Use of androsterone and its combination with fibroblast growth factor for the treatment of hyperglycemia

Abstract

To provide the application of a drug combination to the preparation of a medicine for preventing and / or treating diabetes mellitus.SOLUTION: The invention provides the application of androsterone to the preparation of a medicine for preventing and / or treating hyperglycemia. The invention further provides a drug combination containing fibroblast growth factors and androsterone.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Leydig cells (LCs) synthesize and secrete androgens in a physiological rhythm regulated by the hypothalamic-pituitary-gonadal axis and are the primary source of androgens in males. During male development, two distinct types of Leydig cells exist: fetal LCs (FLCs) and adult Leydig cells. The latter are formed in the male testes during puberty and can be divided into four stages during differentiation: Leydig stem cells (SLCs), progenitor LCs (PLCs), immature LCs (ILCs), and adult LCs (ALCs).

[0003] Because the expression timing of different steroidogenic enzymes in Leydig cells differs, the androgen end products synthesized by Leydig cells at different developmental stages also differ. Leydig stem cells cannot synthesize androgens, Leydig progenitor cells mainly synthesize androsterone, immature Leydig cells mainly synthesize dihydrotestosterone, and mature Leydig cells mainly synthesize testosterone.

[0004] The development and differentiation of Leydig cells is generally limited to embryogenesis and early puberty. Once ALCs are formed, their numbers do not change significantly in healthy populations, even as males enter senescence. However, their hormone synthesis function gradually declines with age. However, existing mature Leydig cells in the testes can undergo specific apoptosis induction (e.g., with the compound ethane dimethane sulfonate (EDS)). Subsequently, dormant Leydig stem cells can be activated, proliferate, and ultimately differentiate into new mature Leydig cells. These regenerative Leydig cells similarly pass through four stages: Leydig stem cells, Leydig progenitor cells, immature testicular cells, and mature testicular cells. The androgen end products synthesized by regenerative cells at each stage are the same as those of the corresponding pre-existing Leydig cells. For example, in rats, EDS-induced apoptosis of existing ALCs occurred, and the apoptosis process lasted for 4 days. At the same time, EDS-induced SLCs began to proliferate and differentiate from days 1 to 7. From days 7 to 28, the regenerated cells were mainly at the PLC stage. From days 35 to 56, the regenerated cells were mainly at the ILC stage. After 56 days, the regenerated cells differentiated to the ALC stage. Summary of the Invention [Problem to be solved by the invention]

[0005] However, the functions of androgens remain poorly understood. [Means for solving the problem]

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

[0007] In one specific embodiment, the factors causing hyperglycemia include prediabetes, diabetes, At least one of diabetes, obesity, and senility. For example, either senility or obesity can cause mild hyperglycemia.

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

[0009] In one specific embodiment, the androsterone is used to prevent and / or treat hyperglycemia in males.

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

[0011] In one 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 one specific embodiment, the mass ratio of the fibroblast growth factor to the androsterone is 1:(1 to 800).

[0013] In one 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 cells that simultaneously express a first factor and a second factor, wherein the first factor is a fibroblast growth factor and the second factor is steroidogenic factor-1 (also known as NR5A1) and / or liver receptor homolog-1 (Liver Receptor Homolog-1, LRH-1; also known as NR5A2), and the cells synthesize and secrete androsterone upon expression of the second factor. As is well known to those skilled in the art, expression of steroidogenic factor-1 can activate key enzymes in the androsterone synthesis pathway, thereby inducing cells to differentiate into Leydig cells capable of synthesizing androsterone. As is further well known to those skilled in the art, expression of LRH-1 can also induce cells to differentiate into Leydig cells, thereby enabling cells to synthesize androsterone.

[0015] In one 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 one specific embodiment, the cells are transdifferentiated cells obtained by activating the expression of the first factor and the expression of the second factor therein.

[0017] In one specific embodiment, the transdifferentiated cells are obtained by transforming a first recipient cell with a CRISPR / dCas9-activated gene expression system for activating expression of the first factor and expression of the second factor, The CRISPR / dCas9-activated gene expression system includes at least one dCas9 expression cassette, at least one guide ribonucleic acid (GUID) of the first factor promoter region, and For example, the CRISPR / dCas9-activated gene expression system may include a dCas9 expression cassette, at least one guide ribonucleic acid (guild RNA, gRNA) expression cassette for a first factor promoter region, and at least one guide ribonucleic acid (guild RNA, gRNA) expression cassette for a second factor promoter region, all contained in the same plasmid, i.e., a dCas9 expression cassette, at least one guide ribonucleic acid (guild RNA, gRNA) expression cassette for a first factor promoter region, and The CRISPR / dCas9-activated gene expression system may include at least one guide ribonucleic acid (gRNA) expression cassette for the second factor promoter region, or may be distributed across two or three plasmids, e.g., three expression cassettes may be located on three plasmids.

