Use of prolyl hydroxylase inhibitors for ameliorating symptoms of hepatolenticular degeneration diseases
阅读说明：本技术 脯氨酰羟化酶抑制剂在改善肝豆状核变性疾病症状中的用途 (Use of prolyl hydroxylase inhibitors for ameliorating symptoms of hepatolenticular degeneration diseases ) 是由 芈肖肖 施军平 于 2020-05-19 设计创作，主要内容包括：本发明公开了脯氨酰羟化酶抑制剂在改善肝豆状核变性疾病症状中的用途,本发明所使用的脯氨酰羟化酶抑制剂可以稳定HIF-1α,并激活其转录活性；肝脏灰度减弱,脂质代谢因子的表达显著降低,肝脏H&E染色显示脯氨酰羟化酶抑制剂可以有效改善肝脏病理,减轻肝脏铜积累；同时检测到与γ-氨基丁酸能神经元信号以及多巴胺能神经元信号相关因子的表达得到改善,运动能力得到改善。(The invention discloses an application of prolyl hydroxylase inhibitor in improving symptoms of hepatolenticular degeneration diseases, the prolyl hydroxylase inhibitor used in the invention can stabilize HIF-1 alpha and activate the transcriptional activity thereof; the gray level of the liver is weakened, the expression of lipid metabolism factors is obviously reduced, and H & E staining of the liver shows that the prolyl hydroxylase inhibitor can effectively improve liver pathology and reduce liver copper accumulation; meanwhile, the expression of the factor related to the GABAergic neuron signal and the dopaminergic neuron signal is improved, and the motor ability is improved.)
1. Use of a prolyl hydroxylase inhibitor for ameliorating symptoms of a hepatolenticular degeneration disease.
2. The use of a prolyl hydroxylase inhibitor according to claim 1 for ameliorating the symptoms of a hepatolenticular degeneration disease, characterized in that: the prolyl hydroxylase inhibitor can obviously enhance the transcriptional activity of HIF-1 alpha, obviously reduce the expression of lipid metabolism factors, improve liver pathology and reduce liver copper accumulation.
3. The use of a prolyl hydroxylase inhibitor according to claim 1 in a medicament for ameliorating conditions of hepatolenticular degeneration disease characterized in that: the prolyl hydroxylase inhibitor can improve the expression of GABAergic neuron signals and dopaminergic neuron signal related factors and improve the motor ability.
4. The use of a prolyl hydroxylase inhibitor according to claim 1 for ameliorating the symptoms of a hepatolenticular degeneration disease, characterized in that: the prolyl hydroxylase inhibitor is one of FG-4592 and DMOG.
5. A pharmaceutical composition for ameliorating symptoms of hepatolenticular degeneration disease, comprising: comprising the prolyl hydroxylase inhibitor according to claim 1, a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient.
6. The method of claim 1, wherein the prolyl hydroxylase inhibitor on a hepatolenticular degeneration zebra fish model:
1) constructing a hepatolenticular degeneration zebra fish model by a CRISPR/Cas9 gene editing technology, and culturing and constructing ATP7B-/-A zebrafish line;
2) by ATP7B in step 1)-/-Zebra fish, and ATP7B is constructed by using CRISPR/Cas9-/-HepG2 cell stable strain;
3) collecting the zebrafish in the step 1) and the HepG2 cells in the step 2), and respectively culturing the zebrafish and the HepG2 cells with the prolyl hydroxylase inhibitor for 24 hours;
4) mixing the zebra fish specimen cultured in the step 3) with ATP7B-/-HepG2 cells were individually identified by phenotypic analysis.
7. The method of claim 6, wherein the prolyl hydroxylase inhibitor on a hepatolenticular degeneration zebra fish model: the prolyl hydroxylase inhibitor of 3) comprises one of FG-4592 or DMOG.
8. The method of claim 6, wherein the prolyl hydroxylase inhibitor on a hepatolenticular degeneration zebra fish model: and 4) performing phenotype analysis and identification on the zebra fish specimen in the step 4, wherein the phenotype analysis and identification comprise mutant liver histology analysis, mutant copper deposition analysis, mutant motion trail detection and fluorescence quantitative QPCR.
9. The method of claim 6, wherein the prolyl hydroxylase inhibitor on a hepatolenticular degeneration zebra fish model: the step 4) ATP7B-/-Phenotypic analysis and identification of HepG2 cells include gene knockout detection, copper accumulation, cell oil red staining and fluorescence quantitative QPCR.
Technical Field
The invention relates to the technical field of medical treatment, in particular to application of prolyl hydroxylase inhibitors in improving symptoms of hepatolenticular degeneration diseases.
