Application of IGF-1R gene expression inhibitor in preparation of medicine for delaying senescence and improving senile diseases

Abstract

The invention relates to an application of an IGF-1R (Insulin-like Growth Factor-1 Receptor) gene expression inhibitor in preparation of a medicine for delaying senescence and improving senile diseases, siRNA (Small Interference Ribonucleic Acid) aiming at an IGF-1R gene is designed by using a gene interference technology and is chemically modified, and expression of the gene and a downstream signal channel of the gene are inhibited by inhibiting transcription of the IGF-1R gene. Compared with a middle-aged control mouse, under the condition of inhibiting the IGF-1R, the hair condition, the athletic ability and the learning and memory functions of the middle-aged mouse are obviously improved, and the middle-aged mouse is even similar to an adult mouse, so that the IGF-1R gene is an important factor for delaying senescence, and therefore, the IGF-1R gene can be used as an anti-aging mouse. A medicine for inhibiting the expression of the IGF-1R gene is designed by taking the IGF-1R gene as a target, a new direction can be provided for research and development of anti-aging and senile disease prevention medicines, and the IGF-1R gene has a very good anti-aging target application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of an IGF-1R gene expression inhibitor in the preparation of drugs for delaying aging and improving senile diseases. Background Art

[0002] The global population is aging rapidly. Delaying aging, preventing age-related diseases, improving the quality of life of the elderly and promoting healthy aging have become urgent needs for a new health revolution. However, the current chemical drugs used to delay aging and prevent age-related diseases (such as metformin, nicotinamide mononucleotide and resveratrol) have not been effective in clinical trials, and effective aging intervention drugs are urgently needed.

[0003] Aging is a biological process regulated by genes. The conserved insulin / insulin-like growth factor 1 (Insulin / IGF-1) signaling pathway regulates the aging process and lifespan of organisms. The loss or mutation of multiple genes in the insulin / IGF-1 signaling pathway can significantly extend the lifespan of various model animals. The loss of the insulin-like growth factor 1 receptor gene (IGF-1R) in nematodes can double their lifespan; compared with mice homozygous for the IGF-1R gene, female mice heterozygous for the IGF-1R gene can extend their lifespan by 30%; in addition, there are point mutations in the IGF-1R gene in centenarians of the Ashkenazi Jewish family. Therefore, inhibiting the IGF-1R gene is of great significance for delaying aging and extending healthy lifespan.

[0004] With the help of gene interference technology, the expression of a gene in an organism can be specifically silenced. It has obvious advantages in the treatment of genetic diseases and gene-related diseases. Currently, there are many small interfering RNA (siRNA) drugs on the market. Since siRNA is easily degraded, it is often necessary to modify siRNA or use a carrier for protection. N-acetylgalactosamine (GalNAc) coupling modification is the most commonly used small nucleic acid drug delivery system. GalNAc has a high affinity for the asialoglycoprotein receptor on the surface of hepatic parenchymal cells. GalNAc modification of siRNA can achieve specific delivery of siRNA to the liver.

[0005] However, there have been no reports on inhibiting the Insulin / IGF-1 signaling pathway based on gene interference technology to delay aging and improve elderly diseases. Summary of the invention

[0006] The purpose of the present invention is to provide an application of an IGF-1R gene expression inhibitor in the preparation of a drug for delaying aging and improving senile diseases. The present invention designs Igf1r siRNA based on the IGF-1R gene and delivers it into the body to silence the IGF-1R gene expression to inhibit the Insulin / IGF-1 signaling pathway, thereby achieving the regulation of aging and preventing age-related diseases.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The invention discloses an application of an IGF-1R gene expression inhibitor in the preparation of a drug for delaying aging and improving elderly diseases.

[0009] Furthermore, the IGF-1R gene expression inhibitor includes an IGF-1R gene knockout agent.

[0010] Furthermore, the IGF-1R gene knockout agent is siRNA, the sense sequence of the siRNA is shown as SEQ ID NO.1, and the antisense sequence of the siRNA is shown as SEQ ID NO.2.

[0011] Furthermore, the 3' end of the sense sequence of the siRNA is modified with trivalent GalNAc, all pyrimidines are modified with 2-methoxy, the entire chain is modified with thio, and the 3' ends of the sense sequence and the antisense sequence are both terminated with deoxythymidine dinucleotide (dTdT).

