Use of an IGF-1R gene expression inhibitor in the preparation of a medicament for delaying aging and improving senile diseases

Abstract

This invention relates to the application of an IGF-1R gene expression inhibitor in the preparation of drugs for delaying aging and improving age-related diseases. Using gene interference technology, siRNA targeting the IGF-1R gene is designed and chemically modified to inhibit its expression by suppressing transcription and downstream signaling pathways. Compared with middle-aged control mice, the hair condition, motor ability, and learning and memory functions of the middle-aged mice were significantly improved under IGF-1R inhibition, even resembling those of adult mice. This indicates that the IGF-1R gene is an important factor in delaying aging. Therefore, designing drugs that inhibit IGF-1R expression can provide a new direction for the development of anti-aging and age-related disease prevention drugs, showing great promise for anti-aging target applications.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the application of an IGF-1R gene expression inhibitor in the preparation of drugs for delaying aging and improving age-related diseases. Background Technology

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

[0003] Aging is a genetically regulated biological process, and the conserved insulin / insulin-like growth factor 1 (IGF-1) signaling pathway regulates the aging process and lifespan in organisms. Deletion or mutation of multiple genes in the insulin / IGF-1 signaling pathway can significantly extend the lifespan of various model organisms. For example, the deletion of the insulin-like growth factor 1 receptor gene (IGF-1R) in nematodes can double their lifespan; compared to homozygous IGF-1R mice, heterozygous female IGF-1R mice can have a 30% longer lifespan; furthermore, German Jewish centenarians exhibit point mutations in the IGF-1R gene. Therefore, inhibiting the IGF-1R gene is of great significance for delaying aging and extending healthy lifespan.

[0004] Gene interference technology can specifically silence the expression of a particular gene in an organism. It has significant advantages in treating hereditary and gene-related diseases, and several small interfering RNA (siRNA) drugs are currently on the market. Because siRNA is easily degraded, it often needs to be modified or protected using vectors. N-acetylgalactosamine (GalNAc) conjugation is currently the most commonly used small nucleic acid drug delivery system. GalNAc has a high affinity for the desialyl glycoprotein receptor on the surface of hepatocytes; modifying siRNA with GalNAc enables specific delivery of siRNA to the liver.

[0005] However, there have been no reports on using gene interference technology to inhibit the insulin / IGF-1 signaling pathway to delay aging and improve age-related diseases. Summary of the Invention

[0006] The purpose of this invention is to provide an application of an IGF-1R gene expression inhibitor in the preparation of drugs for delaying aging and improving age-related diseases. This invention designs Igf1r siRNA based on the IGF-1R gene and delivers it into the body. By silencing IGF-1R gene expression, it inhibits the insulin / IGF-1 signaling pathway, thereby regulating aging and preventing age-related diseases.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] Application of an IGF-1R gene expression inhibitor in the preparation of drugs to delay aging and improve age-related diseases.

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

[0010] Furthermore, the IGF-1R gene knockout reagent is siRNA, the sense sequence of which is shown in SEQ ID NO.1, and the antisense sequence of which is shown in 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, and the entire chain is modified with thio, and the 3' ends of both the sense sequence and the antisense sequence are ended with deoxythymidine dinucleotide (dTdT).

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

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

[0014] Furthermore, the drug described above 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 the S6K protein.

[0017] Furthermore, the drug is a drug that alters the transcriptional state of liver genes, upregulates multiple longevity signaling pathways, and downregulates multiple age-related disease signaling pathways.

[0018] Furthermore, the drug is a drug that prevents the fur of middle-aged mice from turning gray and improves the mice's motor ability and learning and memory function.

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

[0020] This invention delivers siRNA subcutaneously into adult female mice. Using biochemical and molecular biological methods, it was confirmed that GalNAc-Igf1r siRNA effectively inhibits the IGF-1 signaling pathway in the mouse liver without affecting glucose metabolism or liver function. Subsequently, to avoid sex differences, siRNA was delivered into adult male mice to further verify the effectiveness of GalNAc-Igf1r siRNA in inhibiting the IGF-1 signaling pathway in the mouse liver. Next, siRNA was delivered to middle-aged male mice, with adult mice as controls, and behavioral methods were used to clarify the effects of Igf1r siRNA on motor ability and learning / memory function during the aging process in mice.

