Application of nucleoside reverse transcriptase inhibitor in preparation of medicine for preventing and treating premature delivery
By targeting the inhibition of LINE1 retroposal in amniotic cells, tenofovir inhibits the S100A9 nuclear translocation and cGAS-STING pathway, the etiology complexity and treatment difficulties of premature birth were solved, and effective prevention and treatment of premature birth was achieved, significantly Reduced the disability and mortality rate of premature birth.
Patent Information
- Application Number
- CN202411331146.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2024-12-27
AI Technical Summary
The causes of premature birth are complex, the existing prevention and treatment methods are limited, and the lack of effective targets for delivery initiation mechanisms lead to the difficulties of reducing the incidence of premature birth and improving the level of clinical intervention.
Tenofovir, a nucleoside reverse transcriptase inhibitor targeted inhibition of amniotic LINE1 retrorelation, blocks cellular senescence and type I interferon response by inhibiting the LINE1 activation and cGAS-STING pathway induced by S100A9 nuclear translocation, thereby blocking cellular senescence and type I interferon response. Prevent and treat premature birth.
Effectively delaying the delivery time and blocking the occurrence of premature birth, providing new targets and methods to prevent and treat premature birth, significantly reducing the disability and mortality rate of premature birth.
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Figure CN119185331A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and in particular to use of tenofovir, a nucleoside reverse transcriptase inhibitor that targets and inhibits amniotic LINE1 retrotransposition, in the preparation of a drug for preventing and treating premature birth. Background Art
[0002] Premature birth refers to births before 37 weeks of gestation, and its incidence accounts for 4%-16% of all births. Premature birth is not only the leading cause of death for infants under 5 years old, but also has a high disability rate. Therefore, premature birth is a major public issue affecting population quality around the world, including in my country. Reducing the incidence of premature birth and improving the level of clinical intervention for premature birth are urgent needs facing the obstetric community. Since the cause of premature birth is difficult to elucidate, various prevention and treatment methods still have great limitations. There is still an urgent need to explore the molecular mechanism of the initiation of premature / normal delivery in order to find more critical therapeutic targets.
[0003] Tenofovir, a nucleoside reverse transcriptase inhibitor, is an important drug used clinically to treat HIV. It can inhibit the reverse transcription of HIV viral RNA, thereby controlling viral replication. Tenofovir has high maternal and infant safety and is certified as a Class B drug by the US FDA. In addition, tenofovir has also been shown to effectively inhibit the reverse transcription of retrotransposon LINE1. Its molecular formula is: C9H 14 N5O4P, the structural formula is:
[0004] LINE1 is the only active, autonomous transposable element in the human genome, accounting for about 17% of the human genome. When activated, LINE1 will transcribe and translate to produce ORF1 and ORF2 proteins; ORF1 is an RNA-binding protein that can bind to LINE1 RNA, and ORF2 has endonuclease and reverse transcriptase activities. ORF1, ORF2 and LINE1 RNA will combine to form a ribonucleoprotein complex (RNP). When LINE1 ribonucleoprotein enters the cell nucleus, ORF2 protein will target and cut DNA, and reverse transcribe LINE1 RNA into DNA. This process is called targeted reverse transcription. The transposition of transposons in the genome is a double-edged sword. It may cause gene mutations or genomic instability and lead to the occurrence of diseases, but it can also promote genomic diversity and genomic and species evolution. At present, there are no reports on the role of LINE1 in the initiation of premature and normal labor, and whether selective inhibitors of LINE1 reverse transposition can prevent and treat premature labor.
[0005] The present invention found that compared with non-labor, the levels of LINE1 mRNA and cDNA formed by reverse transcription in human amniotic fibroblasts increased significantly during labor, and were induced by S100A9 nuclear translocation. LINE1 reverse transposition forms cDNA, which activates the cGAS-STING pathway in amniotic cells, leading to type I interferon response and promoting cell senescence, thereby participating in the initiation of labor. Animal experiments have shown that the levels of LINE1 mRNA and cDNA in mouse fetal membranes also increase significantly with the progression of gestational age, and premature birth can be induced in mice by inducing LINE1 activation and reverse transposition by overexpressing S100A9 in the nuclei of mouse fetal membrane cells. The administration of the nucleoside reverse transcription inhibitor tenofovir not only causes delayed labor in mice, but also blocks the increase in LINE1 reverse transcription and the occurrence of premature birth caused by overexpression of S100A9 in the nucleus. Summary of the invention
[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides the use of nucleoside reverse transcriptase inhibitors in the preparation of drugs for preventing and treating premature birth.