[0012] Furthermore, the drug is a drug that inhibits, silences or knocks out the IGF-1R gene and delays biological aging.

[0013] Furthermore, the drug is a drug that inhibits the IGF-1 downstream signaling pathway.

[0014] Furthermore, the drug is a drug that reduces the expression level of IGF-1R.

[0015] Furthermore, the drug is a drug that reduces the phosphorylation level of AKT protein.

[0016] Furthermore, the drug is a drug that reduces the phosphorylation level of S6K protein.

[0017] Furthermore, the drug is a drug that changes the transcription state of liver genes, upregulates multiple longevity signaling pathways and downregulates multiple geriatric disease signaling pathways.

[0018] Furthermore, the drug is a drug for preventing the graying of hair of middle-aged mice and improving the motor ability and learning and memory functions of mice.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention uses subcutaneous injection to deliver siRNA to adult female mice, and uses biochemical chemistry and molecular biology research methods to confirm that GalNAc-Igf1r siRNA can effectively inhibit the IGF-1 signaling pathway in mouse liver without affecting mouse glucose metabolism and liver function. Thereafter, in order to avoid gender differences, siRNA is delivered to adult male mice, and the effectiveness of GalNAc-Igf1r siRNA in inhibiting the IGF-1 signaling pathway in mouse liver is verified again. Then, siRNA is delivered to middle-aged male mice, and adult mice are used as controls, and behavioral methods are used to clarify the effects of Igf1r siRNA on motor ability and learning and memory functions in the aging process of mice.

[0021] The present invention uses gene interference technology to design siRNA for the IGF-1R gene, and chemically modifies it, inhibits the expression of the gene by inhibiting the transcription of the IGF-1R gene, and inhibits its downstream signaling pathway. Compared with middle-aged control mice, the hair condition, motor ability and learning and memory function of middle-aged mice are significantly improved when IGF-1R is inhibited, and are even similar to those of adult mice. The present invention finds that IGF-1R siRNA can significantly change the gene expression status in the liver of adult mice, and significantly improve the motor ability and learning and memory function of middle-aged mice, indicating that the IGF-1R gene is an important factor in delaying aging. By designing drugs that inhibit its expression with the IGF-1R gene as a target, a new direction can be provided for the research and development of anti-aging and prevention of senile diseases drugs, and it has a good application prospect for anti-aging targets. The present invention relates to the field of biomedicine, and specifically to the application of the IGF-1R gene in the preparation of drugs for delaying aging and improving senile diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 The figure is a schematic diagram of the results of GalNAc-Igf1r siRNA on body weight, glucose metabolism and liver function of adult female mice; A: change in mouse body weight, B: blood glucose curve of oral glucose test of mice, C: area under the blood glucose curve of oral glucose test of mice, D: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicators of mouse liver function; Note: CON is the blank control without any treatment, Gal-NC is the negative control, and Gal-Igf1r is the Igf1r siRNA treatment group.

[0024] Figure 2Schematic diagram of the results of ELISA kit and western blotting to verify the inhibitory effect of GalNAc-Igf1r siRNA on the IGF-1 signaling pathway in adult female mice; A: serum IGF-1 level detected by ELISA kit, B: IGF-1R protein content and phosphorylation levels of downstream AKT and S6K proteins detected by western blotting.

[0025] Figure 3 The results of liver transcript analysis of adult female mice; A: principal component analysis of liver transcripts of mice in the three groups of CON, Gal-NC and Gal-Igf1r, B: heat map of all differentially expressed genes among the three groups, C: KEGG enrichment results of significantly down-regulated genes in the Gal-Igf1r group compared with CON and Gal-NC, D: KEGG enrichment results of significantly up-regulated genes in the Gal-Igf1r group compared with CON and Gal-NC.

[0026] Figure 4 Schematic diagram of the results of ELISA kit and western blotting to verify the inhibitory effect of GalNAc-Igf1r siRNA on the IGF-1 signaling pathway in adult male mice; A: serum IGF-1 level detected by ELISA kit, B: IGF-1R protein content and phosphorylation levels of downstream AKT and S6K proteins detected by western blotting.

[0027] Figure 5 Schematic diagram of hair color and scoring results of 3-month-old young mice (Young-3m) and 10-month-old middle-aged mice (CON, Gal-NC and Gal-Igf1r).

