulin, hedgehog pathway agonist SAG, cyclic adenosine monophosphate, luteinizing hormone and rosiglitazone.

[0025] In a specific embodiment, the insulin is used at a concentration of 1 μg / mL to 100 μg / mL, the hedgehog pathway agonist SAG is used at a concentration of 0.1 μM to 1 μM, the cyclic AMP is used at a concentration of 0.1 mM to 10 mM, the transforming growth factor β signaling pathway inhibitor LY2109761 is used at a concentration of 0.1 μM to 10 μM, the fibroblast growth factor 2 is used at a concentration of 1 ng / mL to 100 ng / mL, the luteinizing hormone is used at a concentration of 1 ng / mL to 100 ng / mL, and the rosiglitazone is used at a concentration of 1 μg / mL to 100 μg / mL.

[0026] In a specific embodiment, the first recipient cell and the second recipient cell are independently human cells or murine cells.

[0027] In a specific embodiment, the first receptor cell and the second receptor cell are independently one of mesenchymal stem cells, induced pluripotent stem cells (iPSCs) and fibroblasts. The first receptor cell or the second receptor cell can also be independently a fibroblast obtained by inducing differentiation of embryonic stem cells or iPSCs by genetic engineering methods.

[0028] In a specific embodiment, the fibroblasts are one of skin fibroblasts, embryonic fibroblasts, adult fibroblasts and visceral fibroblasts.

[0029] In a specific embodiment, the sequence of the deoxyribonucleic acid corresponding to the guide RNA in the promoter region of the first factor is at least one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4.

[0030] In a specific embodiment, there are four guide RNA expression cassettes in the promoter region of the first factor.

[0031] In a specific embodiment, the sequence of the deoxyribonucleic acid corresponding to the guide RNA in the promoter region of the second factor is at least one of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8.

[0032] In a specific embodiment, there are four guide RNA expression cassettes in the promoter region of the second factor.

[0033] The fourth aspect of the present invention provides the use of the combination drug described in any one of the second aspect of the present invention or the cell described in any one of the third aspect of the present invention in the preparation of a drug for preventing and / or treating hyperglycemia (i.e., a blood sugar level higher than normal).

[0034] In one embodiment, the factor causing the hyperglycemia is at least one of prediabetes, diabetes, aging and obesity. For example, aging or obesity can cause mild hyperglycemia.

[0035] In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0036] In a specific embodiment, the androsterone is used to prevent and / or treat hyperglycemia in males (or expressed as men).

[0037] In the present invention, the term "expression cassette" means, according to specific needs, generally including a promoter, a target gene (such as a first factor gene or a second factor gene) and a terminator.

[0038] Beneficial effects of the present invention:

[0039] The present invention first discovered that androsterone can be used to prevent and / or treat male hyperglycemia, wherein the factor causing the hyperglycemia is at least one of prediabetes, diabetes, aging and obesity. Furthermore, the present invention also discovered that by combining fibroblast growth factor with androsterone to prevent and / or treat male hyperglycemia, a synergistic effect can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 .Contents of sex hormones in serum of type 2 diabetic rats at different times after EDS treatment, *p<0.05, **p<0.01, ***p<0.001, vs. normal control group.

[0041] Figure 2 .Chart of fasting blood glucose levels in type 2 diabetic rats at four differentiation development stages of testicular Leydig cell regeneration.

[0042] Figure 3 .Insulin levels in serum of type 2 diabetic rats at different times after EDS treatment, **p<0.01vs normal control group.

[0043] Figure 4 .A single intraperitoneal injection of androsterone improves the blood glucose content of a self-administered diet in diabetic rats.

[0044] Figure 5 .The improving effect of FGF21 on fasting blood glucose levels in diabetic rats, *p<0.05, **p<0.01, ***p<0.001, vs. diabetic model group.

[0045] Figure 6 .The improvement effect of combined use of androstenone and FGF21 on hyperglycemia in rats, *p<0.05, **p<0.01, vs diabetic model group.

[0046] Figure 7 . The combined use of androstenone and FGF21 at different doses improves hyperglycemia in diabetic animals. ## p<0.01, ### p<0.001, vs normal control group.

[0047] Figure 8 : Gene expression analysis of FGF21 and SF-1 in transdifferentiated cells, **p<0.01, ***p<0.001, vs. Leydig cells; n=3, n represents the number of experimental repetitions.