[0018] The recombinant vector for the CRISPR / dCas9-activated gene expression system may be a commercially available vector, such as PB-TRE-dCas9-VPR (Addgene; catalog number: 63800) or PB-SAM (Addgene; catalog number: 102559). PB-TRE-dCas9-VPR and pX330A-1x4 may be used in combination, or PB-SAM and pX330A-1x4 may be used in combination. Of these, pX330A-1x4 requires simple modification to remove the Cas9 gene.

[0019] In one specific embodiment, the cell is a transdifferentiated cell obtained by activating and regulating the expression of the first factor and the expression of the second factor therein with a first signal pathway regulator.

[0020] In one specific embodiment, the cell is a transdifferentiated cell obtained by transforming the CRISPR / dCas9-activated gene expression system into the first recipient cell and modulating it with the first signal pathway regulator.

[0021] In a specific embodiment, the cell is a modified cell obtained by transforming an expression vector carrying an expression cassette for the first factor and an expression cassette for the second factor into the second receptor cell and regulating it with the second signal pathway regulator.

[0022] In one specific embodiment, the first signaling pathway regulator and the second signaling pathway regulator independently comprise at least one of insulin, a hedgehog signaling pathway agonist SAG, cyclic adenosine monophosphate, a transforming growth factor beta signaling pathway inhibitor LY2109761, fibroblast growth factor 2, luteinizing hormone, and rosiglitazone.

[0023] In one specific embodiment, the regulation by the first signal pathway regulator and the regulation by the second signal pathway regulator are independently divided into two stages, the signal pathway regulators in the first stage include insulin, a hedgehog signal pathway active substance SAG, cyclic adenosine monophosphate, transforming growth factor β signal pathway inhibitor LY2109761, fibroblast growth factor 2, and luteinizing hormone, and the signal pathway regulators in the second stage include insulin, a hedgehog signal pathway active substance SAG, cyclic adenosine monophosphate, luteinizing hormone, and rosiglitazone.

[0024] In one specific embodiment, the insulin is used at a concentration of 1 μg / mL to 100 μg / mL, the hedgehog signal pathway active substance SAG is used at a concentration of 0.1 μM to 1 μM, the cyclic adenosine monophosphate is used at a concentration of 0.1 mM to 10 mM, the transforming growth factor β signal 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.

[0025] In one specific embodiment, the first receptor cell and the second receptor cell are independently of human or murine origin.

[0026] In one specific embodiment, the first and second recipient cells are independently one of mesenchymal stem cells, induced pluripotent stem cells (iPSCs), and fibroblasts. In particular, the first and second recipient cells may be independently fibroblasts obtained by inducing differentiation of embryonic stem cells or iPSCs by genetic engineering methods.

[0027] In one specific embodiment, the fibroblasts are one of skin fibroblasts, embryonic fibroblasts, adult fibroblasts and organ fibroblasts.

[0028] In one specific embodiment, the deoxyribonucleic acid sequence corresponding to the guide ribonucleic acid of the first factor promoter region is at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0029] In one specific embodiment, there are four guide ribonucleic acid expression cassettes in the first factor promoter region.

[0030] In one specific embodiment, the deoxyribonucleic acid sequence corresponding to the guide ribonucleic acid of the second factor promoter region is at least one of the sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0031] In one specific embodiment, there are four guide ribonucleic acid expression cassettes in the second factor promoter region.

[0032] The fourth aspect of the present invention provides use of a combination drug according to any one of the second aspects of the present invention or a cell according to any one of the third aspects of the present invention in the manufacture of a drug for preventing and / or treating hyperglycemia (i.e., a situation in which blood glucose levels are higher than normal levels).

[0033] In one specific embodiment, the factor causing hyperglycemia is at least one of prediabetes, diabetes, senility, and obesity. For example, senility or obesity can both cause mild hyperglycemia.

[0034] In a specific embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0035] In one specific embodiment, the androsterone is used to prevent and / or treat hyperglycemia in males.

[0036] In the present invention, the term "expression cassette" generally includes a promoter, a target gene (eg, a gene for the first factor or a gene for the second factor) and a terminator, depending on specific needs.