[0028] Figure 6 The results are schematic diagrams of the motor ability of mice on the rotarod; A: the latency of young mice (Young-3m) and 10-month-old middle-aged mice (CON, Gal-NC and Gal-Igf1r) to fall on the rotarod. B: the distance the mice moved on the rotarod.

[0029] Figure 7 is the latency for mice to find the escape platform during water maze training ( Figure 7 A, B) and the number of times the platform is crossed after the platform is removed ( Figure 7 C). DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0031] In the following examples, the sources of the materials are as follows:

[0032] The mice in the examples were of C57BL / 6J strain and were raised in the Experimental Animal Center of Shanghai Jiao Tong University.

[0033] If no specific techniques or conditions are specified in the examples, the procedures are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0034] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0035] Example 1

[0036] This embodiment provides a method for designing and modifying siRNA that interferes with the IGF-1R gene, and the specific steps are as follows:

[0037] According to the IGF-1R gene sequence published by NCBI (NCBI Gene: 16001), this embodiment designs a pair of siRNA sequences targeting the IGF-1R gene. The siRNA sequences are shown in Table 1.

[0038] Table 1 siRNA sequences targeting IGF-1R gene

[0039]

[0040] The 3' end of the sense strand of the above two groups of siRNA was modified with trivalent GalNAc, all pyrimidines were modified with 2-methoxy, and the entire strand was modified with thiolation. The 3' end of both the sense strand and the antisense strand was terminated with deoxythymidine dinucleotide (dTdT) to improve the stability of siRNA (sequence dTdT was omitted in the ST.26 standard sequence nucleotide or amino acid sequence table).

[0041] Example 2

[0042] This example provides a method for detecting the ability of Gal-Igf1r siRNA to silence the IGF-1 signaling pathway in adult female mice, and the specific steps are as follows:

[0043] Seven-week-old C57BL / 6J female mice were randomly divided into three groups after one week of adaptive feeding, namely, blank control group CON, negative control group Gal-NC (injection of non-targeted negative control Gal-NC siRNA), and IGF-1R siRNA treatment group Gal-Igf1r (injection of Gal-Igf1r siRNA). On the 0th day and 15th day of the experiment, the mice in the Gal-NC and Gal-Igf1r groups were subcutaneously injected with 1 mg / kg GalNAc-NC siRNA and Gal-Igf1r siRNA, respectively, and the mice in the CON group were not treated. The weight changes of the mice were recorded during the 30-day experimental period, and the mice were tested for oral glucose on the 24th day. The mice were killed on the 30th day to collect tissue samples.

[0044] The results are as follows Figure 1 As shown, GalNAc-Igf1r siRNA had no significant effect on mouse body weight, blood glucose curve, and liver function indicators of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and was safe.

[0045] At the same time, the serum IGF-1 level was detected by enzyme-linked immunosorbent assay kit, the IGF-1R protein expression level in the mouse liver, kidney, heart and spleen was detected by protein immunoblotting, and the phosphorylation levels of AKT and S6K proteins downstream of IGF-1R in the liver were detected.

[0046] The results are as follows Figure 2 As shown in Figure 2, GalNAc-Igf1r siRNA significantly decreased the level of IGF-1 in mouse serum ( Figure 2 A), and decreased the expression level of IGF-1R in the liver, and significantly decreased the phosphorylation levels of AKT and S6K proteins ( Figure 2 B), confirming that GalNAc-Igf1r siRNA can effectively silence IGF-1R gene expression in the liver. However, GalNAc-Igf1r siRNA did not affect other tissues, such as the heart, kidney, and spleen.

[0047] Example 3

[0048] This example provides a method for detecting the effect of GalNAc-Igf1r siRNA on gene expression by liver transcript sequencing, and the specific steps are as follows:

[0049] Mouse liver samples were obtained from Example 2. On the 30th day, 20 mg of liver samples were collected from each mouse. The RNA in the liver was extracted using the TRIZOL method, and its concentration and quality level were tested. After the quality was qualified, transcriptomic sequencing was performed.