[0021] This invention utilizes gene interference technology to design siRNA targeting the IGF-1R gene and chemically modify it. This inhibits IGF-1R gene expression by suppressing transcription and its downstream signaling pathways. Compared to middle-aged control mice, the inhibition of IGF-1R significantly improved hair condition, motor skills, and learning and memory functions in middle-aged mice, even resembling those in adult mice. This invention reveals that IGF-1R siRNA can significantly alter gene expression in the liver of adult mice and significantly improve motor skills and learning and memory functions in middle-aged mice, indicating that the IGF-1R gene is an important factor in delaying aging. Designing drugs to inhibit IGF-1R gene expression can provide a new direction for the development of anti-aging and age-related disease prevention drugs, showing great promise for anti-aging target applications. This invention relates to the field of biomedicine, specifically to the application of the IGF-1R gene in the preparation of drugs for delaying aging and improving age-related diseases. Attached Figure Description

[0022] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used to explain the invention but do not constitute a limitation thereof. In the drawings:

[0023] Figure 1 This diagram illustrates the effects of GalNAc-Igf1r siRNA on body weight, glucose metabolism, and liver function in adult female mice. A: Mouse body weight change; B: Blood glucose curve from oral glucose test; C: Area under the blood glucose curve from oral glucose test; D: Liver function indicators of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Note: CON is the blank control (no treatment), Gal-NC is the negative control, and Gal-Igf1r is the Igf1r siRNA treatment group.

[0024] Figure 2This diagram illustrates the results of using an enzyme-linked immunosorbent assay (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 represents the serum IGF-1 level detected by the ELISA kit, and B represents the IGF-1R protein content and phosphorylation levels of downstream AKT and S6K proteins detected by Western blotting.

[0025] Figure 3 The results of the analysis of liver transcripts from adult female mice are shown in the figure. Among them, A: principal component analysis of liver transcripts from the three groups of mice: CON, Gal-NC, and Gal-Igf1r; B: heatmap of all differentially regulated genes among the three groups; C: KEGG enrichment results of genes that were significantly downregulated in the Gal-Igf1r group compared with CON and Gal-NC; D: KEGG enrichment results of genes that were significantly upregulated in the Gal-Igf1r group compared with CON and Gal-NC.

[0026] Figure 4 This diagram illustrates the results of using an enzyme-linked immunosorbent assay (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 represents the serum IGF-1 level detected by the ELISA kit, and B represents the IGF-1R protein content and phosphorylation levels of downstream AKT and S6K proteins detected by Western blotting.

[0027] Figure 5 This is a schematic diagram showing the results of hair color and scoring for 3-month-old young mice (Young-3m) and 10-month-old middle-aged mice (CON, Gal-NC, and Gal-Igf1r).

[0028] Figure 6 This diagram illustrates the results of mouse movement on the rotarod; where A represents the latency of young mice (Young-3m) and 10-month-old middle-aged mice (CON, Gal-NC, and Gal-Igf1r) falling off the rotarod. B represents the distance the mouse travels on the rotarod.

[0029] Figure 7 The latency period for mice to find an escape platform during water maze training ( Figure 7 A, B) and the number of times the platform was traversed after its removal ( Figure 7 C). Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

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

[0032] The mice used in this example were of the C57BL / 6J strain and were housed at the Experimental Animal Center of Shanghai Jiao Tong University.