[0037] The beneficial effects of the present invention are as follows: The present inventors have first discovered that androsterone can be used to prevent and / or treat hyperglycemia in males, and that the causes of hyperglycemia are at least one of prediabetes, diabetes, senility, and obesity. Furthermore, the present inventors have further discovered that a synergistic effect can be achieved by using fibroblast growth factor and androsterone in combination to prevent and / or treat hyperglycemia in males. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1 shows the serum sex hormone contents at different times in type 2 diabetic rats after EDS treatment, *p<0.05, **p<0.01, ***p<0.001, vs. normal control group. [Figure 2] FIG. 2 shows the curves of fasting blood glucose content in type 2 diabetic rats at four differentiation and development stages of Leydig cell regeneration. [Figure 3]Figure 3 shows the insulin content in serum of type 2 diabetic rats at different times after EDS treatment, **p<0.01 vs. normal control group. [Figure 4] FIG. 4 shows the effect of a single intraperitoneal injection of androsterone on the voluntary blood glucose content of diabetic rats. [Figure 5] FIG. 5 shows the effect of FGF21 on improving fasting blood glucose levels in diabetic rats, where * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001, versus the diabetic model group. [Figure 6] FIG. 6 shows the ameliorative effect of combined use of androsterone and FGF21 on hyperglycemia in rats, where * indicates p<0.05 and ** indicates p<0.01, vs. the diabetic model group. [Figure 7] Figure 7 shows the ameliorative effect of androsterone and FGF21 in combination at different doses on hyperglycemia in diabetic animals, where ## indicates p<0.01 and ### indicates p<0.001, versus the normal control group. [Figure 8] FIG. 8 shows gene expression analysis of FGF21 and SF-1 in transdifferentiated cells, where ** indicates p<0.01 and *** indicates p<0.001, versus Leydig cells, n=3, and n indicates the number of repeated tests. [Figure 9] FIG. 9 shows gene expression analysis of androgen synthase in transdifferentiated cells, where ** indicates p<0.01 and *** indicates p<0.001, versus fibroblasts, n=3, and n indicates the number of repeated tests. [Figure 10] 10 shows a Western blot of FGF21 in the supernatant of a transdifferentiated cell culture medium. Lanes 1 and 2 are the supernatant of a transdifferentiated cell culture medium, lane 3 is the supernatant of a fibroblast culture medium, and lane M is a protein molecular weight marker. [Figure 11] FIG. 11 shows the androsterone synthesis levels of transdifferentiated cells at different culture times. [Figure 12]FIG. 12 shows the effect of transdifferentiated cells on fasting blood glucose content in hyperglycemic rats, where ** indicates p<0.01 and *** indicates p<0.001, versus the diabetic model group. [Figure 13] FIG. 13 is an analysis of collagen fiber proliferation in rat liver tissue sections, with the sections magnified 200 times. [Figure 14] FIG. 14 is an analysis of lipid accumulation in rat liver tissue sections, with the sections magnified 200 times. DETAILED DESCRIPTION OF THE INVENTION

[0039] The above content of the present invention will be explained in more detail below in the form of preferred examples, but the present invention is not limited thereto.

[0040] Unless otherwise stated, all reagents in the examples of the present invention may be purchased commercially.

[0041] Example 1 Construction of a type 2 diabetes rat model Three-month-old adult Sprague-Dawley rats (male SD rats) were purchased and housed in a constant temperature of 24±2°C, relative humidity of 50%-70%, and a day / night rhythm of 12:12. The rats were divided into two groups: a type 2 diabetes model group fed a high-fat, high-sugar diet, and a normal control group fed a normal diet. After 30 consecutive days of housekeeping, the rats were treated with streptozotocin at a dose of 35 mg / kg (streptozotocin dissolved in citrate buffer (0.1 mM, pH=4.4) in an ice bath, shaded, and dry environment). A type 2 diabetes model group of rats was constructed by intraperitoneally injecting streptozotocin (completed use within 5 minutes). A normal control group was injected with the same volume of citrate buffer (0.1 mM, pH 4.4). Four days later, blood was collected from the rats' tails, and fasting blood glucose concentrations of the streptozotocin-injected rats were measured using a blood glucose meter. Rats whose blood glucose concentrations reached 198 mg / dL or higher were considered to have successfully constructed the model. Rats whose blood glucose concentrations did not reach the target level were given an additional intraperitoneal injection of streptozotocin at a dose of 15 mg / kg. Blood glucose levels were measured again four days later. If the blood glucose concentration reached 198 mg / dL or higher, the construction of a type 2 diabetes rat model was considered successful.

[0042] The rats in the model group that reached the above blood glucose concentration index were subsequently fed a high-fat, high-sugar diet, while the rats in the normal control group were fed a normal diet. After 4 weeks, the changes in fasting blood glucose levels were measured. The final results showed that the mean fasting blood glucose level of the type 2 diabetic rats in the model group was over 308.84±24.2mg / dL, while the mean fasting blood glucose level of the healthy rats in the normal control group was 89.5±9.50mg / dL.

[0043] Example 2 Androsterone synthesis in regenerated Leydig cells can improve hyperglycemia in type 2 diabetic animals Type 2 diabetic rats were divided into two groups: a diabetic model group and an EDS-treated diabetic animal group.

[0044] Healthy rats were similarly divided into two groups: a normal control group and an EDS-treated control group.

[0045] Each group consisted of 10 rats, all of which were male.

