Application of creatine phosphate in preparation of medicine for treating type 2 diabetes and nephropathy complications
阅读说明：本技术 磷酸肌酸在制备用于治疗2型糖尿病肾病并发症的药物中的用途 (Application of creatine phosphate in preparation of medicine for treating type 2 diabetes and nephropathy complications ) 是由 唐泽耀 杨晓云 牛梦月 王宏焱 艾杰 王福韩 韩国柱 唐中元 彭金咏 马晓东 张 于 2019-11-24 设计创作，主要内容包括：本发明公开了磷酸肌酸在制备用于治疗2型糖尿病肾病并发症的药物中的用途。磷酸肌酸能够有效改善糖尿病肾脏损伤,其机制是通过抑制ERK的磷酸化激活,调控线粒体凋亡途径和ERK/Nrf2/HO-1通路,进而抑制细胞凋亡和缓解氧化应激损伤。本发明为临床上糖尿病肾脏并发症的预防和治疗提供了有价值的参考信息。一定程度上为磷酸肌酸对2型糖尿病肾病并发症的保护作用防治提供新的思路和理论基础。(The invention discloses an application of creatine phosphate in preparing a medicament for treating type 2 diabetic nephropathy complications. The creatine phosphate can effectively improve diabetic kidney injury, and the mechanism is that the creatine phosphate can regulate and control a mitochondrial apoptosis pathway and an ERK/Nrf2/HO-1 pathway by inhibiting phosphorylation activation of ERK, so that apoptosis is inhibited and oxidative stress injury is relieved. The invention provides valuable reference information for clinically preventing and treating diabetic renal complications. Provides a new thought and theoretical basis for the protective effect of the creatine phosphate on the complications of type 2 diabetic nephropathy to a certain extent.)
1. Use of creatine phosphate in the manufacture of a medicament for the treatment of complications of type 2 diabetic nephropathy.
2. The use according to claim 1, wherein the creatine phosphate has the chemical structure shown in formula (1) below:
3. the use of claim 1 or 2, wherein the medicament is for treating type 2 diabetic nephropathy complications by alleviating kidney tissue, cytopathic effects, improving oxidative stress status of kidney cells, inhibiting apoptotic damage of kidney cells in a subject suffering from type 2 diabetic nephropathy complications.
4. The use of claim 3, wherein alleviating renal tissue, cytopathy in a subject suffering from complications of type 2 diabetic nephropathy is a reduction in the subject's renal hypertrophy index.
5. The use of claim 3, wherein the medicament is for improving the oxidative stress status of kidney cells by reducing malondialdehyde levels in kidney tissue of a subject, increasing reduced glutathione levels and superoxide dismutase activity, inhibiting an increase in ROS levels in kidney cells of a subject with complications of type 2 diabetic nephropathy.
6. The use of claim 3, wherein the medicament is effective in reducing the level of intracellular Ca in kidney cells by up-regulating the level of expression of Pro-Caspase-9 in its inactive form, down-regulating the levels of expression of Caspase-3 and Caspase-9, increasing the Bcl-2/Bax ratio2+Accumulating and increasing mitochondrial membrane potential of kidney cells to reduce apoptosis of kidney cells.
7. The use of claim 3, wherein the medicament is for treating type 2 diabetic nephropathy complications by inhibiting p-ERK activation in renal cells of a subject suffering from type 2 diabetic nephropathy complications, and downregulating ERK phosphorylation levels to modulate the ERK/Nrf2/HO-1 signaling pathway.
8. The use according to claim 1 or 2, wherein the type 2 diabetic nephropathy complication is type 2 diabetic nephropathy complication in a mammal.
9. The use of claim 8, wherein the mammal is a human.
10. The use according to claim 3, wherein said type 2 diabetic nephropathy complication is type 2 diabetic nephropathy complication in a mammal.
Technical Field
The invention belongs to the field of new medical indications, and particularly relates to application of creatine phosphate in preparation of a medicine for treating nephropathy complications of
Background
Diabetes Mellitus (DM) is a relatively common disorder of endocrine metabolism. Diabetes causes a lot of complications, and nephropathy complications are more serious and common, and are also the main reasons for end-stage renal failure. The health-care tea has become an increasingly serious public health problem worldwide and seriously affects the life quality of people. At present, oral hypoglycemic drugs which can be used for treating diabetes are not ideal for preventing and treating nephropathy complications, and adverse side effects are accompanied after the drugs are taken or the reaction is weakened after the drugs are used for a long time. Therefore, there is a need to find new drugs for the treatment of
The Chinese medicinal composition is clinically used for protecting cardiac muscle by being added into cardioplegia solution during cardiac surgery, and can also be applied to myocardial metabolic abnormality under an ischemic state. Previous studies by the inventors have demonstrated that creatine phosphate (PCr) can ameliorate oxidative damage associated with ox-LDL induced atherosclerosis, methylglyoxal induced vascular complications of diabetes.