[0033] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or the product instructions shall be followed. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0034] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0035] Example 1

[0036] This embodiment provides a method for designing and modifying siRNA to interfere with the IGF-1R gene. The specific steps are as follows:

[0037] Based on 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 the IGF-1R gene

[0039]

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

[0041] Example 2

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

[0043] Seven-week-old female C57BL / 6J mice were randomly divided into three groups after one week of acclimatization: a blank control group (CON), a negative control group (Gal-NC, injected with the non-targeted negative control Gal-NC siRNA), and an IGF-1R siRNA treatment group (Gal-Igf1r, injected with Gal-Igf1r siRNA). On days 0 and 15 of the experiment, mice in the Gal-NC and Gal-Igf1r groups were subcutaneously injected with 1 mg / kg GalNAc-NC siRNA and Gal-Igf1r siRNA, respectively. Mice in the CON group received no treatment. Body weight changes were recorded over the 30-day experimental period. Mice underwent an oral glucose tolerance test on day 24, and were sacrificed on day 30 for tissue sample collection.

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

[0045] Serum IGF-1 levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit, and the expression levels of IGF-1R protein in mouse liver, kidney, heart, and spleen were detected using Western blotting. The phosphorylation levels of downstream IGF-1R proteins AKT and S6K in the liver were also detected.

[0046] The results are as follows Figure 2 As shown, GalNAc-Igf1r siRNA significantly reduced the level of IGF-1 in mouse serum ( Figure 2 A), and it leads to a decrease in IGF-1R expression levels in the liver and a significant decrease in the phosphorylation levels of AKT and S6K proteins. Figure 2 B) confirmed that GalNAc-Igf1r siRNA can effectively silence IGF-1R gene expression in the liver. However, GalNAc-Igf1r siRNA does not affect other tissues, such as the heart, kidneys, and spleen.

[0047] Example 3

[0048] This embodiment provides a method for detecting the effect of GalNAc-Igf1r siRNA on gene expression through liver transcript sequencing. The specific steps are as follows:

[0049] Mouse liver samples were obtained from Example 2. On day 30, 20 mg of liver sample was taken from each mouse. RNA was extracted from the liver using the TRIZOL method, and its concentration and quality level were detected. After passing the quality test, transcriptomics sequencing was performed.

[0050] The results are as follows Figure 3As shown, principal component analysis revealed a significant separation between the overall gene transcripts of the Gal-Igf1r group mice and the two control groups. Figure 3 A). Compared with the blank control CON and the negative control Gal-NC, 1727 and 3298 genes in the Gal-Igf1r siRNA group showed significant differences (FDR < 0.05). A heatmap of all differentially expressed genes showed they could be divided into 6 clusters: TR-C1 represented genes significantly downregulated in the Gal-Igf1r group compared to both control groups, and TR-C4 represented genes significantly upregulated in the Gal-Igf1r group compared to both control groups. Figure 3 B). KEGG enrichment analysis of these genes showed that genes downregulated in the Gal-Igf1r group were associated with various human diseases, cholesterol metabolism, and amino acid metabolism. Figure 3 C), while genes upregulated in the Gal-Igf1r group are associated with autophagy, the FoxO signaling pathway, the AMPK signaling pathway, and longevity regulatory signaling pathways. Figure 3 D).

[0051] Example 4

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

[0053] To avoid sex differences, following 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 days 0 and 15 of the experiment. Mice were sacrificed on day 30, and tissue samples were collected. The effects of GalNAc-Igf1r siRNA on the IGF-1 signaling pathway in adult male mice were detected using an enzyme-linked immunosorbent assay (ELISA) kit and Western blotting.

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

[0055] Example 5

[0056] This embodiment 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] Two-month-old male C57BL / 6J mice were fed at baseline for four months, and then divided into three groups (CON, Gal-NC, and Gal-Igf1r) at six 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, at 6, 7, 8, and 9 months of age. At 10 months of age, the mice's hair grayness, motor ability, and learning and memory functions were assessed (three-month-old mice served as young controls).

[0058] Mouse hair grayscale assessment: The color of the hair on the back of the mice was scored, with 0 points indicating no gray hair and higher scores indicating more gray hair. Results are as follows: Figure 5 As shown, aging leads to a gradual graying of fur in mice. Three-month-old young mice showed no gray hair, while ten-month-old CON and Gal-NC group mice exhibited partial graying of fur on their backs, indicating aging. However, graying of fur on the backs of middle-aged mice in the Gal-Igf1r group was not obvious, and their scores were significantly lower than those in the CON and Gal-NC groups. Treatment with Gal-Igf1r siRNA significantly reduced the aging phenotype of graying fur in middle-aged mice.