[0048] Fig. 9 .Expression analysis of androgen synthase gene in transdifferentiated cells, **p<0.01, ***p<0.001, vs fibroblasts; n=3, n represents the number of experimental repetitions.

[0049] Fig.10 . Western Blot of FGF21 in the culture supernatant of transdifferentiated cells. Lanes 1 and 2: culture supernatant of transdifferentiated cells; Lane 3: culture supernatant of fibroblasts; Lane M: protein molecular weight marker

[0050] Fig.11 .The level of androstenone synthesized by transdifferentiated cells at different culture time points.

[0051] Fig.12 .Effects of transdifferentiated cells on fasting blood glucose levels in hyperglycemic rats, **p<0.01, ***p<0.001, vs. diabetic model group.

[0052] Fig.13 .Analysis of collagen fiber proliferation in rat liver tissue sections, the sections were magnified 200 times.

[0053] Fig.14 .Analysis of lipid accumulation in rat liver tissue sections, sections magnified 200 times. DETAILED DESCRIPTION

[0054] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0055] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0056] Example 1

[0057] Establishment of rat model of type 2 diabetes mellitus

[0058] Adult Sprague-Dawley rats (SD rats) aged 3 months were purchased, male, and were raised in an environment with a constant temperature of 24 ± 2 ° C, a relative humidity of 50%-70%, and a day and night regularity of L: D = 12: 12. The rats were divided into two groups, wherein the type 2 diabetes model group was fed with a high-fat and high-sugar diet, and the normal control group was fed with a normal diet, and they were raised for 30 consecutive days. After that, the rats in the type 2 diabetes model group were intraperitoneally injected with streptozotocin at a dose of 35 mg / kg (streptozotocin was dissolved in a citric acid buffer (0.1 mM, pH = 4.4) in an ice bath, light-proof and dry environment, and used within 5 minutes), while the normal control group was injected with an equal volume of citric acid buffer (0.1 mM, pH = 4.4). After 4 days, blood was collected from the rat tail, and the fasting blood glucose concentration of the rats injected with streptozotocin was measured with a blood glucose meter. The model was considered successful when the blood glucose concentration reached 198 mg / dL or above. For rats whose blood glucose concentration did not meet the standard, streptozotocin was injected intraperitoneally at a dose of 15 mg / kg, and the blood glucose was tested again after 4 days until the blood glucose level reached or exceeded 198 mg / dL, indicating that the type 2 diabetes rat model was successfully established.

[0059] The rats in the model group that reached the above blood sugar concentration index continued to be fed with high-fat and high-sugar feed, while the rats in the normal control group were fed with ordinary feed. After 4 weeks, the changes in their fasting blood sugar levels were monitored. The final results showed that the average fasting blood sugar level of type 2 diabetic rats in the model group was higher than 308.84±24.2mg / dL, while the average fasting blood sugar level of healthy rats in the normal control group was 89.5±9.50mg / dL.

[0060] Example 2

[0061] Regenerating Leydig cells to synthesize androsterone can improve hyperglycemia in type 2 diabetic animals

[0062] The type 2 diabetic rats were divided into two groups: the diabetes model group and the EDS-treated diabetic animal group.

[0063] The healthy rats were also divided into two groups: normal control group and EDS-treated control group.

[0064] There were 10 male rats in each group.

[0065] For the rats in the EDS-treated diabetic animal group and the EDS-treated control group, a buffer solution containing 75 mg / kg EDS was injected once to induce the regeneration of testicular interstitial cells; at the same time, the rats in the diabetic model group and the normal control group were injected with the same volume of buffer solution without EDS. Afterwards, the four groups of rats were fed ordinary feed for 91 consecutive days. Starting from the day of injection, blood was collected through the tail vein on the 7th, 14th, 21st, 35th and 56th days, and then the radioactive enzyme-linked immunosorbent assay was used to determine the levels of androsterone, dihydrotestosterone, testosterone and estrogen in the serum at each time point. The results are shown in Table 1. Figure 1 At the same time, the fasting blood glucose level of rats after fasting for 8 hours was measured using a blood glucose meter every 7 days to observe the relationship between the four differentiation and development stages of testicular interstitial cells and the fasting blood glucose level. The curve is shown in Figure 2 .