Background
Hepatolenticular degeneration (also known as Wilsondsease, OMIM #277900) is a frequently stained, recessively inherited metabolic disease caused by mutations in the ATP7B gene. The ATP7B gene mutation can cause excessive accumulation of copper ions in liver cells, and excessive copper ions can be deposited on the lenticular nucleus of brain, cornea and kidney, thus causing damage to corresponding organs. The global incidence of the disease is about 1/30,000, the incidence rate of Asian people is higher than that of European and American people, and the incidence rate of Chinese people is about 1/17,000. The disease is a common cause of liver cirrhosis of unknown juvenile causes. The clinical manifestations of patients with hepatolenticular degeneration are mainly liver lesions and neuropsychiatric symptoms, with early pathological and clinical manifestations of the liver being mainly steatosis, which can subsequently progress to hepatitis and cirrhosis.
Patients with hepatolenticular degeneration predominate in young and middle-aged years, while more and more preschool children are reported in succession. The disease must be discovered and treated early, and the existing copper expelling therapeutic drugs bring serious side effects of neurodegeneration and poor patient compliance, so that the treatment of the disease still has a great bottleneck at present.
We have successfully constructed ATP7B by using CRISPR/Cas9 gene editing technology-/-The hepatolenticular degeneration zebra fish model can simulate hepatolenticular degeneration liver and neurological symptoms. Since the digestive organ maturation can be achieved after 5 days of development, the model can shorten the study period compared with rodent models.
In the early days we found the HIF-1 signal to be in a significantly altered line by transcriptome analysis of hepatolenticular degeneration disease models in the database. HIF (Hypoxia-indelibactor) is a transcription complex sensitive to oxygen levels and regulates hepatic glucose and lipid metabolism. HIF is a heterodimer comprising a regulatory alpha subunit and a constitutive beta subunit.
Downstream factors of HIF-1 α transcriptional regulation include two classes, one is heme-producing factor and angiogenesis factor, which can increase oxygen utilization; another class is energy metabolism regulators, which can reduce oxygen consumption. Knockout of hepatocyte HIF-1 α exacerbates alcohol-induced liver lipid accumulation in mice. In vitro studies of the human hepatoma cell line HepG2 cell line also revealed that knocking down HIF-1 α exacerbates lipid accumulation, while over-expressing HIF-1 α decreases lipid deposition. HIF-1 α can interfere with fatty acid β oxidation in liver cancer cells. The liver cell specificity knockout of HIF-1 alpha can intensify the liver fatty degeneration and the liver triglyceride content of the choline-deficient diet-induced mice, and block the rise of the intranuclear lpin 1.
Disclosure of Invention
The invention provides the use of a prolyl hydroxylase inhibitor for ameliorating symptoms of a hepatolenticular degeneration disease.
The scheme of the invention is as follows:
use of a prolyl hydroxylase inhibitor for ameliorating symptoms of a hepatolenticular degeneration disease.
Preferably, the prolyl hydroxylase inhibitor can significantly enhance the transcriptional activity of HIF-1 alpha, significantly reduce the expression of lipid metabolism factors, improve liver pathology, and reduce liver copper accumulation.
Preferably, the prolyl hydroxylase inhibitor improves the expression of GABAergic neuronal signals and dopaminergic neuronal signal related factors.
Preferably, the prolyl hydroxylase inhibitor is one of FG-4592 and DMOG.
The invention also discloses a pharmaceutical composition for improving symptoms of hepatolenticular degeneration diseases, which contains the prolyl hydroxylase inhibitor of claim 1, pharmaceutically acceptable salts and pharmaceutically acceptable auxiliary materials.
The invention also discloses a method for preparing the prolyl hydroxylase inhibitor on the hepatolenticular degeneration zebra fish model, which comprises the following steps:
1) constructing a hepatolenticular degeneration zebra fish model by a CRISPR/Cas9 gene editing technology, and culturing and constructing ATP7B-/-A zebrafish line;
2) by ATP7B in step 1)-/-Zebra fish, and ATP7B is constructed by using CRISPR/Cas9-/-HepG2 cell stable strain;
3) collecting the zebrafish in the step 1) and the HepG2 cells in the step 2), and respectively culturing the zebrafish and the HepG2 cells with the prolyl hydroxylase inhibitor for 24 hours;
4) mixing the zebra fish specimen cultured in the step 3) with ATP7B-/-HepG2 cells were individually identified by phenotypic analysis.
Preferably, the prolyl hydroxylase inhibitor in the step 3) comprises one of FG-4592 or DMOG.
Preferably, the phenotypic analysis and identification of the zebra fish specimen in the step 4) comprises mutant liver histology analysis, mutant copper deposition analysis, mutant movement track detection and fluorescence quantitative QPCR.
Preferably, the step 4) ATP7B-/-Phenotypic analysis and identification of HepG2 cells include gene knockout detection, copper accumulation, cell oil red staining and fluorescence quantitative QPCR.
Due to the adoption of the technical scheme, the prolyl hydroxylase inhibitor can be used for improving the symptoms of the hepatolenticular degeneration disease.