[0050] The results are as follows Figure 3As shown, principal component analysis showed that the overall gene transcripts of the Gal-Igf1r group of mice were significantly separated from the two control groups ( Figure 3 A). Compared with the blank control CON and the negative control Gal-NC, 1727 and 3298 genes were significantly different in Gal-Igf1r siRNA, respectively (FDR<0.05). All differentially expressed genes were plotted as heat maps, and these genes can be divided into 6 clusters. TR-C1 is the gene that was significantly down-regulated in the Gal-Igf1r group compared with the two control groups, and TR-C4 is the gene that was significantly up-regulated in the Gal-Igf1r group compared with the two control groups ( Figure 3 B). KEGG enrichment analysis of these genes showed that the downregulated genes in the Gal-Igf1r group were related to a variety of human diseases, cholesterol metabolism, and amino acid metabolism ( Figure 3 C), while the upregulated genes in the Gal-Igf1r group were related to autophagy, FoxO signaling pathway, AMPK signaling pathway, and longevity regulation signaling pathway ( Figure 3 D).

[0051] Example 4

[0052] This example provides a method for testing the effect of GalNAc-Igf1r siRNA on the IGF-1 signaling pathway of adult male mice, and the specific steps are as follows:

[0053] To avoid gender differences, referring to Example 2, mice in the Gal-NC and Gal-Igf1r groups were subcutaneously injected with 1 mg / kg GalNAc-NC siRNA and Gal-Igf1r siRNA, respectively, on day 0 and day 15 of the experiment. Mice were killed on day 30 to collect tissue samples. The effect of GalNAc-Igf1r siRNA on the IGF-1 signaling pathway of adult male mice was detected by enzyme-linked immunosorbent assay and protein immunoblotting.

[0054] The results are as follows Figure 4 As shown in Figure 2, GalNAc-Igf1r siRNA significantly decreased the level of IGF-1 in mouse serum ( Figure 4 A), and significantly decreased the expression level of IGF-1R in the liver, and significantly decreased the phosphorylation levels of AKT and S6K proteins ( Figure 4 B) without affecting heart, kidney, and spleen tissue.

[0055] Example 5

[0056] This example provides a method for testing the hair condition, motor ability, and learning and memory function of middle-aged male mice with silenced IGF-1R. The specific steps are as follows:

[0057] After 4 months of baseline feeding of 2-month-old male C57BL / 6J mice, 6-month-old mice were divided into 3 groups (CON, Gal-NC and Gal-Igf1r). At 6, 7, 8 and 9 months of age, mice in the Gal-NC and Gal-Igf1r groups were subcutaneously injected with 2 mg / kg GalNAc-NC siRNA and GalNAc-Igf1r siRNA, respectively, and the hair grayness, motor ability and learning and memory function of mice were evaluated at 10 months of age (3-month-old mice were used as young controls).

[0058] Mouse hair grayscale assessment: The hair color on the back of the mouse was scored, with 0 points indicating no gray hair on the mouse, and a higher score indicating more gray hair on the mouse. Figure 5 As shown in the figure, aging causes the hair of mice to gradually turn gray. Young mice at 3 months old have no gray hair, while the hair on the back of middle-aged mice in the CON and Gal-NC groups at 10 months old partially turns gray, showing an aging state. However, the gray hair on the back of middle-aged mice in the Gal-Igf1r group is not obvious, and the score is significantly lower than that of the CON and Gal-NC groups. Treatment with Gal-Igf1r siRNA significantly reduced the aging phenotype of gray hair in middle-aged mice.

[0059] Assessment of exercise capacity: The rotarod instrument was used to assess the exercise endurance and balance of mice. The rotarod instrument has 6 channels. Once the mouse falls from the rotating rod, the instrument records the fall time and fall distance. The rotating rod is set to record from 4 rpm and gradually increase to 40 rpm within 300 seconds. If the mouse falls within the first 30 seconds, it is considered an accidental slip and repeated. Training is performed three times a day for three consecutive days, and the fourth day is the formal experimental day.

[0060] The results are as follows Figure 6 As shown, aging leads to a decline in the sustained exercise capacity of mice. The latency of CON group mice to fall off the rotating rod was significantly lower than that of young mice. Treatment with GalNAc-Igf1r siRNA can reverse this age change and enhance the exercise capacity of middle-aged mice.