[0066] according to Figure 1 It can be seen that the androstenone content in the EDS-treated diabetic animal group during the period from 7 to 35 days was significantly higher than that on the 56th day, and was also significantly higher than that in the diabetic model group and the normal control group; the dihydrotestosterone content in the EDS-treated diabetic animal group was the highest on the 35th day, followed by its contents on the 21st day and the 56th day, while its content on the 14th day was equivalent to that in the diabetic model group and the normal control group, and was significantly higher than that in the EDS-treated diabetic animal group on the 7th day; the testosterone content in the EDS-treated diabetic animal group on the 56th day was equivalent to that in the diabetic model group and the normal control group, and was significantly higher than that in the periods from 7 to 35 days; the estrogen content in the EDS-treated diabetic animal group from 7 to 56 days was equivalent to that in the diabetic model group and the normal control group. Based on the changes in the contents of the three sex hormones, androstenone, dihydrotestosterone and testosterone, it can be judged that EDS induces the apoptosis of the original ALC in type 2 diabetic rats and activates the dormant testicular interstitial stem cells. The whole process is the same as that of healthy rats. Similarly, SLC proliferates and differentiates from days 1 to 7 induced by EDS. Therefore, the regenerated cells are mainly in the PLC stage from days 7 to 28; and during days 28-35, the regenerated cells gradually differentiate into the ILC stage; from days 35 to 56, the regenerated interstitial cells are mainly in the ILC stage, and after day 56, the regenerated cells are in the ALC stage.

[0067] according to Figure 2The results of the study showed that by comparing the diabetic model group and the EDS-treated diabetic animal group, the fasting blood glucose content of type 2 diabetic rats gradually decreased with the increase of EDS induction time, until it reached the lowest value around the 28th day after induction, and then the fasting blood glucose content of type 2 diabetic rats gradually increased until it was equivalent to that of the diabetic model group rats. Therefore, EDS had a good improvement effect on blood glucose content from the 7th to the 35th day after induction, and was better than the effect from the 1st to the 7th day after induction and after the 35th day after induction; by comparing the normal control group and the EDS-treated control group, the blood glucose content of both was at normal levels at different time points, indicating that EDS-induced regeneration of testicular interstitial cells would not cause hypoglycemia in healthy rats.

[0068] In summary, androstenone can effectively lower the blood sugar content of animals with hyperglycemia, and its effect is better than dihydrotestosterone and testosterone, but it will not cause hypoglycemia in healthy animals. Therefore, androstenone has good safety.

[0069] Example 3

[0070] Type 2 diabetic rats were divided into two groups: diabetic model group and EDS treatment group. Meanwhile, healthy rats injected with the same volume of buffer were used as normal control group.

[0071] There were 10 male rats in each group.

[0072] For the rats in the EDS-treated group, a buffer solution containing 75 mg / kg EDS was injected once to induce the regeneration of interstitial cells; at the same time, the rats in the diabetes model group and the normal control group were injected with the same volume of buffer solution without EDS. Afterwards, the three groups of rats were fed with ordinary feed for 56 consecutive days. Among them, starting from the day of injection, blood was collected through the tail vein on the 7th, 14th, 21st, 35th and 56th days, and the ELISA method was used to determine the insulin content in the serum of the rats after fasting for 8 hours. The results are shown in Figure 3 .

[0073] according to Figure 3 The results showed that the fasting serum insulin levels of type 2 diabetic rats in the five time periods after EDS induction were comparable to those of the diabetic model group, and were significantly lower than those of the normal control group.

[0074] The above results indicate that androstenone cannot promote the synthesis of insulin by pancreatic islet cells.

[0075] Example 4

[0076] Injection of androsterone improves hyperglycemia in animals with type 2 diabetes

[0077] Androstenone was prepared into a 40 mg / mL stock solution in 95% ethanol and diluted for injection. The volume injected into each rat was the same, and the stock solution was diluted to the required concentration based on the injection volume.

[0078] Type 2 diabetic rats were divided into three groups: 10 mg androsterone / kg rat body weight group, 40 mg androsterone / kg rat body weight group and 80 mg androsterone / kg rat body weight group. Each group had 10 rats, all of which were male.