[0046] Leydig cell regeneration was induced in rats in the EDS-treated diabetic and EDS-treated control groups by a single injection of a buffer solution containing 75 mg / kg EDS. Simultaneously, rats in the diabetic model and normal control groups were injected with the same volume of buffer solution without EDS. All four groups of rats were then fed a normal diet for 91 consecutive days. Blood samples were taken from the tail vein on days 7, 14, 21, 35, and 56, starting from the day of injection. Serum androsterone, dihydrotestosterone, testosterone, and estrogen levels were measured at each time point using a radioactive enzyme-linked immunosorbent assay (RII). The results are shown in Figure 1. Fasting blood glucose levels were also measured in rats after an 8-hour fast every seven days. The relationship between fasting blood glucose levels and the four stages of Leydig cell differentiation and development was observed. The curves are shown in Figure 2.

[0047] As can be seen from Figure 1, the androsterone content in the EDS-treated diabetic animals was significantly higher from day 7 to day 35 than on day 56, and was also significantly higher than that in the diabetic model and normal control groups. The dihydrotestosterone content in the EDS-treated diabetic animals was highest on day 35, followed by those on days 21 and 56. The dihydrotestosterone content on day 14 was comparable to that in the diabetic model and normal control groups and significantly higher than that in the EDS-treated diabetic animals on day 7. The testosterone content in the EDS-treated diabetic animals on day 56 was comparable to that in the diabetic model and normal control groups and significantly higher than that in the EDS-treated diabetic animals on days 7 to 35. The estrogen content in the EDS-treated diabetic animals from day 7 to day 56 was comparable to that in the diabetic model and normal control groups. The changes in the contents of the three sex hormones, androsterone, dihydrotestosterone, and testosterone, indicated that EDS induces apoptosis of existing ALCs and activates dormant Leydig stem cells in type 2 diabetic rats. The overall process is the same as in healthy rats. Similarly, from day 1 to day 7 after EDS induction, SLCs proliferate and differentiate, so that from day 7 to day 28, regenerative cells are mainly in the PLC stage, and from day 28 to day 35, regenerative cells are mainly in the PLC stage. During the 35-day period, regenerative cells differentiate sequentially to the ILC stage. From day 35 to day 56, regenerative Leydig cells are primarily at the ILC stage, and after day 56, regenerative cells are at the ALC stage.

[0048] As shown in Figure 2, comparing the diabetic model group with the EDS-treated diabetic animal group, the fasting blood glucose levels of type 2 diabetic rats gradually decreased with increasing EDS induction time, reaching a minimum at approximately day 28 after induction. Thereafter, the fasting blood glucose levels of type 2 diabetic rats gradually increased until they reached a level comparable to that of the diabetic model group. Therefore, EDS had a significant effect on blood glucose levels from days 7 to 35 after induction, and was superior to the effects on days 1 to 7 and day 35 after induction. Comparing the normal control group with the EDS-treated control group, the blood glucose levels of both groups remained normal at different time points, indicating that EDS-induced Leydig cell regeneration did not cause hypoglycemia in healthy rats.

[0049] From the above, androsterone can effectively reduce the blood glucose content in hyperglycemic animals, and its effect is superior to that of dihydrotestosterone and testosterone, but it does not cause hypoglycemia in healthy animals, so androsterone has good safety.

[0050] Example 3 Type 2 diabetic rats were divided into two groups: a diabetic model group and an EDS-treated group. At the same time, healthy rats injected with the same volume of buffer solution served as normal controls.

[0051] Each group consisted of 10 rats, all of which were male.

[0052] The rats in the EDS-treated group were injected with a buffer solution containing 75 mg / kg EDS once to induce Leydig cell regeneration. At the same time, the rats in the diabetic model and normal control groups were injected with the same volume of buffer solution without EDS. All three groups of rats were then fed a normal diet for 56 consecutive days. Blood samples were taken from the tail vein on days 7, 14, 21, 35, and 56 after the injection, and serum insulin levels were measured using ELISA after an 8-hour fast. The results are shown in Figure 3.

[0053] As can be seen from the results in Figure 3, the fasting serum insulin content of type 2 diabetic rats at five time points after EDS induction was comparable to that of the diabetic model group, and was significantly lower than that of the normal control group.

[0054] These results demonstrate that androsterone cannot stimulate insulin synthesis in pancreatic islet cells.

[0055] Example 4 Androsterone injections can improve hyperglycemia in type 2 diabetic animals Androsterone was prepared in 95% ethanol to a 40 mg / mL solution and diluted before injection. The same volume was injected into each rat, and the solution was diluted to the desired concentration depending on the injection volume.

[0056] Type 2 diabetic rats were divided into three groups: 10 mg androsterone / kg body weight, 40 mg androsterone / kg body weight, and 80 mg androsterone / kg body weight. Each group contained 10 male rats.