[0007] The present invention provides the use of tenofovir, a nucleoside reverse transcriptase inhibitor, in the preparation of a medicine for preventing and treating premature birth.
[0008] Furthermore, in the drug, the active ingredient is tenofovir, the chemical name of which is R-9-(2-methoxypropyl phosphate) adenine, and the molecular formula of which is C9H 14 N5O4P, the structural formula is as follows:
[0009]
[0010] Specifically, the control is prevention and / or treatment.
[0011] Furthermore, tenofovir prevents and treats premature birth by targeted inhibition of LINE1 retrotransposition.
[0012] Furthermore, in the drug, the active ingredient is tenofovir.
[0013] Furthermore, the drug also includes a pharmaceutically acceptable carrier.
[0014] Furthermore, the weight percentage of tenofovir in the components of the drug is 100%.
[0015] The present invention provides use of a nucleoside reverse transcriptase inhibitor for targeted inhibition of amniotic LINE1 retrotransposition in the preparation and screening of drugs for preventing and treating premature birth.
[0016] The present invention also provides the use of a compound that targets and inhibits the cGAS-STING pathway induced by LINE1 retrotransposition in amniotic cells in the preparation and screening of drugs for preventing and treating premature birth.
[0017] The present invention also provides the use of a compound that targets and inhibits the nuclear translocation of S100A9 that activates LINE1 retrotransposition in amniotic cells in the preparation and screening of drugs for preventing and treating premature birth.
[0018] The present invention also provides the use of LINE1 activation and retrotransposition induced by S100A9 nuclear translocation at the onset of labor as targets in the preparation and screening of drugs for preventing and treating premature birth.
[0019] Compared with the existing technology, the technical effect of the present invention is positive and obvious. Studies have shown that fetal membrane cell aging and aseptic inflammation are important links in the normal delivery initiation mechanism. The present invention found that S100A9 nuclear translocation can induce the activation and retrotransposition of LINE1 in amniotic fibroblasts. The cDNA formed by its retrotransposition promotes the aging of amniotic fibroblasts and the occurrence of type I interferon response by activating the cGAS-STING pathway. Therefore, LINE1 retrotransposition can be used as a new target for the prevention and treatment of premature birth, and nucleoside reverse transcriptase inhibitors can prevent and treat premature birth by inhibiting LINE1 retrotransposition. The present invention provides new ideas for the study of the pathogenesis of premature birth and prevention and treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 : Changes of LINE1 mRNA and cDNA levels in human amniotic fibroblasts before and after the onset of labor.
[0021] Figure 2 : Changes of S100A9 nuclear translocation in human amniotic fibroblasts before and after the onset of labor.
[0022] Figure 3 :S100A9 nuclear translocation promotes the increase of LINE1 mRNA, ORFp1 and ORFp2 proteins and LINE1 cDNA levels and reverse transcription efficiency in amniotic fibroblasts.
[0023] Figure 4 :S100A9 nuclear translocation activates LINE1 and induces senescence and type I interferon response in amniotic fibroblasts.
[0024] Figure 5 :The nucleoside reverse transcriptase inhibitor tenofovir blocks the induction of S100A9 nuclear translocation on LINE1 activation, cell senescence and type I interferon response.
[0025] Figure 6 : cGAS-STING pathway inhibitors RU521 and H151 blocked the induction of cellular senescence and type I interferon response after LINE1 activation.
[0026] Figure 7:The levels of LINE1, cell senescence, and type I interferon response-related genes in mouse fetal membranes vary in a gestational day-dependent manner.
[0027] Figure 8 : Effects of the nucleoside reverse transcriptase inhibitor tenofovir on the time of delivery in mice. DETAILED DESCRIPTION
[0028] The specific implementation and technical scheme of the present invention are further described in detail below in conjunction with the accompanying drawings and specific examples, and the specific examples provide preferred implementations. In the present invention, unless otherwise specified, the raw materials can be purchased from conventional commercial products, and the equipment and testing methods are conventional equipment and methods in the art.
[0029] Example 1: Increased LINE1 mRNA and cDNA levels in human amniotic fibroblasts at the onset of labor
[0030] Amniotic tissues were collected from full-term cesarean section (TNL) and full-term vaginal delivery (TL). Primary amniotic fibroblasts were isolated and purified by trypsin and collagenase digestion. Total RNA and total DNA were extracted using RNA extraction kit and DNA extraction kit, respectively. Extragenomic cDNA was extracted using the modified hirt method. Real-time fluorescence quantitative PCR was used to detect the expression of LINE1 mRNA in amniotic fibroblasts ( Figure 1 a) Total DNA ( Figure 1 b) and extragenomic cDNA ( Figure 1 c) The levels increased significantly.