[0079] The three groups were injected with androsterone subcutaneously once, and then fed with ordinary feed and free diet for 18 consecutive days. Among them, the free diet blood glucose (random blood glucose) content of the rats was measured every day with a blood glucose meter starting before the injection of androsterone. Among them, the average blood glucose content of type 2 diabetic rats before androsterone treatment was as high as 612.38±23.5mg / dL. The 18-day curve of the free diet blood glucose content of the three groups of rats is shown in Figure 4 .

[0080] according to Figure 4 The results show that 24 hours after the injection of androsterone, the free diet content of the three groups of type 2 diabetic rats began to decline, and further improved with the extension of treatment time, until the 9th-11th day after injection to achieve the optimal hypoglycemic effect, and this effect was positively correlated with the dose of androsterone. Among them, the high-concentration treatment group achieved the best hypoglycemic effect. After 10 days of treatment, the free diet blood sugar of the rats dropped to 45% before treatment. 11-18 days after injection, the blood sugar of the three groups of rats showed a gradual upward trend and eventually returned to the level before androsterone treatment. These results show that androsterone can effectively improve the hyperglycemia symptoms of diabetic animals, and a single injection can effectively relieve the symptoms for up to about 2 weeks.

[0081] Example 5

[0082] Injection of fibroblast growth factor 21 (FGF21) improves hyperglycemia in type 2 diabetic animals

[0083] Recombinant human fibroblast growth factor 21 (FGF21, purchased from Peprotech, catalog number: 100-42) was prepared into a 1 mg / mL stock solution with normal saline and diluted for injection. The volume injected into each rat was the same, and the stock solution was diluted to the required concentration based on the injection volume.

[0084] Type 2 diabetic rats were divided into four groups, namely, low-dose FGF21 treatment group (injection dose of 0.1 mg FGF21 / kg body weight), medium-dose FGF21 treatment group (injection dose of 0.5 mg FGF21 / kg body weight), high-dose FGF21 treatment group (injection dose of 1 mg FGF21 / kg body weight) and diabetes model group (injection of the same volume of saline). At the same time, healthy rats injected with the same volume of saline were used as the normal control group. There were 10 rats in each group, all of which were male.

[0085] FGF21 was injected through the tail vein once a day for 4 weeks. During this period, the rats were fed with normal feed and had free access to food. Before the experimental rats were killed, they were fasted for 8 hours and the fasting blood glucose level of the rats was measured with a blood glucose meter. The results are shown in Figure 5 .

[0086] according to Figure 5 The results showed that the average fasting blood glucose level of healthy rats in the normal control group was 99.03 mg / dL, and the average fasting blood glucose level of rats in the diabetic model group was 354.81 mg / dL; compared with the diabetic model group, the average fasting blood glucose levels of the three groups of type 2 diabetic rats treated with FGF21 were significantly decreased in a dose-dependent manner, among which the average blood glucose level of rats in the high-dose FGF21 treatment group decreased to 137.94 mg / dL.

[0087] Example 6

[0088] Combination of androstenone and FGF21 can improve hyperglycemia in type 2 diabetic animals

[0089] Androstenone was prepared into a 40 mg / mL stock solution with 95% ethanol and diluted for injection.

[0090] FGF21 (Peprotech) was prepared into a 1 mg / mL stock solution with physiological saline and diluted for injection.

[0091] Each rat was injected with the same volume, and both stock solutions were diluted to the desired concentration based on the injection volume.

[0092] Type 2 diabetic rats were divided into four groups, namely, diabetes model group, androsterone treatment group (20 mg androsterone / kg body weight, once a week), FGF21 treatment group (0.2 mg FGF21 / kg body weight, once a day), combined treatment group (10 mg androsterone / kg body weight, once a week; 0.1 mg FGF21 / kg body weight, once a day). At the same time, a normal control group (healthy rats, injected with an equal volume of 95% ethanol, once a week; an equal volume of normal saline, once a day) was set up. Each group included 10 male rats.

[0093] The rats were injected with androstenone by intraperitoneal injection and FGF21 by tail vein injection for 2 weeks. During this period, they were fed with ordinary feed and had free access to food. Before the experimental rats were killed, they were fasted for 8 hours and the fasting blood glucose level of the rats was measured with a blood glucose meter. The results are shown in Figure 6 The results showed that the combined use of drugs can significantly improve the blood sugar content of diabetic model animals, and compared with the single drug group, it has a significant synergistic effect.