[0057] The three groups received a single subcutaneous injection of androsterone, and were then fed normal chow and allowed to eat and drink ad libitum for 18 consecutive days. Before androsterone injection, the rats' random blood glucose levels were measured daily using a blood glucose meter. The mean blood glucose level of the type 2 diabetic rats before androsterone treatment was 612.38±23.5 mg / dL, which was significantly higher. The 18-day blood glucose curves of the rats in the three groups are shown in Figure 4.

[0058] As shown in Figure 4, 24 hours after androsterone injection, the free-feeding blood glucose levels of all three groups of type 2 diabetic rats began to decrease. This continued to improve over time, reaching optimal hypoglycemic effects 9-11 days after injection, and this effect was positively correlated with the androsterone dose. The high-dose treatment group achieved the optimal hypoglycemic effect, with the rats' free-feeding blood glucose levels decreasing to 45% of pre-treatment levels 10 days after treatment. From 11-18 days after injection, the blood glucose levels of rats in all three groups gradually increased, eventually returning to pre-androsterone levels. These results indicate that androsterone can effectively ameliorate hyperglycemic symptoms in diabetic animals, and the alleviating effect of a single injection can last for approximately two weeks.

[0059] Example 5 Injection of fibroblast growth factor 21 (FGF21) can improve hyperglycemia in type 2 diabetic animals Recombinant human fibroblast growth factor 21 (FGF21, purchased from Peprotech, catalog number 100-42) was diluted with saline to a 1 mg / mL stock solution and used for injection. The injection volume for each rat was the same, and the stock solution was diluted to the desired concentration depending on the injection volume.

[0060] Type 2 diabetic rats were divided into four groups: a low-dose FGF21 treatment group (injected at 0.1 mg FGF21 / kg body weight), a medium-dose FGF21 treatment group (injected at 0.5 mg FGF21 / kg body weight), a high-dose FGF21 treatment group (injected at 1 mg FGF21 / kg body weight), and a diabetic model group (injected with the same volume of saline). At the same time, healthy rats injected with the same volume of saline served as normal controls. Each group consisted of 10 male rats.

[0061] FGF21 was administered intravenously into the tail vein (once daily) for 4 weeks. During this period, the rats were fed a normal diet and allowed to eat and drink freely. Before sacrifice, the rats were fasted for 8 hours, and their fasting blood glucose levels were measured using a blood glucose meter. The results are shown in Figure 5.

[0062] As can be seen from the results in Figure 5, the average fasting blood glucose content of healthy rats in the normal control group was 99.03 mg / dL, and the average fasting blood glucose content of rats in the diabetic model group was 354.81 mg / dL. Compared with the diabetic model group, the average fasting blood glucose content of type 2 diabetic rats in the three groups treated with FGF21 was significantly reduced and dose-dependently. In particular, the average blood glucose content of rats in the high-dose FGF21 treatment group was reduced to 137.94 mg / dL.

[0063] Example 6 A combined drug of androsterone and FGF21 can improve hyperglycemia in type 2 diabetic animals Androsterone was prepared in 95% ethanol to a stock solution of 40 mg / mL and used for injection after dilution.

[0064] FGF21 (Peprotech) was diluted with saline to a 1 mg / mL stock solution. After dilution, it was used for injection.

[0065] The injection volume for each rat was the same, and the two mother solutions were diluted to the desired concentrations according to the injection volume.

[0066] Type 2 diabetic rats were divided into four groups: a diabetic model group, an androsterone-treated group (20 mg androsterone / kg body weight, once per week), an FGF21-treated group (0.2 mg FGF21 / kg body weight, once per day), and a combined treatment group (10 mg androsterone / kg body weight (once per week) and 0.1 mg FGF21 / kg body weight (once per day)). A normal control group (healthy rats) was also injected with the same volume of 95% ethanol (once per week) and the same volume of saline (once per day). Each group contained 10 male rats.

[0067] Androsterone was injected intraperitoneally, and FGF21 was injected intravenously into the tail vein for 2 weeks. During this period, the rats were fed normal diet and allowed to eat and drink freely. Before being sacrificed, the rats were fasted for 8 hours, and their fasting blood glucose levels were measured using a blood glucose meter. The results are shown in Figure 6. The results showed that the combination drug significantly improved the blood glucose levels of the diabetic model animals, and had a significant synergistic effect compared to the single-drug groups.

[0068] Subsequently, a study was designed to further analyze the hypoglycemic effects of different doses of the combination treatment groups. Type 2 diabetic rats were divided into four groups: a low-dose combination treatment group (10 mg androsterone / kg body weight once per week, 0.1 mg FGF21 / kg body weight once per day), a medium-dose combination treatment group (40 mg androsterone / kg body weight once per week, 0.5 mg FGF21 / kg body weight once per day), a high-dose combination treatment group (80 mg androsterone / kg body weight once per week, 1 mg FGF21 / kg body weight once per day), and a diabetic model group (injected with the same volume of 95% ethanol once per week and the same volume of saline once per day). Simultaneously, a normal control group (healthy rats, injected with the same volume of 95% ethanol once per week and the same volume of saline once per day) contained 10 male rats per group.