[0059] Motor ability assessment: A rotarod apparatus was used to assess the mice's exercise endurance and balance. The rotarod apparatus has 6 channels; once a mouse falls from the rotating bar, the instrument records the fall time and distance. The rotation speed was set to start recording at 4 revolutions per minute, gradually increasing to 40 revolutions per minute over 300 seconds. If a mouse falls within the first 30 seconds, it is considered an accidental slip, and the process is repeated. Training was conducted three times a day for three consecutive days, with the fourth day being the formal experimental day.

[0060] The results are as follows Figure 6 As shown, aging leads to a decline in the sustained motor ability of mice. The latency of mice in the CON group to fall off the rotarod was significantly shorter than that of younger mice. Treatment with GalNAc-Igf1r siRNA can reverse this age-related change and enhance the motor ability 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 160cm and a depth of 50cm was prepared and artificially divided into four quadrants. A curtain separated the pool from the external environment. Spatial cues of different shapes and colors were placed inside the pool. The escape platform was a cylindrical platform located in the southwest quadrant. A camera was mounted above the pool and connected to a computer with a behavior detection system to record the mice's movements. Day 1 was the visible platform test, with the escape platform 1cm above the water surface. The mice's swimming ability and vision were assessed. Days 2-5 were the hidden platform test, with the escape platform 1cm below the water surface. If a mouse successfully reached the platform within 60 seconds and remained there for 5 seconds, the computer automatically stopped the timer and recorded it as the escape latency. If the mouse failed to find the platform within 60 seconds, the computer stopped the timer 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 the mice crossed the escape platform and their movement trajectory were observed within 60 seconds.

[0062] The results are as follows Figure 7 As shown, the latency of finding the escape platform was not shortened in the blank control group (CON) and the negative control group (Gal-NC) during the training period, indicating a decline in learning and memory abilities in middle-aged mice. However, the latency of finding the escape platform gradually shortened in young mice and the Gal-Igf1r group, indicating that GalNAc-Igf1r siRNA can improve the learning and memory function of mice. By day five of training, the latency of finding the escape platform in the Gal-Igf1r group was significantly lower than that in the CON and Gal-NC groups, and similar to that in young mice. Furthermore, after the escape platform was removed, the number of times the Gal-Igf1r group mice crossed the platform was similar to that of young mice, and significantly higher than that in the CON and Gal-NC groups.

[0063] These results indicate that GalNAc-Igf1r siRNA can help middle-aged mice resist aging-related changes, improve their motor skills and learning and memory functions, and is of great significance for healthy aging in mice. Using the IGF-1R gene as a target for delaying aging and promoting healthy aging shows great promise for future applications.

[0064] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. The application of an IGF-1R gene expression inhibitor in the preparation of drugs for delaying aging, characterized in that, The IGF-1R gene expression inhibitor is an IGF-1R gene interference agent, specifically IGF-1R siRNA. The IGF-1R siRNA is used in the preparation of drugs that significantly improve motor ability and learning and memory function by inhibiting the downstream signaling pathway of IGF-1, reducing IGF-1R expression, and reducing the phosphorylation level of AKT and S6K proteins. The sense sequence of the siRNA is shown in SEQ ID NO.1, and the antisense sequence of the siRNA is shown in SEQ ID NO.2; The siRNA's sense sequence has a 3' end modified with trivalent GalNAc, all pyrimidines modified with 2-methoxy, and the entire strand modified with thio. Both the sense and antisense sequences end with deoxythymidine dinucleotide at their 3' ends.

2. The application of the IGF-1R gene expression inhibitor according to claim 1 in the preparation of drugs for delaying aging, characterized in that, The application of the IGF-1R siRNA in the preparation of drugs that silence the IGF-1R gene and delay biological aging.

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