[0094] Subsequently, the experiment was designed to further analyze the hypoglycemic effect of the combined drug groups at different doses. The type 2 diabetic rats were divided into four groups, namely, the combined treatment low-dose group (10 mg androsterone / kg body weight, once a week; 0.1 mg FGF21 / kg body weight, once a day), the combined treatment medium-dose group (40 mg androsterone / kg body weight, once a week; 0.5 mg FGF21 / kg body weight, once a day), the combined treatment high-dose group (80 mg androsterone / kg body weight, once a week; 1 mg FGF21 / kg body weight, once a day), and the diabetes model group (injection of an equal volume of 95% ethanol, once a week; an equal volume of normal saline, once a day). At the same time, a normal control group (healthy rats, injected with an equal volume of 95% ethanol, once a week; an equal volume of normal saline, once a day) was set up. Each group included 10 male rats.

[0095] The rats were injected with androstenone by intraperitoneal injection and FGF21 by tail vein injection for 2 weeks. During this period, they were fed with ordinary feed and had free access to food. Before the experimental rats were killed, they were fasted for 8 hours and the fasting blood glucose level of the rats was measured with a blood glucose meter. The results are shown in Figure 7 .

[0096] according to Figure 7 The results show that low-dose androstenone and fibroblast growth factor 21 combined use for 2 weeks can quickly improve the hyperglycemia of model animals; and in the combined treatment of medium and high dose groups, the blood sugar of diabetic animals has dropped to the same level as the normal control group. This result shows that the combined use of the two has a synergistic effect on improving hyperglycemia and is concentration-dependent.

[0097] Example 7

[0098] Obtaining transdifferentiated cells expressing fibroblast growth factor 21 and androsterone

[0099] PB-TRE-dCas9-VPR (Cat. No. 63800) and a kit containing pX330A-1x4 and all reagents required to construct a gRNA expression vector (Cat. No. #1000000055) were purchased from Addgene.

[0100] 1. Recombinant plasmid expressing FGF21 and steroidogenic factor-1 (SF-1) promoter region-directed RNA

[0101] The pX330A-1x4 vector was double-digested with EcoRV and Pmll to cut out a fragment of about 936 bps in the Cas9 gene in the plasmid. The backbone fragments were recovered by agarose gel electrophoresis and ligated with T4 ligase to obtain the recombinant plasmid pX330A-1x4-mut.

[0102] The guide RNA (gRNA) of the FGF21 promoter region was designed, and 4 guide RNAs were screened. The sequences of the deoxyribonucleic acids corresponding to these 4 guide RNAs are shown in SEQ ID Nos. 1-4. After the four sequences were synthesized, they were cloned into the expression plasmid pX330A-1x4-mut to obtain the recombinant plasmid pX330A-FGF21-gRNA1-4 with 4 gRNA expression cassettes in series, wherein the 5' end of each gRNA was a U6 promoter.

[0103] Assume that the guide RNA (gRNA) in the SF-1 promoter region is selected, and 4 guide RNAs are screened. The sequences of the deoxyribonucleic acids corresponding to these 4 guide RNAs are shown in SEQ ID Nos.5-8. After the four sequences are synthesized, they are cloned into the expression plasmid pX330A-1x4-mut to obtain the recombinant plasmid pX330A-SF1-gRNA1-4 with 4 gRNA expression cassettes in series, wherein the 5' end of each gRNA is a U6 promoter.

[0104] 2. Isolation of Skin Fibroblasts

[0105] Take the foreskin tissue, peel off the epidermis and dermis, and cut the remaining part into 1mm pieces with scissors. 3 Transfer the tissue block to a cell culture dish, add 6 mL of culture medium, and culture for 5 days. After the fibroblasts migrate out of the tissue block, remove the tissue block with sterile tweezers, replace with fresh culture medium, and continue to culture until the confluence is 90%. Discard the old culture medium, wash the cells twice with PBS buffer, digest with trypsin for 1 min, add 3 mL of serum-containing culture medium to terminate digestion, blow to make a cell suspension, collect the cell suspension in a 15 mL centrifuge tube, centrifuge for 5 min, and the collected cells are skin fibroblasts.