[0069] Androsterone was injected intraperitoneally, and FGF21 was injected intravenously into the tail vein for 2 weeks. During this period, the rats were fed normal diet and allowed to eat and drink freely. Before the rats were sacrificed, they were fasted for 8 hours, and the fasting blood glucose level of the rats was measured using a blood glucose meter. The results are shown in Figure 7.

[0070] As can be seen from the results in Figure 7, after two weeks of combined administration of low doses of androsterone and fibroblast growth factor 21, hyperglycemia in the model animals could be rapidly improved. In addition, the blood glucose level of the diabetic animals in the high-dose combined treatment group was reduced to a level equivalent to that of the normal control group. This indicates that the combined use of the two drugs has a synergistic effect on improving hyperglycemia and exhibits a concentration-dependent relationship.

[0071] Example 7 Obtaining transdifferentiated cells expressing fibroblast growth factor 21 and transdifferentiated cells expressing androsterone PB-TRE-dCas9-VPR (catalog number: 63800) and a kit containing pX330A-1x4 and all the reagents required for constructing a gRNA expression vector (catalog number: #1000000055) are both purchased from Addgene.

[0072] 1. Recombinant plasmid expressing guide RNA of FGF21 and steroidogenic factor-1 (SF-1) promoter region The pX330A-1x4 vector was cleaved with EcoRV and Pmll enzymes to extract a 936-bp fragment of the Cas9 gene in the plasmid. The backbone fragment was recovered by agarose gel electrophoresis and ligated with T4 ligase to obtain the recombinant plasmid pX330A-1x4-mut.

[0073] Guide ribonucleic acids (gRNAs) for the FGF21 promoter region were designed, and four guide ribonucleic acids were selected from them. The deoxyribonucleic acid sequences corresponding to these four guide ribonucleic acids are shown in SEQ ID NOs: 1 to 4. The four sequences were synthesized and then cloned into the expression plasmid pX330A-1x4-mut to obtain the recombinant plasmid pX330A-FGF21-gRNA1-4, which contains four gRNA expression cassettes in tandem, each of which is attached to a U6 promoter at the 5' end.

[0074] Guide ribonucleic acids (gRNAs) were designed for the SF-1 promoter region, and four guide ribonucleic acids were selected from them. The deoxyribonucleic acid sequences corresponding to these four guide ribonucleic acids are shown in SEQ ID NOs: 5 to 8. The four sequences were synthesized and then cloned into the expression plasmid pX330A-1x4-mut to obtain the recombinant plasmid pX330A-SF1-gRNA1-4, which contains four gRNA expression cassettes in tandem, in which the 5' end of each gRNA is a U6 promoter.

[0075] 2. Skin Fibroblast Isolation Human foreskin tissue was taken, and the epidermis and dermis layers were peeled off. The remaining part was then cut into 1mm pieces with scissors. 3 The tissue was cut into 100 mm tissue blocks. The tissue blocks were transferred to cell culture dishes, 6 mL of medium was added, and cultured for 5 days. After the fibroblasts had migrated out of the tissue block, the tissue block was discarded with sterile tweezers and replaced with fresh medium. Cultures were continued until the cells reached 90% confluency. The old medium was discarded, and the cells were washed twice with PBS buffer, digested with pancreatin for 1 minute, and 3 mL of serum-containing medium was added to stop the digestion. A cell suspension was prepared by repeated aspiration and ejection. The cell suspension was collected in a 15 mL centrifuge tube and centrifuged for 5 minutes. The collected cells were skin fibroblasts.

[0076] 3. Transdifferentiation of skin fibroblasts into cells that express FGF21 and synthesize androsterone The transdifferentiation process can be divided into two stages.

[0077] Step 1: 3 × 10 skin fibroblasts 5 The cells were seeded at a density of 10 cells / well in Dulbecco's Modified Eagle Medium (DMEM / F12-Dulbecco's Modified Eagle Medium, purchased from Sigma, catalog number D8437) containing 10% fetal bovine serum and cultured for 12 hours. Subsequently, the dermal fibroblasts were transfected with the PB-TRE-dCas9-VPR, pX330A-FGF21-gRNA1-4, and pX330A-SF1-gRNA1-4 recombinant vectors (1.8 μg, 0.35 μg, and 0.35 μg, respectively) using a liposomal transfection reagent (Lipofectamine 3000) according to the manufacturer's instructions. After 6 hours of culture at 37°C, the cells were transfected with 10% fetal bovine serum and 0.5 μM Smoothened agonist (Smoothened agonist) at a final concentration of 10%. The cells were cultured in fresh DMEM / F12 medium containing 1000mg / ml luteinizing hormone (LH), 100mg / ml cyclic adenosine monophosphate (SAG), 100mg / ml fibroblast growth factor 2 (bFGF), 10µg / ml insulin, and 5µM transforming growth factor-β signaling pathway inhibitor LY2109761. The medium was replaced with fresh medium containing the same components at the same concentrations once every two days, and the cells were cultured for six consecutive days to complete the first stage.