[0061] Learning and memory function assessment: A water maze was used to assess the learning and memory functions of mice. Before the experiment, a circular pool with a diameter of 160 cm and a depth of 50 cm was prepared and divided into four quadrants. The pool was separated from the outside environment by a curtain, and spatial cues of different shapes and colors were placed inside the pool. The escape platform was a cylindrical platform located in the southwest quadrant. The camera was installed above the pool and connected to a computer with a behavior detection system to record the movements of the mice. The first day was a visible platform test, and the escape platform was located 1 cm above the water surface to determine whether the mice could swim normally and whether their vision was normal. Days 2-5 were hidden platform tests, and the escape platform was located 1 cm below the water surface. If the mouse successfully reached the platform within 60 seconds and stayed for 5 seconds, the computer automatically stopped timing and recorded it as the escape latency. If the mouse failed to find the platform within 60 seconds, the computer stopped timing at 60 seconds and recorded the escape latency as 60 seconds; on the sixth day of the experiment, the platform was removed, and the number of times and movement trajectories of the mouse crossing the escape platform within 60 seconds were observed.

[0062] The results are as follows Figure 7 As shown in the figure, during the training period, the latency of the blank control group CON and the negative control group Gal-NC mice to find the escape platform did not shorten, indicating that the learning and memory ability of middle-aged mice declined; while the latency of the young mice and the Gal-Igf1r group mice to find the escape platform gradually shortened, indicating that GalNAc-Igf1r siRNA can improve the learning and memory function of mice. On the fifth day of training, the latency of the Gal-Igf1r group mice to find the escape platform was significantly lower than that of the CON and Gal-NC groups, and was similar to that of young mice. At the same time, after the escape platform was removed, the number of times the Gal-Igf1r group mice crossed the platform was close to that of young mice, and significantly higher than that of the CON and Gal-NC groups.

[0063] These results indicate that GalNAc-Igf1r siRNA can help middle-aged mice resist changes caused by aging, improve their motor ability and learning and memory functions, and is of great significance for the healthy aging of mice. Using the IGF-1R gene as a target for designing anti-aging and promoting healthy aging has a good application prospect.

[0064] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of an IGF-1R gene expression inhibitor in the preparation of drugs for delaying aging and improving elderly diseases.

2. The use of an IGF-1R gene expression inhibitor according to claim 1 in the preparation of drugs for delaying aging and improving senile diseases, characterized in that: The IGF-1R gene expression inhibitor includes an IGF-1R gene knockout agent.

3. Use of an IGF-1R gene expression inhibitor according to claim 2 in the preparation of drugs for delaying aging and improving elderly diseases, characterized in that: The IGF-1R gene knockout reagent is siRNA, the sense sequence of the siRNA is shown as SEQ ID NO.1, and the antisense sequence of the siRNA is shown as SEQ ID NO.

2.

4. Use of an IGF-1R gene expression inhibitor according to claim 3 in the preparation of drugs for delaying aging and improving elderly diseases, characterized in that: The 3' end of the sense sequence of the siRNA is modified with trivalent GalNAc, all pyrimidines are modified with 2-methoxy, and the entire chain is modified with thiolation. The 3' ends of the sense sequence and the antisense sequence both end with deoxythymidine dinucleotide.

5. Use of an IGF-1R gene expression inhibitor according to claim 1 in the preparation of drugs for delaying aging and improving senile diseases, characterized in that: The drug is a drug that inhibits, silences or knocks out the IGF-1R gene and delays biological aging.

6. Use of an IGF-1R gene expression inhibitor according to claim 1 in the preparation of drugs for delaying aging and improving elderly diseases, characterized in that: The drug is a drug that inhibits the IGF-1 downstream signaling pathway.

7. Use of an IGF-1R gene expression inhibitor according to claim 6 in the preparation of drugs for delaying aging and improving senile diseases, characterized in that: The drug is a drug that reduces the expression level of IGF-1R.

8. Use of an IGF-1R gene expression inhibitor according to claim 7 in the preparation of drugs for delaying aging and improving elderly diseases, characterized in that: The drug is a drug that reduces the phosphorylation level of AKT protein.

9. Use of an IGF-1R gene expression inhibitor according to claim 7 in the preparation of drugs for delaying aging and improving elderly diseases, characterized in that: The drug is a drug that reduces the phosphorylation level of S6K protein.

10. Use of an IGF-1R gene expression inhibitor according to claim 1 in the preparation of drugs for delaying aging and improving elderly diseases, characterized in that: The drug is a drug that changes the transcription state of liver genes, upregulates multiple longevity signaling pathways and downregulates multiple geriatric disease signaling pathways.

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