[0106] 3. Transdifferentiation of skin fibroblasts into cells expressing FGF21 and synthesizing androsterone

[0107] The transdifferentiation process is divided into 2 stages:

[0108] Stage 1: Skin fibroblasts were cultured at 3×10 5The cells were inoculated into Dulbecco's Modified Eagle Medium (DMEM / F12-Dulbecco's Modified Eagle Medium, purchased from Sigma, catalog number: D8437) containing 10% fetal bovine serum at a density of 10 cells / well and cultured for 12 hours. Then, according to the instructions of the liposome transfection reagent (Lipofectamine 3000), the PB-TRE-dCas9-VPR, pX330A-FGF21-gRNA1-4 and pX330A-SF1-gRNA1-4 recombinant vectors (masses of 1.8 μg, 0.35 μg, and 0.35 μg, respectively) were transfected into skin fibroblasts. After incubation at 37°C for 6 hours, the medium was replaced with fresh DMEM / F12 medium containing a final concentration of 10% fetal bovine serum, 0.5 μM smoothened agonist (SAG), 10 ng / ml luteinizing hormone, 1 mM cyclic AMP, 10 ng / ml fibroblast growth factor 2 (bFGF), 10 μg / ml insulin, and 5 μM transforming growth factor-β signaling pathway inhibitor LY2109761 for culture. The medium containing the same components at the same concentration was replaced every 2 days. The culture was continued for 6 days to complete the first stage.

[0109] The second stage: the culture medium was replaced with fresh Dulbecco's modified Eagle's medium containing a final concentration of 10% fetal bovine serum, 0.5 μM SAG, 10 ng / ml luteinizing hormone, 1 mM cyclic adenosine monophosphate, 10 μg / ml insulin, and 10 μg / ml rosiglitazone. The culture medium containing the same components at the same concentration was replaced every 2 days. The culture was continued for 12 days to obtain transdifferentiated cells that express FGF21 and synthesize androsterone.

[0110] 4. Transcription and expression analysis of FGF21 and androgen synthase in transdifferentiated cells

[0111] Leydig cells were isolated from human testis using the method of Chen X et al. (Cell Death & Disease, 2019, 10(3): 220).

[0112] The skin fibroblasts were isolated using the method described in Section 2 of this example.

[0113] 4.1 Transcriptional analysis

[0114] The primers used for FGF21 RT-PCR analysis were FGF21-F (SEQ ID No. 9) and FGF21-R (SEQ ID No. 10).

[0115] The primers used for SF-1 RT-PCR analysis were SF-1-F (SEQ ID No. 11) and SF-1-R (SEQ ID No. 12).

[0116] The primers used for luteinizing hormone receptor RT-PCR analysis were LHCGR-F (SEQ ID No. 13) and LHCGR-R (SEQ ID No. 14).

[0117] The primers used for RT-PCR analysis of steroidogenic acute regulatory protein (StAR) were StAR-F (SEQ ID No. 15) and StAR-R (SEQ ID No. 16).

[0118] The primers used for RT-PCR analysis of cholesterol side chain cleavage enzyme (Cytochrome P450, family 11, subfamily A, polypeptide 1, CYP11A1) were CYP11A1-F (SEQ ID No. 17) and CYP11A1-R (SEQ ID No. 18).

[0119] The primers used for RT-PCR analysis of Hydroxy-Delta-5-SteroidDehydrogenase, 3Beta And Steroid Delta-Isomerase 2 (HSD3B2) were HSD3B2-F (SEQ ID No. 19) and HSD3B2-R (SEQ ID No. 20).

[0120] The primers used for RT-PCR analysis of cytochrome P450 17A1 (CYP17A1) were CYP17A1-F (SEQ ID No. 21) and CYP17A1-R (SEQ ID No. 22).

[0121] The primers used for RT-PCR analysis of the internal reference gene ribosomal protein S16 (RPS16) were PRS16-F (SEQ ID No. 23) and PRS16-R (SEQ ID No. 24)

[0122] The expression levels of SF-1 and FGF21 in transdifferentiated cells induced for 12 days were analyzed by RT-PCR, with skin fibroblasts and testicular Leydig cells as controls. Figure 8 .from Figure 8 It can be seen that skin fibroblasts do not express SF-1 and FGF21; Leydig cells can express SF-1 and a small amount of FGF21; transdifferentiated cells can express SF-1 and FGF21 in large quantities, and the expression level is significantly higher than that of Leydig cells, indicating that the endogenous genes SF-1 and FGF21 of transdifferentiated cells are activated.