[0031] The above results suggest that LINE1 is activated and retrotransposed in amniotic fibroblasts when labor starts.
[0032] Example 2: Increased nuclear translocation of S100A9 in human amniotic fibroblasts at the onset of labor
[0033] The amniotic tissues of full-term cesarean section (TNL) and full-term vaginal delivery (TL) were collected. One part was fixed with paraformaldehyde and embedded in paraffin for immunofluorescence staining, and the other part was used for primary fibroblast culture. The immunofluorescence staining experiment of amniotic tissues showed that S100A9 was located in the nucleus of human amniotic fibroblasts in addition to the cytoplasm, and the nuclear S100A9 level in the TL group was significantly higher than that in the TNL group ( Figure 2 a).
[0034] The isolated amniotic fibroblasts were further separated and extracted for cytoplasmic and nuclear proteins using a nuclear protein isolation kit, and Western blotting revealed that the level of S100A9 in the nucleus of the TL group was significantly higher than that of the TNL group ( Figure 2 b).
[0035] In addition, we also performed immunofluorescence staining on amniotic fibroblasts in the TL and TNL groups and found that the level of S100A9 in the nucleus of the TL group was significantly higher than that in the TNL group ( Figure 2 c).
[0036] The above results suggest that the nuclear translocation of S100A9 in amniotic fibroblasts increases when labor starts.
[0037] Example 3: S100A9 nuclear translocation induces activation and retrotransposition of LINE1 in amniotic fibroblasts
[0038] To explore whether there is a correlation between S100A9 nuclear translocation and LINE1 activation at the onset of labor, we constructed an adenoviral vector that specifically overexpresses S100A9 in the cell nucleus by adding nuclear localization sequences at both ends of S100A9, and transfected primary cultured human amniotic fibroblasts to explore whether nuclear S100A9 can activate LINE1.
[0039] First, immunofluorescence and Western blotting experiments of nuclear and cytoplasmic proteins confirmed that S100A9 was specifically overexpressed in the nuclei of amniotic cells ( Figure 3 a, 3b). On this basis, we further collected RNA, extrachromosomal DNA and protein from amniotic fibroblasts that overexpressed S100A9 in the nucleus (OE) or were transfected with a control blank vector (NC). Real-time fluorescence quantitative PCR and Western blotting methods confirmed that overexpression of S100A9 in the nucleus could promote the expression of LINE1 mRNA in amniotic fibroblasts ( Figure 3 c), extrachromosomal cDNA levels ( Figure 3 d) and the protein levels of ORFp1 and ORFp2, two proteins encoded by LINE1 and essential for retrotransposition ( Figure 3 e).
[0040] In addition, we constructed a vector to detect the efficiency of LINE1 retrotransposition and transfected amniotic cells, confirming that nuclear S100A9 overexpression can also significantly enhance the efficiency of LINE1 retrotransposition ( Figure 3 f).
[0041] The above results suggest that S100A9 nuclear translocation can induce the activation and retrotransposition of LINE1.
[0042] Example 4: S100A9 nuclear translocation induces type I interferon response and cell senescence in amniotic fibroblasts
[0043] Previous studies have shown that LINE1 activation and reverse transcription of cDNA lead to type I interferon response and promote cell senescence. Senescence of fetal membrane cells plays an important role in the initiation of labor. On the one hand, it can aggravate the local inflammatory response of the fetal membrane, and on the other hand, it can also cause the rupture of the fetal membrane to release labor signals. Therefore, we speculate that the activation of LINE1 is likely to induce the senescence of amniotic fibroblasts and thus initiate labor.
[0044] We first extracted RNA from amniotic fibroblasts with or without nuclear overexpression of S100A9 and detected type I interferon response, SASP (senescence-associated secretory phenotype) and senescence-related genes by real-time fluorescence quantitative PCR. We found that nuclear overexpression of S100A9 (OE) significantly promoted the expression of these genes ( Figure 4 a, 4b), and further extracted cell proteins and used Western blotting to confirm that overexpression of S100A9 (OE) in the nucleus significantly promoted the protein levels of two classic markers of cell senescence, p21 and p53 ( Figure 4 c).
[0045] In addition, β-galactosidase staining further confirmed that nuclear overexpression of S100A9 (OE) could significantly increase the number of human amniotic fibroblasts ( Figure 4 d), and immunofluorescence staining of DNA damage response molecule γH2AX in amniotic fibroblasts showed that nuclear overexpression of S100A9 (OE) could significantly increase the number of γH2AX-positive cells ( Figure 4 e).