Based on the background data, the creatine phosphate has a protective effect on apoptosis damage of diabetic kidney complications under oxidative stress. Specifically, the present invention establishes a diabetic rat model by disrupting islet cells with Streptozotocin (STZ) and interfering with insulin production. The method adopts Methylglyoxal (MGO) to induce the injury of the renal tubular epithelial cells (NRK-52E) of the rat to establish the diabetic nephropathy in-vitro model, discovers the protective effect of the PCRs on the renal injury of the diabetic rat, and provides scientific basis for reasonable use in the field.
Disclosure of Invention
In view of the above-described needs in the art, in some embodiments of the present invention, there is provided a use of creatine phosphate in the manufacture of a medicament for the treatment of complications of
In a specific embodiment, the chemical structure of phosphocreatine is shown in formula (1) below:
in a specific embodiment, the medicament treats the
In a specific embodiment, ameliorating renal tissue, cytopathy in a subject suffering from complications of
In a specific embodiment, the medicament improves the oxidative stress state of kidney cells by reducing the level of malondialdehyde in kidney tissues of a subject, increasing the content of reduced glutathione and the activity of superoxide dismutase, and inhibiting the increase of the level of ROS in kidney cells of a subject with complications of
In a specific embodiment, the medicament is used for increasing the Bcl-2/Bax ratio and reducing Ca in kidney cells by up-regulating the expression level of unactivated Pro-Caspase-9, down-regulating the expression levels of Caspase-3 and Caspase-9, and2+accumulating and increasing mitochondrial membrane potential of kidney cells to reduce apoptosis of kidney cells.
In a specific embodiment, the medicament is used for treating the
In a specific embodiment, the
In a more specific embodiment, the mammal is a human.
The invention has the beneficial effects that: the mechanism of the PCr is that the phosphorylation activation of ERK is inhibited, and the mitochondrial apoptosis pathway and the ERK/Nrf2/HO-1 pathway are regulated, so that the apoptosis is inhibited and the oxidative stress injury is relieved. The invention provides valuable reference information for clinically preventing and treating diabetic renal complications. Provides a new thought and theoretical basis for the prevention and treatment of the protective effect of the PCRs on the
Drawings
Fig. 1A and 1B: protective effects of PCr on body weight and renal index in diabetic rats. FIG. 1A: rat body weight (g). FIG. 1B: rat/KW/BW values (kidney weight/body weight, mg/g). Data are presented as mean ± SD (n ═ 8). P compared to control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
FIG. 2: and (3) the protective effect of the PCRs on the kidney injury caused by the H & E staining (200 times, amplification) STZ of the kidney tissue of the diabetic nephropathy rat.
Fig. 3A to 3C: effect of PCr on levels of renal tissue MDA (fig. 3A), GSH (fig. 3B), and SOD (fig. 3C) in diabetic nephropathy rats. Data are expressed as averagesThe value ± SD (n ═ 8). Comparison with control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 4A to 4C: effect of PCr on apoptosis in diabetic nephropathy rats. FIG. 4A: TUNEL stained rat kidney tissue fluorescence image, green fluorescence shows positive cells (200-fold magnification). FIG. 4B: western blot was used to detect tissue protein expression of rat Bcl-2, Bax, Caspase-3, Caspase-9 and Pro-Caspase-9. FIG. 4C is a statistical chart of the result of Western blot electrophoresis in FIG. 4B. The value of the control is related to the value of actin. Data are presented as mean ± SD (n ═ 3). P compared to control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 5A to 5C: effect of PCR on the ERK/Nrf2/HO-1 signaling pathway in diabetic nephropathy rats. FIG. 5A: effect of PCr on p-ERK levels in renal tissue based on immunofluorescence (200-fold magnification). FIG. 5B: representative Western blot analysis of renal tissue phosphorylation and total ERK, Nrf2 and HO-1 protein expression. FIG. 5C is a statistical chart of the result of Western blot electrophoresis in FIG. 5B. The value of the control is related to the value of actin. Data are presented as mean ± SD (n ═ 3). P compared to control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 6A to 6C: protective effects of PCRs on MGO-induced NRK-52E cell damage. FIG. 6A shows that the survival rate of NRK-52E cells treated with MGO for 24 hours was gradually decreased from 0.4 to 1.0mM in response to increasing MGO concentration, and the maximum dose of 1.0mM MGO inhibited the survival activity of cells to 51.4%. FIG. 6B was pretreated with PCRs (10, 20, 30mM, 4h) followed by stimulation with MGO (0.8mM) for 24 h. When MGO (0.8mM) alone was stimulated for 24 hours, the cell survival activity reached 63.6%, whereas pre-treatment with PCr (10, 20, 30mM, 4h) followed by MGO (0.8mM) for 24 hours, the cell survival activity gradually recovered dose-dependently, showing a significant difference, with 30mM PCr pretreatment, the cell survival activity recovered to 84.3%. FIG. 6C survival Activity of NRK-52E cells treated with PCR for 24 hours, cells treated with different concentrations of PCR (10, 20, 30mM, 4h)No significant change in survival activity occurred. Data are expressed as mean ± SD (n ═ 5). P compared to control group<0.05 and x p<0.01; compared with the MGO group with only 0.8mM,#p<0.05 and##p<0.01。
FIG. 7: morphology and fluorescence images of NRK-52E cells. The first row is the effect of PCr on cell morphology in bright field (200 x). The second row is fluorescence images of DAPI stained NRK-52E apoptotic changes observed by inverted fluorescence microscopy (200-fold magnification).