[0123] The expression levels of LHCGR, StAR, CYP11A1, HSD3B2 and CYP17A1 in transdifferentiated cells induced for 12 days were analyzed by RT-PCR, and skin fibroblasts and Leydig cells were used as controls. Fig. 9 .from Fig. 9 It can be seen that transdifferentiated cells can express a large amount of key enzymes in the androgen synthesis pathway: LHCGR, StAR, CYP11A1, HSD3B2 and CYP17A1, indicating that the androgen synthesis pathway in fibroblasts was successfully activated by the above treatment method.

[0124] 4.2 Expression and synthesis analysis

[0125] After 12 days of culture of transdifferentiated cells, the culture supernatant was collected for Western Blot, and the antibody used was FGF21 antibody (Abcam, Catalog No.: ab66564). Fig.10 .from Fig.10 It can be seen that transdifferentiated cells can express and secrete FGF21.

[0126] After culturing the transdifferentiated cells for 3, 6, 12 and 18 days, the culture supernatant was collected and the androgen content in the culture supernatant was determined by radioactive enzyme-linked immunosorbent assay. Skin fibroblasts were used as a control. Fig.11 The results showed that androsterone could be detected in the culture supernatant of transdifferentiated cells after 3 days of culture, and the androsterone content gradually increased with the extension of culture time. On the 18th day, the androsterone content in the culture supernatant was 1.4ng / 1 million cells / 24 hours; while in the culture supernatant of skin fibroblasts used as a control, only a very small amount of androsterone below the detection threshold could be detected.

[0127] Example 8

[0128] Transdifferentiated cells can improve hyperglycemia in type 2 diabetic animals

[0129] 1. Transplantation of transdifferentiated cells to treat type 2 diabetes

[0130] Type 2 diabetic rats were divided into two groups, namely the transdifferentiation cell treatment group and the diabetes model group. At the same time, healthy rats treated with skin fibroblasts were used as the normal control group. There were 6 rats in each group, all of which were male.

[0131] For the transdifferentiated cell treatment group, 2×10 6 For the diabetes model group, 2×10 6 For the normal control group, 2×10 6 The rats were inoculated with skin fibroblasts at the amount of 10 cells / rat. Then they were fed with normal feed and free diet for 21 days. Starting from the day of inoculation, the fasting blood glucose level of the rats was measured every 7 days after fasting for 8 hours using a blood glucose meter. The results are shown in Fig.12 .

[0132] from Fig.12 The results showed that the fasting blood glucose levels of the diabetic model group and the normal control group remained stable, one maintained at a high level, with an average fasting blood glucose level of 311.35 mg / dL for 6 rats on 21 days, and the other maintained at a normal low level, with an average fasting blood glucose level of 93.24 mg / dL for 6 rats on 21 days; while the fasting blood glucose level of the rats in the transdifferentiation cell therapy group continued to decrease with time, and at 21 days, its blood glucose level was 119.2 mg / dL, close to that of the normal control group.

[0133] 2. Pathological examination of liver tissue of treated rats

[0134] Masson staining is mainly used for differential staining of collagen fibers and muscle fibers, and is of great value in the diagnosis of fibrous tissue hyperplasia in the lobular portal area caused by various liver diseases.

[0135] Similar to Section 1 of this example, a transdifferentiation cell treatment group, a diabetes model group and a normal control group were set up.

[0136] According to the instructions of Masson's Trichrome Stain Kit (Solarbio, catalog number: G1340), liver tissue sections of type 2 diabetic rats transplanted with transdifferentiated cells on days 7, 14, and 21 were stained and analyzed; for the diabetes model group and the normal control group, liver tissue sections were also stained on day 21 after transplantation of skin fibroblasts. The results are shown in Fig.13 .

[0137] The results showed that in the livers of animals in the normal control group, the lobule structure was intact without fibrous tissue hyperplasia; in the livers of animals in the diabetic model group, fibrosis was observed in the portal area, periportal area, and perisinusoidal areas, and fibrous tissue hyperplasia was obvious in the fibrous septa, with inflammatory cell infiltration, but no pseudolobules were formed; in the livers of animals in the treatment group, 7 days after cell transplantation, a certain degree of fibrous tissue hyperplasia was observed, with inflammatory cell infiltration in some places, but the fibrosis was milder than that in the diabetic model group; after 14 days of treatment, liver fibrosis was significantly alleviated compared with that in the diabetic model group, with fibrous tissue hyperplasia still observed, but the fibrous septa were significantly thinned, and the infiltration of connective tissue and inflammatory cells was significantly reduced; after 21 days of treatment, the degree of liver fibrosis was significantly reduced, with only a small number of inflammatory cells infiltrating.