[0046] The above results indicate that nuclear overexpression of S100A9 can induce type I interferon response and cell senescence in amniotic fibroblasts.
[0047] Therefore, targeted inhibition of S100A9 nuclear translocation can block the senescence of amniotic cells and thus effectively prevent premature birth.
[0048] Example 5: The nucleoside reverse transcriptase inhibitor tenofovir can block the induction of S100A9 nuclear translocation on LINE1 activation, cell senescence and type I interferon response
[0049] In order to clarify whether the promotion of S100A9 nuclear translocation on amniotic cell senescence is achieved by activating LINE1 and its reverse transcription, we overexpressed S100A9 in the nucleus of amniotic fibroblasts and simultaneously added the LINE1 reverse transcription inhibitor tenofovir (Tenofovir, Tenof; 5 μM). The results showed that tenofovir not only blocked the reverse transcription effect of nuclear S100A9 (OE) on LINE1 ( Figure 5 a), and blocked the nuclear S100A9(OE) response to type I interferon ( Figure 5 b) and cellular senescence ( Figure 5 c,5d) induction effect.
[0050] These data suggest that nuclear-localized S100A9 can induce type I interferon response and cell senescence by promoting LINE1 reverse transcription into cDNA, while the nucleoside reverse transcription inhibitor tenofovir can block its reverse transcription effect on LINE1 and its induction of type I interferon response and cell senescence.
[0051] Therefore, nucleoside reverse transcriptase inhibitors that target and inhibit LINE1 retrotransposition, such as tenofovir, can block the senescence of amniotic cells and thus effectively prevent premature birth.
[0052] Example 6: cGAS-STING pathway inhibitors RU521 and H151 can block the induction of genes related to cell senescence and type I interferon response after LINE1 activation
[0053] Previous studies have shown that the cDNA formed after LINE1 activation can recognize cGAS in the cell, thereby activating the cGAS-STING pathway, further leading to type I interferon response and cell senescence. Therefore, we further confirmed the role of this pathway in LINE1 activation-induced amniotic cell senescence.
[0054] We used cGAS-specific inhibitor RU521 (1 μM) and STING-specific inhibitor H151 (1 μM) to treat amniotic fibroblasts that overexpressed S100A9 in the nucleus to activate LINE1. The results showed that both RU521 and H151 could significantly block the effects of nuclear overexpression of S100A9 (OE) on the cGAS-STING pathway (protein levels of p-TBK1 and p-IRF3) ( Figure 6 a) Type I interferon response ( Figure 6 b) and cellular senescence ( Figure 6 c) induction effect.
[0055] The above results indicate that nuclear-localized S100A9 can promote the reverse transcription of LINE1 in amniotic fibroblasts into cDNA and activate the cGAS-STING pathway, thereby promoting type I interferon response and cell senescence.
[0056] Therefore, inhibitors targeting the cGAS-STING pathway can also block the senescence of amniotic cells, thereby effectively preventing premature birth.
[0057] Example 7: Changes in LINE1 mRNA, cell senescence, and type I interferon response-related gene levels in mouse fetal membranes as a function of gestational day
[0058] The above in vitro experiments on human primary amniotic cells confirmed the role of LINE1 activation and reverse transcription induced by S100A9 nuclear translocation in the aging of amniotic cells. Next, in order to further clarify whether LINE1 activation induced by S100A9 nuclear translocation can induce premature birth in mice, we further studied the role of LINE1 activation-mediated cell senescence in the initiation of labor in a mouse model.
[0059] We first separated and collected mouse fetal membrane tissue at 17.5 days gestation (17.5 dpc) and 19 days gestation (19 dpc) under a stereoscope. Part of it was formaldehyde-fixed and paraffin-embedded for sectioning for immunofluorescence staining, part of it was used for total RNA and total protein extraction, and part of it was used for nuclear cytoplasm separation protein extraction.
[0060] Through immunofluorescence staining of mouse fetal membranes and Western blotting of nuclear-cytoplasmic separated proteins, it was confirmed that the level of S100A9 in the nuclei of mouse fetal membrane cells at 19 dpc was significantly higher than that at 17.5 dpc ( Figure 7 a, 7b); Real-time fluorescence quantitative PCR analysis of mouse fetal membrane RNA revealed that LINE1 mRNA ( Figure 7 c) and levels of genes related to type I interferon response ( Figure 7 d) was significantly higher than that at 17.5dpc; and Western blotting analysis of total mouse fetal membrane protein confirmed that the p21 protein level in the fetal membrane of 19dpc mice was significantly higher than that at 17.5dpc ( Figure 7 e).