Fig. 8A and 8B: protective effect of PCRs on MGO-induced apoptosis of NRK-52E cells. Figure 8A flow cytometry detects apoptosis. The early and late apoptosis rates represent the percentage of Annexin V single positive and Annexin V/PI double positive cells. Fig. 8B is a statistical plot of the apoptosis detected by flow cytometry and data are presented as mean ± SD (n ═ 3). P compared to control group<0.05 and x p<0.01; compared with the MGO group with only 0.8mM,#p<0.05 and##p<0.01。
fig. 9A to 9C: effect of PCr on MGO induced apoptosis of NRK-52E cells. FIG. 9A: TUNEL staining showed NRK-52E positive cells, and the fluorescence image was green fluorescent (200-fold magnification). FIG. 9B: western blot was used to detect tissue protein expression of Bcl-2, Bax, Caspase-3, Caspase-9 and Procaspase-9 in NRK-52E cells. FIG. 9C is the statistical chart of FIG. 9B, with respect to actin, the values associated with actin. Data are presented as mean ± SD (n ═ 3). P compared to control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 10A to 10B: the protective effect of PCr on Mitochondrial Membrane Potential (MMP). MMP was measured using the JC-1 probe and fluorescence images were taken using a fluorescence microscope (FIG. 10A). Red fluorescence shows normal intramitochondrial membrane polarization, green fluorescence is emitted by cytoplasmic JC-1 monomer, MMP dissipates. The red and green fluorescence intensity ratios were used to calculate MMP levels for each group. Fig. 10B is the statistical plot of fig. 10A, with data expressed as mean ± SD (n-3). Comparison with control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 11A and 11B: flow cytometer detection of PCr pairsProtection of intracellular calcium levels of NRK-52E. Data are presented as mean ± SD (n ═ 3). P compared to control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 12A to 12C: effect of PCr on NRK-52E cell MDA (fig. 12A), GSH (fig. 12B), SOD (fig. 12C) levels. Data are presented as mean ± SD (n ═ 3). Comparison with control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 13A to 13B: effect of PCr on MGO induced reactive oxygen levels in NRK-52E cells. Data are presented as mean ± SD (n ═ 3). P compared to control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 14A to 14C: effect of PCR on ERK/Nrf2/HO-1 signaling pathway in NRK-52E cells. FIG. 14A: effect of PCr on p-ERK levels based on immunofluorescence (200-fold magnification). FIG. 14B: representative Western blot analysis of NRK-52E cell phosphorylation and total ERK, Nrf2 and HO-1 protein expression. FIG. 14C is the statistical chart of FIG. 14B, with respect to actin, the values associated with actin. Data are presented as mean ± SD (n ═ 3). P compared to control group<0.05，**p<0.01; in comparison with the set of models,#p<0.05，##p<0.01。
fig. 15A and 15B: cDNA-induced overexpression of ERK abolished the effect of PCr on MGO-induced apoptosis of NRK-52E cells. Data are presented as mean ± SD (n ═ 3). Comparison with the control cDNA group, p<0.05 and x p<0.01; p compared to MGO + control cDNA group<0.05 and x p<0.01; compared with the PCr + MGO + control cDNA group,#p<0.05 and##p<0.01。
fig. 16A and 16B: c-DNA-induced overexpression of ERK1/2 eliminates the influence of PCRs on expression change of ERK/Nrf2/HO-1 signaling pathway-related proteins of NRK-52E cells and MGO-induced apoptosis. Data are presented as mean ± SD (n ═ 3). Comparison with control cDNA group<0.05 and x p<0.01; p compared to MGO + control cDNA group<0.05 and x p<0.01; compared with the PCr + MGO + control cDNA group,#p<0.05 and##p<0.01。
Detailed Description
The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In vivo experiments
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