[0078] Second stage: The cells were cultured in fresh Dulbecco's modified Eagle's medium containing 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 medium was replaced with the same components at the same concentrations once every two days, and the cells were cultured for 12 consecutive days to obtain transdifferentiated cells that express FGF21 and synthesize androsterone.

[0079] 4. Analysis of transcription and expression of FGF21 and androgen synthase in transdifferentiated cells Leydig cells were isolated from human testis using the method of Chen X et al. (Cell Death & Disease, 2019, 10(3): 220).

[0080] The resulting skin fibroblasts were isolated by the method described in Section 2 of this Example.

[0081] 4.1 Transcription analysis The primers used for FGF21 RT-PCR analysis were FGF21-F (SEQ ID NO: 9) and FGF21-R (SEQ ID NO: 10).

[0082] 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).

[0083] The primers used for luteinizing hormone receptor RT-PCR analysis were LHCGR-F (SEQ ID NO: 13) and LHCGR-R (SEQ ID NO: 14).

[0084] 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).

[0085] 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).

[0086] Hydroxy-Delta-5-Steroid Dehydrogenase-3β Steroid Delta Isomerase 2 The primers used for RT-PCR analysis of Beta And Steroid Delta-Isomerase 2 (HSD3B2) were HSD3B2-F (SEQ ID NO: 19) and HSD3B2-R (SEQ ID NO: 20).

[0087] 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).

[0088] The primers used for RT-PCR analysis of the reference gene ribosomal protein S16 (RPS16) were PRS16-F (SEQ ID NO: 23) and PRS16-R (SEQ ID NO: 24).

[0089] The expression levels of SF-1 and FGF21 in the transdifferentiated cells on day 12 of the induction were analyzed by RT-PCR, with dermal fibroblasts and Leydig cells serving as controls. The results are shown in Figure 8. As can be seen from Figure 8, dermal fibroblasts did not express SF-1 or FGF21, Leydig cells could express SF-1 and at the same time, small amounts of FGF21, and transdifferentiated cells could express large amounts of SF-1 and FGF21, and the expression levels were significantly higher than those of Leydig cells, indicating that the endogenous genes SF-1 and FGF21 in transdifferentiated cells were activated.

[0090] On day 12 of induction, the expression levels of LHCGR, StAR, CYP11A1, HSD3B2, and CYP17A1 in the transdifferentiated cells were analyzed by RT-PCR, with skin fibroblasts and Leydig cells used as controls. The results are shown in Figure 9. As can be seen from Figure 9, the transdifferentiated cells were able to express large amounts of LHCGR, StAR, CYP11A1, HSD3B2, and CYP17A1, key enzymes in the androgen synthesis pathway, indicating that the above treatment method successfully activated the androgen synthesis pathway in fibroblasts.

[0091] 4.2 Expression and synthesis analysis After culturing the transdifferentiated cells for 12 days, the culture supernatant was collected and subjected to Western blotting. The antibody used was FGF21 antibody (Abcam, catalog number: ab66564). The results are shown in Figure 10. Figure 10 indicates that the transdifferentiated cells can express and secrete FGF21.

[0092] After culturing the transdifferentiated cells for 3, 6, 12, and 18 days, the culture supernatant was collected and the androsterone content in the culture supernatant was measured by radioactive enzyme-linked immunosorbent assay. Dermal fibroblasts were used as a control. The results are shown in Figure 11. The results show that after culturing the transdifferentiated cells for 3 days, androsterone was detectable in the culture supernatant, and the androsterone content gradually increased over time, reaching 1.4 ng / million cells / 24 hours on day 18. In contrast, only trace amounts of androsterone were detected in the culture supernatant of the control dermal fibroblasts, below the detection threshold.

[0093] Example 8 Transdifferentiated cells can improve hyperglycemia in type 2 diabetic animals 1. Treating type 2 diabetes with transdifferentiated cell transplantation Type 2 diabetic rats were divided into two groups: a transdifferentiated cell therapy group and a diabetic model group. Healthy rats treated with skin fibroblasts served as normal controls. Each group consisted of six male rats.