The invention has the advantages that:
can stabilize HIF-1 alpha and activate its transcription activity; the gray level of the liver is weakened, the expression of lipid metabolism factors is obviously reduced, and H & E staining of the liver shows that liver pathology is effectively improved and liver copper accumulation is reduced; the expression of the factor related to GABAergic neuron signals and dopaminergic neuron signals is improved, and the motor ability is improved.
Drawings
FIG. 1 shows prolyl hydroxylase inhibitor vs ATP7B-/-The liver gray scale shown by a bright field microscope influenced by the zebra fish liver symptoms;
FIG. 2 shows prolyl hydroxylase inhibitor vs ATP7B-/-Quantitatively comparing the liver gray levels influenced by the zebra fish liver symptoms;
FIG. 3 shows prolyl hydroxylase inhibitor vs ATP7B-/-QPCR affected by liver symptoms of zebrafish shows transcriptional activity of HIF-1 signaling;
FIG. 4 shows prolyl hydroxylase inhibitor vs ATP7B-/-QPCR of zebra fish liver symptom influence shows lipid metabolism factor change;
FIG. 5 shows prolyl hydroxylase inhibitor vs ATP7B-/-Zebrafish liver symptom affected H&E staining shows liver pathology;
FIG. 6 shows prolyl hydroxylase inhibitor vs ATP7B-/-ICP-MS of zebra fish liver symptom influence quantifies liver copper deposition amount;
FIG. 7 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-Zebra fish nervous symptom shadowA sound motion trail tracking diagram;
FIG. 8 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-Counting the movement distance influenced by the zebra fish nervous symptoms;
FIG. 9 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-QPCR of zebrafish neurological symptom influence shows GABAergic signaling factor change and dopaminergic signaling factor change;
FIG. 10 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-CRISPR/Cas9 establishment of ATP7B for the effects of HepG2 cell lipid metabolism-/-HepG2 cells;
FIG. 11 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-Genotyping of mutant cell lines affected by lipid metabolism of HepG2 cells;
FIG. 12 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-QPCR of the effects of lipid metabolism in HepG2 cells showed mutant and wild-type strain ATP7B expression;
FIG. 13 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-The genotype comparison result of the mutant cell strain influenced by the lipid metabolism of the HepG2 cell;
FIG. 14 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-QPCR of HepG2 cell lipid metabolism effects shows lipid metabolism factor changes;
FIG. 15 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-Oil red staining of HepG2 cells affected by lipid metabolism revealed intracellular lipid deposition;
FIG. 16 shows the addition of prolyl hydroxylase inhibitor to ATP7B-/-Intracellular copper content changes affected by lipid metabolism of HepG2 cells.
Detailed Description
In order to remedy the above deficiencies, the present invention provides the use of a prolyl hydroxylase inhibitor for ameliorating the symptoms of a hepatolenticular degeneration disease to solve the problems of the background art described above.
Use of a prolyl hydroxylase inhibitor for ameliorating symptoms of a hepatolenticular degeneration disease.
The prolyl hydroxylase inhibitor can obviously enhance the transcriptional activity of HIF-1 alpha, obviously reduce the expression of lipid metabolism factors, improve liver pathology and reduce liver copper accumulation.
The prolyl hydroxylase inhibitors can improve the expression of GABAergic neuron signals and dopaminergic neuron signal-related factors and improve the motor ability.
The prolyl hydroxylase inhibitor is one of FG-4592 and DMOG.
The invention also discloses a pharmaceutical composition for improving symptoms of hepatolenticular degeneration diseases, which contains the prolyl hydroxylase inhibitor of claim 1, pharmaceutically acceptable salts and pharmaceutically acceptable auxiliary materials.
The invention also discloses a method for preparing the prolyl hydroxylase inhibitor on the hepatolenticular degeneration zebra fish model, which comprises the following steps:
1) constructing a hepatolenticular degeneration zebra fish model by a CRISPR/Cas9 gene editing technology, and culturing and constructing ATP7B-/-A zebrafish line;
2) by ATP7B in step 1)-/-Zebra fish, and ATP7B is constructed by using CRISPR/Cas9-/-HepG2 cell stable strain;
3) collecting the zebrafish in the step 1) and the HepG2 cells in the step 2), and respectively culturing the zebrafish and the HepG2 cells with the prolyl hydroxylase inhibitor for 24 hours;
4) mixing the zebra fish specimen cultured in the step 3) with ATP7B-/-HepG2 cells were individually identified by phenotypic analysis.
The prolyl hydroxylase inhibitor of 3) comprises one of FG-4592 or DMOG.
And 4) performing phenotype analysis and identification on the zebra fish specimen in the step 4, wherein the phenotype analysis and identification comprise mutant liver histology analysis, mutant copper deposition analysis, mutant motion trail detection and fluorescence quantitative QPCR.
The step 4) ATP7B-/-Phenotypic analysis and identification of HepG2 cells include gene knockout detection, copper accumulation, cell oil red staining and fluorescence quantitative QPCR.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.