[0138] 3. Liver lipid content of treated rats

[0139] Oil Red O staining is mainly used to analyze lipid status in tissue samples.

[0140] Similar to Section 1 of this example, a transdifferentiation cell treatment group, a diabetes model group and a normal control group were set up.

[0141] According to the instructions of the Oil Red O Staining Kit (Sigma-Aldrich, Catalog No. MAK194), the liver tissue sections of type 2 diabetic rats transplanted with transdifferentiated cells on the 7th, 14th and 21st day were stained and analyzed; for the diabetes model group and the normal control group, the liver tissue sections were also stained on the 21st day after the transplantation of skin fibroblasts. The results are shown in Fig.14 .

[0142] The experimental results showed that in the livers of animals in the normal control group, the lobule structure was intact, the hepatocyte cords were arranged regularly, and they were radially distributed around the central vein, and no obvious Oil Red O staining was observed. In the livers of animals in the diabetic model group, there was obvious fatty degeneration, cell enlargement, compression and narrowing of the hepatic sinusoids, and disordered arrangement of the hepatic cords, and large areas of red lipid droplets were observed in the liver tissue. In the livers of animals in the treatment group, lipid droplets were observed in some places after 7 and 14 days of treatment, but the staining area was significantly reduced compared with that in the diabetic model group; after 21 days of treatment, there was little or no Oil Red O staining, indicating that the accumulation of lipids in the liver was significantly improved. Sequence Listing <110> Wenzhou Medical University Su Zhijian <120> Application of androsterone and its combination with fibroblast growth factor in the treatment of hyperglycemia <130> LHA2260327 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 tgctcctgaa ataacctggg gg 22 <210> 2 <211> twenty three <212> DNA <213> Artificial Sequence <400> 2 gcctaccctt gcacccttag ggg 23 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <400> 3 ttggctgcca actccagtca ggg 23 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 gcccctaagg gtgcaagggt agg 23 <210> 5 <211> twenty three <212> DNA <213> Artificial Sequence <400> 5 cccgcttgtt agaaaccggg tgg 23 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 gagaccaatt gggcctccgg tgg 23 <210> 7 <211> twenty three <212> DNA <213> Artificial Sequence <400> 7 ccgcttgtta gaaaccgggt ggg 23 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence <400> 8 cacccggttt ctaacaagcg ggg 23 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <400> 9 ctgtgggttt ctgtgctgg 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 ccggcttcaa ggctttcag 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 ccggctacca ctacggact 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 ctgcgtcttg tcgatcttgc 20 <210> 13 <211> twenty three <212> DNA <213> Artificial Sequence <400> 13 ggtctcactc gactatcact tgc 23 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <400> 14 ctccgggctc aatgtatctc a 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 gggagtggaa ccccaatgtc 20 <210> 16 <211> twenty two <212> DNA <213> Artificial Sequence <400> 16 ccagctcgtg agtaatgaat gt 22 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <400> 17 gcagtgtctc gggacttcg 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 ggcaaagcgg aacaggtca 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 agaacggcca cgaagaagag 20 <210> 20 <211> twenty one <212> DNA <213> Artificial Sequence <400> 20 tgggtcttaa cgcacaagtg t 21 <210> twenty one <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty one gctgcttacc ctagcttatt tgt 23 <210> twenty two <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty two accgaataga tggggccata ttt 23 <210> twenty three <211> 19 <212> DNA <213> Artificial Sequence <400> twenty three tcggacgcaa gaagacagc 19 <210> twenty four <211> twenty one <212> DNA <213> Artificial Sequence <400> 24 agcagcttgt actgtagcgt g 21

Claims

1. Use of androsterone in the preparation of a medicament for preventing and / or treating type 2 diabetes.

2. A combined drug for preventing and / or treating type 2 diabetes, comprising fibroblast growth factor 21 and androsterone; the mass ratio of the fibroblast growth factor 21 to the androsterone is 1:(1-800).

3. The combined drug according to claim 2, characterized in that: The mass ratio of the fibroblast growth factor 21 to the androsterone is 1:(8-100).

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