[0094] For the transdifferentiation cell therapy group, 2 × 10 cells were injected subcutaneously into the armpits of type 2 diabetic rats. 6 Transdifferentiated cells were seeded at a dose of 2 × 10 cells / rat. For the diabetic model group, 2 × 10 cells were seeded subcutaneously in the armpits of type 2 diabetic rats. 6 Dermal fibroblasts were seeded at a dose of 2 × 10 cells / rat in the subcutaneous tissue of the armpits of healthy rats for the normal control group. 6Dermal fibroblasts were seeded at a dose of 100 cells / rat. The rats were then fed a normal diet with free access to food and water for 21 days. Starting on the day of seeding, the rats were fasted for 8 hours, and their fasting blood glucose levels were measured using a blood glucose meter every 7 days. The results are shown in Figure 12.

[0095] As can be seen from the results in Figure 12, the fasting blood glucose levels of both the diabetic model group and the normal control group remained stable, with one group maintaining a high level, with the fasting blood glucose levels of the six rats averaging 311.35 mg / dL on day 21, and the other group maintaining a normal low level, with the fasting blood glucose levels of the six rats averaging 93.24 mg / dL on day 21. Furthermore, the fasting blood glucose levels of the transdifferentiated cell therapy rats continued to decrease over time, with the blood glucose levels reaching 119.2 mg / dL on day 21, close to that of the normal control group.

[0096] 2. Pathological detection of liver tissue from treated rats Masson staining is primarily used to distinguish collagen fibers and muscle fibers, and is of great value in diagnosing lobular and portal tract fibrous hyperplasia due to various liver diseases.

[0097] As in Section 1 of this Example, a transdifferentiation cell therapy group, a diabetes model group, and a normal control group were established.

[0098] Using the Masson's Trichrome Stain Kit (Solarbio, catalog number G1340) according to the manufacturer's instructions, liver tissue sections from type 2 diabetic rats were stained and analyzed on days 7, 14, and 21 after transplantation of the transdifferentiated cells. Liver tissue sections from the diabetic model and normal control groups were similarly stained on day 21 after transplantation of skin fibroblasts. The results are shown in Figure 13.

[0099] Results showed that the livers of normal control animals had intact lobule structures and no fibrous tissue hyperplasia. In the livers of diabetic model animals, portal, periportal, and perisinusoidal fibrosis was observed, with clear fibrous tissue hyperplasia within fibrous septa and inflammatory cell infiltration, but no pseudolobule formation. Seven days after cell transplantation, the livers of treated animals showed some degree of fibrous tissue hyperplasia and inflammatory cell infiltration in some areas, but the fibrosis was less severe than in the diabetic model. After 14 days of treatment, liver fibrosis was significantly reduced compared to the diabetic model, with fibrous tissue hyperplasia still present, but with a significant thinning of fibrous septa, connective tissue, and inflammatory cell infiltration. After 21 days of treatment, the degree of liver fibrosis was significantly reduced, with inflammatory cell infiltration observed in only a few areas.

[0100] 3. Liver oil content of treated rats Oil Red O staining is primarily used to analyze lipid profiles in tissue samples.

[0101] As in Section 1 of this Example, a transdifferentiation cell therapy group, a diabetes model group, and a normal control group were established.

[0102] Lipid (Oil Red O) Staining Kit The procedure was performed according to the manufacturer's instructions (Kit, Sigma-Aldrich, Catalog No. MAK194), and liver tissue sections from type 2 diabetic rats were stained and analyzed on days 7, 14, and 21 after transdifferentiation cell transplantation. Liver tissue sections from the diabetic model group and normal control group were similarly stained on day 21 after skin fibroblast transplantation. The results are shown in Figure 14.

[0103] The test results clearly showed that in the livers of normal control animals, the lobule structure was intact, the hepatic cell cords were regularly arranged and radially distributed around the central vein, and no obvious Oil Red O staining was observed. In the livers of diabetic model animals, obvious steatosis, cellular swelling, compression and narrowing of hepatic sinusoids, and disorganized hepatic cord arrangement were observed, and large areas of red lipid droplets were deposited in the liver tissue. In the livers of treated animals, lipid droplet deposition was observed in some areas after 7 and 14 days of treatment, but the stained area was significantly smaller than in the diabetic model group. After 21 days of treatment, little or no Oil Red O staining was observed, indicating a clear improvement in lipid accumulation in the liver.

Claims

1. Use of androsterone in the manufacture of a medicament for preventing and / or treating hyperglycemia.

2. The use according to claim 1, wherein the factor causing hyperglycemia is at least one of prediabetes, diabetes, obesity and senility.

3. The use according to claim 2, characterized in that the diabetes is type 1 diabetes or type 2 diabetes.

4. A drug combination for preventing and / or treating hyperglycemia, comprising fibroblast growth factor 21 and androsterone.

5. The combination drug according to claim 4, wherein the mass ratio of said fibroblast growth factor 21 to said androsterone is 1:(1-800).

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