[0061] The above results suggest that similar to humans, S100A9 nuclear translocation, LINE1 activation, type I interferon response and cell senescence in the fetal membrane of mice also increase significantly when labor starts.
[0062] Example 8: Effect of the nucleoside reverse transcriptase inhibitor tenofovir on the delivery time of mice
[0063] Based on the above, we further explored whether the activation of LINE1 induced by S100A9 nuclear translocation can directly lead to premature birth in mice, and whether the nucleoside reverse transcriptase inhibitor tenofovir can block premature birth in mice caused by LINE1 activation.
[0064] In order to specifically activate or block the reverse transcription of LINE1 in the fetal membrane, we used intra-amniotic injection ( Figure 8 a), The pregnant mice were first randomly divided into four groups according to the random table method: empty group (NC), nuclear overexpression of S100A9 group (OE), tenofovir alone group (Tenof), nuclear overexpression of S100A9 and tenofovir group (OE+Tenof).
[0065] At 15.5 days of gestation, mice were opened in the abdomen under anesthesia for intra-amniotic injection. The NC group was injected with the corresponding empty vector scAAV (1x 10 10 TU / 10 μl / each amniotic sac), and the OE group was injected with scAAV (1x 10 10 TU / 10μl / each amniotic sac), and the Tenof group was injected with empty scAAV+tenofovir (1x10 10 TU+2nmol / 10μl / each amniotic sac), OE+Tenof injected with nuclear localized overexpressed S100A9 scAAV and tenofovir (1x 10 10 TU + 2nmol / 10μl / each amniotic sac).
[0066] The results showed that the delivery time of mice with nuclear overexpression of S100A9 (OE group) was significantly advanced, with a premature birth rate of 81.8%, and this effect could be significantly blocked by tenofovir ( Figure 8 b,8c).
[0067] In addition, tenofovir alone (Tenofovir group) can also significantly cause delayed delivery in mice, with a delayed delivery rate of 27.3% ( Figure 8 b,8c).
[0068] The above results indicate that tenofovir administration can effectively prevent and treat premature birth. In addition, we collected the fetal membranes of mice 60 hours after drug injection to detect type I interferon response and cell senescence. The results showed that, as expected, compared with the NC group, the expression of LINE1 mRNA ( Figure 8 d) and cDNA levels ( Figure 8 e), Expression levels of genes related to type I interferon response ( Figure 7 d) and cell senescence level ( Figure 8f, 8g) were significantly increased. Tenofovir can significantly block the cDNA level of mouse fetal membrane LINE1, the expression level of type I interferon response-related genes and the level of cell senescence caused by nuclear overexpression of S100A9 ( Figure 8 dg).
[0069] Animal experiments have shown that tenofovir has a significant therapeutic effect on premature birth. In addition, in addition to tenofovir, other inhibitors that target the inhibition of S100A9 nuclear translocation-LINE1 activation and retrotransposition-cGAS-STING pathways can also be used as new targets in the prevention and treatment of premature birth, providing new ideas and methods for the prevention and treatment of premature birth in the future.
Claims
1. Use of the nucleoside reverse transcriptase inhibitor tenofovir in the preparation of a drug for preventing and treating premature birth.
2. The use according to claim 1, characterized in that: The active ingredient in the drug is tenofovir, whose chemical name is R-9-(2-methoxypropyl phosphate) adenine and whose molecular formula is C9H 14 N5O4P, the structural formula is as follows:
3. The use according to claim 1, characterized in that: When labor starts, nuclear translocation of S100A9 in amniotic cells induces activation and retrotransposition of LINE1, and tenofovir prevents and treats premature birth by targeted inhibition of retrotransposition of amniotic LINE1.
4. The use according to any one of claims 1 to 3, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.
5. The use according to claim 4, characterized in that: The weight percentage of tenofovir in the medicine is 100%.
6. Use of nucleoside reverse transcriptase inhibitors that target and inhibit amniotic LINE1 retrotransposition in the preparation and screening of drugs for preventing and treating premature birth.
7. Use of compounds that target and inhibit the cGAS-STING pathway induced by LINE1 retrotransposition in amniotic cells in the preparation and screening of drugs for preventing and treating premature birth.
8. Use of a compound that targets and inhibits the nuclear translocation of S100A9 that induces LINE1 activation and retrotransposition in amniotic cells in the preparation and screening of drugs for preventing and treating premature birth.
9. The application of LINE1 activation and retrotransposition induced by S100A9 nuclear translocation at the onset of labor as targets in the preparation and screening of drugs for the prevention and treatment of premature birth.